Humanized MUC1 antibodies and antibody drug conjugates

Abstract

The present invention relates to humanized MUC1 antibodies, antibody drug conjugates (ADCs) comprising such antibodies, and their use for the treatment of disorders such as cancer, in particular in the case of MUC1 overexpression.

Description

[0001] This application claims the benefit of Indian Patent Application No. 202321051338, filed on July 31, 2023, which is hereby incorporated by reference.

[0002] The computer-readable sequence list is submitted electronically along with this application and is hereby incorporated in its entirety by reference. The ST26 sequence list is contained in a file named “159818-02601_SL.xml” created on July 31, 2024. Technical Field

[0003] This invention relates to humanized MUC1 antibodies, antibody-drug conjugates (ADCs) containing such antibodies, and their use in treating conditions such as cancer, particularly in cases of MUC1 overexpression. Background Technology

[0004] MUC1 glycoprotein is overexpressed by a variety of high-incidence, high-mortality human epithelial malignancies (including breast cancer, prostate cancer, pancreatic cancer, ovarian cancer, lung cancer, and colon cancer), as well as by malignant plasma cells of multiple myeloma and myeloid cells of acute myeloid leukemia. Due to its preferential high expression on malignant cells and its naked expression on the cell surface, MUC1 has been investigated as a target for targeted cancer therapy and a marker of disease progression. (See Rivalland et al., Expert Opinion on Biol Therapy, 15(2015) 1773-1787; Taylor-Papadimitriou et al., Journal of the Biochem Society, 46(2018) 659-668.)

[0005] Structurally, the MUC1 molecule is a transmembrane glycoprotein (called MUC-TM). MUC-TM is a heterodimer consisting of an extracellular domain of 20 to 125 or more repeats (called variable-number tandem repeats, VNTRs) containing a 20-amino acid-long sequence, a transmembrane domain, and a short cytoplasmic tail region mediating intracellular signal transduction. The MUC1 molecule is self-cleaved within the SEA (sperm protein, enterokinase, and aggregate protein) module, a highly conserved domain of 110 amino acids. This results in a large extracellular α subunit containing the array of tandem repeats binding to a transmembrane β subunit containing both the transmembrane and cytoplasmic domains of the molecule via strong non-covalent interactions. The binding of the α chain to the β chain is discontinuous: the α chain binds to the β chain intermittently. While the β chain remains consistently on the cell surface, the α chain, possessing its VNTR, maintains cell binding only intermittently.

[0006] Numerous anti-MUC1 antibodies have been reported in the literature, most of which target the highly immunogenic VNTR of the α chain. Although anti-VNTR antibodies can successfully bind to MUC1+ cells in vitro, the shedding of the VNTR-containing MUC1 α chain into the peripheral circulation in vivo severely impairs the clinical ability of anti-VNTR antibodies to affect MUC1-expressing tumors. The shedding of the α chain from the tumor cell surface not only significantly reduces the number of MUC1 targets for anti-α chain antibodies, but also, the freely circulating MUC1 α chain in the peripheral circulation can bind to and neutralize anti-VNTR antibodies or anti-glycosylated VNTR antibodies, thereby limiting their ability to reach even MUC1-expressing tumors.

[0007] Antibodies that recognize cancer-specific truncated O-glycoforms of VNTRs (such as antibodies PankoMab-Gex, 5E5, SM3, and VU-2-G7) have been proposed as a possible way to overcome the potential toxicity of targeting MUC1 expressed in normal tissues. However, limitations on targeting α-chain VNTRs, namely their detachment from the cell surface and their ability to bind to therapeutically administered anti-MUC1 antibodies, remain. Burchell et al., J Mammary Gland Biol Neoplasia, 6(2001) 355-364; Fiedler et al., Eur J Cancer, 63 (2016) 55-63; Ryuko et al., Tumour Biol, 21 (2000) 197-210; Tarp et al., Glycobiology, 17 (2007) 197-209; Zhou et al., Molecules, 23 (6) (2018) 1326.

[0008] Due to the instability of the antibody target, as mentioned above, the target binds to tumor cells intermittently only through a discontinuous mechanism; therefore, no anti-MUC1 VNTR antibody has yet been proven to be clinically effective. (Wu et al., Cancer Cell International (2022) 22:417; Fiedler, ibid.; Kimura et al., Expert Opinion on Biotherapy, 13 (2013) 35-49; Ibrahim et al., Clin CancerRes, 17 (2011) 6822-6830.)

[0009] In contrast to the α chain and its VNTR, the MUC1SEA domain, formed by the interaction of the extracellular portions of the α and β subunits, is a stable, cell membrane-fixed molecular component. Anti-MUC1 α / β conjugate antibodies have been disclosed in Rubinstein et al., Cancer Research, 66 (2006) 11247-11253; Pichinuk et al., Cancer Research, 72 (2012) 3324-3336; and Rubinstein et al., International Journal of Cancer, 124 (2009) 46-54.

[0010] U.S. Patent No. 8,648,172 discloses certain antibodies that bind to both the α and β subunits of the intact MUC1 protein (“anti-MUC1 α / β antibodies”, which bind to the MUC1 α / β subunit junction but substantially do not bind to either the MUC1 α subunit or the MUC1 β subunit in the absence of the other).

[0011] International Publication No. WO 2021 / 186427 discloses certain isolated monoclonal antibodies that specifically target the junction of the α and β chains of MUC1 (which contain the SEA domain).

[0012] Singh et al. published in the Journal of Pharmacokinetics and Pharmacodyn the characterization of the bystander effect of antibody-drug conjugates such as trastuzumab-vc-MMAE.

[0013] There is a continued need for MUC1 antibody and MUC1 antibody therapy to improve cancer outcomes. Summary of the Invention

[0014] The inventors have developed a humanized monoclonal antibody that binds to the MUC1 SEA domain.

[0015] One embodiment is a humanized antibody that binds to an epitope in the SEA domain of MUC1, preferably having a binding affinity KD of less than 1000 pM (e.g., less than 500 pM, less than 100 pM, or less than 30 pM). In one embodiment, the humanized antibody binds to an epitope in the SEA domain of MUC1 (SEQ ID NO: 41), the epitope being preferably formed by arginine at position 1108, glutamic acid at position 1109, asparagine at position 1113, and glutamic acid at position 1118 of MUC1. In another embodiment, the humanized antibody binds to the same epitope as a chimeric antibody (e.g., chimeric antibody 5F3), the chimeric antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising the amino acid sequences of SEQ ID NO: 1 and 2, respectively. In another embodiment, the humanized antibody binds to the same epitope as the chimeric antibody 5F3, wherein the sequences of the heavy and light chains are provided as in SEQ ID NO: 42 and 43, respectively. In yet another embodiment, the antibody is a humanized form of any of the chimeric antibodies DMB4F4 (4F4), DMB7F3 (7F3), or DMB10F10 (10F10), each of which is described in International Publication No. WO 2021 / 186427, which is hereby incorporated by reference. In yet another embodiment, the humanized antibody binds to the same epitope as the chimeric antibody 4F4, 7F3, or 10F10.

[0016] Another embodiment is a humanized antibody comprising means for binding to an epitope in the SEA domain (SEQ ID NO:41) of MUC1, the epitope being formed by arginine at position 1108, glutamic acid at position 1109, asparagine at position 1113, and glutamic acid at position 1118 of MUC1.

[0017] Another embodiment is a humanized antibody that binds to the same epitope as a chimeric antibody (e.g., chimeric antibody 5F3), the chimeric antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising the amino acid sequences of SEQ ID NO: 1 and 2, respectively. In another embodiment, the humanized antibody binds to the same epitope as chimeric antibody 5F3, wherein the sequences of the heavy chain and light chain are provided as in SEQ ID NO: 42 and 43, respectively.

[0018] Another embodiment is a humanized monoclonal antibody that binds to the MUC1 SEA domain, wherein the antibody comprises:

[0019] (a) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NO. 7-11, 45, and 46; and

[0020] (b) Light chain variable region, wherein the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO. 12-15.

[0021] Another embodiment is a humanized monoclonal antibody that binds to the MUC1 SEA domain, wherein the antibody comprises:

[0022] (a) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NO. 7-11; and

[0023] (b) Light chain variable region, wherein the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO. 12-15.

[0024] In one embodiment, the humanized antibody comprises:

[0025] (a) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12;

[0026] (b) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0027] (c) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14;

[0028] (d) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13;

[0029] (e) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14;

[0030] (f) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0031] (g) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14;

[0032] (h) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13;

[0033] (i) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; or

[0034] (j) A heavy chain variable region and a light chain variable region, wherein the heavy and light chain variable regions contain the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 15.

[0035] In a preferred embodiment, the humanized monoclonal antibody binding to the MUC1 SEA domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy and light chain variable regions comprise the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15.

[0036] In one embodiment of any humanized antibody described herein, the constant domain of the heavy chain of the humanized antibody comprises the amino acid sequence of SEQ ID NO: 39. In another embodiment of any humanized antibody described herein, the constant domain of the light chain of the humanized antibody comprises the amino acid sequence of SEQ ID NO: 40. In yet another embodiment of any humanized antibody described herein, the constant domain of the heavy chain of the humanized antibody comprises the amino acid sequence of SEQ ID NO: 39, and the constant domain of the light chain of the humanized antibody comprises the amino acid sequence of SEQ ID NO: 40.

[0037] In one embodiment of any humanized antibody described herein, the humanized antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and the light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0038] In one embodiment, the antigen-binding fragment is Fv, single-chain Fv (scFv), single-chain Fv-Fc (scFv-Fc), Fab', Fab, F(ab')2, or F(ab)2.

[0039] Another embodiment is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) containing a nucleotide sequence encoding a monoclonal antibody or an antigen-binding fragment thereof as described herein.

[0040] Another embodiment is an expression vector that contains any nucleic acid molecule as described herein (e.g., isolated nucleic acid molecule).

[0041] Another embodiment is a host cell that is transfected with any expression vector as described herein.

[0042] Another embodiment is an immunoconjugate comprising any antibody as described herein (e.g., isolated antibody) and an additional cytotoxic agent or therapeutic agent. In one embodiment, the cytotoxic agent is selected from: alkylating agents, anthracycline drugs, pyrimidine derivatives, vinca alkaloids, photodynamic drugs, platinum-containing compounds, taxanes, topoisomerase inhibitors, ribosome inactivators, DNA damage-inducing agents, tubulin inhibitors, antimitotic agents, radioisotopes, cytotoxic antibodies, and bacterial toxins. In another embodiment, the cytotoxic agent is a Pseudomonas exotoxin. In yet another embodiment, the cytotoxic agent is monomethylolpropamine E (MMAE).

[0043] In one embodiment, the antibody is conjugated to maleimide-hexanoyl-valine-citrullinated-p-aminobenzyloxycarbonyl (where PABC is p-aminobenzyloxycarbonyl) or mc-vc-PABC-MMAE, also written as maleimide-hexanoyl-Val-Cit-PABC-MMAE. Another embodiment is an immunoconjugate comprising a humanized monoclonal antibody that binds to the MUC1 SEA domain, wherein (i) the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, the light chain variable region comprising the amino acid sequence of SEQ ID NO: 15, and (ii) the antibody is conjugated to maleimide-hexanoyl-Val-Cit-PABC-MMAE.

[0044] Another embodiment is an immunoconjugate comprising an antibody conjugated with maleimide hexanoyl-Val-Cit-PABC-MMAE as described herein, wherein the antibody (i) binds to the MUC1 SEA domain and (ii) comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and the light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0045] In one embodiment, the immunoconjugate reduces tumor volume after being administered to a subject with cancer.

[0046] In one embodiment, the antibody is bispecific or trispecific. A bispecific antibody may have two distinct heavy / light chain pairs and two distinct binding sites. A bispecific antibody may include the heavy chain variable region described herein (e.g., a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs 7-11, 45, and 46) and the light chain variable region described herein (e.g., a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs 12-15). A bispecific antibody may include an antigen-binding fragment that does not bind to the MUC1 SEA domain.

[0047] Another embodiment is a pharmaceutical composition comprising (a) a humanized monoclonal antibody as described herein, an immunoconjugate as described herein, and (b) a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent.

[0048] Another embodiment is a method of treating or improving a disease or condition, the method comprising administering to a subject in need at least one therapeutically effective amount of a humanized monoclonal antibody, an immunoconjugate, or a pharmaceutical composition as described herein. In one embodiment, the disease or condition is cancer, such as cancer expressing MUC1. For example, cancer may be selected from: lung cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, pancreatic cancer, multiple myeloma, and acute myeloid leukemia and other cancers expressing MUC1. In another embodiment, the disease or condition is an autoimmune or inflammatory disease, such as rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, amyloidosis, or autoimmune pancreatitis. In yet another embodiment, the disease or condition is a non-malignant, clinically significant abnormal growth symptom, such as cysts, such as renal cysts, thyroid cysts and thyroid nodules, or liver cysts. In one embodiment, the method further comprises administering an additional therapeutic agent to a subject in need.

[0049] Another embodiment is the use of a monoclonal antibody, an immunoconjugate, or a pharmaceutical composition as described herein in a method of treating or improving a disease or condition, the method comprising administering a therapeutically effective amount of the monoclonal antibody, the immunoconjugate, or the pharmaceutical composition to a subject in need. In one embodiment, the disease or condition is cancer. In another embodiment, the disease or condition is a non-malignant abnormal growth condition, such as a cyst, for example a kidney cyst, a thyroid cyst and a thyroid nodule, or a liver cyst. In yet another embodiment, the method further comprises administering an additional therapeutic agent to a subject in need.

[0050] Another embodiment is a method for diagnosing a disease or condition in a subject, wherein the disease or condition is associated with MUC-1 expression, the method comprising:

[0051] (a) Contacting a biopsy obtained from a patient with at least one monoclonal antibody according to any one of claims 1 to 4; and

[0052] (b) Detect the monoclonal antibody,

[0053] The detection of cells overexpressing MUC1 SEA in the biopsy indicates that the subject has been diagnosed with the disease or condition. In one embodiment, the disease or condition is cancer. In another embodiment, the disease or condition is an autoimmune or inflammatory disease, or the disease or condition is a non-malignant abnormal growth condition, such as a cyst, such as a kidney cyst, thyroid cyst and thyroid nodule, or a liver cyst. In yet another embodiment, a monoclonal antibody is detectably labeled.

[0054] Another embodiment is a method for imaging a disease or symptom, the method comprising:

[0055] (a) Introducing at least one monoclonal antibody as described herein into a subject, wherein said antibody is labeled with a radioactive isotope or other suitable agent, such as a fluorescent dye, for detection; and

[0056] (b) Visualize detectable labeled monoclonal antibodies.

[0057] The detection of cells and / or tissues labeled with the isotope or detection agent indicates the presence and / or location and / or extent and / or presence of the disease or condition in the subject. In one embodiment, the disease or condition is cancer. In another embodiment, the disease or condition is an autoimmune or inflammatory disease, or the disease or condition is a non-malignant abnormal growth condition, such as a cyst, for example a kidney cyst, thyroid cyst and thyroid nodule, or a liver cyst. Attached Figure Description

[0058] Figure 1 This is a graph showing the binding affinity of chimeric antibody 5F3 and humanized antibody SP66 to SKOV3 cell lines expressing human MUC1, as determined by flow cytometry as described in Example 1.

[0059] Figure 2AThis is a graph showing the dose-dependent binding assessment of different variants of the humanized anti-MUC1 antibody SP66 (HM1 LM2, HM2 LM1, HM2 LM2, HM3 LM2, HM4 LM2, HM4 LM3, and HM5 LM2) compared to chimeric 5F3, performed by flow cytometry on the DA3-TM cell line expressing human MUC1.

[0060] Figure 2B This is a graph showing the dose-dependent binding assessment of different variants of the humanized anti-MUC1 antibody 5F3 (i.e., HM5 LM3 and HM5 LM4) compared to chimeric 5F3 by flow cytometry on the DA3-TM cell line expressing human MUC1.

[0061] Figure 3A This is a graph showing the geometric mean fluorescence plotted against humanized anti-MUC1 5F3 variants and different concentrations of chimeric 5F3, analyzed by flow cytometry on the DA3-TM cell line expressing human MUC1.

[0062] Figure 3B This is a graph showing the geometric mean fluorescence plots against variants of humanized anti-MUC1 5F3 (i.e., HM5LM3 and HM5LM4 of humanized anti-MUC1 antibody 5F3) and different concentrations of chimeric 5F3, analyzed by flow cytometry on the DA3-TM cell line expressing human MUC1.

[0063] Figures 4A-4D This is a representative flow cytometry histogram of chimeric IgG1 anti-MUC1-SEA mAb (5F3) (red) on various human cancer cell lines, relative to MUC1-SEA surface expression on the unbound rituximab (blue) as an allotype control. Below each histogram is a list of human cancer cell lines evaluated and identified as MUC1-SEA (+, ++, +++, or -) compared to the allotype control.

[0064] Figures 5A-5E This is a flow cytometry histogram showing the expression of humanized IgG1 anti-MUC1-SEA mAb (HM5, LM4) (blue) on various human cancer cell lines, namely HT-29 (A), MCF7 (B), Calu-3 (C), SKOV3 (D), and COLO357 (E), relative to the expression of unbound rituximab (red) on the surface of MUC1-SEA as an isotype control.

[0065] Figures 6A-6C are histograms showing the dose-dependent binding of humanized IgG1 anti-MUC1-SEA mAb (HM5 LM4) to human cancer cell lines COLO-357 (A), SKOV-3 (B), and HT-29 (C).

[0066] Figure 6D This is a graph showing the concentration of humanized IgG1 anti-MUC1-SEA mAb (HM5 LM4) versus the geometric mean of fluorescence, plotted against MUC1-SEA expression in COLO-357, SKOV-3, and HT-29 cells.

[0067] Figure 7 The surface and cytoplasmic staining of humanized IgG1 anti-MUC1-SEA mAb (red) on the MUC1-expressing COLO-357 cell line are shown, indicating binding and internalization. Hoechst was used for reverse staining of the cell nuclei (blue). Magnification: 40X.

[0068] Figure 8 The chemical structure of the antibody-drug conjugate HM5LM4-mc-vc-PABC-MMAE is shown.

[0069] Figures 9A-9D The histogram shows the comparable dose-dependent binding of HM5LM4-ADC (A) and humanized IgG1 anti-MUC1-SEA mAb (B) in the COLO-357 cell line. Unbound rituximab conjugated with MMAE was used as an allotype ADC control (C), and rituximab was used as an allotype mAb (D).

[0070] Figure 9E This is a graph showing the concentration versus fluorescence geometric mean of HM5LM4-ADC, humanized IgG1 anti-MUC1-SEA mAb (HM5 LM4), isotype ADC, and isotype on COLO-357.

[0071] Figure 10A and 10B This is a graph showing the single-cycle kinetics of humanized IgG1 anti-MUC1-SEA mAb (HM5 LM4) (10A) and HM5LM4-ADC (10B) on recombinant human MUC1-SEA on SPR.

[0072] Figure 11A and 11B It is used on HT-29 cells with HM5LM4-ADC or isotype ADC ( Figure 11A ) or free MMAE ( Figure 11B A graph showing the percentage of cell survival after contact.

[0073] Figure 12A and 12B It is used on COLO-357 pancreatic cancer cells with HM5LM4-ADC or isotype ADC ( Figure 12A ) or free MMAE ( Figure 12BA graph showing the percentage of cell survival after contact.

[0074] Figure 13A and 12B It is used on BxPC3 pancreatic cancer cells with HM5LM4-ADC or isotype ADC ( Figure 13A ) or free MMAE ( Figure 13B A graph showing the percentage of cell survival after contact.

[0075] Figure 14A and 14B It is used on SKOV3 cells with HM5LM4-ADC or isotype ADC ( Figure 14A ) or free MMAE ( Figure 14B A graph showing the percentage of cell survival after contact.

[0076] Figure 15A and 15B It is used on T47D breast cancer cells with HM5LM4-ADC or isotype ADC ( Figure 15A ) or free MMAE ( Figure 15B A graph showing the percentage of cell survival after contact.

[0077] Figure 16A This is a diagram of the wild-type MUC1-X extracellular domain (MUC1-X-FL-WT), showing the subsequences responsible for forming the loop, β-sheet, and α-helix, as described in Example 2.

[0078] Figure 16B Wild-type MUC1-X-FL and its mutants MUC1-X-FL-1116 and MUC1-X-FL-1118, generated by epitope screening, are shown. The location and mutated amino acid residue are mentioned in the boxes. The downward-facing arrow between the glycine and serine residues indicates the cleavage site in the MUC1-SEA domain.

[0079] Figure 17 The bar chart shows the binding of chimeric antibodies DMB5F3 (also known as 5F3), DMB4F4 (4F4), DMB7F3 (7F3), and DMB10F10 (10F10), as well as SP66 (the humanized form of the 5F3 antibody) and anti-CD20 IgG antibody to MUC1-X-FL-WT (left side of each individual antibody bar chart), MUC1-X-FL-1116 (middle), and MUC1-X-FL-1118 (right side) (as measured by optical density at 450 nm), as described in Example 2.

[0080] Figure 18This illustrates the tumor volume (mm²) over time in an ovarian cancer xenograft model following administration of the mediator described in Example 4 (10 ml / kg, intravenous, Q4D x 6), HM5 / LM4 (6 mg / kg, intravenous, Q4D x 6), HM5LM4-ADC (1.5, 3, or 6 mg / kg, intravenous, Q4D x 6), or 0.12 mg / kg of free MMAE payload (equivalent to 6 mg / kg HM5LM4-ADC). 3 (The image is shown.)

[0081] Figure 19 This shows the tumor volume (mm²) over time in a pancreatic cancer xenograft model following administration of the mediator described in Example 4 (10 ml / kg, intravenous, Q4D x 6), HM5LM4-ADC (1, 1.5, 2 or 3 mg / kg, intravenous, Q4D x 6), or rituximab ADC (3 mg / kg, intravenous, Q4D x 6). 3 (The image is shown.)

[0082] Figure 20 This illustrates the tumor volume (mm²) over time in a breast cancer xenograft model following administration of the mediator described in Example 4 (10 ml / kg, intravenous, Q4D x 6), HM5LM4-ADC (1, 2, or 3 mg / kg, intravenous, Q4D x 6), rituximab ADC (3 mg / kg, intravenous, Q4D x 6), or free MMAE (0.06 mg / kg). 3 (The image is shown.)

[0083] Figure 21 This shows the tumor volume (mm²) over time in a head and neck cancer xenograft model following administration of the mediator described in Example 4 (10 ml / kg, intravenous, Q4D x 6), HM5LM4-ADC (1, 2, or 3 mg / kg, intravenous, Q4D x 6), rituximab ADC (3 mg / kg, intravenous, Q4D x 6), or free MMAE (0.06 mg / kg). 3 (The image is shown.)

[0084] Figure 22 This is a graph showing the total flux (in photons per second) over time in a pancreatic cancer xenograft model after administration of the mediator as described in Example 4 (10 ml / kg, intravenous, Q4D x 6), HM5LM4-ADC (3 mg / kg, intravenous, Q4D x 6), rituximab ADC (3 mg / kg, intravenous, Q4D x 6), or free MMAE (0.06 mg / kg).

[0085] Figure 23 This shows the tumor volume (mm²) over time in a lung cancer xenograft model following administration of the mediator described in Example 4 (10 ml / kg, intravenous, Q4D x 6), HM5LM4-ADC (3 mg / kg, intravenous, Q4D x 6), or rituximab ADC (3 mg / kg, intravenous, Q4D x 6). 3 (The image is shown.) Detailed Implementation

[0086] This invention provides the sequence of a monoclonal antibody (mAb) targeting the MUC1 SEA α-β linker (referred to as the SEA domain), which has potent anticancer activity in vivo.

[0087] This invention provides the sequence of an antibody targeting the MUC1 SEA domain.

[0088] The monoclonal antibodies and immunoconjugates of the present invention can be used to treat various cancers, including lung cancer, prostate cancer, breast cancer, ovarian cancer, head and neck cancer, colon cancer, and pancreatic cancer. For example, the monoclonal antibodies and immunoconjugates of the present invention can be used to treat COLO-357 and BxPC3 pancreatic cancer cells and T47D breast cancer cells.

[0089] Therefore, the present invention provides an antibody for treating malignant tumors expressing MUC1.

[0090] Therefore, in its first aspect, the present invention provides a monoclonal antibody (e.g., an isolated monoclonal antibody) that binds to the MUC1 SEA domain.

[0091] The term "MUC1 SEA domain" (also referred to herein as the "MUC1 SEA module") refers to a highly conserved 110-amino acid domain formed through the interaction of the extracellular portions of the MUC1 α subunit and the MUC1 β subunit. Therefore, it is located at the MUC1 α-β junction and is a cell membrane fixation site.

[0092] The SEA domain, as known in the art, is defined as the region located between amino acids 1039 and 1148 of the human MUC1 protein (UniprotKB-P15941 MUC1_HUMAN).

[0093] MUC1 transmembrane glycoprotein (MUC-TM) is a heterodimer composed of an extracellular domain, a transmembrane domain, and a short cytoplasmic tail region mediating intracellular signal transduction, consisting of 20 to 125 repeats (called variable-number tandem repeats, VNTRs) of a 20-amino acid-long sequence. MUC1 is cleaved by autoproteolytic cleavage within the SEA module. This results in a large extracellular α subunit containing the array of tandem repeats binding to the transmembrane β subunit containing both the transmembrane and cytoplasmic domains of the molecule via strong non-covalent interactions.

[0094] In some embodiments, the humanized antibody has a binding affinity (K0.05) of less than 200 pM or 100 pM. D Humanized antibodies bind to epitopes within the MUC1SEA domain. In some cases, humanized antibodies exhibit binding affinities (K0.05) of less than 200 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or 40 pM. D Humanized antibodies bind to epitopes in the MUC1 SEA domain. In some cases, humanized antibodies exhibit binding affinity (K0.05) of less than 30 pM. D The humanized antibody binds to the epitope in the MUC1 SEA domain. In some cases, the humanized antibody binds with a binding affinity of less than 20 pM (K0). D ) is combined with the tabletop in the MUC1 SEA structural domain.

[0095] Specifically, the present invention provides a monoclonal antibody (e.g., an isolated monoclonal antibody) that binds to the MUC1 SEA domain, wherein the antibody comprises:

[0096] (a) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NO. 7-11, 45, and 46; and

[0097] (b) Light chain variable region, wherein the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO. 12-15.

[0098] In one embodiment, the humanized antibody comprises:

[0099] (a) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NO. 7-11; and

[0100] (b) Light chain variable region, wherein the light chain variable region comprises the amino acid sequence of any one of SEQ ID NO. 12-15.

[0101] In one embodiment, the humanized antibody comprises:

[0102] (a) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12;

[0103] (b) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0104] (c) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14;

[0105] (d) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13;

[0106] (e) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14;

[0107] (f) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13;

[0108] (g) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14;

[0109] (h) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13;

[0110] (i) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 14;

[0111] (j) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 15.

[0112] In another embodiment, the humanized antibody comprises:

[0113] (k) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15;

[0114] (l) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12;

[0115] (m) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 15;

[0116] (n) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 10 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12;

[0117] (o) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15;

[0118] (p) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 12;

[0119] (q) a heavy chain variable region and a light variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light variable region comprises the amino acid sequence of SEQ ID NO: 12;

[0120] (r) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13;

[0121] (s) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 14 or 7.

[0122] (t) Heavy chain variable region and light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 15.

[0123] In a preferred embodiment, the humanized monoclonal antibody binding to the MUC1 SEA domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy and light chain variable regions comprise the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15.

[0124] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene that specifically binds to and recognizes an antigen (in the case of this invention, the MUC1 SEA domain).

[0125] As defined herein, the terms "monoclonal antibody" or "mAb" refer to a population of homologous antibodies, meaning that the individual antibodies comprising this population are identical except for rare mutations that may occur naturally. Monoclonal antibodies target a single antigenic site (epitope).

[0126] Monoclonal antibodies can be prepared and purified by any method known in the art. For example, monoclonal antibodies can be isolated from antibody fragments displayed on phage coat proteins or prepared from B cells taken from the spleen or lymph nodes of immunized animals such as rabbits, rats, mice, or monkeys.

[0127] The purification of monoclonal antibodies can be performed using any method known in the art, such as hydrophobic interaction, ion exchange, and / or size exclusion chromatography, for example by affinity chromatography, i.e., by using an affinity column conjugated to a specific epitope (or antigen). Alternatively, antibody purification can be based on the use of protein A, protein G, and protein L column chromatography.

[0128] As is known in the art, exemplary antibody structural units comprise tetramers. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one “light chain” and one “heavy chain”. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen (epitope) recognition.

[0129] Therefore, the terms "heavy chain variable region" (VH) and "light chain variable region" (VL) refer to these heavy and light chains, respectively. More specifically, the variable region is further subdivided into hypervariable regions and framework (FR) regions. A hypervariable region has a high proportion of different amino acids at a specific position relative to the most common amino acid at that position. Four FR regions with more stable amino acid sequences separate the hypervariable regions. The hypervariable region directly contacts a portion of the antigen surface. Therefore, the hypervariable region is referred to herein as a "complementarity-determining region" or "CDR". The CDR is located at the heavy chain of the antibody ("heavy chain complementarity-determining region") and the light chain of the antibody ("light chain complementarity-determining region").

[0130] From the N-terminus to the C-terminus, both the light and heavy chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs are primarily responsible for binding epitopes of the antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (numbered sequentially from the N-terminus) and are usually identified by the chain in which the CDR is located.

[0131] Therefore, complementarity-determining regions CDRH1, CDRH2, and CDRH3 refer to the three complementarity-determining regions starting from the N-terminus of the antibody heavy chain (also referred to as heavy chain complementarity-determining regions in this paper), while complementarity-determining regions CDRL1, CDRL2, and CDRL3 refer to the three complementarity-determining regions starting from the N-terminus of the antibody light chain (also referred to as light chain complementarity-determining regions in this paper).

[0132] This invention covers antigen-binding fragments of the monoclonal antibodies against the MUC1 SEA domain of the present invention. As used herein, the term "antigen-binding fragment" refers to a fragment of the full-length antibody that retains the specificity of the antibody in binding to the MUC1 SEA domain. Antigen-binding fragments include, but are not limited to, Fv, single-chain Fv (scFv), single-chain Fv-Fc (scFv-Fc), Fab', Fab, F(ab')2, and F(ab)2. Such fragments can be generated by any method known in the art, such as by proteolytic cleavage using enzymes such as papain (to generate the Fab fragment) or pepsin (to generate the F(ab')2 fragment). In some embodiments, the antibody fragment is selected from: single-chain Fv-Fc (scFv-Fc) molecules, single-chain Fv (scFv), Fv, Fab', Fab, F(ab')2, and F(ab)2. These fragments can be generated using recombinant DNA technology.

[0133] In a specific embodiment, the monoclonal antibody or its antigen-binding fragment of the present invention binds to the MUC1 SEA domain.

[0134] In specific embodiments, the monoclonal antibody or its antigen-binding fragment of the present invention can effectively reduce the tumor volume of a subject. In the context of this invention, the term "reduction" of tumor volume means, as measured by any means known in the art, that the monoclonal antibody or its antigen-binding fragment of the present invention reduces the tumor size by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% compared to the tumor size in the absence of the antibody or its antigen-binding fragment of the present invention. In some embodiments, the term "reduction" means a reduction of at least about 50%, 60%, 70%, 80%, or 90%.

[0135] As used herein, the term "humanized antibody" refers to an antibody based on the structure of a non-human species (e.g., mouse), the amino acid sequence of which has been modified to increase its similarity to naturally occurring antibody variants in humans. Methods for preparing humanized antibodies are known in the art.

[0136] To prepare large quantities of antibodies, transient or stable cell lines expressing antibodies can be prepared by transfecting mammalian cells (e.g., CHO cells) with an Ig expression vector containing the heavy and light chain DNA sequences of the antibody. These antibodies can then be manufactured, for example, in a bioreactor system. Antibodies can be purified to clinical grade using established monoclonal antibody purification methods. Clones producing high levels of anti-MUC1 SEA domain antibodies can then be selected and amplified based on antibody levels in the supernatant, as tested by any method known in the art (e.g., a MUC1 SEA domain-specific ELISA assay). Master cell libraries developed for specific clones can serve as starting growth material for all clinical-grade batches.

[0137] In some embodiments, the present invention provides an anti-MUC1 SEA domain monoclonal antibody (e.g., an isolated monoclonal antibody) or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is encoded by a nucleic acid sequence that is at least 70%, or 75%, or 80%, or 85%, or 90% or higher percentage identical to the nucleic acid sequence represented by any one of SEQ ID NO: 7-11, 45 and 46, and wherein the light chain variable region is encoded by a nucleic acid sequence that is at least 70%, or 75%, or 80%, or 85%, or 90% or higher percentage identical to the nucleic acid sequence represented by any one of SEQ ID NO: 12-15.

[0138] In other embodiments, the present invention provides an anti-MUC1 SEA domain monoclonal antibody (e.g., an isolated monoclonal antibody) or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is encoded by a nucleic acid sequence identical to at least 70%, or 75%, or 80%, or 85%, or 90% or higher percentage of the nucleic acid sequence represented by SEQ ID NO: 11, and wherein the light chain variable region is encoded by a nucleic acid sequence identical to at least 70%, or 75%, or 80%, or 85%, or 90% or higher percentage of the nucleic acid sequence represented by SEQ ID NO: 15.

[0139] In some embodiments, the present invention provides an anti-MUC1 SEA domain monoclonal antibody (e.g., an isolated monoclonal antibody) or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence or a variant thereof represented by any one of SEQ ID NO: 7-11, 45, 46, and the light chain variable region comprising an amino acid sequence or a variant thereof represented by any one of SEQ ID NO: 12-15.

[0140] In some embodiments, the present invention provides an anti-MUC1 SEA domain monoclonal antibody (e.g., an isolated monoclonal antibody) or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 11 or a variant thereof, and the light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 15 or a variant thereof.

[0141] This invention also covers variants of the heavy chain variable region and the light chain variable region. These variants may include mutations in the complementarity-determining regions of the heavy and light chains that do not alter the activity of the antibody described herein, or they may include mutations in the frame region.

[0142] The term "variant" means an amino acid or nucleotide sequence that differs from the sequence specifically identified herein, wherein one or more amino acid residues or nucleotides are deleted, substituted, or added. It should be understood that, as used herein, the term "addition" means the addition of any amino acid residue to the sequence described herein. Variants encompass a variety of amino acid substitutions. An amino acid "substitution" is the result of replacing one amino acid with another amino acid having similar or different structural and / or chemical properties. Amino acid substitutions can be based on similarity in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. Generally, variants encompass conserved amino acid substitutions. Conservative representations of functionally similar amino acids are well known in the art. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Each of the following eight groups contains other exemplary amino acids that are conserved substitutes for each other:

[0143] 1) Alanine (A), glycine (G);

[0144] 2) Aspartic acid (D), glutamic acid (E);

[0145] 3) Asparagine (N), glutamine (Q);

[0146] 4) Arginine (R), Lysine (K);

[0147] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0148] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0149] 7) Serine (S), threonine (T); and

[0150] 8) Cysteine ​​(C), Methionine (M).

[0151] Conserved nucleic acid substitution is a nucleic acid substitution that results in a conserved amino acid substitution as defined above.

[0152] Variants according to the invention also encompass nonpolar to polar amino acid substitutions, as well as polar to nonpolar amino acid substitutions.

[0153] In some cases, variants include alanine to valine mutations. In some cases, variants include serine to threonine mutations. In some cases, variants include alanine to isoleucine mutations. In some cases, variants include leucine to isoleucine mutations. In some cases, variants include valine to arginine mutations. In some cases, variants include alanine to phenylalanine mutations. In some cases, variants include methionine to isoleucine mutations. In some cases, variants include tyrosine to lysine mutations. In some cases, variants include phenylalanine to tyrosine mutations. In some cases, variants include arginine to tyrosine mutations. In some cases, variants include valine to isoleucine mutations.

[0154] As used herein, the term "amino acid" or "amino acid residue" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids.

[0155] A variant sequence is an amino acid or nucleic acid sequence that can be characterized by the percentage of identity between its amino acid or nucleotide sequence and the amino acid or nucleotide sequence described herein (e.g., the amino acid or nucleotide sequence of the heavy and light chains of the antibody described herein).

[0156] In some embodiments, variant sequences as defined herein refer to nucleic acid sequences encoding heavy chain variable regions and light chain variable regions, each having at least 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity compared to the sequences of the heavy chain variable regions and light chain variable regions described herein.

[0157] In some other embodiments, the variant sequences as defined herein refer to the amino acid sequences of the heavy chain variable region and the light chain variable region, each of which has at least 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequences of the heavy chain variable region and the light chain variable region described herein.

[0158] The term "antibody activity" means the ability of an antibody to bind to the MUC1 SEA domain and, preferably alone or as part of an immunoconjugate having a cytotoxic moiety, to mediate cytotoxicity. Antibody activity can be measured in vivo or in vitro using methods known in the art.

[0159] The binding of the antibody of the present invention to its target protein can be measured, for example, using ELISA, surface plasmon resonance (SPR), biolayer interferometry (BLI), Western blotting, or immunofluorescence assay (IFA).

[0160] The biological activity of antibodies can be measured, for example, in in vivo cancer models, as detailed in the following examples.

[0161] Another aspect of the invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof according to the invention.

[0162] As defined herein, the term “nucleic acid” or “nucleic acid molecule” refers to a polymer of nucleotides, which can be single-stranded or double-stranded polynucleotides, such as deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) where appropriate. These terms should also be understood to include, as equivalents, RNA or DNA analogs prepared from nucleotide analogs, and (where applicable to the described embodiments) single-stranded (e.g., sense or antisense) and double-stranded polynucleotides. The term “DNA” as used herein also encompasses cDNA, i.e., complementary or copy DNA produced from an RNA template by the action of reverse transcriptase (RNA-dependent DNA polymerase).

[0163] The present invention further provides expression vectors comprising nucleic acid molecules (e.g., isolated nucleic acid molecules) as defined herein.

[0164] As used herein, "expression vector" is sometimes referred to as an expression medium or expression construct, encompassing vectors such as plasmids, viruses, bacteriophages, integrative DNA fragments, and other mediums capable of integrating DNA fragments into the host genome. Expression vectors are typically self-replicating DNA or RNA constructs containing a desired gene or a fragment thereof, and operatively linked genetic control elements that are recognized in suitable host cells and enable the expression of the desired gene. These control elements enable expression in a suitable host. Expression vectors according to the invention are capable of expression in bacterial, yeast, or mammalian host cells (to name just a few).

[0165] Another embodiment uses a host cell transfected with a nucleic acid molecule according to the invention (e.g., an isolated nucleic acid molecule) or an expression vector according to the invention.

[0166] As used herein, the term "host cell" refers to a cell sensitive to the introduction of the isolated nucleic acid molecule according to the invention or the expression vector according to the invention. Preferably, the cell is a mammalian cell, such as a CHO cell or an NSO cell. The isolated nucleic acid molecule or expression vector according to the invention can be transfected into the host cell by any method known in the art.

[0167] Another embodiment is an immunoconjugate comprising an antibody or antigen-binding fragment thereof according to the invention and an additional cytotoxic agent or therapeutic agent as described herein. The term "immunoconjugate" refers to an antibody or antigen-binding fragment thereof according to the invention conjugated (linked or joined) to an additional pharmaceutical agent. Immunoconjugates can be prepared by any method known in the art, such as by crosslinking the additional pharmaceutical agent with an antibody according to the invention or by recombinant DNA methods. The antibody can be linked to the additional pharmaceutical agent (e.g., a drug, such as a cytotoxic agent) via a linker. The linker can be, for example, a protease-cleavable group or a non-cleavable group. In one embodiment, the linker is a maleimide-hexanoyl-valine-citrullinated-p-aminobenzyloxycarbonyl (mc-val-cit-PABC) linker. Without being bound by any particular theory, the immunoconjugate binds to the surface of tumor cells and is internalized. The linker (e.g., a protease-cleavable linker) is then cleaved, thereby releasing the additional pharmaceutical agent (e.g., a cytotoxic agent).

[0168] In some embodiments, the immunoconjugate is an immunotoxin, thereby conjugating an antibody or its antigen-binding fragment according to the invention to a cytotoxic agent. As used herein, the term "cytotoxic agent" refers to any agent that exerts a cytotoxic effect on cells upon contact. Examples of cytotoxic agents that can be used in the immunoconjugates of the invention include, but are not limited to: alkylating agents, anthracyclines, pyrimidine derivatives, vinca alkaloids, photodynamic agents, platinum-containing compounds, taxanes, topoisomerase inhibitors, ribosome inactivators (e.g., white tree toxin), DNA-damaging agents (e.g., chalcogenide), microtubule inhibitors (e.g., emtansine), antimitotic agents (e.g., monomethylaurestatin), or bacterial toxins. The cytotoxic agent may also be a radioisotope or a cytotoxic antibody. In one embodiment, the cytotoxic agent is a Pseudomonas exotoxin, such as ZZ-PE38 (a ZZ IgG-binding protein fused to a Pseudomonas exotoxin). In another embodiment, the cytotoxic agent is monomethylaurestatin E (MMAE).

[0169] In some embodiments, the immunoconjugate is an immunotoxin, thereby conjugating an antibody or its antigen-binding fragment according to the invention to a drug or payload. Suitable drugs include, but are not limited to, camptothecin derivatives (such as camptothecin, topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, rubitecan, belotetcan, deruxtecan, and SN-38), topoisomerase inhibitors, maytansine alkaloids, Calicheamycin, duocarmycin, tubulysin, amatoxin, dolastatin, and oliquistatins such as monomethyloliquistatin E (MMAE) and monomethyloliquistatin F (MMAF), pyrrolobenzodiazepine dimers, indolinobenzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTAC, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogues or prodrugs. Other suitable drugs include calicheamicin, docamycin A (such as CC-1065, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, and taxol and its derivatives), platinum-based antitumor agents (such as cisplatin or its derivatives), tobrexin (such as tobrexin A), amatoxins (such as α-amatoxin, β-amatoxin, γ-amatoxin, ε-amatoxin, amatoxins, and amanullinic acid).Acid, amaninamide, amanitin and proamanullin (non-toxic cyclic peptides), scabra (such as scabra 10 or scabra 15), radioactive isotopes, cytokines (such as interleukins, ricin, diphtheria toxin, Pseudomonas exotoxin PE38), kinase inhibitors (such as imatinib, nilotinib, dasatinib, bosutinib, ponatinib, gefitinib, erlotinib, afatinib), and afatinib. Afatinib, osimertinib, lapatinib, neratinib, sorafenib, sunitinib, pazopanib, axitinib, lenvatinib, cabozatinib, vandetanib, regorafenib, vemurafenib, dabrafenib, trametinib Rametinib, Cobimetinib, Crizotinib, Certinib, Alectinib, Brigatinib, Lorlatinib, Ibrutinib, Acalibrutinib, Midostaurin, Ruxolitinib, Idelalisib, Copanlisib, Palbociclib Ribociclib and abemaciclib, MEK inhibitors (such as MEK1 and / or MEK2 inhibitors, trametinib (GSK1120212), cobimetinib or XL518, binimetinib (MEK162), selumetinib, PD-325901, CI-1040, PD035901 and TAK-733), and KSP (kinin spindle protein) inhibitors (such as ispinesib (SB-715992), SB743921, AZ 3146, GSK923295, BAY1217389, MPI-0479605 and ARQ)621). In one embodiment, the drug is a nucleic acid. In another embodiment, the drug is beloteccan, derutecan, or SN-38.

[0170] One specific embodiment is an immunoconjugate comprising: (a) an isolated monoclonal antibody or an antigen-binding fragment thereof that binds to the MUC1 SEA domain and (b) the cytotoxic agent monomethyl guanylate E (MMAE).

[0171] Another embodiment is an immunoconjugate comprising: (a) an antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 15; (ii) a cytotoxic agent, such as MMAE; and optionally (c) a linker group (such as maleimide hexanoyl-Val-Cit-PABC-). In one embodiment, the immunoconjugate reduces tumor volume after administration to a subject with cancer.

[0172] The anti-MUC1 SEA domain antibody of the present invention can be administered in combination with at least one other therapeutic agent.

[0173] As used in this article, the term "another therapeutic agent" refers to any medicine that can be used to treat a disease or condition, such as cancer.

[0174] In some embodiments, the additional therapeutic agent is an additional antibody. The term "additional antibody" as defined herein refers to the antibodies of the present invention (i.e., a combination of at least two antibodies of the present invention) as well as antibodies not of the present invention, which may be combined with the antibodies of the present invention for the treatment of diseases or conditions (e.g., cancer). Such other antibodies include, but are not limited to, anti-CD22 antibodies, anti-CD30 antibodies, anti-HER2 receptor antibodies, anti-VEGF antibodies, anti-EGFR antibodies, anti-tumor-associated antigen (TAA) antibodies, and anti-checkpoint inhibitors.

[0175] The other therapeutic agents may also be chemotherapeutic agents or anti-inflammatory agents.

[0176] Another embodiment is a pharmaceutical composition comprising at least one anti-MUC1SEA antibody of the present invention (e.g., isolated anti-MUC1 SEA antibody) or its antigen-binding fragment or immunoconjugate as defined herein, as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluent.

[0177] In a specific embodiment, the pharmaceutical composition is used to treat diseases or conditions associated with overexpression of MUC1.

[0178] The term "disease or condition associated with MUC1 overexpression" is used herein in its broadest sense and refers to any disease characterized by abnormal expression of MUC1. In one specific embodiment, the disease or condition associated with MUC1 overexpression is cancer. Examples include, but are not limited to: lung cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, small bowel cancer, pancreatic cancer, gastric cancer, liver cancer, multiple myeloma, or acute myeloid leukemia. In one embodiment, the disease or condition is an advanced and / or metastatic solid tumor. In another embodiment, the disease or condition is breast cancer (e.g., ER+ breast cancer). In yet another embodiment, the disease or condition is non-small cell lung cancer. In yet another embodiment, the disease or condition is epithelial ovarian cancer.

[0179] In other embodiments, the disease or condition associated with MUC1 overexpression is an autoimmune or inflammatory disease. Non-limiting examples of autoimmune or inflammatory diseases include rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, amyloidosis, and autoimmune pancreatitis.

[0180] In other embodiments, the disease or condition is a non-malignant abnormal growth symptom, such as a cyst, for example a clinically significant renal cyst, a large non-functional thyroid cyst and thyroid nodule, or a liver cyst.

[0181] The “pharmaceutical composition” of the present invention may comprise an antibody or any antigen-binding fragment thereof as defined herein and a buffer (a pharmaceutical agent for adjusting the osmotic pressure of the composition), and optionally one or more pharmaceutically acceptable carriers, excipients and / or diluents known in the art.

[0182] As used herein, the term "pharmaceutically acceptable carrier, excipient, or diluent" includes any solvent, dispersion medium, coating, antibacterial agent, and antifungal agent known in the art. A carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of compatibility with other components and harmlessness to the subject. Its use in a therapeutic composition should be considered, except where any conventional medium or agent is incompatible with the active ingredient. For example, for a pharmaceutical composition intended for intravenous administration, a pharmaceutically acceptable carrier may be a 0.9% sodium chloride injection solution.

[0183] In other embodiments, the pharmaceutical composition according to the invention further comprises additional therapeutic agents. Non-limiting examples of additional therapeutic agents include anti-MUC1 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-HER2 receptor antibodies, anti-VEGF antibodies, anti-EGFR antibodies, anti-TAA antibodies, and checkpoint inhibitors.

[0184] Another embodiment is a method for treating or improving diseases or conditions (e.g., cancer) associated with MUC1 overexpression, the method comprising administering to a subject in need a therapeutically effective amount of a monoclonal antibody of the present invention (e.g., an isolated monoclonal antibody) or an antigen-binding fragment thereof, or an immunoconjugate comprising an antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition comprising an isolated monoclonal antibody of the present invention or an antigen-binding fragment thereof or an immunoconjugate thereof.

[0185] The terms "subject" and "patient" are used interchangeably and refer to a subject who may benefit from the present invention, such as a mammal (e.g., a dog, cat, sheep, pig, horse, cow, or human). In one specific embodiment, the subject or patient is a human. Diagnosis of diseases or conditions associated with MUC1 overexpression can be performed by a skilled physician using methods known in the art.

[0186] In the context of this invention, the term "subject in need" refers in particular to mammals, especially human subjects, suffering from diseases or conditions associated with overexpression of MUC1 as defined herein.

[0187] It should be understood that, as used herein, the term "treat / treating / treatment" or variations thereof means to reduce, prevent, cure, reverse, improve, weaken, alleviate, minimize, suppress, or stop the harmful effects of a disease or condition, or delay the onset of one or more clinical indications of a disease or symptom (e.g., cancer) associated with MUC1 overexpression as defined herein. In some embodiments of the method according to the invention, said method further comprises administering additional therapeutic agents as defined herein to a subject in need.

[0188] According to the invention, administration can be carried out by any of the following routes: oral administration; intravenous injection, intramuscular injection, intraperitoneal injection, intrathecal injection or subcutaneous injection; rectal administration; intranasal administration, ocular administration or topical administration.

[0189] In a specific embodiment, the application according to the present invention is performed intravenously.

[0190] An antibody or antibody fragment as defined herein, any pharmaceutical composition comprising said antibody or antibody fragment, or any conjugate comprising said antibody or antibody fragment may be administered to a subject in a single or multiple doses.

[0191] The "therapeutic effective amount" of the isolated monoclonal antibody or any antigen-binding fragment thereof according to the invention, or the pharmaceutical composition according to the invention, used for the purposes defined herein, is determined by considerations known in the art for curing, preventing, or at least alleviating or improving medical symptoms. For any preparation used in the methods of the invention, the dosage or therapeutic effective amount may initially be estimated from in vitro cell culture assays or based on suitable animal models.

[0192] In some embodiments, the therapeutically effective dose according to the invention is in the range of about 10 pg / kg to about 50 mg / kg.

[0193] In other embodiments, the therapeutically effective dose according to the invention is in the range of 0.1 mg / kg to 40 mg / kg, 1 mg / kg to 10 mg / kg, or 5 mg / kg to 10 mg / kg.

[0194] Specific exemplary doses include, but are not limited to, 0.25 mg / kg, 0.75 mg / kg, 2.5 mg / kg, 5 mg / kg, or 10 mg / kg, which may be administered as a daily dose, or every three days, or weekly, or every three weeks, at the physician's discretion. In one embodiment, the dose is administered intravenously.

[0195] The present invention further provides an anti-MUC1 SEA antibody (e.g., isolated anti-MUC1 SEA antibody) or any antigen-binding fragment thereof according to the present invention, or an immunoconjugate or pharmaceutical composition according to the present invention, for use in methods of treating or improving diseases or conditions (e.g., cancer) associated with overexpression of MUC1 as defined herein.

[0196] The present invention further provides the use of the monoclonal antibody (e.g., isolated monoclonal antibody) or its antigen-binding fragment, immunoconjugate or pharmaceutical composition thereof in the preparation of a medicament for treating or improving a disease or condition (e.g., cancer) associated with overexpression of MUC1 as defined herein.

[0197] It should be understood that the terms "purified" or "isolated" refer to molecules that have been removed, isolated, or separated from their natural environment, such as amino acid or nucleic acid sequences, peptides, polypeptides, or antibodies. Therefore, "isolated antibody" is a purified antibody. As used herein, the terms "purified" or "purified" also refer to the removal of contaminants from a sample.

[0198] Another embodiment is a method for diagnosing a disease or condition (e.g., cancer) from a biopsy obtained from a subject, the method comprising:

[0199] (a) Contacting the biopsy with at least one isolated monoclonal antibody or its antigen-binding fragment of the present invention; and

[0200] (b) Detect the isolated monoclonal antibody or any antigen-binding fragment thereof;

[0201] Cells overexpressing MUC1 SEA were detected in the biopsy as an indication of the disease or condition (e.g., cancer).

[0202] The ability of the isolated antibodies of the present invention to detect MUC1-SEA expression can be evaluated by any method known in the art, such as immunohistochemistry or flow cytometry. Immunohistochemistry can be performed on formaldehyde-fixed sections from fresh frozen (FF) tissue and on paraffin-embedded formaldehyde-fixed (PEFF) tissue. The antibodies are capable of recognizing cells expressing MUC1 using flow cytometry. In various embodiments, the isolated antibodies according to the present invention can be labeled according to any method known in the art. In other embodiments, detection can be based on the identification of said antibodies using secondary antibodies.

[0203] The term "biopsy" is used herein in its broadest sense to refer to any biopsy taken from a subject in which cells overexpressing MUC1 SEA can be detected, as defined herein. Biopsies can be obtained from mammals, including humans, and encompass fluid samples containing cells and tissue samples. In some embodiments, the fluid sample is blood, plasma, serum, lymph, or urine. In some embodiments, a biopsy is a tissue sample suspected of containing cancer cells.

[0204] On the other hand, the present invention provides a method for imaging diseases or symptoms, the method comprising:

[0205] (a) Introducing at least one isolated anti-MUC1 SEA monoclonal antibody or its antigen-binding fragment of the present invention into a subject, wherein the antibody or its antigen-binding fragment is detectably labeled with a radioisotope or with a visualization agent (i.e., a drug that can be visualized, for example, by scanning); and

[0206] (b) Visualize the detectably labeled isolated anti-MUC1 SEA monoclonal antibody or any antigen-binding fragment thereof;

[0207] The detection of cells and / or tissues labeled with the isotope or the visualization agent indicates the presence and / or location and / or extent and / or presence of the disease or condition in the subject.

[0208] As used herein, the term "about" means that a value may deviate from the mentioned value by up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases by a percentage higher or lower than 20%, the deviation range including integer values ​​and, where applicable, non-integer values ​​constituting a continuous range. Although the invention has been disclosed and described, it should be understood that the invention is not limited to the specific embodiments, method steps, and compositions disclosed herein, as these method steps and compositions may vary slightly. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be defined only by the appended claims and their equivalents.

[0209] It should be noted that, as used in this specification and the appended claims, the singular forms “a / an” and “the” include plural references unless the content clearly indicates otherwise.

[0210] Throughout this specification and in the accompanying examples and claims, unless the context otherwise requires, the word “comprise” and its variations (e.g., “comprises” or “comprising”) should be understood to imply inclusion of the integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps. The term “comprise” will also be understood to include “consisting of” and “substantially consisting of”.

[0211] Example

[0212] Example 1

[0213] Humanized anti-MUC1 antibody

[0214] The chimeric antibody 5F3 (also known as chimeric DMB5F3) described in International Publication No. WO 2021 / 186427 was humanized as follows. A human framework was used as a template to produce a humanized version of anti-MUC1α / β mAb (i.e., 5F3). The sequences of the variable regions of the heavy and light chains of chimeric antibody 5F3 are provided in SEQ ID NO: 42 and 43, respectively.

[0215] CDR residues from the VH- (SEQ ID NO: 1) and VL- (SEQ ID NO: 2) domains of the mouse anti-MUC1 antibody (m5F3) were identified using three antibody numbering systems and grafted onto the human frame region sequences VH- (SEQ ID NO: 3) and VL- (SEQ ID NO: 4) domains, respectively. CDRs from 5F3 were chemically synthesized and subsequently cloned into mammalian expression vectors. Expression vectors containing humanized 5F3 heavy and light chains were named SP66-HC and SP66-LC, respectively. The VH domain of SP66 is SEQ ID NO: 5, and the VL domain is SEQ ID NO: 6. Antibodies expressed using SP66-HC and SP66-LC were named SP66. SP66 was transiently expressed in CHO cells and purified by protein A affinity chromatography. Its binding to the human MUC1 SEA domain was assessed by flow cytometry on an SKOV3 cell line expressing human MUC1. Figure 1 The results of this study clearly demonstrate that, despite having the same CDR, the SP66 IgG product cannot bind to the human MUC1 SEA domain using this technique.

[0216] In addition, SP66 was tested on human cancer cell lines COLO357, SKOV-3, and HT29. Anti-CD20 IgG antibody was used as a control. No growth inhibition was observed in any cell line after treatment with SP66.

[0217] Some vernier region residues of mouse 5F3 mAb were reverse-mutated to SP66. Numerous constructs were generated, in which different combinations of mutations were introduced into the frame regions of the SP66-VH and -VL domains. For the variants of the SP66-VH domain, these constructs were named HM1 (SEQ ID NO: 7), HM2 (SEQ ID NO: 8), HM3 (SEQ ID NO: 9), HM4 (SEQ ID NO: 10), HM5 (SEQ ID NO: 11), HM6 (SEQ ID NO: 45), and HM7 (SEQ ID NO: 46), and for the variants of the SP66-VL domain, these constructs were named LM1 (SEQ ID NO: 12), LM2 (SEQ ID NO: 13), LM3 (SEQ ID NO: 14), and LM4 (SEQ ID NO: 15) (Table 1).

[0218] Table 1

[0219] List of mutations introduced into the VH and VL domains of humanized anti-MUC1 mAb (SP66) to restore binding affinity

[0220]

[0221] These variants were expressed, purified, and their binding to MUC1 was assessed by surface plasmon resonance (SPR) or flow cytometry. Binding data indicated that the various variants of SP66 exhibited improved binding to MUC1 compared to SP66 (Tables 2 and 3 below). Figure 2A , 2B (3A and 3B). The final version of the humanized antibody was named HM5 / LM4. Combined data showed that HM5 / LM4 had a similar binding affinity to MUC1 compared to the parental 5F3 antibody.

[0222] Table 2

[0223] Binding affinity of different variants of humanized anti-MUC1 5F3 mAb determined by SPR

[0224]

[0225] Table 3

[0226] The binding of different variants of humanized and chimeric anti-MUC1 antibodies to MUC1-positive DA3-TM cell lines was evaluated by flow cytometry.

[0227]

[0228] method

[0229] The VH and VL domains were cloned into a mammalian expression vector.

[0230] The gene fragments (SEQ ID NO: 5 and SEQ ID NO: 6) were chemically synthesized. The DNA sequence of the VH domain (SEQ ID NO: 5) was amplified by PCR using primers SP190-FP and SP191-RP (SEQ ID NO: 16 and 17, Table A) and Vent DNA polymerase (NEB Corporation (NEB)). The resulting PCR product was digested with BSSHII and NheI restriction enzymes and inserted into the pMAZIgH vector digested with the same enzymes. Similarly, the VL domain (SEQ ID NO: 6) was amplified by PCR using primers SP192-FP and SP193-RP (SEQ ID NO: 18 and 19, respectively) and inserted into the pMAZIgL vector after digestion with BSSHII and BSiWI restriction enzymes. All constructs were confirmed by DNA sequencing.

[0231] Construction of a humanized version of anti-MUC1 antibody 5F3

[0232] The vernier residues in the VH and VL domains of the SP66 antibody were mutated to corresponding residues present in the mouse anti-human MUC1 antibody m5F3. Amino acid mutations were performed using the Q5 site-directed mutagenesis kit (NEB) according to the manufacturer's instructions. Primer sequences for each of these mutations are provided at SEQ ID NO: 16-36 and SEQ ID NO: 44 in Table A. Different versions of templates and primers used to generate humanized anti-MUC1 (i.e., 5F3) are given in Table 4. The positions of the mutated amino acids are based on the Kabat numbering system.

[0233] Table 4

[0234] Different versions of the VH and VL domains were used to generate humanized anti-MUC1 mAb (SP66) to recover the template and primer lists for binding affinity.

[0235]

[0236]

[0237] Example 2

[0238] Human MUC1 SEA epitope map

[0239] The sequence of the wild-type MUC1-X extracellular domain (MUC1-X-FL-WT) (a subsequence of MUC1, SEQ ID NO:41) was used to generate various mutants in the SEA domain. Mutations were performed at various sites in the MUC1 SEA domain using site-directed mutagenesis. Figure 16B As shown, mutants named MUC1-X-FL-1116 (mutations of R1108A, E1109A, N1113A, and D1116A) and MUC1-X-FL-1118 (mutations of R1108A, E1109A, N1113A, and E1118A) were generated and cloned into the pET30a(+) expression vector. These recombinant proteins were expressed in *E. coli* and isolated using a Ni-NTA column. The binding of the 5F3 antibody to these mutants was determined using an ELISA method.

[0240] Figure 16A This is a visual representation of the wild-type MUC1-X extracellular domain (MUC1-X-FL-WT, a subsequence of human MUC1, SEQ ID NO: 41).

[0241] The chimeric antibodies DMB5F3 (also known as 5F3), DMB4F4 (4F4), DMB7F3 (7F3), and DMB10F10 (10F10), as well as SP66 and HM5 LM4 (humanized forms of the 5F3 antibody) and anti-CD20 IgG antibodies were tested for binding to MUC1-X-FL-WT, MUC1-X-FL-1116, and MUC1-X-FL-1118. Each of the chimeric antibodies is described in International Publication No. WO 2021 / 186427. The results are shown in Figure 17 In the study, the 5F3 antibody and its humanized version, HM5 LM4, lost binding affinity to MUC1 when a mutation was present at position 1118 (MUC1-X-FL-1118), but binding was maintained when a mutation was present at position 1116 (MUC1-X-FL-1116). Conversely, the 7F3 antibody lost its binding affinity to MUC1 when a mutation was present at position 1116 (MUC1-X-FL-1116), but binding affinity was maintained when a mutation was present at position 1118 (MUC1-X-FL-1118). The binding affinity of the 10F10 antibody was impaired when mutations were present at positions 1116 or 1118.

[0242] Example 3

[0243] Humanized anti-MUC1 antibody drug conjugate

[0244] Expression of Humanized Anti-MUC1 SEA Antibody in CHO Cells: High-level transient expression of humanized anti-MUC1 SEA antibody protein was achieved using plastic non-adhesive CHO-S cells suitable for growth under serum-free conditions. CHO-S cells were transfected with plasmid DNA encoding the humanized anti-MUC1 SEA antibody sequence at 0.8 µg / mL to 1.5 µg / mL using the Expi-CHO Expression System Kit, following the manufacturer's instructions (Thermo Fisher Scientific (Waltham, MA), catalog A29133). Twenty-four hours post-transfection, the transfected cell culture was diluted to 16% V / V using Expi-CHO feed and enhancer (Thermo Fisher Scientific, catalog A29129) and incubated for an additional 14 days in baffle-free flasks at 32°C and 120 rpm in a humidified 5% CO2 shaker incubator. Fourteen days later, cell-free supernatant was collected and the antibody was purified using appropriate methods.

[0245] Purification: At the end of the production phase, CHO medium containing secretory mAb was collected and centrifuged, followed by 0.2 µm filtration to remove cells, any debris, and insoluble material. EDTA was then added to this cell-free culture supernatant at a final concentration of 2 mM. This culture supernatant was then purified by protein A column chromatography using an AKTA Avant (GE Healthcare Ltd., Little Chalfont, Buckinghamshire, UK) system. In short, pre-packaged protein A columns (5 mL or multiples thereof) were equilibrated by passing through 3–5 bed volumes of equilibration buffer containing 25 mM Tris-Cl (pH 7.0) and 100 mM NaCl. MabSelect Sure TM (GE Healthcare, Little Charfont, Buckinghamshire, UK). Based on the antibody titer and considering the resin's ≤ 35 mg / mL dynamic binding capacity (according to the manufacturer's instructions), the readjusted culture supernatant was loaded onto the equilibrated protein A affinity column at a controlled flow rate to achieve the optimal residence time (RT of 2–3 min). The column was then washed with 3–5 bed volumes of equilibration buffer to remove any unbound proteins. Additionally, the column was washed with 3–5 bed volumes of a high-ionic-strength buffer containing 25 mM Tris-Cl (pH 7.0) and 1 M NaCl to remove any non-specifically bound proteins or culture medium components. The column was then washed with 3–5 bed volumes of a low-pH buffer containing 50 mM sodium acetate (pH 6.0). Elution of bound proteins was then performed at an acidic pH using 5–7 bed volumes of an elution buffer containing 25 mM sodium acetate (pH 3.5) and 100 mM NaCl. The eluted protein fractions were incubated at room temperature for 45–60 minutes for low-pH virus inactivation, and then the pH was adjusted to 5.5 with 2M Tris solution. Finally, the Protein A column was cleaned by passing it through 0.1 N NaOH solution followed by Milli-Q water, as per the manufacturer's instructions, and stored at 2–8°C by passing it through 20% ethanol. Protein A purified protein samples were prepared by adding sucrose (250 mM final concentration) and polysorbate 20 (0.01% v / v final concentration), followed by 0.2 μm filtration. The purified protein was stored for short periods at 2–8°C or long periods at -20°C.

[0246] Antibody conjugation for monomethyl ozretamine E (MMAE) generated by ADC: Humanized IgG1 anti-MUC1-SEAmAb HM5-LM4 buffer was exchanged for conjugation buffer (20 mM sodium phosphate, 2 mM EDTA, pH 7.4) and partially reduced at 37°C with a 2.2 mole excess of TCEP for approximately 120 min. After reduction, it was buffer-exchanged with conjugation buffer via 30 kDa centrifugation ultrafiltration, tangential flow filtration (TFF), or Sephadex G25 resin (gel filter medium). The generated thiols were quantified using Ellman's reagent 5,5'-dithiobis(2-nitrobenzoic acid) [DTNB]. A 10-fold molar excess of maleimide hexanoyl-Val-Cit-PABC-MMAE (vcMMAE) was added to the reducing antibody. The conjugation reaction was carried out for 60 minutes at 22 °C ± 3 °C in the presence of approximately 10% DMF and 100 mM sodium octanoate. Following conjugation, an excess of cysteine ​​relative to 20 moles of vcMMAE was added to quench unreacted vcMMAE. The conjugate was subjected to 30 kDa centrifugation and ultrafiltration or buffer exchange with HIC (hydrophobic interaction chromatography) binding buffer via TFF or Sephadex G25 resin (gel filter medium), and further purified by preparative HIC chromatography to enrich substances with drug-antibody ratios (DAR) of 2 and 4. The HIC-purified ADC was concentrated and transferred to succinate buffer containing 9.0% (w / v) sucrose and 0.04% Tween 80, pH 5.5 via 30 kDa MWCO centrifugation and ultrafiltration or TFF buffer exchange, and stored at 4 °C or -20 °C for further use. The resulting ADC was designated HM5LM4-ADC.

[0247] Flow cytometry was used to screen for surface MUC1 expression in human cancer cells: Human cancer cell lines (including A253, COLO357, HT29, SKOV3, etc.) were collected enzymatically using Dulbecco's phosphate-buffered saline (D-PBS) containing 0.25% trypsin and 0.2% EDTA. Cells were washed with excess cell culture medium and cultured in cell line-specific medium at 1–2 × 10⁻⁶. 6Resuspend cells at a density of / mL. Add anti-MUC1-SEAmAb (chimeric or humanized IgG) or reference IgG antibody at a specific concentration to the cells. Incubate cells at 4 °C for 1 hour and wash with excess cell culture medium. Further stain these cells at 4 °C for 1 hour with goat anti-human IgG and Fcγ conjugated to Alexa Fluor 647 (Jackson Immuno Research, West Grove, PA, USA). Concentrations of goat anti-human IgG and Fcγ were as per the manufacturer's instructions. Wash cells further with excess cell culture medium and resuspend in 100 µl of cell culture medium. Detect and record cell-related fluorescence using flow cytometry (Beckman Coulter, Cytoflex). A logarithmic shift to the right of the autofluorescence peak toward isotype control cells is considered a logarithmic shift (or MUC1+), and subsequent shifts are considered two shifts (or MUC1++), three shifts (or MUC1+++), etc.

[0248] Dose-dependent binding of surface MUC1 expression on human cancer cells was determined by flow cytometry: Human cancer cell lines (COLO357, HT29, and SKOV3) or mouse cancer cell lines expressing human MUC1 (DA3-TM) were collected enzymatically using Duchenne phosphate-buffered saline (D-PBS) containing 0.25% trypsin and 0.2% EDTA, washed with excess cell culture medium, and cultured in cell line-specific medium at 1–2 × 10⁻⁶. 6 Resuspend cells at a density of 100 µl / mL. Add anti-MUC1-SEA mAb (chimeric or humanized IgG) / ADC or reference IgG antibody / ADC to the cells at concentrations ranging from 0–50 nM. Incubate cells at 4 °C for 1 hour and wash with excess cell culture medium. Further stain these cells at 4 °C for 1 hour with goat anti-human IgG and Fcγ conjugated with Alexa Fluor 647 (Jackson Immunological Research, Sigrove, PAN, USA). Concentrations of goat anti-human IgG and Fcγ were as per the manufacturer’s instructions. Further wash cells with excess cell culture medium and resuspend in 100 µl of cell culture medium. Detect and record cell-associated fluorescence using flow cytometry (Beckman Coulter, Cytoflex). Mean fluorescence intensity was obtained from FlowJo software, and a graph of concentration versus mean fluorescence intensity was plotted in Graphpad Prism.

[0249] Internalization of cell-bound anti-MUC1-SEA antibody was determined by flow cytometry: EG Kim et al., *Biomolecules*, 2020, 10, 955; doi:10.3390 / biom10060955. The procedure was slightly modified, where the percentage of intracellularly bound antibody to the cell surface was determined by comparing the net decrease in the geometric mean fluorescence (MFI) of the secondary antibody relative to the primary antibody signal normalized at 10 µg / ml at 37°C with the corresponding control MFI relative to the primary antibody signal normalized at 0 min. The relative MFI of the sample relative to the control at each time point x (t = x = 0, 0.5, 1, 2, and 4 hours) was calculated as follows:

[0250] Normalized MFI (surface / total) = MFI from secondary antibody / MFI from primary antibody

[0251] Relative MFI (% relative to control) = (MFI surface area / total) t= x / MFI surface / total t= 0 ) X 100, where

[0252] MFI t = 0 MFI is the control at time 0.

[0253] MFI t = x It is the MFI of the matched sample at 37°C at the indicated time point x.

[0254] Then the percentage of antibody internalization at each time point x is calculated as follows:

[0255] Internalization (%) = 100% - relative MFI (% relative to control)

[0256] Internalization assessment of anti-MUC1-SEA antibody was performed using microscopy: 10,000 Colo357 cells seeded in 8-well slides were treated with either 10 µg / ml Alexa647-labeled antibody or an Alexa647-labeled isotype control and incubated at 37°C for 24 h in a humidified incubator set to 5% CO2. After 24 hours of treatment, the cells were washed with complete culture medium and then fixed with 2% paraformaldehyde at room temperature (RT) for 10–15 min. After fixation, the cells were washed, the nuclei were reverse stained with Hoechst dye for 5 min, and the slides were mounted with coverslips using Fluoromount-G. The cells were imaged using a fluorescence microscope at a 40X objective, and the images were processed in ImageJ software. The Alexa647 signal is shown in red, while the nuclear counterstain is shown in blue.

[0257] Binding strength was assessed using surface plasmon resonance (SPR): MUC1 antigen (Acrobiosystems, catalog MU1-H52H9) was captured for 60 seconds on anti-His immobilized on a CM5 chip at a flow rate of 5 μl / min. Antibody or ADC in this concentration range of 0–50 nM was passed through the MUC1 antigen at a flow rate of 30 µl / min for 120 seconds, followed by a dissociation time of 1800 seconds at 25°C. The resulting dose response was evaluated on BIAevaluation software to calculate the antibody affinity for the antigen, as measured by the dissociation constant (KD).

[0258] Growth inhibition of human cancer cell lines using MMAE-based ADCs

[0259] Human cancer cell lines (such as Colo357, HT29, SKOV3, BxPC3, and T47D) were seeded at 5000 cells / well in 96-well flat-bottomed clear plates and allowed to adhere overnight. The next day, antibodies / ADCs were added to these cells at the mentioned concentration or within the mentioned concentration range. After 5–7 days of treatment, 20 μl of presto blue dye was added to the wells, and the absorbance was recorded at 570 nm (600 nm reference wavelength). The survival percentage was calculated for wells containing untreated cells in complete culture medium, and the IC50 was calculated in Graphpad Prism.

[0260] result

[0261] MUC1 is expressed in various human cancer cell lines: such as Figures 4A-4D As shown, a series of human cancer cell lines were screened for surface MUC1 expression using chimeric IgG1 anti-MUC1-SEA mAb. These cancer cell lines were then classified as MUC1+, MUC1++, and MUC1+++ based on the logarithmic shift of MUC1 fluorescence compared to a reference negative control. Among all screened cell lines, Colo357 and ZR75 were found to have the highest surface MUC1 expression and were designated as MUC1+++; while HS766T, Jurkat, and Raji were found to have no detectable surface MUC1. After humanization of the chimeric IgG1 anti-MUC1-SEA mAb, the binding of humanized IgG1 anti-MUC1-SEA mAb (HM5, LM4) to human cancer cell lines with different MUC1 expression levels was also examined. Figures 5A-5E As shown in the image.

[0262] Dose-dependent binding of humanized IgG1 anti-MUC1-SEA mAb (HM5 LM4) in human cancer cell lines: Dose-dependent binding of humanized IgG1 anti-MUC1-SEA mAb (HM5 LM4) in the range of 0 to 50 nM was performed using flow cytometry in human cancer cell lines expressing high, intermediate, and low / no MUC1, as shown in Figures 6A-6C. Colo357 (MUC1+++ cell line), SKOV3 (MUC1++ cell line), and HT29 (MUC1- / + cell line) showed a dose-dependent increase in MUC1-related fluorescence signal. Dose response plotted relative to mean fluorescence intensity was obtained. Figure 6D It is displayed in the middle.

[0263] Humanized IgG anti-MUC1-SEA mAb was internalized in MUC1-expressing cell lines: Approximately 40% of the humanized IgG1 anti-MUC1-SEA mAb (HM5 LM4) was internalized in the MUC1-SEA-expressing COLO357 cell line, as shown in Table 4 below. Internalization was further confirmed by microscopic imaging of MUC1-SEA-expressing COLO357 cells using Alexa647-labeled humanized IgG anti-MUC1-SEA mAb, as shown in Table 4. Figure 7 As shown. Humanized anti-MUC1 SEA IgG1 (red) staining on the surface and cytoplasm of COLO-357 cell lines expressing MUC1 indicates binding and internalization.

[0264] Table 4

[0265]

[0266] The binding strength of HM5LM4-ADC (humanized IgG anti-MUC1-SEA mAb conjugated with MC-Val-Cit-PABC-MMAE) is similar to that of unconjugated humanized IgG anti-MUC1-SEA mAb: MC-Val-Cit-PABC-MMAE linker - payload ( Figure 8 After conjugating humanized IgG1 anti-MUC1-SEA mAb to prepare an antibody-drug conjugate (ADC) called HM5LM4-ADC, the binding strength remained similar to that of the unconjugated parental antibody. This was demonstrated by comparing the dose-response of HM5LM4-ADC and the unconjugated antibody with COLO357 cells using flow cytometry. Figures 9A-9E Similarly, the dissociation constant of HM5LM4-ADC has been verified using SPR, and the unconjugated antibody remains comparable, i.e., approximately 40 pM antigen, as shown. Figure 10A and 10B And as shown in Table 5.

[0267] Table 5

[0268]

[0269] HM5LM4-ADC specifically exerts its cytotoxic effect on cell lines highly expressing MUC1: Target-specific cytotoxicity of HM5LM4-ADC was investigated in human cancer cell lines exhibiting different levels of MUC1. Data showed that in vitro cytotoxicity of HM5LM4-ADC was observed only in the COLO-357, BxPC3, and T47D cell lines highly expressing MUC1-SEA. Figure 12A-12B 13A-13B and 15A-15B). However, in cell lines that do not express and moderately express MUC1, namely HT-29 ( Figure 11A and 11B ) and SKOV-3 ( Figure 14A and 14B No cytotoxic effects were observed in the cells.

[0270] Table 6-8 below shows the IC50 values ​​of HM5LM4-ADC and allotype control ADC, as well as their corresponding equivalent MMAE and free MMAE, against COLO-357 and BxPC3 pancreatic cancer cells and T47D breast cancer cells. 50 .

[0271] Table 6

[0272]

[0273] Table 7

[0274]

[0275] Table 8

[0276]

[0277] Example 4

[0278] Efficacy of HM5LM4-ADC in xenograft mouse models

[0279] To evaluate the antitumor efficacy of HM5LM4-ADC, a human tumor xenograft mouse model was used. Human cancer cell lines suspended in a mixture of serum-free growth medium and matrix gel (1:1 ratio) were subcutaneously seeded into the right lateral region. After palpable tumors formed, animals were randomly assigned to different groups to ensure a uniform distribution of animals with comparable mean tumor volumes within each group. Treatment was then administered to the animals. Animal treatment and intravenous treatment were performed in a sterile environment throughout the study duration. Tumor size, body weight, and animal health signs were collected twice weekly during the study period. Response to treatment was interpreted by calculating tumor growth inhibition (TGI), tumor regression, partial or complete response, changes in body weight, and general health signs.

[0280] Calculate the TGI percentage using the following formula: (1 - (processing mm) 3 最终 -Process mm 3 初始 ) / (Comparison mm 3 最终 -Compare with mm 3 初始 )) × 100. Calculate the tumor regression percentage using the following formula: ((treatment) 初始 -deal with 最终 ) / deal with 初始 ) × 100

[0281] The criteria for partial response (PR) are as follows: a reduction of at least 30% compared to the initial tumor volume.

[0282] The criteria for complete response (CR) are as follows: complete disappearance of the tumor.

[0283] All values ​​are expressed as mean ± SEM (standard error of the mean) and power statistics were analyzed using two-way ANOVA and Dunnett's multiple comparisons test using GraphPad Prism (version 9.3.1). The statistics shown in the figure are for the last time point.

[0284] In vivo antitumor efficacy of HM5LM4-ADC in a mouse ovarian cancer xenograft model

[0285] The antitumor efficacy of HM5LM4-ADC was evaluated in a xenograft model of the SKOV3-derived ovarian cancer cell line expressing MUC1. Three days prior to cell seeding, animals were supplemented with subcutaneously implanted estrogen deposits. Female Balb / c athymic nude mice with subcutaneous tumors were intravenously administered (a) 1.5, 3, or 6 mg / kg HM5LM4-ADC, (b) HM5-LM4 (5F3) antibody, or (c) 0.12 mg / kg free MMAE payload (equivalent to 6 mg / kg HM5LM4-ADC) every four days for a total of six doses, and tumor growth and health signs were monitored. Results are shown in… Figure 18 In the middle. HM5LM4-ADC in Figure 18 The drug is referred to as 5F3-HM5-LM4 ADC. Treatment with HM5LM4-ADC at 6 mg / kg yielded a tumor growth inhibition (TGI) of 99 ± 5% (p < 0.0001), and at 3 mg / kg, the TGI was 63 ± 14% (p < 0.0001), while treatment with 1.5 mg / kg HM5LM4-ADC resulted in a marginal TGI of 45 ± 17% (p < 0.05). A free MMAE payload of 0.12 mg / kg also showed a TGI of 106 ± 10% (p < 0.0001). In the xenograft model, all dosing regimens of the drug and HM5LM4-ADC were well tolerated; however, death was observed in 3 of the 8 animals that received free MMAE.

[0286] In vivo antitumor efficacy of HM5LM4-ADC in a mouse pancreatic cancer xenograft model

[0287] The antitumor efficacy of HM5LM4-ADC was evaluated in a COLO 357-derived xenograft model of pancreatic cancer cells expressing MUC1. Female Balb / c athymic nude mice with subcutaneous tumors were administered HM5LM4-ADC at doses of 1, 1.5, 2, and 3 mg / kg or rituximab ADC (3 mg / kg as a non-binding control) intravenously every four days for a total of six doses, and tumor growth and health signs were monitored. Results are shown in… Figure 19 In the xenograft model, the tumor growth inhibition (TGI) achieved with treatment of 3 mg / kg HM5LM4-ADC was 76 ± 6% (p < 0.0001), and the TGI was 60 ± 8% (p < 0.0001) with 2 mg / kg, while the TGI was 43 ± 7% (p = 0.001) with 1.5 mg / kg HM5LM4-ADC. The 3 mg / kg rituximab ADC was ineffective (TGI = 12 ± 11). All dosing regimens of the catalyst and HM5LM4-ADC were well tolerated in the xenograft model.

[0288] In vivo antitumor efficacy of HM5LM4-ADC in a mouse xenograft model of breast cancer

[0289] The antitumor efficacy of HM5LM4-ADC was evaluated in a MCF7-derived xenograft model of breast cancer cell line expressing MUC1. Three days prior to cell seeding, animals were supplemented with subcutaneously implanted estrogen deposits. Female Balb / c athymic nude mice with subcutaneous tumors were intravenously administered 1, 2, or 3 mg / kg HM5LM4-ADC, 3 mg / kg rituximab ADC (as a non-binding control), or 0.06 mg / kg free MMAE payload every four days for a total of six doses, while tumor growth and health signs were monitored. Results are shown in… Figure 20 In the free MMAE treatment group, the tumor growth inhibition (TGI) was 109 ± 8% (p < 0.0001) for 3 mg / kg HM5LM4-ADC treatment and 104 ± 9% (p < 0.0001) for 2 mg / kg treatment. HM5LM4-ADC treatment also induced tumor regression of 12 ± 20% in the 2 mg / kg group and 16 ± 16% in the 3 mg / kg group. Rituximab ADC administered at 3 mg / kg also showed efficacy with a TGI of 60 ± 14% (p < 0.001). In the free MMAE treatment group, 3 out of 8 animals died on day 22. The 2 mg / kg HM5LM4-ADC regimen completely inhibited the growth of the MCF7 xenograft.

[0290] In vivo antitumor efficacy of HM5LM4-ADC in a mouse head and neck cancer xenograft model

[0291] The antitumor efficacy of HM5LM4-ADC was evaluated in a xenograft model derived from the Detroit 562 head and neck cancer cell line expressing MUC1. Female Balb / c athymic nude mice with subcutaneous tumors were administered intravenously at doses of 1, 2, or 3 mg / kg HM5LM4-ADC, 3 mg / kg rituximab ADC (as a non-binding control), or 0.06 mg / kg free MMAE payload for a total of six doses every four days, while tumor growth and health signs were monitored. Results are shown in… Figure 21 In the xenograft model, the tumor growth inhibition (TGI) achieved with treatment of 3 mg / kg HM5LM4-ADC was 72 ± 5% (p < 0.001), and the TGI at 2 mg / kg was 60 ± 11% (p < 0.05). Rituximab ADC administered at 3 mg / kg also showed a TGI of 70 ± 7% (p < 0.01). All dosing regimens of the rutabsid and HM5LM4-ADC were well tolerated in the xenograft model.

[0292] In vivo antitumor efficacy of HM5LM4-ADC in a mouse pancreatic cancer xenograft model

[0293] The antitumor efficacy of HM5LM4-ADC was evaluated in a xenograft model derived from the MUC1-expressing cell line BxPc3-Luc. Female Balb / c athymic nude mice carrying orthotopically established tumors were administered intravenously at 3 mg / kg HM5LM4-ADC, rituximab ADC (as a non-binding control), or an equivalent free MMAE payload of 0.06 mg / kg every four days for a total of six doses. Within 15 minutes of injection of the D-fluorescein substrate (150 mg / kg, intraperitoneally, single dose / mouse), whole-animal imaging in an IVIS imaging system revealed tumor growth with increasing bioluminescence imaging (BLI) signal measured in photons per second. Results are shown in… Figure 22 In all groups, treatment with HM5LM4-ADC initially reduced tumor growth; however, tumor regrowth occurred with a marginal tumor growth inhibition (TGI) of 65 ± 25%. All treatments were well tolerated in the orthotopic xenograft model.

[0294] In vivo antitumor efficacy of HM5LM4-ADC in a mouse lung cancer xenograft model

[0295] The antitumor efficacy of HM5LM4-ADC was evaluated in a xenograft model derived from the A549 cell line, which expresses low MUC1. Male Balb / c athymic nude mice with subcutaneous tumors were administered 3 mg / kg HM5LM4-ADC or rituximab ADC (as a non-binding control) intravenously every four days for a total of six doses. Results are shown in... Figure 23 Treatment with HM5LM4-ADC resulted in a moderate tumor growth inhibition (TGI) of 50 ± 7% (p < 0.0001). All treatments were well tolerated in the xenograft model.

[0296] Example 5

[0297] Stability and Pharmacokinetics of HM5LM4-ADC

[0298] Stability of HM5LM4-ADC in mouse and human plasma

[0299] The stability of HM5LM4-ADC in mouse and human plasma was evaluated by monitoring MMAE release. HM5LM4-ADC was incorporated into plasma at an equivalent MMAE concentration of 5000 nM and incubated at 37°C for a maximum of 48 hours at predefined time points. At the end of 48 hours, MMAE release in human plasma was < 5 nM, while in mouse plasma it was 214 nM, corresponding to < 0.1% and 4.5% MMAE release in human and mouse plasma, respectively.

[0300] Stability of HM5LM4-ADC in purified cathepsin B enzyme assay

[0301] The stability of HM5LM4-ADC in purified cathepsin B was evaluated by monitoring MMAE release. HM5LM4-ADC was incubated at a predefined time point at 37°C with an equivalent concentration of 5000 nM MMAE for up to 6 hours. At the end of incubation, 2800 nM of MMAE was released, corresponding to 56% MMAE release. The data indicate that cathepsin B mediates payload release by acting on the val-cit bond.

[0302] Pharmacokinetics of HM5LM4-ADC in tumor-bearing mice

[0303] 1. Single-dose pharmacokinetic studies of HM5LM4-ADC administered intravenously and intraperitoneally in mice carrying COLO357 tumors.

[0304] The aim of this study was to evaluate the plasma and tumor pharmacokinetics of HM5LM4-ADC, total antibody (TAB), and unconjugated MMAE (uMMAE) after a single intravenous (IV) or intraperitoneal (IP) administration of 10 mg / kg to mice carrying COLO357 tumors.

[0305] Blood samples were collected from mice via the retroorbital plexus at 0.017, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 168, and 336 hours post-drug administration (n = 3 mice / time point; sparse sampling design), and plasma was collected immediately. Tumors were collected at 24, 48, 72, 96, 168, and 336 hours. Analysis of total antibody (TAB) and conjugated antibody (CAB) was performed using applicable ELISA methods, and analysis of free payload (MMAE) was performed using applicable LC-MS / MS methods. Pharmacokinetic analyses were performed using Phoenix WinNonlin. ® The non-compartmental analysis tool of the software was used. The results are shown in Tables 9 and 10 below.

[0306] Table 9

[0307]

[0308]

[0309] Table 10

[0310]

[0311] Typically, intraperitoneal administration of CAB and TAB results in approximately twice the exposure of intravenous administration. Compared to plasma, MMAE results in significantly higher tumor exposure, which is comparable across both routes of administration.

[0312] 2. Single-dose pharmacokinetic study of HM5LM4-ADC administered intravenously to mice carrying COLO357 tumors.

[0313] The aim of this study was to evaluate the serum, tumor, and hepatic pharmacokinetics of TAB, CAB, and uMMAE after a single intravenous administration of 3 mg / kg HM5LM4-ADC in a mouse model carrying COLO357 tumors, as well as the pharmacokinetics of MMAE after a single intravenous administration of 0.06 mg / kg MMAE.

[0314] Blood samples were collected from mice via the retroorbital plexus at 0.083, 0.5, 1, 2, 4, 8, 24, 48, 72, 96, 168, and 336 hours post-drug administration (n = 3 mice / time point; sparse sampling design), and serum was collected immediately. Tumor and liver samples were collected at 24, 48, 72, 96, 168, and 336 hours. Analysis of total antibody (TAB) and conjugated antibody (CAB) was performed using applicable ELISA methods, and analysis of free payload (MMAE) was performed using applicable LC-MS / MS methods. Pharmacokinetic analyses were performed using Phoenix WinNonlin. ® The non-compartmental analysis tool of the software was used. The results are shown in Tables 11 and 12 below.

[0315] Table 11

[0316]

[0317] Table 12

[0318]

[0319] In mice, a single intravenous administration of HM5LM4-ADC resulted in tumor exposure that was 240 times higher than serum exposure and 6.2 times higher than liver exposure. Similarly, a single intravenous administration of MMAE in mice resulted in tumor exposure that was 66 times higher than serum exposure and 7 times higher than liver exposure.

[0320] 3. Single-dose pharmacokinetic study of HM5LM4-ADC administered intravenously to mice carrying SKOV3 tumors.

[0321] The aim of this study was to evaluate the serum, tumor, and tissue pharmacokinetics of TAB, CAB, and unconjugated MMAE in a mouse model carrying SKOV3 tumors following a single intravenous administration of HM5LM4-ADC at a dose of 3 mg / kg.

[0322] Blood samples were collected from mice via the retroorbital plexus at 0.083, 0.5, 1, 2, 4, 24, 48, 72, 96, and 168 hours post-drug administration (n = 3 mice / time point; sparse sampling design), and serum was collected immediately. Tumor, liver, brain, spleen, lung, and kidney tissues were collected at 24, 48, 72, 96, and 168 hours. Analysis of total antibody (TAB) and conjugated antibody (CAB) was performed using applicable ELISA methods, and analysis of free payload (MMAE) was performed using applicable LC-MS / MS methods. Pharmacokinetic analyses were performed using Phoenix WinNonlin. ® The non-compartmental analysis tool of the software was used. The results are shown in Tables 13 and 14 below.

[0323] Table 13

[0324]

[0325] Table 14

[0326]

[0327] In mice, after a single intravenous administration of HM5LM4-ADC, the exposure of uMMAE in tumors was 71 times that in serum, and the tissue / plasma exposure ratio of uMMAE in descending order was: tumor (71) > liver (39) > kidney (31) > spleen (16) > lung (10) > brain (1.8).

[0328]

[0329] Table A below provides the sequences described above. SEQ ID NO: 1-15 are in the form FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Bold text in the sequences indicates CDR regions. Underlined text indicates mutations.

[0330] Table A

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338] All publications, patents, and patent applications cited herein are hereby incorporated by reference as if they were fully set forth herein. Although the invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of the illustrative embodiments and other embodiments of the invention will be apparent to those skilled in the art during the reference to this description. Therefore, the appended claims are intended to cover such modifications and enhancements.

Claims

1. A humanized monoclonal antibody that binds to the MUC1 SEA domain, wherein the antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NO. 7-11, 45, and 46; and (b) A light chain variable region comprising the amino acid sequence of any one of SEQ ID NO. 12-15.

2. The humanized antibody according to claim 1, wherein the antibody comprises: (a) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 12; (b) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; (c) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; (d) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 9 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13; (e) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 9 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; (f) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 13; (g) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; (h) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO: 13; (i) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 11 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14; or (j) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 11 and the light chain variable region contains the amino acid sequence of SEQ ID NO:

15.

3. The humanized antibody according to claim 1 or 2, wherein the constant domain of the heavy chain comprises the amino acid sequence of SEQ ID NO: 39, and the constant domain of the light chain comprises the amino acid sequence of SEQ ID NO:

40.

4. A humanized monoclonal antibody that binds to the MUC1 SEA domain, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and the light chain variable region comprising the amino acid sequence of SEQ ID NO:

15.

5. A humanized monoclonal antibody that binds to the MUC1 SEA domain, wherein the antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 37, and the light chain comprising the amino acid sequence of SEQ ID NO:

38.

6. The humanized antibody according to claim 5, wherein a portion The light chain of the humanized antibody is conjugated to its maleimide terminus.

7. The humanized monoclonal antibody according to claim 5 or 6, wherein the heavy chain has the amino acid sequence of SEQ ID NO: 37 and the light chain has the amino acid sequence of SEQ ID NO:

38.

8. The humanized monoclonal antibody according to any one of the preceding claims, wherein its antigen-binding fragment is Fv, single-chain Fv (scFv), single-chain Fv-Fc (scFv-Fc), Fab', Fab, F(ab')2 or F(ab)2.

9. A humanized antibody that binds to an epitope in the MUC1 SEA domain, preferably having a binding affinity K of less than 100 pM. D .

10. The humanized antibody of claim 9, wherein the humanized antibody comprises means for binding to an epitope in the SEA domain (SEQ ID NO: 41) of MUC1, the epitope being formed by arginine at position 1108, glutamic acid at position 1109, asparagine at position 1113, and glutamic acid at position 1118 of MUC1.

11. The humanized antibody according to claim 9 or 10, wherein the humanized antibody binds to the same epitope as the chimeric antibody, the chimeric antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region comprising the amino acid sequences of SEQ ID NO: 1 and 2, respectively.

12. The humanized antibody according to claim 9 or 10, wherein the antibody is a humanized form of any one of the chimeric antibodies DMB4F4 (4F4), DMB7F3 (7F3), or DMB10F10 (10F10).

13. A humanized antibody that binds to an epitope in the SEA domain (SEQ ID NO: 41) of MUC1, said epitope being formed by arginine at position 1108, glutamic acid at position 1109, asparagine at position 1113, and glutamic acid at position 1118 of MUC1.

14. A humanized antibody that binds to the same epitope as a chimeric antibody, said chimeric antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region and light chain variable region comprising the amino acid sequences of SEQ ID NO: 1 and 2, respectively.

15. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody according to any one of the preceding claims.

16. An expression vector comprising the isolated nucleic acid molecule according to claim 15.

17. A host cell transfected with the expression vector according to claim 16.

18. An immunoconjugate comprising an antibody according to any one of claims 1 to 14, and additional cytotoxic or therapeutic agents.

19. The immunoconjugate according to claim 18, wherein the cytotoxic agent is selected from the group consisting of: alkylating agents, anthracycline drugs, pyrimidine derivatives, vinca alkaloids, photodynamic drugs, platinum-containing compounds, taxanes, topoisomerase inhibitors, ribosome inactivators, DNA damage-inducing agents, tubulin inhibitors, antimitotic agents, radioisotopes, cytotoxic antibodies, and bacterial toxins.

20. The immunoconjugate of claim 18, wherein the cytotoxic agent is a pseudomonas exotoxin.

21. The immunoconjugate of claim 18, wherein the cytotoxic agent is monomethyloretine E (MMAE).

22. The immunoconjugate of claim 21, wherein the antibody is conjugated with maleimide hexanoyl-Val-Cit-PABC-MMAE.

23. An immunoconjugate comprising a humanized monoclonal antibody conjugated with maleimide hexanoyl-Val-Cit-PABC-MMAE, wherein the antibody (i) binds to the MUC1 SEA domain and (ii) comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and the light chain comprising the amino acid sequence of SEQ ID NO:

38.

24. The immunoconjugate according to any one of claims 18 to 23, wherein the immunoconjugate reduces tumor volume after being administered to a subject with cancer.

25. A pharmaceutical composition comprising (a) a humanized monoclonal antibody according to any one of claims 1 to 14, an immunoconjugate according to any one of claims 18 to 24, and (b) a pharmaceutically acceptable carrier, excipient, or diluent.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition further comprises an additional therapeutic agent.

27. A method of treating or improving a disease or condition, the method comprising administering to a subject in need a therapeutically effective amount of at least one humanized monoclonal antibody according to any one of claims 1 to 14, or an immunoconjugate according to any one of claims 18 to 24, or a pharmaceutical composition according to claim 25 or 26.

28. The method of claim 27, wherein the disease or condition is cancer.

29. The method of claim 28, wherein the cancer is a cancer expressing MUC1.

30. The method of claim 28, wherein the cancer is selected from the following: lung cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, pancreatic cancer, multiple myeloma, and acute myeloid leukemia.

31. The method of claim 27, wherein the disease or condition is an autoimmune or inflammatory disease.

32. The method of claim 31, wherein the autoimmune or inflammatory disease is selected from the following: rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, amyloidosis, and autoimmune pancreatitis.

33. The method of claim 27, wherein the disease or symptom is a non-malignant, clinically significant abnormal growth condition.

34. The method according to any one of claims 27 to 33, wherein the method further comprises administering an additional therapeutic agent to a subject in need.

35. The monoclonal antibody according to any one of claims 1 to 14, the immunoconjugate according to any one of claims 18 to 24, or the pharmaceutical composition according to claim 25 or 26, used in a method of treating or improving a disease or condition, the method comprising administering a therapeutically effective amount of the monoclonal antibody, the immunoconjugate, or the pharmaceutical composition to a subject in need.

36. The monoclonal antibody, immunoconjugate, or pharmaceutical composition for use according to claim 35, wherein the disease or condition is cancer.

37. The monoclonal antibody, immunoconjugate, or pharmaceutical composition for use according to claim 35, wherein the disease or condition is an autoimmune or inflammatory disease, or wherein the disease or condition is a non-malignant abnormal growth condition.

38. The monoclonal antibody, immunoconjugate, or pharmaceutical composition for use according to any one of claims 35 to 37, wherein the method further comprises administering an additional therapeutic agent to a subject in need.

39. A method for diagnosing a disease or condition in a subject, wherein the disease or condition is associated with MUC-1 expression, the method comprising: (a) Contacting a biopsy obtained from a patient with at least one monoclonal antibody according to any one of claims 1 to 14; and (b) Detect the monoclonal antibody, The detection of cells overexpressing MUC1 SEA in the biopsy indicates that the subject has been diagnosed with the disease or condition.

40. The method of claim 39, wherein the disease or condition is cancer.

41. The method of claim 39, wherein the disease or condition is an autoimmune or inflammatory disease, or wherein the disease or condition is a non-malignant abnormal growth condition, such as a cyst, such as a kidney cyst, a thyroid cyst and a thyroid nodule, or a liver cyst.

42. The method according to any one of claims 39 to 41, wherein the monoclonal antibody is detectably labeled.

43. A method for imaging a disease or symptom, the method comprising: (a) Introducing at least one monoclonal antibody according to any one of claims 1 to 14 into a subject, wherein the antibody is detectably labeled with a radioisotope or a visual agent; and (b) Visualize detectable labeled monoclonal antibodies. The detection of cells and / or tissues labeled with the isotope or visualization agent indicates the presence and / or location and / or extent and / or presence of the disease or condition in the subject.

44. The method of claim 43, wherein the disease or condition is cancer.

45. The method of claim 43, wherein the disease or condition is an autoimmune or inflammatory disease, or wherein the disease or condition is a non-malignant abnormal growth condition, such as a cyst, such as a kidney cyst, a thyroid cyst and a thyroid nodule, or a liver cyst.

Images