Methods of Treating Disease Using Gremlin 1 Antagonists

Abstract

The present disclosure provides herein a method of treatment for GREM1-associated diseases or conditions characterized by PTEN and / or p53 deficiency. A method of treatment for castration-resistant prostate cancer is also provided by the present disclosure.

Description

[Technical field]

[0001]

[0001] This disclosure relates generally to novel methods of treating GREM1-associated diseases using GREM1 antagonists. [Background technology]

[0002]

[0002] Gremlin 1 is a highly conserved secreted protein in the DAN family of BMP antagonists. It was reported that Gremlin 1 binds to BMP-2, BMP-4 or BMP-7 to form heterodimers, preventing BMP ligands from interacting with the corresponding BMP receptors, and subsequently inhibiting the activation of BMP signaling. Gremlin 1 is a central protein during embryogenesis and is closely related to tissue fibrotic lesions as well as glioma and colon cancer. However, our understanding of Gremlin 1 as a secreted protein is by no means detailed. Except for the BMP signaling pathway, it has not been clarified whether Gremlin 1 exerts its function through non-BMP mechanisms. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0003] Therefore, there is a need to investigate new medical applications of Gremlin 1 targeting agents. [Means for solving the problem]

[0004]

[0004] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means "one antibody" or "more than one antibody."

[0005]

[0005] In one aspect, the disclosure provides a method of treating a GREM1-expressing disease or condition in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of a GREM1 antagonist to the subject, wherein the disease or condition is characterized by reduced or inhibited androgen receptor (AR) signaling.

[0006]

[0006] In certain embodiments, the subject is receiving or has received an AR inhibitor. In certain embodiments, the disease or condition is resistant to an AR inhibitor.

[0007]

[0007] In certain embodiments, the disease or condition is an AR-associated cancer (e.g., prostate cancer, breast cancer, glioblastoma, melanoma, bladder cancer, renal cell carcinoma, pancreatic cancer, hepatocellular carcinoma, ovarian cancer, endometrial cancer, mantle cell lymphoma, or salivary gland cancer), or an AR-associated non-cancer condition (e.g., hair loss, acne, hirsutism, ovarian cysts, polycystic ovarian disease, precocious puberty, spinal and bulbar muscular atrophy, or age-related macular degeneration).

[0008]

[0008] In one aspect, the present disclosure provides a method of treating a GREM1-expressing cancer in a subject in need of such treatment, the method comprising administering a therapeutically effective amount of a GREM1 antagonist to the subject, wherein the cancer is characterized by reduced androgen receptor (AR) signaling.

[0009]

[0009] In some embodiments, the cancer is an AR-expressing cancer or an AR-negative cancer.

[0010]

[0010] In some embodiments, the cancer is prostate cancer, breast cancer, lung cancer, head and neck cancer, testicular cancer, endometrial cancer, ovarian cancer, and skin cancer.

[0011]

[0011] In some embodiments, the subject is undergoing or has undergone androgen deprivation therapy, or is resistant to androgen deprivation therapy.

[0012]

[0012] In some embodiments, the cancer is further determined to be deficient in PTEN and / or p53.

[0013] In some embodiments, the cancer is metastatic. In some embodiments, the cancer is metastatic prostate cancer.

[0014] In some embodiments, the cancer is lung metastasis of cancer. In some embodiments, the cancer is lung metastasis of prostate cancer.

[0015] In some embodiments, the cancer is prostate cancer. In some embodiments, the prostate cancer is: a) negative for androgen receptor (AR) expression; b) negative for both androgen receptor (AR) expression and neuroendocrine (NE) differentiation; c) resistant to androgen deprivation therapy, optionally castration resistant; d) exhibits a prostate specific antigen (PSA) level lower than a reference level; or e) any combination of a)-d).

[0016]

[0016] In some embodiments, the cancer is characterized by GREM1 overexpression.

[0017]

[0017] In one aspect, the disclosure provides a method for increasing the sensitivity of an AR-expressing cancer to androgen deprivation therapy in a subject, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist.

[0018]

[0018] In one aspect, the present disclosure provides a method for treating a GREM1-related disease or condition characterized by PTEN and / or p53 deficiency in a subject in need of such treatment, or for inhibiting FGFR1 activation in a subject in need of such inhibition, or for inhibiting MAPK signaling in a subject in need of such inhibition, comprising the step of administering a therapeutically effective amount of a GREM1 antagonist to the subject.

[0019]

[0019] In some embodiments, the deficiency of PTEN and / or p53 is characterized by the absence of functional PTEN and / or p53.

[0020]

[0020] In some embodiments, the deficiency of PTEN and / or p53 is characterized by the presence of an inactivating mutation in PTEN and / or p53.

[0021]

[0021] In some embodiments, PTEN and / or p53 deficiency is characterized by the absence of PTEN and / or p53 expression.

[0022]

[0022] In some embodiments, the GREM1-associated disease or condition is characterized by GREM1 expression or overexpression.

[0023]

[0023] In some embodiments, the GREM1-related disease or condition is selected from the group consisting of cancer, fibrotic disease, angiogenesis, glaucoma or retinal disease, renal disease, pulmonary arterial hypertension, and osteoarthritis (OA).

[0024]

[0024] In some embodiments, the GREM1-associated disease or condition is cancer.

[0025]

[0025] In some embodiments, the cancer is prostate cancer, breast cancer, glioma, liposarcoma, hepatocellular carcinoma, lung cancer, cervical cancer, endometrial cancer, uterine leiomyosarcoma, squamous cell carcinoma of the head and neck, thyroid cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, esophageal cancer, bile duct cancer, osteosarcoma, glioblastoma, ovarian cancer, gastric cancer, triple-negative breast cancer (TNBC), small cell lung cancer or melanoma.

[0026]

[0026] In some embodiments, the cancer is prostate cancer.

[0027]

[0027] In some embodiments, the prostate cancer a) is negative for androgen receptor (AR) expression, b) is negative for both androgen receptor (AR) expression and neuroendocrine (NE) differentiation; c) is resistant to androgen deprivation therapy, optionally castration resistant, d) exhibits a prostate-specific antigen (PSA) level lower than a reference level, or e) any combination of a)-d).

[0028]

[0028] In some embodiments, the cancer is breast cancer.

[0029]

[0029] In some embodiments, the breast cancer is triple-negative breast cancer.

[0030]

[0030] In some embodiments, the fibrotic disease is pulmonary fibrosis, dermal fibrosis, diabetic nephropathy, or ischemic kidney injury.

[0031]

[0031] In some embodiments, the GREM1 antagonist reduces GREM1 levels or activity.

[0032]

[0032] In some embodiments, the GREM1 antagonist reduces GREM1 activity selectively in cancer cells relative to non-cancer cells.

[0033]

[0033] In some embodiments, the GREM1 antagonist includes an anti-GREM1 antibody or an antigen-binding fragment thereof, an inhibitory GREM1 mimetic peptide, an inhibitory nucleic acid targeting GREM1 RNA or DNA, a polynucleotide encoding the inhibitory nucleic acid, a compound that inhibits the interaction between gremlin and BMP, or a compound that inhibits GREM1 activity.

[0034]

[0034] In some embodiments, the inhibitory nucleic acid targeting GREM1 RNA or DNA includes a small hairpin RNA (shRNA), a microinterfering RNA (miRNA), a double-stranded RNA (dsRNA), a small interfering RNA (siRNA), a guide RNA, or an antisense oligonucleotide.

[0035]

[0035] In some embodiments, the GREM1 antagonist comprises a GREM1-FGFR1 axis inhibitor.

[0036]

[0036] In some embodiments, the GREM1-FGFR1 axis inhibitor inhibits GREM1-dependent FGFR1 signaling.

[0037]

[0037] In some embodiments, the GREM1-FGFR1 axis inhibitor blocks the binding between GREM1 and FGFR1.

[0038]

[0038] In some embodiments, the GREM1-FGFR1 axis inhibitor comprises an FGFR1 binding inhibitor.

[0039]

[0039] In some embodiments, the FGFR1 binding inhibitor binds to the extracellular domain 2 of FGFR1, and optionally binds to FGFR1 at an epitope including residue Glu160, where the residue numbers are according to SEQ ID NO:75.

[0040]

[0040] In some embodiments, the GREM1-FGFR1 axis inhibitor binds to hGREM1 at an epitope including residues Lys123 and / or Lys124, the residue numbers being according to SEQ ID NO: 69; or blocks binding of FGFR1 to residues Lys123 and / or Lys124 of hGREM1.

[0041]

[0041] In some embodiments, the GREM1 antagonist or GREM1-FGFR1 axis inhibitor comprises an antibody or antigen-binding fragment thereof against hGREM1.

[0042]

[0042] In some embodiments, the antibody has the following characteristics: a) is capable of reducing hGREM1-mediated inhibition of BMP signaling selectively in cancer cells compared to non-cancer cells; b) exhibits less than 50% reduction in hGREM1-mediated inhibition of BMP signaling in non-cancer cells; c) is capable of binding to a chimeric hGREM1 comprising the amino acid sequence of SEQ ID NO: 68; d) is capable of binding to hGREM1 but is unable to specifically bind to mouse Gremlin 1; e) has an epitope comprising residues Gln27 and / or Asn33. The present invention includes at least one of the following: f) binding to hGREM1, where the residue numbers are according to SEQ ID NO: 69, or binding to an hGREM1 fragment including residue Gln27 and / or residue Asn33, optionally the hGREM1 fragment having a length of at least 3 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acid residues; f) being able to reduce hGREM1-mediated activation of MAPK signaling; and / or g) being able to bind to hGREM1 with a KD of 1 nM or less as measured by Fortebio.

[0043]

[0043] In some embodiments, the antibody comprises a linear epitope or a conformational epitope.

[0044]

[0044] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the heavy chain variable region comprises a) a heavy chain complementarity determining region 1 (HCDR1) comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21 and 31, b) an HCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22 and 32, and c) an HCDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 23 and 33, and / or the light chain variable region comprises d) a light chain complementarity determining region 1 (LCDR1) comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 24 and 34, e) an LCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 5, 15, 25 and 35, and f) an LCDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 6, 16, 26 and 36.

[0045]

[0045] In some embodiments, the heavy chain variable region is selected from the group consisting of: a) a heavy chain variable region comprising HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:2, and HCDR3 comprising the sequence of SEQ ID NO:3; b) a heavy chain variable region comprising HCDR1 comprising the sequence of SEQ ID NO:11, HCDR2 comprising the sequence of SEQ ID NO:12, and HCDR3 comprising the sequence of SEQ ID NO:13; c) a heavy chain variable region comprising HCDR1 comprising the sequence of SEQ ID NO:21, HCDR2 comprising the sequence of SEQ ID NO:22, and HCDR3 comprising the sequence of SEQ ID NO:23; and d) a heavy chain variable region comprising HCDR1 comprising the sequence of SEQ ID NO:31, HCDR2 comprising the sequence of SEQ ID NO:32, and HCDR3 comprising the sequence of SEQ ID NO:33.

[0046]

[0046] In some embodiments, the light chain variable region is selected from the group consisting of: a) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 4, an LCDR2 comprising the sequence of SEQ ID NO: 5, and an LCDR3 comprising the sequence of SEQ ID NO: 6; b) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 14, an LCDR2 comprising the sequence of SEQ ID NO: 15, and an LCDR3 comprising the sequence of SEQ ID NO: 16; c) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 24, an LCDR2 comprising the sequence of SEQ ID NO: 25, and an LCDR3 comprising the sequence of SEQ ID NO: 26; and d) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 34, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 36.

[0047] In some embodiments, a) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:2, and an HCDR3 comprising the sequence of SEQ ID NO:3; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:4, an LCDR2 comprising the sequence of SEQ ID NO:5, and an LCDR3 comprising the sequence of SEQ ID NO:6; b) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:11, an HCDR2 comprising the sequence of SEQ ID NO:12, and an HCDR3 comprising the sequence of SEQ ID NO:13; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:15, and an LCDR3 comprising the sequence of SEQ ID NO:16. c) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:21, an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:23; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:24, an LCDR2 comprising the sequence of SEQ ID NO:25, and an LCDR3 comprising the sequence of SEQ ID NO:26; or d) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:31, an HCDR2 comprising the sequence of SEQ ID NO:32, and an HCDR3 comprising the sequence of SEQ ID NO:33; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:34, an LCDR2 comprising the sequence of SEQ ID NO:35, and an LCDR3 comprising the sequence of SEQ ID NO:36.

[0048]

[0048] In some embodiments, the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55 and SEQ ID NO:57, and homologous sequences thereof having at least 80% sequence identity but retaining specific binding specificity or affinity for Gremlin.

[0049]

[0049] In some embodiments, the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, and SEQ ID NO:61, and homologous sequences thereof having at least 80% sequence identity but retaining specific binding specificity or affinity for Gremlin.

[0050] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises: a) a heavy chain variable region comprising the sequence of SEQ ID NO:7 and a light chain variable region comprising the sequence of SEQ ID NO:8; or b) a heavy chain variable region comprising the sequence of SEQ ID NO:17 and a light chain variable region comprising the sequence of SEQ ID NO:18; or c) a heavy chain variable region comprising the sequence of SEQ ID NO:27 and a light chain variable region comprising the sequence of SEQ ID NO:28; or d) a heavy chain variable region comprising the sequence of SEQ ID NO:37 and a light chain variable region comprising the sequence of SEQ ID NO:38; or e) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45, and a light chain variable region selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:49. or f) a pair of heavy chain variable region sequences and light chain variable region sequences selected from the group consisting of SEQ ID NO: 41 / 47, 41 / 49, 43 / 47, 43 / 49, 45 / 47, and 45 / 49; or g) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55 and SEQ ID NO: 57, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 61; or h) a pair of heavy chain variable region sequences and light chain variable region sequences selected from the group consisting of SEQ ID NO: 51 / 59, 51 / 61, 53 / 59, 53 / 61, 55 / 59, 55 / 61, 57 / 59, and 57 / 61.

[0051]

[0051] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof further comprises one or more amino acid residue substitutions or modifications, but retains specific binding specificity or affinity for GREM1.

[0052]

[0052] In some embodiments, at least one of the substitutions or modifications is present in one or more of the CDR sequences and / or in one or more non-CDR regions of the VH or VL sequences.

[0053]

[0053] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region.

[0054]

[0054] In some embodiments, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0055]

[0055] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is humanized.

[0056]

[0056] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody, and bivalent domain antibody.

[0057]

[0057] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is bispecific.

[0058]

[0058] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to a first and a second epitope of Gremlin, or capable of specifically binding to both hGREM1 and a second antigen.

[0059] In one aspect, the disclosure provides an antigen-binding fragment thereof, wherein the second antigen comprises an immune-related target.

[0060]

[0060] In one aspect, the disclosure provides an antigen-binding fragment thereof, wherein the second antigen comprises PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD47, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16 or CD83.

[0061] In one aspect, the disclosure provides the antigen-binding fragment, wherein the second antigen comprises a tumor antigen.

[0062]

[0062] In one aspect, the disclosure provides an antigen-binding fragment thereof, wherein the tumor antigen comprises a tumor-specific antigen or a tumor-associated antigen.

[0063] In one aspect, the disclosure provides an antigen-binding fragment thereof, wherein the tumor antigen is prostate specific antigen (PSA), CA-125, ganglioside G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptide, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, Ki-67, cancer associated mucin, VEGF, VEGFR (e.g., VEGFR-1, VEGFR-2, , VEGFR-3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2, GPC-3, nectin-4, ROR1, mesothelin, PCMA, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, BCR-ABL, E2APRL, H4-RET, IGH-IGK, MYL-RAR, IL-2R, CO17-1A, TROP2, or LIV-1.

[0064]

[0064] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is not cross-reactive to mouse GREM1.

[0065]

[0065] In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is cross-reactive to mouse GREM1.

[0066] In some embodiments, the method further comprises administering a therapeutically effective amount of a second therapeutic agent.

[0067]

[0067] In some embodiments, the second therapeutic agent comprises an anti-cancer therapeutic agent, optionally selected from chemotherapy agents, radiation therapy, immunotherapy agents, anti-angiogenesis agents (e.g., antagonists of VEGFR such as VEGFR-1, VEGFR-2, and VEGFR-3), targeted therapy agents, cell therapy agents, gene therapy agents, hormonal therapy agents, cytokines, palliative care, surgery for the treatment of cancer (e.g., tumor removal), one or more antiemetic agents, treatments for complications resulting from chemotherapy, or diet supplements for cancer patients (e.g., indole-3-carbinol).

[0068]

[0068] In some embodiments, the anti-cancer therapeutic agent comprises an anti-prostate cancer agent, optionally an androgen deprivation therapy agent.

[0069]

[0069] In some embodiments, the anti-prostate cancer drug comprises an androgen axis inhibitor, an androgen synthesis inhibitor, a PARP inhibitor, or a combination thereof.

[0070]

[0070] In some embodiments, the androgen axis inhibitor is selected from the group consisting of a luteinizing hormone releasing hormone (LHRH) agonist, an LHRH antagonist, and an androgen receptor antagonist.

[0071]

[0071] In some embodiments, the androgen axis inhibitor is degarelix, bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, or abiraterone.

[0072] In some embodiments, the anti-prostate cancer drug is abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), darolutamide, degarelix, docetaxel, Eligard (leuprolide acetate), enzalutamide, Erida (apalutamide), Firmagon (degarelix), flutamide, goserelin acetate, histrelin (Bantas), Jevtana (cabazitaxel), leuprolide acetate, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lynparza (olaparib), Kesel (Ezetamide), or cetearyl alcohol (Cialis). Selected from the group consisting of toconazole (Nizoral), mitoxantrone hydrochloride, nilandrone (nilutamide), nilutamide, Nubequo (darolutamide), olaparib, Provenzi (sipuleucel-T), radium-223 dichloride, relugolix (Orgovix), Rubraca (rucaparib cansylate), rucaparib cansylate, sipuleucel-T, taxotere (docetaxel), triptorelin (Trelstar), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Zoladex (goserelin acetate) and Zytiga (abiraterone acetate).

[0073]

[0073] In one aspect, the disclosure provides a method for determining the likelihood of responsiveness to a GREM1 antagonist in a subject having or suspected of having cancer, the method comprising the steps of (a) detecting androgen receptor (AR) expression or signaling in a biological sample from the subject, and (b) determining the likelihood of responsiveness based on the AR expression or signaling detected in step (a).

[0074]

[0074] In some embodiments, if a subject is detected as having no AR expression or signaling, or detected as having reduced AR expression or signaling compared to a reference level, the subject is determined to be likely responsive to a GREM1 antagonist.

[0075]

[0075] In some embodiments, the method further comprises detecting GREM1 expression in a biological sample from the subject.

[0076]

[0076] In some embodiments, if the subject is detected as having GREM1 expression, the subject is determined to be likely to respond to a GREM1 antagonist.

[0077]

[0077] In one aspect, the present disclosure provides a method for detecting the presence or amount of GREM1 in a sample determined to have no AR expression or reduced androgen receptor (AR) signaling, the method comprising the steps of contacting the sample with a detection reagent for detecting GREM1, and determining the presence or amount of GREM1 in the sample.

[0078]

[0078] In one aspect, the disclosure provides a method for determining the likelihood of responsiveness to a GREM1 antagonist in a subject having or suspected of having a disease or condition, the method comprising the steps of (a) detecting PTEN and / or p53 deficiency in a biological sample from the subject, and (b) determining the likelihood of responsiveness based on the PTEN and / or p53 deficiency detected in step (a).

[0079]

[0079] In some embodiments, if the subject is detected as deficient in PTEN and / or p53, the subject is determined to be likely responsive to a GREM1 antagonist.

[0080]

[0080] In some embodiments, the method further comprises detecting GREM1 expression in a biological sample from the subject.

[0081]

[0081] In some embodiments, if the subject is detected as having GREM1 expression, the subject is determined to be likely responsive to a GREM1 antagonist.

[0082]

[0082] In one aspect, the present disclosure provides a method for detecting the presence or amount of GREM1 in a sample determined to be deficient in PTEN and / or p53, the method comprising the steps of contacting the sample with a detection reagent for detecting GREM1, and determining the presence or amount of GREM1 in the sample.

[0083]

[0083] In some embodiments, the sample is obtained from a subject having or suspected of having a GREM1-related disease or condition.

[0084]

[0084] In some embodiments, the GREM1-associated disease or condition is cancer, a fibrotic disease, angiogenesis, glaucoma or retinal disease, renal disease, pulmonary arterial hypertension, or osteoarthritis (OA).

[0085]

[0085] In some embodiments, the cancer is prostate cancer, breast cancer, glioma, liposarcoma, hepatocellular carcinoma, lung cancer, cervical cancer, endometrial cancer, uterine leiomyosarcoma, squamous cell carcinoma of the head and neck, thyroid cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, esophageal cancer, bile duct cancer, osteosarcoma, glioblastoma, ovarian cancer, gastric cancer, triple-negative breast cancer (TNBC), small cell lung cancer or melanoma.

[0086]

[0086] In some embodiments, the cancer is prostate cancer or breast cancer, and the prostate cancer is a) resistant to androgen deprivation therapy, optionally castration resistant, and / or b) exhibits a prostate specific antigen (PSA) level lower than a reference level.

[0087]

[0087] In some embodiments, the method further comprises administering to the subject determined to be likely responsive a therapeutically effective amount of a GREM1 antagonist. [Brief description of the drawings]

[0088] [Figure 1A]

[0088] Figure 1A shows immunohistochemical staining images of Gremlin 1 in hormone-sensitive PCa (HSPC) (n=474) and castration-resistant prostate cancer (CRPC) (n=60). Representative images are presented. Scale bar=100 μm. [Figure 1B]

[0089] Figure 1B shows that GREM1 staining intensity is significantly higher in CRPC than in HSPC. Cytoplasmic H-scores are analyzed with Aperio ScanScope software. [Figure 1C]

[0090] Figure 1C shows images of immunohistochemical staining of PSA and Gremlin 1 in CRPC. Scale bar = 200 μm. [Figure 1D]

[0091] FIG. 1D shows that GREM1 mRNA transcription is downregulated by AR activation by R1881 (1 nM) and significantly increased by AR inhibition by enzalutamide (10 μg / ml) in LNCaP cells. [Figure 1E]

[0092] FIG. 1E shows that GREM1 protein levels are downregulated by AR activation by R1881 (1 nM) and significantly increased by AR inhibition by enzalutamide (10 μg / ml) in LNCaP cells. [Figure 1F]

[0093] Figure 1F shows that GREM1 promoter-driven luciferase activity was downregulated by AR activation with R1881 (1 nM) and significantly increased by AR inhibition with enzalutamide (10 μg / ml) in LNCaP cells. (Two-tailed Student's t test was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are presented as mean ± SEM.) [Figure 1G]

[0094] FIG. 1G shows the results of a chromatin immunoprecipitation (ChIP) assay showing enrichment levels of AR on the Gremlin 1 promoter in LNCaP cells upon treatment with R1881 or enzalutamide. Enz: enzalutamide. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 1H]

[0095] FIG. 1H shows that PCa patients with higher Gremlin 1 expression had shorter overall survival (p<0.05). [Figure 1I]

[0096] FIG. 1I shows that GREM1 was most highly expressed in prostate cancer tissues compared with other organs from Pbsn-Cre4;PTENfl / fl;Trp53fl / fl mice (n=3). [Figure 2A]

[0097] FIG. 2A shows that LNCaP castration-resistant cells (LNCaP-R) exhibit higher expression of Gremlin 1 compared to parental LNCaP cells. [Figure 2B]

[0098] FIG. 2B shows immunoblot analysis of GREM1 expression in AR-overexpressing LNCaP cells. [Figure 2C]

[0099] Figure 2C shows q-PCR analysis of GREM1 expression in AR-overexpressing LNCaP cells. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 2D]

[0100] FIG. 2D shows immunoblot analysis of GREM1 expression in AR knockout LNCaP cells. [Figure 2E]

[0101] Figure 2E shows q-PCR analysis of GREM1 expression in AR knockout LNCaP cells. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 3A]

[0102] FIG. 3A shows immunoblots confirming the efficiency of GREM1 knockdown or overexpression in PC3 cells. [Figure 3B]

[0103] Figure 3B shows that GREM1 knockdown suppresses the sphere-forming ability in PC3 cells, and GREM1 overexpression or addition of exogenous Gremlin 1 protein exhibits a promoting effect. Experiments were performed in triplicate. [Figure 3C]

[0104] Figure 3C shows that GREM1 knockdown suppresses cell proliferation in PC3 cells, and GREM1 overexpression or addition of exogenous Gremlin 1 protein exhibits a promoting effect. Experiments were performed in triplicate. [Figure 3D]

[0105] FIG. 3D shows that knockdown of GREM1 increases cell apoptosis in PC3 cells. [Figure 3E]

[0106] FIG. 3E shows that GREM1 knockdown suppresses PC3 xenograft growth in vivo. [Figure 3F]

[0107] Figure 3F shows that GREM1 overexpression promotes PC3 xenograft formation frequency and tumor growth in vivo. Scale bar = 1 cm. [Figure 3G]

[0108] FIG. 3G shows that overexpression of Grem1 is confirmed by immunoblot in Himyc mouse PCa-derived organoids. [Figure 3H]

[0109] Figure 3H shows that Gremlin 1 promotes organoid formation and androgen deprivation therapy (ADT) tolerance of Himyc PCa organoids. (Two-tailed Student's t-test and two-way ANOVA analysis were used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 4A]

[0110] FIG. 4A shows immunoblots confirming the efficiency of GREM1 knockdown and overexpression in LNCaP cells. [Figure 4B]

[0111] FIG. 4B shows that GREM1 knockdown inhibits the ability of LNCaP cells to form spheres, while GREM1 overexpression or exogenous addition of Gremlin 1 protein exerts the opposite effect. [Figure 4C]

[0112] Figure 4C shows that Gremlin 1 promotes the growth of LNCaP PCa cells under androgen deprivation therapy. ADT, androgen deprivation therapy. Experiments were performed in triplicate. [Figure 4D]

[0113] Figure 4D shows that knockdown of GREM1 increases cell apoptosis in LNCaP cells with androgen deprivation therapy. GREM1 expression and addition of exogenous Gremlin 1 protein suppress cell apoptosis in LNCaP cells treated with ADT. (Two-tailed Student's t test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 5A]

[0114] FIG. 5A shows an immunoblot confirming the efficiency of GREM1 overexpression in LAPC4 cells. [Figure 5B]

[0115] FIG. 5B shows that Gremlin 1 enhances sphere formation of LAPC4. [Figure 5C]

[0116] FIG. 5C shows that Gremlin 1 promotes the growth of LAPC4 cells under ADT treatment. [Figure 5D]

[0117] Figure 5D shows that GREM1 overexpression prevented cell death upon treatment with enzalutamide, characterized by reduced Annexin V / PI staining. (Two-tailed Student's t test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 6A]

[0118] FIG. 6A shows gene set enrichment analysis of RNA-seq data demonstrates that FGFR1 and MAPK signaling pathways are the most enriched signaling pathways in GREM1-overexpressing LNCaP sublines. [Figure 6B]

[0119] FIG. 6B shows gene set enrichment analysis of RNA-seq data demonstrates that FGFR1 and MAPK signaling pathways are the most enriched signaling pathways in GREM1-overexpressing LNCaP sublines. [Figure 6C]

[0120] Figure 6C shows that the FGFR / MEK / ERK signaling pathway is activated by Gremlin 1 protein in a dose-dependent manner in PC3 cells and LNCaP-resistant cells. FGF (20 ng / ml) is used as a positive control to stimulate FGFR. [Figure 6D]

[0121] Figure 6D shows that activation of the MEK / ERK signaling pathway by Gremlin 1 is independent of BMP4. PC3 cells and LNCaP-resistant cells were treated with Gremlin 1 protein in the presence or absence of BMP4 (20 ng / ml). [Figure 6E]

[0122] FIG. 6E shows that Gremlin 1 (100 ng / ml) treatment resulted in a prolonged stimulation of FGFR / MEK / ERK signaling activation compared with FGF (20 ng / ml) in PC3 cells. [Figure 6F]

[0123] FIG. 6F shows that Gremlin 1 (100 ng / ml) treatment results in prolonged stimulation of FGFR / MEK / ERK signaling activation compared with FGF (20 ng / ml) in LNCaP cells. [Figure 6G]

[0124] FIG. 6G shows that activation of the FGFR / MEK / ERK signaling pathway is suppressed by CRISPR / Cas9-mediated FGFR1 knockout. [Figure 6H]

[0125] Figure 6H shows that Gremlin 1 activates the MEK / ERK signaling pathway through FGFRs PC3 and LNCaP resistant cells were treated with Gremlin 1 (100 ng / ml), FGF1 (20 ng / ml), or the FGFR1 / 2 / 3 inhibitor BGJ398 (1 μM) as indicated. [Figure 6I]

[0126] Figure 6I shows that activation of the MEK / ERK signaling pathway by Gremlin 1 is independent of EGFR, PC3 and LNCaP resistant cells were treated with Gremlin 1 (100 ng / ml), EGF (20 ng / ml), or the EGFR inhibitor erlotinib (1 μM) as indicated. [Figure 7A]

[0127] FIG. 7A shows that LNCaP castration-resistant cells (LNCaP R) exhibit robust activation of the FGFR1 / MEK / ERK signaling pathway compared to parental AR-dependent LNCaP cells. [Figure 7B]

[0128] FIG. 7B shows that the FGFR1 / MEK / ERK signaling pathway is upregulated in GREM1-overexpressing mouse HiMyc PCa organoids compared to control organoids. [Figure 8A]

[0129] FIG. 8A shows that the promoting effect of Gremlin 1 protein on LNCaP cell proliferation (n=6) was suppressed by FGFR1 or MEK inhibitors but was unaffected by the addition of BMP4; BGJ398 is an FGFR1 inhibitor and trametinib is a MEK inhibitor. [Figure 8B]

[0130] FIG. 8B shows that the promoting effect of Gremlin 1 protein on sphere formation (n=3) was suppressed by FGFR1 or MEK inhibitors but was not affected by the addition of BMP4; BGJ398 is an FGFR1 inhibitor and trametinib is a MEK inhibitor. [Figure 8C]

[0131] FIG. 8C shows immunoblot results demonstrating the efficiency of FGFR1 knockout in LNCaP cells. [Figure 8D]

[0132] Figure 8D shows that FGFR1 knockout significantly attenuates the positive effect of Gremlin 1 on LNCaP cell proliferation. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 8E]

[0133] Figure 8E shows that FGFR1 knockout significantly attenuates the positive effect of Gremlin 1 on sphere formation. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 9A]

[0134] FIG. 9A shows that FGFR1 binds to Gremlin1-biotin immobilized on a streptavidin sensor (ForBio) (Kd=1.06E-8M). [Figure 9B]

[0135] Figure 9B shows the protein structure of Gremlin 1 and the predicted interactions of FGFR1 extracellular domain mimicked by Z-dock (http: / / zdock.umassmed.edu / ). Protein structure is generated from PDB (http: / / www.rcsb.org / ). Gremlin 1: 5AEJ; FGFR1: 3ojv. [Figure 9C]

[0136] FIG. 9C shows that Gremlin 1 co-immunoprecipitates with FGFR1 in 293T and LNCaP-resistant cells transfected with Flag-tagged Gremlin 1 and HA-tagged FGFR1 expression plasmids. [Figure 9D]

[0137] FIG. 9D shows that endogenous Gremlin 1 co-immunoprecipitates with FGFR1 in LNCaP-resistant cells. [Figure 9E]

[0138] FIG. 9E shows that Gremlin 1, but not Gremlin 2 or other members of the DAN protein family, binds to FGFR1 as measured by enzyme-linked immunosorbent assay (ELISA). [Figure 9F]

[0139] FIG. 9F shows that the interaction between purified Gremlin 1 and soluble FGFR1 protein is demonstrated by pull-down experiments. [Figure 9G]

[0140] FIG. 9G shows that soluble FGFR1 competitively inhibits activation of FGFR1 / MEK / ERK signaling by Gremlin 1 in PC3. [Figure 9H]

[0141] FIG. 9H shows that BiFC assay reveals colocalization between Gremlin 1 and FGFR1 in LNCaP-resistant cells. [Figure 9I]

[0142] Figure 9I shows immunofluorescence staining images of Gremlin 1 and FGFR1 in LNCaP-R cells. Cells were treated with Gremlin 1 (100 ng / ml) or PBS at 37°C for 10 minutes. [Figure 9J]

[0143] FIG. 9J shows a diagram of truncated FGFR1. [Figure 9K]

[0144] FIG. 9K shows the results of a Co-IP assay between truncated FGFR1 and Gremlin 1 (left panel) or FGF1 (right panel). [Figure 9L]

[0145] Figure 9L shows the Gremlin 1 mutagenesis strategy. Point mutations are in bold and underlined. [Figure 9M]

[0146] FIG. 9M shows that Gremlin 1 K123A-K124A mutant abolishes co-immunoprecipitation between Gremlin 1 and FGFR1, numbering refers to SEQ ID NO:69. [Figure 9N]

[0147] Figure 9N shows a schematic of FGFR1 mutations, with point mutations in bold and underlined. [Figure 9O]

[0148] FIG. 9O shows that co-immunoprecipitation of FGFR1 and Gremlin 1 is impaired by the FGFR1 E160A mutation. [Figure 9P]

[0149] FIG. 9P shows a schematic diagram of FGFR1 mutations. [Figure 9Q]

[0150] FIG. 9Q shows that FGFR1-C176G or FGFR1-R248Q mutations abolish co-immunoprecipitation of FGF1 and FGFR1 (left panel) but do not affect the formation of a protein complex between Gremlin 1 and FGFR1 (right panel). [Figure 9R]

[0151] Figures 9R-9U show that the binding between Gremlin1 and FGFR1 is not affected by the addition of FGF1 and vice versa, as revealed by Fortebio (R), co-immunostaining (S) and pull-down (T, U) assays. [Figure 9S]

[0151] Figures 9R-9U show that the binding between Gremlin 1 and FGFR1 is not affected by the addition of FGF1 and vice versa, as revealed by Fortebio (R), co-immunostaining (S) and pull-down (T, U) assays. [Figure 9T]

[0151] Figures 9R-9U show that the binding between Gremlin 1 and FGFR1 is not affected by the addition of FGF1 and vice versa, as revealed by Fortebio (R), co-immunostaining (S) and pull-down (T, U) assays. [Figure 9U]

[0151] Figures 9R-9U show that the binding between Gremlin 1 and FGFR1 is not affected by the addition of FGF1 and vice versa, as revealed by Fortebio (R), co-immunostaining (S) and pull-down (T, U) assays. [Figure 9V]

[0152] FIG. 9V shows a docking model highlighting key amino acid residues in the binding pocket between Gremlin 1 and FGFR1. [Figure 10A]

[0153] Figure 10A shows that the binding specificity of anti-mouse gremlin 1 antibody to gremlin 1 is confirmed by enzyme-linked immunosorbent assay. The Ab is anti-mouse gremlin 1 in this figure. [Figure 10B]

[0154] Figure 10B shows that Gremlin 1 is highly expressed in the castrated Pbsn-Cre4;PTENfl / fl;Trp53fl / fl mouse PCa model. Representative images of Gremlin 1 immunostaining are presented. [Figure 10C]

[0155] Figure 10C shows that anti-Gremlin 1 antibody (10 μg / ml) exerts a significant inhibitory effect on PCa growth, with a clear inhibition observed in the marked tumor appearance. [Figure 10D]

[0156] Figure 10D shows that anti-Gremlin 1 antibody (10ug / ml) exerts a significant inhibitory effect on PCa growth. A clear inhibition is found in significant tumor weight. [Figure 10E]

[0157] Figure 10E shows that anti-Gremlin 1 antibody (10ug / ml) exerted a significant inhibitory effect on PCa growth. Obvious inhibition was found in large amounts, and PCNA positive cells were significantly reduced. [Figure 10F]

[0158] FIG. 10F shows that anti-Gremlin 1 treatment significantly suppresses the growth of invasive PCa in castrated Pbsn-Cre;PTENfl / fl;Trp53fl / fl mice. [Figure 10G]

[0159] Figures 10G and 10H reveal that gene set enrichment analysis shows significant suppression of the FGFR signaling pathway in the prostate of the anti-Gremlin 1 treated group. [Figure 10H]

[0159] Figures 10G and 10H reveal that gene set enrichment analysis shows significant suppression of the FGFR signaling pathway in the prostate of the anti-Gremlin 1 treated group. [Figure 10I]

[0160] Figures 10I and 10J reveal that immunostaining and immunoblot analysis show the inhibitory effect of anti-Gremlin 1 antibody on FGFR1 / MAPK signaling pathway in the prostate of Pbsn-Cre;PTENfl / fl;Trp53fl / fl mice. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 10J]

[0160] Figures 10I and 10J show that immunostaining and immunoblot analysis show the inhibitory effect of anti-Gremlin 1 antibody on FGFR1 / MAPK signaling pathway in the prostate of Pbsn-Cre;PTENfl / fl;Trp53fl / fl mice. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean±SEM.) [Figure 10K]

[0161] FIG. 10K shows that antibodies against Gremlin 1 (100 ng / ml) enhance the inhibition of in vitro cell proliferation by enzalutamide (10 μg / ml) (n=3). [Figure 10L]

[0162] FIG. 10L shows that anti-Gremlin 1 treatment suppresses the sphere-forming ability of LNCaP-R cells (n=3). [Figure 10M]

[0163] FIG. 10M shows that activation of the FGFR1 / MEK / ERK signaling pathway is inhibited by Gremlin 1 antibody in LNCaP-R cells. [Figure 10N]

[0164] FIG. 10N shows Annexin-V / DAPI staining demonstrates that anti-Gremlin 1 antibody exhibits synergy with Enzalutamide in inducing cell death. Ab: anti-human-Gremlin 1. ADT: treated with 10 μg / ml Enzalutamide. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 10O]

[0165] Figure 10O shows a schematic illustrating treatment with Pbsn-Cre4;Ptenfl / fl;Trp53fl / fl GEMMs. Mice were castrated at 2 months and received anti-Gremlin 1 antibody (intraperitoneally, 10 mg / kg) or IgG as indicated, three times a week for 2 months. [Figure 11A]

[0166] Figure 11A shows that Gremlin was expressed primarily by epithelial cells in castrated Pbsn-Cre;PTENfl / fl;Trp53fl / fl PCa. ECAD, E-cadherin; VIM, vimentin. [Figure 11B]

[0167] FIG. 11B shows that Gremlin 1 antibody treatment does not induce significant side effects when administered systemically to mice (10 mg / kg twice weekly). No obvious changes in peripheral blood cell counts are detected in antibody-treated mice. Ab: anti-mGREM1 antibody. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean±SEM.) [Figure 11C]

[0168] FIG. 11C shows that Gremlin 1 antibody treatment does not induce significant side effects when administered systemically to mice (10 mg / kg twice weekly). No obvious changes are detected in major organs in mice that received antibody treatment. Ab: anti-mGREM1 antibody. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean±SEM.) [Figure 12A]

[0169] Figure 12A shows that the binding specificity of anti-human gremlin 1 (14E3) to gremlin 1 is confirmed by enzyme-linked immunosorbent assay. The Ab is anti-human gremlin 1 in this figure. [Figure 12B]

[0170] FIG. 12B shows that antibody against human Gremlin 1 (10 ug / ml) inhibits cell proliferation of PC3 cells. [Figure 12C]

[0171] FIG. 12C shows that anti-Gremlin 1 antibody (10 ug / ml) exerts an inhibitory effect on sphere formation of PC3 cells. [Figure 12D]

[0172] FIG. 12D shows that Gremlin 1 antibodies dose-dependently neutralize activation of FGFR1 / MEK / ERK signaling by Gremlin 1 protein in PC3 cells. [Figure 12E]

[0173] Figures 12D and 12F show that anti-Gremlin 1 treatment significantly impedes the in vivo growth of PC3 tumor xenografts in a serial passage experiment. Antibody was given at 10 mg / kg by intraperitoneal injection at the indicated time points (see arrowheads). [Figure 12F]

[0173] Figures 12D and 12F show that anti-Gremlin 1 treatment significantly impedes the in vivo growth of PC3 tumor xenografts in a serial passage experiment. Antibody was given at 10 mg / kg by intraperitoneal injection at the indicated time points (see arrowheads). [Figure 13]

[0174] FIG. 13 shows that treatment with 14E3 reduced tumor burden and increased percent survival in a PC3 CRPC model. [Figure 14A]

[0175] FIG. 14A shows that antibodies against Gremlin 1 (100 ng / ml) enhance the inhibition of in vitro cell proliferation by Enzalutamide (1 ug / ml). [Figure 14B]

[0176] FIG. 14B shows that anti-Gremlin 1 treatment suppresses the sphere-forming ability of LNCaP cells. [Figure 14C]

[0177] FIG. 14C shows that activation of the FGFR1 / MEK / ERK signaling pathway is inhibited by Gremlin 1 antibody in LNCaP cells. [Figure 14D]

[0178] FIG. 14D shows Annexin-V / DAPI staining demonstrates that anti-gremlin 1 antibody synergizes with enzalutamide to induce cell death. Ab: anti-human gremlin 1 14E3. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean±SEM.) [Figure 15]

[0179] FIG. 15 shows that 14E3 reduced GREM1-mediated promotion of cancer cell migration. [Figure 16A]

[0180] 16A-16C show that 14E3 reduced the GREM1-mediated increase in the percentage of the PSA low population independent of the BMP-binding loop. [Figure 16B]

[0180] Figures 16A-16C show that 14E3 reduced the GREM1-mediated increase in the percentage of the PSA low population independent of the BMP-binding loop. [Figure 16C]

[0180] Figures 16A-16C show that 14E3 reduced the GREM1-mediated increase in the percentage of the PSA low population independent of the BMP-binding loop. [Figure 17A]

[0181] FIG. 17A shows immunoblots confirming the efficiency of BMPRII knockout in LNCaP cells. [Figure 17B]

[0182] Figures 17B and 17C reveal that BMPRII knockout does not significantly affect the inhibitory effect of Gremlin 1 antibody on LNCaP cell proliferation and sphere formation. Ab: anti-human Gremlin 1 14E3. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 17C]

[0182] Figures 17B and 17C show that BMPRII knockout does not significantly affect the inhibitory effect of Gremlin 1 antibody on LNCaP cell proliferation and sphere formation. Ab: anti-human Gremlin 1 14E3. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 18A]

[0183] FIG. 18A shows that immunofluorescence staining of AMCAR and DAPI indicates the tumor origin of patient-derived organoids. [Figure 18B]

[0184] Figures 18B and 18C show that the reduced organoid size and number suggest an inhibitory effect of antibody against Gremlin 1 14E3 on PDO formation and growth. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 18C]

[0184] Figures 18B and 18C show that the reduced organoid size and number suggest an inhibitory effect of the antibody against Gremlin 1 14E3 on PDO formation and growth. (Two-tailed Student's t-test was used for statistical analysis. *, P<0.05; **, P<0.01; ***, P<0.001. Data are presented as mean ± SEM.) [Figure 18D]

[0185] Figure 18D shows detailed information about the patient samples used in this patient-derived organoid experiment. [Figure 19A]

[0186] Figure 19A shows heat map images of tumors in each mouse model from the control group (mIgG2a) and the experimental group (14E3), each of which has 16 mice. [Figure 19B]

[0187] FIG. 19B shows that antibodies against Gremlin 1 (eg, 14E3) had no significant effect on body weight in a PCa metastasis mouse model study. [Figure 19C]

[0188] FIG. 19C shows that mean radiant intensity was reduced with Gremlin 1 antibody treatment in a PCa metastasis mouse model study. [Figure 19D]

[0189] FIG. 19D shows an image of a lung tissue section, with arrows indicating the sites of lung metastasis. [Figure 19E]

[0190] FIG. 19E shows statistics of the number of micrometastases in the lungs in a PCa metastasis mouse model study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089]

[0191] The following description of the present disclosure is intended only to describe various embodiments of the present disclosure.Therefore, the specific changes discussed should not be interpreted as limiting the scope of the present disclosure.It is clear to those skilled in the art that various equivalents, changes and modifications may be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments may be included in the present specification.All references cited in this specification, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0090] definition

[0192] As used herein, the terms "a," "an," "the," and similar terms as used in the context of the present invention (particularly in the context of the claims) are to be interpreted as encompassing both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0091]

[0193] The term "inactivating mutation", as used herein with respect to biomarkers provided herein, such as AR, PTEN and / or p53, refers to a mutation or post-transcriptional modification that results in at least partial (or complete) loss of function or activity of the gene or gene product of the biomarker (e.g., AR, PTEN and / or p53), or results in a non-functional gene or gene product. For example, the activity of the gene or gene product affected by the biomarker may be significantly lower than or even eliminated from its wild-type counterpart. Inactivating mutations can be translocations, intragenic chromosomal breaks, transpositions, deletions (e.g., biallelic deletions, heterozygous or homozygous copy number losses), microcopy number changes, insertions, substitutions, aberrant splicing, or any combination thereof, which decrease the biological activity of the biomarker. In certain embodiments, insertions or deletions in a polynucleotide sequence may cause a frameshift, which changes the reading frame of the codons, resulting in a translated gene product that is completely different from the original. This often results in a truncated protein that results in loss of function.

[0092]

[0194] As used herein, the term "deletion" when used as a type of inactivating mutation of a biomarker refers to a mutation in which one or more nucleobase pairs are missing or deleted from a polynucleotide sequence, or one or more amino acid residues are deleted from a polypeptide sequence. For example, the term can refer to the deletion, loss, or removal of the entire coding region of the biomarker, or a portion thereof.

[0093]

[0195] As used herein, a "substitution" refers to a mutation that replaces one nucleobase with another in a polynucleotide sequence, or replaces one amino acid residue with another in a polypeptide sequence. A substitution in a polynucleotide sequence can 1) change a codon to one that codes for a different amino acid residue, thus causing a change in the amino acid sequence of the protein produced, or 2) change a codon to one that codes for the same amino acid residue, thereby causing no change in the protein produced; or 3) change an amino acid-encoding codon to a single "stop" codon, causing an incomplete protein (the incomplete protein is usually non-functional).

[0094]

[0196] As used herein, an "insertion" refers to a mutation in which one or more extra nucleobase pairs are inserted at a location in a polynucleotide sequence, or one or more amino acid residues are inserted into a polypeptide sequence.

[0095]

[0197] As used herein, "translocation" refers to a type of chromosomal abnormality resulting from the exchange of genetic material between two non-homologous chromosomes.Translocations can be balanced or unbalanced, with balanced translocations resulting in neither gain nor loss of material, and unbalanced translocations resulting in trisomy or monosomy of a particular chromosomal segment.Chromosomal translocations are typically found in leukemias, such as acute myeloid leukemia.

[0096]

[0198] The term "level" with respect to biomarkers such as AR, PTEN and / or p53 refers to the content or amount of the biomarker of interest present in a sample.Such content or amount can be expressed in absolute terms, i.e., the total amount of biomarker in a sample, or in relative terms, i.e., the concentration or percentage of biomarker in a sample.The level of biomarker can be measured at DNA level (e.g., expressed by the content or amount of gene in a chromosomal region or copy number), RNA level (e.g., as mRNA content or amount), or protein level (e.g., as the content or amount of protein or protein complex).

[0097]

[0199] As used herein, the term "reference level" in relation to a biomarker refers to a benchmark level that allows comparison. Reference levels can be selected by those skilled in the art according to the desired purpose. Means for determining suitable reference levels are known to those skilled in the art, for example, reference levels can be determined from experience, existing knowledge or data collected from clinical studies.

[0098]

[0200] As used herein, the term "negative" with respect to a biomarker means that the biomarker tests negative or is not present in the test sample. For example, a biomarker that is negative in a test sample may have a level that is comparable to or indistinguishable from a negative control level in a sample lacking such biomarker, or may instead have a level that is lower than a threshold level that defines the presence or positive result.

[0099]

[0201] As used herein, "likelihood" and "probability" in relation to a subject's response to treatment are a measure of how likely it is that a therapeutic response will occur in a subject. This term may be used interchangeably with "probability". A probability is a probability that is above guesswork, but below certainty. Thus, a therapeutic response is likely if a reasonable person using common sense, training, or experience would conclude that, given the circumstances, a therapeutic response is certain.

[0100]

[0202] The term "benefit from" or "responsive" as used in the context of therapy (e.g., treatment with a GREM1 antagonist) refers to a beneficial or favorable response to treatment, as opposed to an unfavorable response, i.e., an adverse event.

[0101]

[0203] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A natural, intact antibody contains two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, and each heavy chain contains a variable region (V H ) and the first, second, and third constant regions (C H1 , C H2 , C H3 ), mammalian light chains are classified as lambda or kappa, and each light chain consists of a variable region (V L) and constant regions. Antibodies have a "Y" shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y contains the variable region and the first constant region of a single heavy chain bound to the variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2, and LCDR3, while heavy chain CDRs include HCDR1, HCDR2, HCDR3). The CDR boundaries of the antibodies and antigen-binding domains disclosed herein may be defined or identified by the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5; 186(3):651-63 (1985); Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987); N.R. Whitelegg et al., Protein Immunol. 1999, 14, 111-115 (1987)). Engineering, v13(12), pp. 819-824 (2000); Chothia, C. et al., Nature. Dec 21-28; 342(6252): pp. 877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, pp. 481-514 (2015)). The three CDRs are located between adjacent stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops.The constant regions of the heavy and light chains are not involved in antigen binding and exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain). In certain embodiments, the antibodies provided herein encompass any antigen-binding fragment thereof.

[0102]

[0204] As used herein, an "antigen-binding fragment" refers to an antibody fragment formed from a fragment of an antibody that contains one or more CDRs, or any other antibody portion that binds to an antigen but does not contain an intact native antibody structure. Examples of antigen-binding fragments include, without limitation, diabodies, Fab, Fab', F(ab')2, Fd, Fv, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen as the parent antibody binds. In certain embodiments, an antigen-binding fragment can contain one or more CDRs from a particular parent antibody.

[0103]

[0205] "Fab" with respect to an antibody refers to a monovalent antigen-binding fragment of an antibody consisting of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain. Fab can be obtained by papain digestion of an antibody at residues proximal to the N-terminus of the inter-heavy chain disulfide bond in the hinge region.

[0104]

[0206] "Fab'" refers to a Fab fragment containing a portion of the hinge region; Fab' can be obtained by pepsin digestion of an antibody at residues proximal to the C-terminus of the inter-heavy chain disulfide bond in the hinge region and therefore differs from Fab by a small number of residues in the hinge region, including one or more cysteines.

[0105]

[0207] "F(ab')2" refers to a dimer of Fab' containing two light chains and part of two heavy chains.

[0106]

[0208] With respect to antibodies, "Fv" refers to the smallest fragment of an antibody that contains an intact antigen-binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0107]

[0209] A "single chain Fv antibody" or "scFv" is an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). An "scFv dimer" is a single chain containing two heavy chain variable regions and two light chain variable regions with a linker. In certain embodiments, an "scFv dimer" is a single chain comprising the V of one part. H The other parts of V L and another V to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L Dimerized V together with the moiety H -V L In another embodiment, the "scFv dimer" is a bivalent diabody or a bivalent ScFv (BsFv) comprising V H1 and V L1 Working together, V H2 and V L2act in concert, with each concerted pair having a different antigen specificity, L1 -V H2 (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0108]

[0210] A "single chain Fv-Fc antibody" or "scFv-Fc" is an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0109]

[0211] "Camelized single domain antibodies", "heavy chain antibodies", "nanobodies" or "HCAbs" are antibodies that consist of two V HHeavy chain antibodies are antibodies that contain heavy chain domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): 93-101 (2003)). The variable domain of heavy-chain antibodies (VHH domains) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)). "Diabodies" refer to small antibody fragments that have two antigen-binding sites, the fragments being V L V connected to the domain H Domain Included (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci US A. Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161). The two domains on the same chain cannot pair because the linker is too short, so the two domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes).

[0110]

[0212] A "domain antibody" is an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In certain embodiments, two or more V H The domains are covalently linked with a peptide linker to form a bivalent or multivalent domain antibody. H The domains may target the same or different antigens.

[0111]

[0213] In certain embodiments, a "(dsFv)2" is a peptide chain consisting of three peptide chains: two Vs linked by a peptide linker and separated by a disulfide bridge. L Two V's joined together H Includes parts.

[0112]

[0214] In certain embodiments, a "bispecific ds diabody" is H1 and V L1 V via a disulfide bridge between L1 -V H2 V bound to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0113]

[0215] In certain embodiments, a "bispecific dsFv" or "dsFv-dsFv" comprises three peptide chains: heavy chains linked by a peptide linker (e.g., a long, flexible linker) and V chains linked via disulfide bridges. L1 and VL2 V paired with part H1 -V H2 Each disulfide-paired heavy and light chain has a different antigen specificity.

[0114]

[0216] The term "humanized" as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human. In certain embodiments, amino acid residues in the variable region framework of a humanized gremlin antibody are replaced for sequence optimization. In certain embodiments, the variable region framework sequences of the humanized gremlin antibody chains are at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to the corresponding human variable region framework sequences.

[0115]

[0217] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy and / or light chain from one species and the remainder of the heavy and / or light chain from a different species. Illustratively, a chimeric antibody may contain a constant region from a human and a variable region from a non-human species, such as from a mouse.

[0116]

[0218] The term "germline sequence" refers to a nucleic acid sequence encoding a variable region amino acid sequence or subsequence that shares the highest predetermined amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other known variable region amino acid sequences encoded by germline immunoglobulin variable region sequences. A germline sequence can also refer to a variable region amino acid sequence or subsequence that has the highest amino acid sequence identity with a reference variable region amino acid sequence or subsequence compared to all other evaluated variable region amino acid sequences. A germline sequence can be framework regions only, complementarity determining regions only, framework and complementarity determining regions, variable segments (as defined above), or other combinations of sequences or subsequences that include variable regions. Sequence identity can be determined using methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or another alignment algorithm known in the art. A germline nucleic acid or amino acid sequence can have at least about 90%, 91, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a reference variable region nucleic acid or amino acid sequence. Germline sequences can be determined, for example, through the publicly available international ImMunoGeneTics database (IMGT) and V-Base.

[0117]

[0219] "Anti-human Gremlin 1 antibody," "anti-hGREM1 antibody," or "antibody to human Gremlin 1," are used interchangeably herein, and refer to an antibody that can specifically bind to human Gremlin 1 with sufficient specificity and / or affinity to provide, for example, therapeutic use.

[0118]

[0220] The term "affinity" as used herein refers to the strength of the non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen.

[0119]

[0221] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein have a binding affinity of ≦10 -6 M (e.g., ≦5×10 -7 M, ≤2×10 -7 M, ≦10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≦10 -8 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M, or ≤ 10 -9 Binding affinity (K D ) specifically binds human and / or non-human Gremlin 1. D is the ratio of the dissociation rate to the association rate (k off / k on ), which may be determined by any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). In certain embodiments, K D Values ​​can be suitably determined by using flow cytometry techniques. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will give a signal that is at least twice the background signal, and more typically at least 10-100 times the background.

[0120]

[0222] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain characteristic of each amino acid. The names of amino acids are also represented in this disclosure as standard one-letter or three-letter codes and are summarized as follows:

[0121] [Table 1]

[0122]

[0223] "Conservative substitution" in the context of amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitution can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions usually do not cause significant changes in protein conformational structure, and therefore are believed to be able to retain the biological activity of the protein.

[0123]

[0224] "Percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical with the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum correspondence. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available on the US National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool or may customize the parameters to suit the alignment, for example, by selecting an appropriate algorithm. In certain embodiments, non-identical residue positions may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art.See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference.

[0124]

[0225] As used herein, a "homologous sequence" refers to a polynucleotide sequence (or its complementary strand) or amino acid sequence that has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence, when optionally aligned.

[0125]

[0226] An "isolated" material has been altered from its natural state by the hand of man. An "isolated" composition or material, if present in nature, has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it is sufficiently separated from the coexisting materials of its natural state such that it exists in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to an isolated sequence of a nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment that has a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by gel electrophoretic methods (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (e.g., ion exchange chromatography or reverse phase HPLC).

[0126]

[0227] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

[0127]

[0228] As used herein, "treating" a condition or "treatment" thereof includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of development of the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, producing a complete or partial regression of the condition, curing the condition, or a combination thereof.

[0128]

[0229] The term "gremlin 1" or "GREM1" refers to variant 1 of gremlin, and encompasses gremlin 1 from different species, e.g., human, mouse, monkey, etc. GREM1 is evolutionarily conserved, and the human gremlin 1 gene (hGREM1) has been mapped to chromosome 15q13-q15 (Topol LZ et al., (1997) Mol. Cell Biol., 17:4801-4810; Topol LZ et al., Cytogenet Cell Genet., 89:79-84). The amino acid sequence of hGREM1 is available in the GenBank database under accession number NP-037504 or the Uniprot database via accession number O60565, and is provided herein as SEQ ID NO: 66. The terms "human gremlin 1" and "hGREM1" are used interchangeably in this disclosure.

[0129]

[0230] As used herein, a "Gremlin 1-associated" or "GREM1-associated" disease or condition refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased expression or activity of GREM1. In some embodiments, the GREM1-associated condition is, for example, glaucoma, cancer, fibrotic disease, angiogenesis, retinal disease, kidney disease, pulmonary arterial hypertension, or osteoarthritis (OA).

[0130]

[0231] As used herein, "cancer" refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal proliferation, invasion or metastasis, and can be benign or malignant, and includes both solid tumors and non-solid tumors such as leukemia (e.g., blood-based tumors). As used herein, a "solid tumor" refers to a solid mass of neoplastic and / or malignant cells.

[0131]

[0232] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient(s), and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0132]

[0233] The term "therapeutically effective amount" or "effective amount" refers to an amount of a pharmaceutical agent that produces some desired local or systemic therapeutic effect at a reasonable benefit / risk ratio applicable to any treatment. When administered to prevent a disease, the amount is sufficient to avoid or delay the onset of the disease. A therapeutically effective amount or effective amount need not be therapeutic or prevent the disease or condition from ever occurring. In certain embodiments, the therapeutically effective amount of a pharmaceutical agent depends on its therapeutic index, solubility, and the like.

[0133]

[0234] Reference herein to "about" a value or parameter includes (and describes) an embodiment directed to that value or parameter per se. For example, a reference to "about X" includes the reference to "X". A numerical range includes the numbers defining that range. Generally speaking, the term "about" refers to the indicated value of the variable and all values ​​of the variable that are within experimental error of the indicated value (e.g., within a 95% confidence interval for the mean) or within 10% of the indicated value, whichever is greater. When the term "about" is used in the context of a period of time (years, months, weeks, days, etc.), the term "about" means that period of time plus or minus one amount of the next subperiod of time, whichever is greater (e.g., about 1 year means 11-13 months; about 6 months means 6 months plus or minus 1 week, about 1 week means 6-8 days, etc.), or within 10% of the indicated value.

[0134]

[0235] The present disclosure provides a novel medical use of Gremlin 1 (GREM1) antagonist. The novel medical use is based in part on the unexpected discovery that the transcription of GREM1 is suppressed by androgen receptor (AR) and deregulated by androgen deprivation therapy (ADT). The novel medical use is based in part on the discovery that PTEN and / or p53 loss promotes GREM1 expression. Furthermore, the present disclosure surprisingly finds that GREM1 is significantly upregulated in advanced prostate cancer, including castration-resistant prostate cancer (CRPC), and is positively correlated with the development of castration resistance and poor overall survival. The inventors have found that GREM1 antagonists are useful for treating related conditions.

[0135]

[0236] Methods of Treating GREM1 Expression States Having Reduced Androgen Receptor Signaling - Patent application

[0237] In one aspect, the disclosure provides a method of treating a GREM1-expressing disease or condition in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist, wherein the disease or condition is characterized by reduced or inhibited androgen receptor (AR) signaling.

[0136]

[0238] In certain embodiments, the subject is receiving or has been receiving AR inhibitor.In certain embodiments, the disease or condition is resistant to AR inhibitor.As used herein, AR inhibitor refers to the therapeutic agent that is useful for suppressing AR activity, for example, the therapeutic agent that is used in androgen deprivation therapy.

[0137]

[0239] In certain embodiments, the disease or condition is an AR-associated cancer (e.g., prostate cancer, breast cancer, glioblastoma, melanoma, bladder cancer, renal cell carcinoma, pancreatic cancer, hepatocellular carcinoma, ovarian cancer, endometrial cancer, mantle cell lymphoma, or salivary gland cancer), or an AR-associated non-cancer condition (e.g., hair loss, acne, hirsutism, ovarian cysts, polycystic ovarian disease, precocious puberty, spinal and bulbar muscular atrophy, or age-related macular degeneration).

[0138]

[0240] In one aspect, the disclosure provides a method of treating a GREM1-expressing cancer in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist, wherein the cancer is characterized by reduced androgen receptor (AR) signaling.

[0139]

[0241] Decreased androgen receptor (AR) signaling

[0242] The androgen receptor (AR) is a member of the steroid and nuclear receptor superfamily and is primarily expressed in androgen target tissues such as the prostate, skeletal muscle, liver, and central nervous system (CNS), with the highest expression levels observed in the prostate, adrenal glands, and epididymis.

[0140]

[0243] AR is a soluble protein that functions as an intracellular transcription factor. Upon binding and activation by androgens, AR mediates the transcription of target genes that regulate the growth and differentiation of prostate epithelial cells. AR signaling is crucial for the development and maintenance of male reproductive organs, including the prostate.

[0141]

[0244] As used herein, the term "reduced androgen receptor (AR) signaling" refers to AR signaling whose level is substantially lower than the normal or baseline level of AR signaling, e.g., the level of AR signaling in a healthy cell or tissue sample, or the average level of AR signaling in an entire cancer patient population or in a cancer patient population of a particular cancer of interest, or in a patient population with AR-dependent prostate cancer.

[0142]

[0245] The cancer with reduced androgen receptor (AR) signaling can be AR-expressing cancer, and AR signaling is inhibited, for example, due to treatment (e.g., pharmacological treatment or surgical treatment), or due to reduced expression level of AR, or due to certain inactivating mutations in AR. Alternatively, the cancer with reduced AR signaling can have negative AR expression, especially in cancers that normally express AR (e.g., prostate cancer).

[0143]

[0246] In some embodiments, cancer is AR-expressing cancer.Different types of cancer are known to express AR.The examples of AR-expressing cancer include, but are not limited to, prostate cancer, breast cancer, lung cancer, head and neck cancer, testicular cancer, endometrial cancer, ovarian cancer and skin cancer.In certain embodiments, AR-expressing cancer is prostate cancer or breast cancer.

[0144]

[0247] In some embodiments, the subject is undergoing or has undergone androgen deprivation therapy (ADT).The term "androgen deprivation therapy" or "ADT" used herein refers to a therapy that suppresses androgens by reducing androgen levels or inhibiting the biological function of androgens, such as by inhibiting AR signal transduction.The main androgens in the body are testosterone and dihydrotestosterone (DHT).

[0145]

[0248] ADT can be achieved through surgical procedures (eg, surgical castration) or drug treatments. Examples of ADT drugs include, without limitation, LHRH agonists (e.g., leuprolide (Lupron, Eligard), goserelin (Zoladex), triptorelin (Trelstar), and histrelin (Bantas)), LHRH antagonists (e.g., degarelix (Filmagon), relugolix (Orgovix)), drugs that reduce androgen levels from the adrenal glands (e.g., abiraterone (Zytiga), ketoconazole (Nizoral)), androgen receptor antagonists (e.g., flutamide (Eulexin), bicalutamide (Casodex), nilutamide (Nilandrone)), and other antiandrogens (e.g., enzalutamide (Xtandi), apalutamide (Arleed) and darolutamide (Nubequo)).

[0146]

[0249] In some embodiments, the subject or cancer is resistant to ADT. "Resistance" means that the disease has no or reduced responsiveness or sensitivity to ADT. Reduced responsiveness can be indicated, for example, by the need to increase the dose to achieve a given efficacy. In certain embodiments, the disease can be non-responsive to ADT. For example, despite treatment with ADT, the cancer cells or tumor size increases, or the disease shows a regression to its previous state, for example, partial recovery followed by a return to previous symptoms. Resistance to ADT can be innate or acquired.

[0147]

[0250] In some embodiments, the subject or cancer has a reduced expression level of AR or has one or more inactivating mutations in AR. Over 800 different AR mutations have been identified in patients with androgen insensitivity syndrome and prostate cancer. Four different types of mutations that inactivate AR have been detected in the AR gene, including: a) single point mutations that cause amino acid substitutions or premature stop codons; b) nucleotide insertions or deletions that cause frameshifts and early rumination; c) complete or partial gene deletions; and d) intronic mutations that cause alternative splicing (see K. Eisermann et al., Transl Androl, Urol. 2013 Sep;2(3):137-147 for details).

[0148]

[0251] In some embodiments, the cancer is negative for androgen receptor (AR) expression, i.e., AR-negative cancer.As used herein, AR-negative cancer refers to cancer that initially has AR expression but becomes AR-negative.In certain embodiments, the AR-negative cancer is prostate cancer or breast cancer.Some prostate cancer cell lines have been found to be AR-negative, such as PC3 cell lines.AR-negative cancer may test negative (or undetectable) for AR expression or AR signaling, or may have a detection level of AR expression that is comparable to the detection level of known AR-negative prostate cancer cells.

[0149]

[0252] In some embodiments, prostate cancer or breast cancer is negative for both androgen receptor (AR) expression and neuroendocrine (NE) differentiation.NE differentiation in prostate cancer is a well-recognized phenotypic change, which causes prostate cancer cells to transdifferentiate into NE-like cells.NE-like cells lack androgen receptor expression and prostate-specific antigen, and are resistant to treatment.NE differentiation can be assessed by measuring the protein or mRNA levels of NE marker chromogranin A (CgA), the ratio of CgA / prostate-specific antigen (PSA), and / or neuron-specific enolase (NSE). See, for example, Hu et al., Front Oncol. 2015;5:90; Berruti et al., Endocr Relat Cancer (2005) 12(1):109-17.10.1677 / erc.1.00876; Khan et al., J Pak Med Assoc (2011) 61(1):108-11; Taplin et al., Urology (2005) 66(2):386-91.10.1016 / j.urology; Sarkar et al., Cancer Biomark (2010) 8(2):81-7.10.3233 / CBM-2011-0198; Burgio et al., Endocr Relat Cancer (2014) 21(3):487-93.10.1530 / ERC-14-0071; Conteduca et al., Prostate (2014) 74(16):1691-6. 10.1002 / pros.22890; Berruti et al., Cancer (2000) 88(11):2590-7. 10.1002 / 1097-0142(20000601) 88:11; Sasaki et al., Eur Urol (2005) 48(2):224-9. 10.1016 / j.eururo.2005.03.017, the disclosures of which are incorporated herein by reference in their entireties.

[0150]

[0253] In some embodiments, the prostate cancer is further characterized by having a level of prostate specific antigen (PSA) lower than a reference level.

[0151]

[0254] PSA is the classical downstream target of AR. Normally, PSA is hardly secreted into the blood. Increased gland size and tissue damage caused by benign prostatic hyperplasia, prostatitis, or prostate cancer can increase circulating PSA levels. Prostate cancer cells in advanced stages that are poorly differentiated or undifferentiated produce less PSA, with low levels of PSA (e.g., less than 4ng / ml). It is believed that prostate cancer cells with low levels of PSA or negative for PSA can be resistant to antiandrogens, chemotherapeutic agents, prooxidants, or radiation, and may be castration-resistant (Skvortsov S. et al., STEM CELLS, Vol. 36, No. 10, pp. 1457-1474).

[0152]

[0255] The reference level of PSA may be the threshold level of PSA that is usually found in PSA-positive prostate cancer.The reference level of PSA may also be the average level of PSA in the entire prostate cancer patient population or in the patient population with prostate cancer before it progresses to advanced stage.A certain reference level of PSA in blood may be, for example, about 2ng / ml, about 4ng / ml, about 6ng / ml, about 8ng / ml or about 10ng / ml, for example, when measured using an immunodetectable assay, for example, Hybritech (San Diego, Calif), Tosoh (Foster City, Calif), Bayer Centaur PSA assay kit (Tarrytown, NY), or Abbott assay (Chicago, Ill). See, e.g., Dan et al., Cancer, Vol. 109, No. 2, 2007. https: / / doi.org / 10.1002 / cncr.22372; and Oesterling et al., J Urol. 1995; 154:1090-1095, the disclosures of which are incorporated herein by reference in their entireties.

[0153]

[0256] In certain embodiments, the prostate cancer is negative for PSA, e.g., the prostate cancer does not express PSA or tests negative for PSA.

[0154]

[0257] In some embodiments, prostate cancer is castration-resistant.Castration-resistant prostate cancer (CRPC) is an advanced prostate cancer that can grow despite low levels of circulating androgens.CRPC may manifest as a continuous rise in PSA levels, progression of pre-existing disease, and / or the appearance of new metastases after ADT, despite serum testosterone levels below 50ng / dL (Toshiyuki Kamoto et al., Nihon Rinsho. 2014 Dec;72(12):2103-7; Fred Saad et al., Can Urol Assoc J. 2010 Dec;4(6):380-384).

[0155]

[0258] Some CRPC may remain dependent on AR signaling despite androgen depletion or reduction. For example, CRPC can be developed by amplifying AR expression, mutating AR gene and / or genes encoding coactivators / corepressors, activating androgen-independent AR pathway, and / or producing alternative androgens so that disease progression remains dependent on AR pathway (Thenappan et al., Transl Androl Urol. 2015 Jun;4(3):365-380). Some CRPC can bypass the need for AR signaling.

[0156]

[0259] In some embodiments, the prostate cancer a) is negative for androgen receptor (AR) expression, b) is negative for both androgen receptor (AR) expression and neuroendocrine (NE) differentiation; c) is resistant to androgen deprivation therapy, optionally, is castration resistant, d) exhibits a level of prostate specific antigen (PSA) lower than a reference level, or e) any combination of a)-d).

[0157]

[0260] In some embodiments, the cancer is further determined to be PTEN and / or p53 deficient.

[0158]

[0261] In certain embodiments, the cancer is metastatic. A metastatic cancer can or has spread from the primary site to another part of the body. The metastatic tumor is the same type of cancer as the primary tumor. A metastatic cancer can spread to an area near the primary site or to a distant part of the body.

[0159]

[0262] GREM1 overexpression

[0263] Without wishing to be bound by any theory, it is believed that AR signaling is negatively correlated with GREM1 expression, and decreased AR signaling is believed to result in increased expression of GREM1.

[0160]

[0264] In some embodiments, the cancer with decreased AR signaling is further characterized by GREM1 expression or overexpression. GREM1 expression or overexpression can be present in disease cells or in the disease microenvironment.

[0161]

[0265] The term "overexpression" as used herein with respect to GREM1 refers to an increased expression level compared to a reference level. The reference level may be a level of GREM1 expression found in normal cells of the same tissue type, optionally normalized to the expression level of another gene (e.g., a housekeeping gene). Alternatively, the reference level may be a level of GREM1 expression found in a healthy subject. The expression level may be determined based on the nucleic acid level or the protein level. In some embodiments, the GREM1-expressing cancer has a GREM1 expression level at least 10% higher (e.g., at least 15%, 20%, 30%, 35%, 40%, 50% or 1-fold, 2-fold, 3-fold or more higher) than the reference level.

[0162]

[0266] Methods for increasing the sensitivity of AR-expressing cancers to androgen deprivation therapy

[0267] In another aspect, the disclosure further provides a method of increasing the sensitivity of an AR-expressing cancer to androgen deprivation therapy (ADT) in a subject, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist.

[0163]

[0268] Without wishing to be bound by any theory, it is believed that decreased AR signaling by ADT can lead to GREM1 expression or increased expression, and the use of GREM1 antagonists can further improve the sensitivity of AR-expressing cancers to ADT.

[0164]

[0269] The term "sensitivity" in relation to cancer refers to the ability of cancer to respond to treatment (e.g., treatment with a GREM1 antagonist). Cancer sensitivity can be measured, for example, in terms of inhibiting cancer cell proliferation or promoting cancer cell death. Increased sensitivity can be determined based on increased efficacy under the same dose, or reduced dose for similar efficacy.

[0165]

[0270] In some embodiments, the methods include administering to the subject a GREM1 antagonist in combination with ADT.

[0166]

[0271] Methods of Treating GREM1-Associated Diseases or Conditions Characterized by Deficiencies of PTEN and / or p53 - Patent application

[0272] In various embodiments, the disclosure provides methods of treating a GREM1-associated disease or condition characterized by deficiencies in PTEN and / or p53 in a subject.

[0167]

[0273] PTEN and / or p53 deficiency

[0274] PTEN and p53 contribute to the regulation of self-renewal and differentiation in prostate precursors and putative tumor-initiating cells of prostate cancer.Terms PTEN and / or p53 provided herein are intended to encompass different forms, including mRNA, protein and DNA (e.g., genomic DNA).Therefore, the level and / or activity and / or mutation status of PTEN and / or p53 can be measured using RNA (e.g., mRNA), protein or DNA (e.g., genomic DNA).

[0168]

[0275] The terms "TP53" and "p53" are used interchangeably herein. TP53 is a transcription factor that can regulate a number of genes, for example, regulating cell cycle and apoptosis. Alternative names for p53 include, for example, antigen NY-CO-13, phosphorylated protein p53, tumor suppressor p53, and cellular tumor antigen p53. As used herein, p53 can refer to TP proteins, including all isoforms and variants, as well as polynucleotides (e.g., DNA or RNA) encoding TP53 proteins. In certain embodiments, the gene for p53 is available in the GenBank database under the NCBI reference sequence NG_017013.2, and an exemplary sequence of human p53 protein is available in the UniProtKB database under the accession number P04637 (P53-HUMAN). In certain embodiments, the protein of p53 comprises the amino acid sequence of SEQ ID NO: 73.

[0169]

[0276] The terms "PTEN", "Pten" and "PTEN tyrosine phosphatase" are used interchangeably herein. PTEN, also known as phosphatase and tensin homolog deleted on chromosome 10, is a tumor suppressor that acts as a dual specificity protein phosphatase that antagonizes the PI3K signaling pathway through its lipid phosphatase activity and negatively regulates the MAPK pathway through its protein phosphatase activity (Pezzolesi et al., Hum. Molec. Genet. 16:1058-1071, 2007). PTEN, as used herein, can refer to the PTEN protein, including all isoforms and variants, as well as the DNA (e.g., coding gene sequence) or RNA encoding PTEN. An exemplary sequence of human PTEN is available in the UniProtKB database under accession number P60484 (PTEN_HUMAN) and has three isoforms: isoform 1 (P60484-1), isoform alpha (P60484-2) and isoform 3 (P60484-3). An exemplary sequence of the gene for PTEN is available in the GenBank database under the NCBI reference sequence NC_000010.11. In certain embodiments, the protein of PTEN comprises the amino acid sequence of SEQ ID NO:74.

[0170]

[0277] As used herein, "deficient" or "deficient" refers to insufficient activity or level, and can include, for example, less than normal activity or level, or no or zero activity or level. For example, deficient activity or level of PTEN and / or p53 means that PTEN and / or p53 have no or less than normal function, or the expression level of PTEN and / or p53 is absent or reduced in a biological sample.

[0171]

[0278] In certain embodiments, the PTEN and / or p53 deficiency is characterized by the absence of functional PTEN and / or p53.

[0172]

[0279] In certain embodiments, a deficiency in PTEN and / or p53 activity or levels can be indicated by the presence of an inactivating mutation in PTEN and / or p53.

[0173]

[0280] It should be understood that the present disclosure is not limited to any particular PTEN or p53 mutation. Any inactivating mutation in PTEN or p53 can be useful in the present disclosure.

[0174]

[0281] In certain embodiments, the deficiency of PTEN and / or p53 activity or level can be indicated by the expression level or copy number of PTEN and / or p53 in a biological sample.Therefore, to determine whether the activity or level of PTEN and / or p53 in a biological sample is defective, the method provided herein can include determining whether the expression level or copy number of PTEN and / or p53 is decreased in the biological sample compared to a reference level.

[0175]

[0282] The mutation status or expression level of PTEN and / or p53 at the DNA or RNA level can be measured by any method known in the art, such as, but not limited to, amplification assay, hybridization assay, or sequencing assay. The mutation status or expression level of PTEN and / or p53 at the protein level can be measured by any method known in the art, such as, but not limited to, immunoassay.

[0176]

[0283] In certain embodiments, PTEN and / or p53 deficiency is characterized by the absence of PTEN and / or p53 expression.

[0177]

[0284] In certain embodiments, defects in PTEN and / or p53 activity or levels can be indicated by epigenetic silencing, transcriptional repression, or microRNA (miRNA) regulation of PTEN and / or p53.

[0178]

[0285] Without wishing to be bound by any theory, it is believed that, for example, loss of p53 / PTEN due to inactivating mutations leads to increased expression of GREM1. In some embodiments, GREM1-associated diseases or conditions characterized by loss of PTEN and / or p53 are further characterized by GREM1 expression or overexpression. GREM1 expression can be determined using the methods provided above.

[0179]

[0286] In certain embodiments, the subject is a human. In certain embodiments, the subject is identified as having GREM1 expression or overexpression, optionally in a biological sample obtained from the subject.

[0180]

[0287] GREM1-Related Diseases or Conditions

[0288] In some embodiments, the GREM1-associated disease or condition is selected from the group consisting of cancer, fibrotic diseases, angiogenesis, glaucoma or retinal diseases, kidney diseases, pulmonary arterial hypertension, and osteoarthritis (OA).

[0181]

[0289] In some embodiments, the GREM1-related disease or condition is cancer.In certain embodiments, the cancer is metastatic cancer.In certain embodiments, the cancer is prostate cancer, breast cancer, glioma, liposarcoma, hepatocellular carcinoma, lung cancer, cervical cancer, endometrial cancer, uterine leiomyosarcoma, squamous cell carcinoma of the head and neck, thyroid cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, esophageal cancer, bile duct cancer, osteosarcoma, glioblastoma, ovarian cancer, gastric cancer, triple-negative breast cancer (TNBC), small cell lung cancer or melanoma.

[0182]

[0290] In some embodiments, the cancer is prostate cancer.

[0183]

[0291] In some embodiments, the prostate cancer a) is negative for androgen receptor (AR) expression, b) is negative for both androgen receptor (AR) expression and neuroendocrine (NE) differentiation; c) is resistant to androgen deprivation therapy, optionally castration resistant, d) exhibits levels of prostate specific antigen (PSA) lower than a reference level, or e) any combination of a)-d).

[0184]

[0292] In some embodiments, the cancer is breast cancer. The breast cancer can be triple-negative breast cancer.

[0185]

[0293] In some embodiments, the fibrotic disease is pulmonary fibrosis, dermal fibrosis, diabetic nephropathy, or ischemic kidney injury.

[0186]

[0294] The method comprises administering to the subject a therapeutically effective amount of a GREM1 antagonist. The GREM1-associated disease or condition can be a disease or condition that would benefit from regulating GREM1 activity (e.g., reducing GREM1 activity). In some embodiments, the GREM1-associated disease or condition is characterized by GREM1 expression or overexpression.

[0187]

[0295] In some embodiments, the GREM1-associated disease or condition characterized by PTEN and / or p53 deficiency can be selected from the group consisting of cancer, fibrotic disease, angiogenesis, glaucoma or retinal disease, kidney disease, pulmonary arterial hypertension, and osteoarthritis (OA). Increased levels of GREM1 have been associated with many of these diseases and conditions, such as scleroderma, diabetic nephropathy, glioma, head and neck cancer, prostate cancer, and colorectal cancer.

[0188]

[0296] Cancer

[0297] In some embodiments, the GREM1-associated disease or condition characterized by defects in PTEN and / or p53 is cancer, in particular a GREM1-expressing cancer.

[0189]

[0298] The treatment methods provided herein are based on the surprising finding of a previously unknown significant upregulation of GREM1 in cancer cells that are deficient in PTEN and / or p53.

[0190]

[0299] In certain embodiments, the cancer is selected from solid tumors or hematological tumors. In certain embodiments, the solid tumor is selected from adrenocortical carcinoma, anal cancer, astrocytoma, pediatric cerebellum or cerebrum, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, Burkitt's lymphoma, cervical cancer, colon cancer, emphysema, endometrial cancer, esophageal cancer, Ewing's sarcoma, retinoblastoma, cancer, stomach cancer, glioma, head and neck cancer, heart cancer, Hodgkin's lymphoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, liver cancer, lung cancer, neuroblastoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, Ewing's family of tumors, skin cancer, stomach cancer, testicular cancer, pharyngeal cancer, thyroid cancer, or vaginal cancer.

[0191]

[0300] The hematological tumor is a leukemia (e.g., acute lymphoma leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoma leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin's lymphoma, or non-Hodgkin's lymphoma (e.g., Van Denstrom's gammopathy (WM))), or myeloma (e.g., multiple myeloma (MM)). In certain embodiments, the cancer is multiple myeloma (MM). GREM1 has been found to be abundantly secreted by a subset of bone marrow (BM) mesenchymal stromal cells and is believed to play a critical role in MM disease development. Analysis of human and mouse BM stromal samples by quantitative PCR showed that GREM1 / Grem1 expression was significantly higher in MM tumor-bearing cohorts compared to healthy subjects. Anti-GREM1 antibodies have been shown to reduce MM tumor burden in mice (K. Clark et al., Cancers 2020, 12, 2149).

[0192]

[0301] In certain embodiments, the cancer is prostate cancer, gastroesophageal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, hepatic bile duct cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, gastrointestinal cancer, skin cancer, pituitary cancer, stomach cancer, cancer), vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, adenocarcinoma, leukemia (e.g., acute lymphoma leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoma leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin's lymphoma, or non-Hodgkin's lymphoma (e.g., Wandenström's hypergammaglobulinemia (WM))), or myeloma (e.g., multiple myeloma (MM)), triple-negative breast cancer (TNBC)), small cell lung cancer, esophageal cancer, osteosarcoma, and gastric cancer.

[0193]

[0302] In certain embodiments, the cancer is selected from the group consisting of prostate cancer, gastroesophageal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, colon cancer, colorectal cancer, glioma, pancreatic cancer, bladder cancer and breast cancer.In certain embodiments, the cancer is triple-negative breast cancer.In certain embodiments, the cancer is multiple myeloma.

[0194]

[0303] In certain embodiments, the cancer is metastatic. In certain embodiments, the present disclosure further provides a method for treating or preventing cancer metastasis using the antibodies provided herein. Cancer metastasis is the process by which cancer cells spread from their original site to another site in the body.

[0195]

[0304] In certain embodiments, the cancer is prostate cancer, breast cancer or liver cancer. The tumor suppressors Pten and p53 are often lost in prostate or breast cancer.

[0196]

[0305] In certain embodiments, breast cancer is triple-negative breast cancer.The term "triple-negative breast cancer" or "TNBC" refers to breast cancer that tests negative for estrogen receptor, progesterone receptor, and excess HER2 protein.TNBC can be non-responsive to hormone therapy or drugs that target HER2.Expression in sample can be detected as described above under the section "method of treating GREM1-associated prostate cancer with reduced androgen receptor signaling."

[0197]

[0306] TNBCs with defective PTEN and / or p53 have a worse prognosis compared to other TNBCs with normal levels of these tumor suppressors (Jeff CL et al., EMBO Mol Med (2014) 6:1542-1560). Mixed Pten-p53 mutations have been found to accelerate the formation of claudin-low triple-negative-like breast cancers (TNBCs), which exhibit hyperactivated AKT signaling and more mesenchymal features compared to Pten or p53 single mutant tumors.

[0198]

[0307] In some embodiments, the GREM1-related disease or condition characterized by PTEN and / or p53 deficiency is liver cancer, for example, hepatocellular carcinoma (HCC). In some embodiments, the liver cancer is Hepatitis B virus (HBV) infection-related HCC. HCC is the second leading cause of cancer-related death worldwide. Persistent HBV infection is one of the major risk factors for HCC development, which accounts for more than 50% of HCC worldwide. HBV infection-related HCC can be developed through CRISPR / Cas9-mediated mutation of p53 and PTEN locus, which leads to PTEN and / or p53 deficiency (Yongzhen L. et al., Scientific Reports (2017) 7:2796). The origin of HCC is considered to be the enhanced proliferation and maturation arrest of liver progenitor / stem cells, which was shown to be promoted by fibrosis via fibroblast-secreted GREM1, which blocks BMP function (Guimei M et al. BMC Res Notes 2012;5:390).

[0199]

[0308] ii. Fibrotic diseases

[0309] The PTEN and / or p53 deficiency disease or condition may be a non-cancer disease, so long as the disease is characterized by a PTEN and / or p53 deficiency further associated with GREM1 upregulation. For example, non-cancer diseases such as lung and skin fibrosis and diabetic and ischemic kidney injury have been reported to involve dysregulation of p53 or PTEN, and these diseases are also known to be associated with GREM1 expression (see, for more information, Rohan Samarakoon et al., Loss of Tumour Suppressor PTEN Expression in Renal Injury Initiates SMAD3 and p53 Dependent Fibrotic Responses, J Pathol. 2015 Aug;236(4):421-432; Nagaraja MR et al., "p53 Expression in Lung Fibroblasts Is Linked to Mitigation of Fibrotic Lung Remodeling," Am J Pathol. 2018 Oct;188(10):2207-2222). The inventors have unexpectedly discovered a correlation between GREM1 and PTEN / p53 and therefore expect that non-cancer diseases or conditions characterized by defects in PTEN and / or p53 may also be treated by administering GREM1 modulators, including GREM1 antagonists.

[0200]

[0310] In some embodiments, the GREM1-associated disease or condition characterized by the deficiency of PTEN and / or p53 is a fibrotic disease.The fibrotic disease is a disease or condition associated with fibrosis.Fibrosis is a scarring process that is a common feature of chronic organ damage, for example in the lung, liver, kidney, skin, heart, intestine or muscle.Fibrosis is characterized by the increased activity of transforming growth factor beta (TGF-β), which leads to increased and altered deposition of extracellular matrix and other fibrosis-associated proteins. Elevated GREM1 expression has been found in many fibrotic diseases, suggesting that GREM1 may be an important marker of fibrosis (Costello et al., 2010, Am. J. Respir. Cell. Mol. Biol. 42:517-523; Lappin et al., 2002, Nephrol. Dial. Transplant. 17:65-67; ​​Boers et al., 2006, J. Biol. Chem. 281:16289-16295).

[0201]

[0311] Fibrotic diseases can include fibrotic diseases of the lung, liver, kidney, eye, skin, heart, intestine or muscle. Examples of fibrotic diseases of the lung include pulmonary fibrosis, cystic fibrosis, pulmonary hypertension, progressive massive fibrosis, bronchiolitis obliterans, airway remodeling associated with chronic asthma or idiopathic pulmonary fibrosis. Examples of fibrotic diseases of the liver include cirrhosis or non-alcoholic steatohepatitis. Examples of fibrotic diseases of the kidney include, for example, renal fibrosis, ischemic kidney injury, tubulointerstitial fibrosis, diabetic nephropathy, nephrosclerosis or nephrotoxicity. Examples of fibrotic diseases of the eye include, for example, corneal fibrosis, subretinal fibrosis. Examples of fibrotic diseases of the skin include, for example, nephrogenic systemic fibrosis, keloid or scleroderma. Examples of fibrotic diseases of the heart include endomyocardial fibrosis or old myocardial infarction.

[0202]

[0312] iii. Other diseases

[0313] In some embodiments, the GREM1-related disease or condition is pulmonary arterial hypertension (PAH). The term "pulmonary arterial hypertension" ("PAH") refers to a progressive lung disorder characterized by persistent elevation of pulmonary arterial pressure. GREM1 has been found to be elevated in the walls of small intrapulmonary blood vessels in mice during hypoxia. Anti-GREM1 antibodies have been found to alleviate or ameliorate one or more symptoms associated with PAH, for example, inhibiting pulmonary arterial thickening, increasing cardiac output and / or cardiac output to end-systolic volume ratio ("SV / ESV"), increasing right ventricular cardiac output and / or cardiac index (CI), and improving other hemodynamic measures, such as right atrial pressure, pulmonary arterial pressure, pulmonary capillary outlet pressure in the presence of end-expiratory pressure, systemic arterial pressure, heart rate, pulmonary vascular resistance, and / or systemic vascular resistance in subjects with PAH (see US Patent Publication No. 20180057580 for details).

[0203]

[0314] In some embodiments, the GREM1-related disease or condition is osteoarthritis (OA). GREM1 has been reported as a mechanical load-inducing factor in chondrocytes, and is detected at high levels in the middle and deep layers of cartilage after cyclic strain or hydrostatic pressure loading. GREM1 has been reported to be upregulated in osteoarthritis, and GREM1 concentrations in serum and synovial fluid correlate with the onset and severity of knee OA (J.Yi et al., Med Sci Monit, 2016; 22:4062-4065). GREM1 activates nuclear factor-κB signaling, which subsequently induces degradative enzymes. Intra-articular administration of GREM1 antibodies or chondrocyte-specific deletion of GREM1 in mice has been reported to slow down the development of osteoarthritis (see SH Chang et al., Nature Communications, (2019) 10:1442).

[0204]

[0315] In some embodiments, the GREM1-related disease or condition is angiogenesis. GREM1 is an agonist of vascular endothelial growth factor receptor-2 (VEGFR-2), a major pro-angiogenic receptor. Heparan sulfate (HS) and heparin, glycosaminoglycans (GAGs) known for their anticoagulant effects, have been shown to bind to GREM1. GREM1 binds to heparin and activates VEGFR-2 in a BMP-independent manner (Chiodelli et al., 2011; Arterioscler.Thromb.Vasc.Biol.31:e116-e127). Anti-GREM1 antibodies have been found to reduce or ameliorate one or more symptoms associated with angiogenesis or heparin-mediated angiogenesis (see US Patent Publication No. 20200157194 for details).

[0205]

[0316] In some embodiments, GREM1-related disease or condition is glaucoma.Glaucoma may be caused by the alteration of the expression of one or more BMP family genes in eye, which causes increased intraocular pressure and / or glaucomatous neuropathy.GREM1 has been found to have increased expression in glaucoma trabecular meshwork cells.GREM1 antagonist has been found to alleviate or ameliorate one or more symptoms related to angiogenesis or glaucoma (see US Patent No. 7,744,873 for details).

[0206]

[0317] In some embodiments, the GREM1 associated disease or condition is a retinal disease. In some embodiments, the GREM1 associated disease or condition is a kidney disease.

[0207]

[0318] GREM1 antagonists

[0319] In some embodiments, the GREM1 antagonist reduces GREM1 levels or activity. For example, the GREM1 antagonist can partially inhibit, i.e., reduce, or completely inhibit, i.e., completely eliminate, the expression and / or activity of GREM1.

[0208]

[0320] A GREM1 antagonist may reduce GREM1 levels or activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0209]

[0321] Any function or activity of GREM1 can be reduced.In certain embodiments, GREM1 antagonist optionally reduces GREM1-mediated inhibition of BMP signaling and / or GREM1-mediated activation of MAPK signaling in cancer cells.In some embodiments, GREM1 antagonist inhibits BMP-independent GREM1 activity.

[0210]

[0322] In certain embodiments, the GREM1 antagonist selectively reduces the function or activity of GREM1 in cancer cells relative to non-cancer cells. The reduction in GREM1 function or activity or level can be measured using any suitable assay performed in the presence and absence of the GREM1 antagonist.

[0211]

[0323] In some embodiments, GREM1 antagonist comprises GREM1-FGFR1 axis inhibitor.The present disclosure surprisingly finds that GREM1 may play a role in the activation of MAPK signaling, which may be independent of BMP, and possibly acts as a novel ligand of FGFR.Therefore, the GREM1-FGFR1 axis inhibitor provided herein refers to any inhibitor that can interfere with or inhibit the signaling of GREM1-dependent FGFR1 signaling, or block the binding between GREM1 and FGFR1.

[0212]

[0324] In some embodiments, the GREM1-FGFR1 axis inhibitor comprises an FGFR1 binding inhibitor.

[0213]

[0325] In some embodiments, the FGFR1 binding inhibitor binds to the extracellular domain 2 of FGFR1, and optionally binds to FGFR1 at an epitope that includes residue Glu160, where the residue numbering is according to SEQ ID NO:75.

[0214]

[0326] In some embodiments, the GREM1-FGFR1 axis inhibitor binds to hGREM1 at an epitope including residues Lys123 and / or Lys124, the residue numbers being according to SEQ ID NO: 69; or blocks FGFR1 binding to residues Lys123 and / or Lys124 of FGFR1.

[0215]

[0327] In some embodiments, the GREM1 antagonist or GREM1-FGFR1 axis inhibitor comprises an antibody to hGREM1, or an antigen-binding fragment thereof, provided herein.

[0216]

[0328] In various embodiments, the GREM1 antagonist may be an anti-GREM1 antibody or antigen-binding fragment thereof, a GREM1 mimetic peptide, a nucleic acid targeting gremlin RNA or DNA, a compound that inhibits the interaction between gremlin and BMP, or a compound that inhibits GREM1-mediated biological activity. GREM1 antagonists can include anti-GREM1 antibodies or antigen-binding fragments thereof, inhibitory GREM1 mimetic peptides, inhibitory nucleic acids targeting GREM1 RNA or DNA, compounds that inhibit the interaction between gremlin and BMP, polynucleotides encoding inhibitory nucleic acids, and compounds that inhibit GREM1 activity.

[0217]

[0329] In some embodiments, inhibitory nucleic acids targeting GREM1 RNA or DNA include small hairpin RNA (shRNA), microinterfering RNA (miRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), guide RNA, or antisense oligonucleotides.

[0218]

[0330] In certain embodiments, the nucleic acid targeting Gremlin RNA or DNA is a non-coding nucleic acid, such as a small hairpin RNA (shRNA), micro-interfering RNA (miRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), guide RNA, antisense oligonucleotide, or a polynucleotide encoding such.

[0219]

[0331] In certain embodiments, GREM1 antagonist may reduce the level of GREM1 at mRNA level or protein level.For example, GREM1 antagonist may promote the degradation of GREM1 at mRNA level or protein level, destroy the DNA encoding GREM1, or reduce the transcription from the DNA encoding GREM1.Such GREM1 antagonist can include non-coding nucleic acid that targets GREM1 mRNA or DNA, such as small hairpin RNA (shRNA), micro-interfering RNA (miRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), guide RNA, antisense oligonucleotide, and polynucleotide that encodes such.GREM1 antagonist can also include agent that promotes the degradation of GREM1 protein.

[0220]

[0332] In certain embodiments, the GREM1 antagonist may be an agent that prevents (e.g., reduces) GREM1 from binding to BMPs, such as BMP2 / 4 / 7. For example, the GREM1 antagonist may be an anti-GREM1 antibody, a GREM1 mimetic peptide, or a chemical compound that reduces or blocks the binding of GREM1 to BMPs, thereby reducing GREM1-mediated inhibition of BMP signaling. Although the GREM1 antagonist may compete with GREM1 for binding to BMPs, the GREM1 antagonist may also bind to a different epitope or binding site that does not directly affect GREM1 binding to BMPs. However, it still reduces the biological function mediated by GREM1.

[0221]

[0333] In certain embodiments, the GREM1 antagonist includes an anti-GREM1 antibody, a compound that inhibits the interaction between GREM1 and BMP, or a compound that inhibits GREM1-mediated biological activity (e.g., activation of MAPK signaling, or inhibition of BMP signaling).

[0222]

[0334] In certain embodiments, the GREM1 antagonist may include any of the anti-GREM1 antibodies provided herein or any existing anti-GREM1 antibody, such as, for example, those disclosed in WO2018 / 115017, WO2019 / 158658, WO2019 / 243801, WO2014159010, the disclosures of which are incorporated herein by reference in their entireties.

[0223]

[0335] GREM1 antibody

[0336] In certain embodiments, the GREM1 antagonist comprises an antibody or antigen-binding fragment thereof against human Gremlin 1 (hGREM1) that binds to a different epitope than other anti-GREM1 antibodies. For example, the antibody or antigen-binding fragment thereof used as the GREM1 antagonist does not bind to the BMP binding loop comprising the amino acid sequence of SEQ ID NO:63.

[0224]

[0337] In certain embodiments, the GREM1 antagonist comprises an antibody or antigen-binding fragment thereof against human Gremlin 1 (hGREM1), comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the heavy chain variable region is: a) HCDR1 comprising the sequence of SEQ ID NO:1, HCDR2 comprising the sequence of SEQ ID NO:2, and HCDR3 comprising the sequence of SEQ ID NO:3; b) HCDR1 comprising the sequence of SEQ ID NO: 11, HCDR2 comprising the sequence of SEQ ID NO: 12, and HCDR3 comprising the sequence of SEQ ID NO: 13; c) HCDR1 comprising the sequence of SEQ ID NO: 21, HCDR2 comprising the sequence of SEQ ID NO: 22, and HCDR3 comprising the sequence of SEQ ID NO: 23; and d) HCDR1 comprising the sequence of SEQ ID NO: 31, HCDR2 comprising the sequence of SEQ ID NO: 32, and HCDR3 comprising the sequence of SEQ ID NO: 33. is selected from the group consisting of:

[0225]

[0338] In certain embodiments, the GREM1 antagonist comprises an antibody or antigen-binding fragment thereof against human Gremlin 1 (hGREM1), comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the light chain variable region is: a) LCDR1 comprising the sequence of SEQ ID NO:4, LCDR2 comprising the sequence of SEQ ID NO:5, and LCDR3 comprising the sequence of SEQ ID NO:6; b) LCDR1 comprising the sequence of SEQ ID NO: 14, LCDR2 comprising the sequence of SEQ ID NO: 15, and LCDR3 comprising the sequence of SEQ ID NO: 16; c) LCDR1 comprising the sequence of SEQ ID NO: 24, LCDR2 comprising the sequence of SEQ ID NO: 25, and LCDR3 comprising the sequence of SEQ ID NO: 26; and d) LCDR1 comprising the sequence of SEQ ID NO: 34, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 36. is selected from the group consisting of:

[0226]

[0339] In certain embodiments, the GREM1 antagonist comprises an antibody or antigen-binding fragment thereof against human Gremlin 1 (hGREM1), comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region; a) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:2, and an HCDR3 comprising the sequence of SEQ ID NO:3; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:4, an LCDR2 comprising the sequence of SEQ ID NO:5, and an LCDR3 comprising the sequence of SEQ ID NO:6; b) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:11, an HCDR2 comprising the sequence of SEQ ID NO:12, and an HCDR3 comprising the sequence of SEQ ID NO:13; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:15, and an LCDR3 comprising the sequence of SEQ ID NO:16; c) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:21, an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:23; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:24, an LCDR2 comprising the sequence of SEQ ID NO:25, and an LCDR3 comprising the sequence of SEQ ID NO:26; or d) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 31, an HCDR2 comprising the sequence of SEQ ID NO: 32, and an HCDR3 comprising the sequence of SEQ ID NO: 33; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 34, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 36.

[0227]

[0340] In certain embodiments, the GREM1 antagonist comprises an antibody or antigen-binding fragment thereof against human Gremlin 1 (hGREM1), comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, and SEQ ID NO:57, and homologous sequences thereof having at least 80% sequence identity but retaining specific binding specificity or affinity for Gremlin.

[0228]

[0341] In certain embodiments, the GREM1 antagonist comprises an antibody or antigen-binding fragment thereof against human Gremlin 1 (hGREM1), comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:28, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59 and SEQ ID NO:61, and homologous sequences thereof having at least 80% sequence identity but retaining specific binding specificity or affinity for Gremlin.

[0229]

[0342] In certain embodiments, the GREM1 antagonist is a) a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and a light chain variable region comprising the sequence of SEQ ID NO: 8; or b) a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18; or c) a heavy chain variable region comprising the sequence of SEQ ID NO: 27 and a light chain variable region comprising the sequence of SEQ ID NO: 28; or d) a heavy chain variable region comprising the sequence of SEQ ID NO: 37 and a light chain variable region comprising the sequence of SEQ ID NO: 38; or e) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 45, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 47 and SEQ ID NO: 49; or f) a pair of heavy chain and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 41 / 47, 41 / 49, 43 / 47, 43 / 49, 45 / 47, and 45 / 49; or g) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:53, and SEQ ID NO:55, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:59 and SEQ ID NO:61; or h) a pair of heavy chain variable region sequences and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 51 / 59, 51 / 61, 53 / 59, 53 / 61, 55 / 59, 55 / 61, 57 / 59, and 57 / 61; Includes.

[0230]

[0343] In certain embodiments, the antibodies provided herein comprise one or more (e.g., one, two, three, four, five, or six) CDR sequences of anti-hGREM1 antibodies 14E3, 69H5, 22F1, 56C11.

[0231]

[0344] As used herein, "14E3" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:7 and a light chain variable region of SEQ ID NO:8.

[0232]

[0345] As used herein, "69H5" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:27, and a light chain variable region of SEQ ID NO:28.

[0233]

[0346] As used herein, "22F1" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:17, and a light chain variable region of SEQ ID NO:18.

[0234]

[0347] As used herein, "56C11" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:37, and a light chain variable region of SEQ ID NO:38.

[0235]

[0348] Table 1 shows the CDR sequences of these hGREM1 antibodies. The heavy and light chain variable region sequences are also provided in Table 2 below.

[0236]

[0349]

[0237] [Table 2]

[0238]

[0350]

[0239] [Table 3]

[0240]

[0351] The anti-hGREM1 antibodies or antigen-binding fragments thereof provided herein can be monoclonal, polyclonal, humanized, chimeric, recombinant, bispecific, labeled, bivalent, or anti-idiotypic antibodies. Recombinant antibodies are antibodies prepared in vitro using recombinant methods rather than in animals.

[0241]

[0352]

[0242] [Table 4-1]

[0243] [Table 4-2]

[0244]

[0353] In certain embodiments, the GREM1 antagonist comprises an anti-human GREM1 antibody or antigen-binding fragment thereof, which a) is capable of binding to hGREM1 at an epitope comprising residues Gln27 and / or Asn33, the residues numbered according to SEQ ID NO: 69; and / or b) is capable of binding to an hGREM1 fragment comprising residues Gln27 and / or Asn33, optionally the hGREM1 fragment having a length of at least 3 (e.g., 4, 5, 6, 7, 8, 9 or 10) amino acid residues; and / or c) is capable of selectively reducing hGREM1-mediated inhibition of BMP signaling in cancer cells compared to non-cancer cells; and / or d) exhibits no more than a 50% reduction in hGREM1-mediated inhibition of BMP signaling in non-cancer cells; and / or e) is capable of binding to a chimeric hGREM1 comprising the amino acid sequence of SEQ ID NO: 68; and / or f) is capable of reducing hGREM1-mediated activation of MAPK signaling; and / or g) has a K of no more than 1 nM as measured by Fortebio. D can bind to hGREM1.

[0245]

[0354] In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof further comprises one or more amino acid residue substitutions or modifications, but retains specific binding specificity or affinity for hGREM1.

[0246]

[0355] In certain embodiments, at least one of the substitutions or modifications is present in one or more of the CDR sequences and / or in one or more non-CDR regions of the VH or VL sequences.

[0247]

[0356] In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a human Ig constant region or a human IgG constant region.

[0248]

[0357] In certain embodiments, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0249]

[0358] In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is humanized.

[0250]

[0359] In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody, and bivalent domain antibody.

[0251]

[0360] In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is bispecific. The term "bispecific" as used herein encompasses molecules with more than two specificities and molecules with more than two specificities, i.e., multispecific. In certain embodiments, the bispecific antibody and antigen-binding fragment thereof provided herein can specifically bind to a first and a second epitope of hGREM1, or can specifically bind to hGREM1 and a second antigen. In certain embodiments, the first epitope and the second epitope of hGREM1 are completely separate or non-overlapping from each other. In certain embodiments, the bispecific antibody and antigen-binding fragment thereof can simultaneously bind to both the first epitope and the second epitope.

[0252]

[0361] In certain embodiments, the second antigen is different from hGREM1. In certain embodiments, the second antigen comprises an immune-related target. In certain embodiments, the second antigen comprises PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD47, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16 or CD83.

[0253]

[0362] In certain embodiments, the tumor antigen comprises a tumor-specific antigen or a tumor-associated antigen. Tumor antigens include prostate-specific antigen (PSA), CA-125, ganglioside G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptide, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFR (e.g., VEGFR-1, VEGFR-2, VEGFR-3), estrogen, and the like. gen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2, GPC-3, nectin-4, ROR1, mesothelin, PCMA, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, BCR-ABL, E2APRL, H4-RET, IGH-IGK, MYL-RAR, IL-2R, CO17-1A, TROP2, or LIV-1.

[0254]

[0363] In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is not cross-reactive to mouse Gremlin 1.

[0255]

[0364] In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof is cross-reactive to mouse Gremlin 1.

[0256]

[0365] In certain embodiments, the GREM1 antagonist can reduce GREM1-mediated activation of MARK signaling.

[0257]

[0366] Combination Treatment Methods

[0367] The treatment methods provided herein may further include providing a second therapeutic agent and administering a therapeutically effective amount of the second therapeutic agent to the subject, thereby treating, preventing, reducing the severity and / or slowing the rate of progression of a GREM1-associated disease or condition in the subject. In some of these embodiments, the GREM1-associated disease or condition may be characterized by PTEN and / or p53 deficiency, and / or is a cancer characterized by reduced androgen receptor (AR) signaling.

[0258]

[0368] In some of these embodiments, the GREM1 antagonist disclosed herein and administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in some of these embodiments, the GREM1 antagonist and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, a GREM1 antagonist administered "in combination" with another therapeutic agent need not be administered simultaneously with or in the same composition as that agent. A GREM1 antagonist administered prior to or after another agent is considered to be administered "in combination" with that agent, as that phrase is used herein, even if the GREM1 antagonist and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with the GREM1 antagonists disclosed herein will be administered according to a schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)) or protocols known in the art.

[0259]

[0369] i) Combination Treatments for Cancer

[0370] In some embodiments, the GREM1 antagonists disclosed herein may be administered to treat cancer in combination with a second anti-cancer agent, e.g., a chemotherapeutic agent (e.g., cisplatin), an anti-cancer drug, radiation therapy, immunotherapy (e.g., immune checkpoint inhibitors, MPDL-3280A), an anti-angiogenic agent, targeted therapy, cell therapy, gene therapy, hormonal therapy, cytokines, palliative care, surgery for cancer treatment (e.g., tumor removal), one or more antiemetic agents, treatment for complications resulting from chemotherapy, or dietary supplements for cancer patients.

[0260]

[0371] The term "immunotherapy" as used herein refers to a type of immune system that stimulates or generally boosts the immune system to fight against diseases such as cancer.Immunotherapy includes passive immunotherapy by delivering agents that have established tumor immune reactivity (e.g., effector cells) and can directly or indirectly mediate antitumor effects, but do not necessarily rely on an intact host immune system (e.g., antibody therapy or CAR-T cell therapy).Immunotherapy can also include active immunotherapy, where treatment relies on the in vivo stimulation of the endogenous host immune system to react against diseased cells with the administration of immune response modifiers.

[0261]

[0372] Examples of immunotherapeutics include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses and therapeutic vaccines.

[0262]

[0373] Checkpoint modulators can hinder the ability of cancer cells to evade immune system attack and aid in a more powerful immune system response against tumors. Immune checkpoint molecules can mediate costimulatory signals to enhance immune responses or co-inhibitory signals to suppress immune responses. Examples of checkpoint modulators include, without limitation, modulators of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD47, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16, and CD83. In certain embodiments, the immune checkpoint modulator comprises a PD-1 / PD-L1 axis inhibitor.

[0263]

[0374] Adoptive cell transfer is a procedure that attempts to boost the natural ability of T cells to fight cancer. In this procedure, T cells are taken from a patient and expanded and activated in vitro. In certain embodiments, the T cells are modified in vitro into CAR-T cells. The T cells or CAR-T cells that are most active against cancer are cultured in vitro in large batches for 2-8 weeks. During this period, the patient undergoes treatments such as chemotherapy and radiation therapy to reduce the body's immunity. After these treatments, the in vitro cultured T cells or CAR-T cells are returned to the patient. In certain embodiments, the immunotherapy is CAR-T therapy.

[0264]

[0375] Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system. The two main types of cytokines used in cancer treatment are interferons and interleukins. Examples of cytokine therapy include, but are not limited to, interferons such as interferon-α, -β, and -γ, colony stimulating factors such as macrophage-CSF, granulocyte-macrophage CSF, and granulocyte-CSF, insulin growth factor (IGF-1), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), fibroblast growth factor (FGF), interleukins such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12, tumor necrosis factors such as TNF-α and TNF-β, or any combination thereof.

[0265]

[0376] Oncolytic viruses are genetically engineered viruses that can kill cancer cells. Oncolytic viruses specifically infect tumor cells, thereby resulting in tumor cell lysis and subsequent release of large amounts of tumor antigens that can trigger the immune system to target and eliminate cancer cells bearing such tumor antigens. Examples of oncolytic viruses include, but are not limited to, talimogene laherparepvec.

[0266]

[0377] Therapeutic vaccines work against cancer by boosting the immune system's response to cancer cells. Therapeutic vaccines can include non-pathogenic microorganisms (e.g., Mycobacterium bovis Bacillus Calmette-Grant, BCG), genetically modified viruses that target tumor cells, or one or more immunogenic components. For example, BCG can be inserted directly into the bladder using a catheter and can elicit an immune response against bladder cancer cells.

[0267]

[0378] Antiangiogenic agents can block the growth of blood vessels that support tumor growth.Some antiangiogenic agents target VEGF or its receptor VEGFR.Examples of antiangiogenic agents include, but are not limited to, axitinib, bevacizumab, cabozantinib, everolimus, lenalidomide, lenvatinib mesylate, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib, thalidomide, vandetanib, and Ziv-aflibercept.

[0268]

[0379] "Targeted therapy" is a type of therapy that acts on specific molecules associated with cancer, such as specific proteins that are present in cancer cells but not present or more abundant in normal cells, or target molecules in the cancer microenvironment that contribute to the growth and survival of the cancer. Targeted therapy directs therapeutic agents to the tumor, thereby sparing normal tissue from the effects of the therapeutic agent.

[0269]

[0380] Target therapy can target, for example, tyrosine kinase receptor and nuclear receptor.Examples of such receptors include erbB1 (EGFR or HER1), erbB2 (HER2), erbB3, erbB4, FGFR, platelet-derived growth factor receptor (PDGFR), and insulin-like growth factor-1 receptor (IGF-1R), androgen receptor (AR), estrogen receptor (ER), nuclear receptor (NR) and PR.

[0270]

[0381] Targeted therapies can target molecules in the tyrosine kinase or nuclear receptor signaling cascades, such as Erk and PI3K / Akt, AP-2α, AP-2β, AP-2γ, mitogen-activated protein kinase (MAPK), PTEN, p53, p19ARF, Rb, Apaf-1, CD-95 / Fas, TRAIL-R1 / R2, caspase-8, forkhead, box 03A, MDM2, IAPs, NF-kB, Myc, P13K, Ras, FLIP, heregulin (HRG) (also known as gp30), Bcl-2, Bcl-xL, Bax, Bak, Bad, Bok, Bik, Blk, Hrk, BNIP3, BimL, Bid, and EGL-1.

[0271]

[0382] Targeted therapies can also target tumor-associated ligands such as estrogen, estradiol (E2), progesterone, estrogen, androgens, glucocorticoids, prolactin, thyroid hormones, insulin, P70 S6 kinase protein (PS6), survivin, fibroblast growth factor (FGF), EGF, Neu differentiation factor (NDF), transforming growth factor alpha (TGF-α), IL-1A, TGF-beta, IGF-1, IGF-II, IGFBP, IGFBP protease, and IL-10.

[0272]

[0383] In some embodiments, GREM1 antagonist disclosed herein can be administered in combination with a second anticancer drug to treat prostate cancer.In certain embodiments, anticancer drug comprises antiprostate cancer drug.In some embodiments, antiprostate cancer drug comprises androgen axis inhibitor; androgen synthesis inhibitor; ADP-ribose polymerase (PARP) inhibitor; or combination thereof.

[0273]

[0384] In certain embodiments, the androgen axis inhibitor is selected from the group consisting of a luteinizing hormone releasing hormone (LHRH) agonist, an LHRH antagonist, and an androgen receptor antagonist.

[0274]

[0385] In certain embodiments, the androgen axis inhibitor is degarelix, bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, or abiraterone.

[0275]

[0386] In certain embodiments, the androgen synthesis inhibitor is abiraterone acetate or ketoconazole.

[0276]

[0387] In certain embodiments, the PARP inhibitor is olaparib or rucaparib.

[0277]

[0388] In certain embodiments, the anti-prostate cancer drug is abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), darolutamide, degarelix, docetaxel, Eligard (leuprolide acetate), enzalutamide, Erleada (apalutamide), Firmagon (degarelix), flutamide, goserelin acetate, Jevtana (cabazitaxel), leuprolide acetate, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate) ), Lynparza (olaparib), mitoxantrone hydrochloride, Nilandrone (nilutamide), nilutamide, Nubequo (darolutamide), olaparib, Provenzi (sipuleucel-T), radium-223 dichloride, Rubraca (rucaparib cansylate), rucaparib cansylate, sipuleucel-T, Taxotere (docetaxel), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Zoladex (goserelin acetate) and Zytiga (abiraterone acetate).

[0278]

[0389] In certain embodiments, dietary supplements for cancer patients can be suitable nutritional supplements that have a protective effect against cancer.In certain embodiments, dietary supplements include indole-3-carbinol or its derivatives that produce indole-3-carbinol after digestion.Indole-3-carbinol is believed to have a protective effect against cancer and may also be preventative against precancerous conditions.

[0279]

[0390] In certain embodiments, the antibody or antigen-binding fragment disclosed herein may be administered in combination with indole-3-carbinol or its derivative that produces indole-3-carbinol after digestion.In certain embodiments, such combination is useful for treating Gremlin-related diseases.In certain embodiments, such combination is useful for treating cancer, such as breast cancer, hepatocellular carcinoma, and colorectal cancer.In certain embodiments, such combination is useful for treating breast cancer, such as triple-negative breast cancer.

[0280]

[0391] ii) Combination treatments for non-cancerous diseases

[0392] In some embodiments, the second therapeutic agent may be administered to manage or treat at least one complication associated with a non-cancer disease (eg, fibrosis) or cancer.

[0281]

[0393] In some embodiments, the second therapeutic agent is an anti-fibrotic drug such as pirfenidone, an anti-inflammatory drug, an NSAID, a corticosteroid such as prednisone, a nutritional supplement, a vascular endothelial growth factor (VEGF) antagonist (e.g., aflibercept, as described in U.S. Pat. No. 7,087,411, or other VEGF inhibitory fusion protein, or an anti-VEGF antibody or its antigen-binding fragment (e.g., bevacizumab, or ranibizumab)), an antibody against a cytokine such as IL-1, IL-6, IL-13, IL-4, IL-17, IL-25, IL-33, or TGF-β, and any other palliative care that is useful for improving at least one symptom associated with fibrosis-related conditions or cancer.In certain embodiments, the second therapeutic agent is an anti-integrin inhibitor.

[0282]

[0394] In another aspect, the disclosure provides a kit or pharmaceutical composition comprising a GREM1 antagonist provided herein and a second therapeutic agent, which may be formulated in one composition or in different compositions, and may further include instructions for use or indication to provide information on how the combination therapy should be carried out.

[0283]

[0395] In certain embodiments, dietary supplements for cancer patients can be suitable nutritional supplements that have a protective effect against cancer.In certain embodiments, dietary supplements include indole-3-carbinol or its derivatives that produce indole-3-carbinol after digestion.Indole-3-carbinol is believed to have a protective effect against cancer and may also be preventative against precancerous conditions.

[0284]

[0396] In certain embodiments, the antibody or antigen-binding fragment disclosed herein may be administered in combination with indole-3-carbinol or its derivative that produces indole-3-carbinol after digestion.In certain embodiments, such combination is useful for treating Gremlin-related diseases.In certain embodiments, such combination is useful for treating cancer, such as breast cancer, hepatocellular carcinoma, and colorectal cancer.In certain embodiments, such combination is useful for treating breast cancer, such as triple-negative breast cancer.

[0285]

[0397] Route of Administration and Dosing Regimens

[0398] The GREM1 antagonist provided herein may be administered in a therapeutically effective dosage.The therapeutically effective amount of the antibody or antigen-binding fragment provided herein depends on various factors known in the art, such as subject's weight, age, past medical history, current drug therapy, health condition, and the possibility of cross-reaction, allergy, sensitivity and adverse side effects, as well as route of administration and the extent of disease development.Dosage may be proportionally reduced or increased by those skilled in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.

[0286]

[0399] In certain embodiments, the GREM1 antagonists (e.g., antibodies or antigen-binding fragments) provided herein may be administered at a therapeutically effective dosage of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose may vary during treatment. In certain embodiments, the administered dose may vary during treatment depending on the subject's response.

[0287]

[0400] Dosage regimens may be adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0288]

[0401] The GREM1 antagonists (e.g., antibodies and antigen-binding fragments) disclosed herein may be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous including intravenous drip, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0289]

[0402] Methods of Detection and / or Diagnosis

[0403] AR expression or signaling determination

[0404] In one aspect, the disclosure provides a method for determining the likelihood of responsiveness to a GREM1 antagonist in a subject having or suspected of having cancer, the method comprising: (a) detecting androgen receptor (AR) expression or signaling in a biological sample from the subject; and (b) determining the likelihood of responsiveness based on the AR expression or signaling detected in step (a).

[0290]

[0405] As used in this disclosure, the terms "likelihood" and "likely" refer to the percentage likelihood of how likely a therapeutic response will occur. In some embodiments, a subject with a disease or condition (e.g., cancer) identified as "likely to respond" is one who has a disease or condition that has a greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% likelihood of responding to treatment with a GREM1 antagonist provided herein.

[0291]

[0406] In patients, a beneficial response can be expressed in terms of a number of clinical parameters, including disappearance of detectable tumor (complete response), a reduction in tumor size and / or tumor cell count (partial response), cessation of tumor growth (stable disease), an enhancement of the anti-tumor immune response, possibly resulting in regression or elimination of the tumor; some alleviation of one or more symptoms associated with the tumor; an increase in the length of survival following treatment; and / or a decrease in mortality at a given time point following treatment.

[0292]

[0407] AR expression can be detected by using any suitable method known in the art.In some embodiments, the method provided herein comprises contacting biological sample with an agent that can detect the presence or level of AR expression in biological sample.Detection of AR expression can be based on the presence or absence of AR expression, and the absence of AR expression indicates that the sample is negative for AR.

[0293]

[0408] AR signal transduction can be detected or determined by any suitable method known in the art, including, but not limited to, by measuring AR-sensitive gene products such as PSA.The level of AR-sensitive gene products can be determined and compared with a reference level, and a detection level significantly lower than the reference level indicates reduced AR signal transduction.The reference level for AR signal transduction can be obtained from one or more reference samples (e.g., samples obtained from a database) that are determined to have a reference level of AR signal transduction in comparable subjects, and this reference level comprises a collection of data, standards, or levels from one or more reference samples.In some embodiments, such collection of data, standards, or levels is normalized.

[0294]

[0409] Reduced AR signal transduction can also be determined based on the treatment with androgen deprivation therapy or the presence of inactivating mutation in AR.The mutation status or expression level of AR at DNA or RNA level can be measured by any method known in the art, for example, but not limited to, amplification assay, hybridization assay, or sequencing assay.The mutation status or expression level of AR at protein level can be measured by any method known in the art, for example, but not limited to, immunoassay.

[0295]

[0410] In some embodiments, a subject is determined to have potential responsiveness to a GREM1 antagonist if the subject is detected as having no AR expression or signaling, or detected as having reduced AR expression or signaling compared to a reference level.

[0296]

[0411] In some embodiments, if the subject is detected as having no AR expression or signaling, or decreased AR expression or signaling compared to a reference level, the method further includes a step of recommending that the subject have GREM1 expression tested.

[0297]

[0412] In some embodiments, the method further comprises detecting GREM1 expression in a biological sample from the subject.

[0298]

[0413] GREM1 expression can be detected by using any suitable method known in the art.In some embodiments, the method provided herein comprises contacting biological sample with an agent that can detect the presence or level of GREM1 expression in biological sample.Detection of GREM1 expression can be based on the presence or absence of GREM1 expression, and the presence of GREM1 expression indicates that the sample is positive for GREM1.

[0299]

[0414] Alternatively, detection can be based on the level of GREM1 expression, and a detection level higher than the reference level indicates GREM1 positivity. The reference level can be obtained from one or more reference samples (e.g., samples obtained from healthy subjects, from healthy tissues or even from precancerous tissues of tumor patients). Detection of GREM1 expression can be performed in parallel in the reference sample and the biological sample of interest. The reference level can also be obtained from a database, which includes a collection of data, standards or levels from one or more reference samples. In some embodiments, such collection of data, standards or levels is normalized.

[0300]

[0415] In some embodiments, if GREM1 expression is not detected in the biological sample, the method further includes monitoring GREM1 expression in the subject after a period of time, for example, after 1 month, 2 months, 3 months, etc.

[0301]

[0416] In some embodiments, if the subject is detected as having GREM1 expression or elevated GREM1 expression compared to a reference level, the subject is determined to have potential responsiveness to a GREM1 antagonist.

[0302]

[0417] In another aspect, the disclosure provides a method for detecting the presence or amount of GREM1 in a sample determined to have no AR expression or reduced androgen receptor (AR) signaling, the method comprising contacting the sample with a detection reagent for detecting GREM1, and determining the presence or amount of GREM1 in the sample.

[0303]

[0418] In some embodiments, the sample is obtained from a subject having or suspected of having cancer as disclosed herein.

[0304]

[0419] In some embodiments, the method further includes administering a therapeutically effective amount of a GREM1 antagonist (e.g., any of the anti-GREM1 antibodies or antigen-binding fragments thereof provided herein) to a subject determined to have potential responsiveness to the GREM1 antagonist.

[0305]

[0420] PTEN / p53 detection

[0421] In another aspect, the disclosure provides a method for determining the likelihood of responsiveness to a GREM1 antagonist in a subject having or suspected of having a disease or condition, the method comprising the steps of (a) detecting PTEN and / or p53 deficiency in a biological sample from the subject, and (b) determining the likelihood of responsiveness based on the PTEN and / or p53 deficiency detected in step (a).

[0306]

[0422] Deficiencies in PTEN and / or p53 activity or levels can result in absent or less than normal function of PTEN and / or p53, or the absence or reduced expression levels of functional PTEN and / or p53 in a biological sample.

[0307]

[0423] In some embodiments, the method further comprises detecting the expression level of functional PTEN and / or p53 using any suitable method known in the art, for example, without limitation, amplification assay, hybridization assay, sequencing assay, or immunoassay. In some embodiments, the method provided herein comprises contacting the biological sample with an agent capable of detecting the presence or level of functional PTEN and / or p53 in the biological sample. The detection of functional PTEN and / or p53 expression can be based on the presence or absence or level of functional PTEN and / or p53, and the absence or reduced level of functional PTEN and / or p53 indicates that the sample lacks PTEN and / or p53 activity or level. In some embodiments, the method further comprises detecting the mutation status of PTEN and / or p53, for example, at the DNA or RNA level.

[0308]

[0424] In some embodiments, if the subject is detected as deficient in PTEN and / or p53, the subject is determined to have the potential for responsiveness to a GREM1 antagonist.

[0309]

[0425] In some embodiments, the method further comprises the step of advising the subject to test for GREM1 expression if the subject is detected as deficient in PTEN and / or p53.

[0310]

[0426] In some embodiments, the method further comprises detecting GREM1 expression in a biological sample from the subject.Similarly, GREM1 expression can be detected and determined using any of the similar methods described above.

[0311]

[0427] In some embodiments, if the subject is detected as having GREM1 expression, the subject is determined to have a likelihood of responsiveness to a GREM1 antagonist.

[0312]

[0428] In some embodiments, if GREM1 expression is not detected in the biological sample, the method further includes monitoring GREM1 expression in the subject after a period of time, for example, after 1 month, 2 months, 3 months, etc.

[0313]

[0429] In one aspect, the disclosure provides a method for detecting the presence or amount of GREM1 in a sample determined to be deficient in PTEN and / or p53, the method comprising the steps of contacting the sample with a detection reagent for detecting GREM1, and determining the presence or amount of GREM1 in the sample.

[0314]

[0430] In some embodiments, the sample is obtained from a subject having or suspected of having a GREM1-related disease or condition as disclosed herein.

[0315]

[0431] In some embodiments, the method further includes administering a therapeutically effective amount of a GREM1 antagonist (e.g., any of the anti-GREM1 antibodies or antigen-binding fragments thereof provided herein) to a subject determined to have potential responsiveness to the GREM1 antagonist.

[0316]

[0432] Biological samples

[0433] The presence and / or expression level and / or mutation status of a biomarker (eg, AR, PTEN, p53, and / or GREM1) can be determined using a suitable biological sample obtained from the subject.

[0317]

[0434] In some embodiments, the biological sample contains or is suspected to contain cancer cells. In some embodiments, the biological sample is obtained from the cancer microenvironment. In some embodiments, the biological sample can be obtained or derived from a subject, for example, as formalin-fixed paraffin-embedded (FFPE) tissue, fresh biopsy, blood (suspected to contain circulating tumor cells), or other bodily fluids. In some embodiments, cancer cells, stromal cells, and / or extracellular matrix can be isolated from the biological sample. In certain embodiments, the biological sample can be further processed to isolate analytes, such as, for example, nucleic acids or proteins.

[0318]

[0435] In certain embodiments, the biological sample comprises cancer cells, stromal cells, stroma or fibrotic cells.

[0319]

[0436] Detection and / or Determination

[0437] As used herein, the terms "determine," "measure," and "detect" can be used interchangeably and refer to both quantitative and semi-quantitative determinations.

[0320]

[0438] The biomarkers AR, PTEN, p53, and / or GREM1 provided herein are intended to encompass different forms, including mRNA, protein, and DNA (e.g., genomic DNA).Thus, the level and / or activity of these biomarkers can be measured using the RNA (e.g., mRNA), protein, or DNA (e.g., genomic DNA) of each biomarker.Similarly, the mutation status of biomarkers can be measured using DNA (e.g., genomic DNA), RNA (e.g., mRNA), or protein (e.g., by measuring the altered protein product encoded by the mutated gene).

[0321]

[0439] Expression levels of biomarkers at the DNA or RNA level can be measured by any method known in the art, including, but not limited to, amplification assays, hybridization assays, or sequencing assays, using techniques including, but not limited to, RNA sequencing (RAN-seq) and RNAscope (Wang, Z., Gerstein, M., & Snyder, M. (2009). RNA-seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics, 10(1), 57-63; Wang et al., RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues, J Mol Diagn. 2012 Jan;14(1):22-9). The expression level of a biomarker at the protein level can be measured by any method known in the art, including, but not limited to, immunoassays (e.g., Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), sandwich assays, competitive assays, immunofluorescence staining and imaging, immunohistochemistry (IHC), and fluorescence-activated cell sorting (FACS)).

[0322]

[0440] The mutation status of a biomarker at the DNA or RNA level can be measured by any method known in the art, including, but not limited to, amplification assays, hybridization assays, or sequencing assays. The mutation status of a biomarker at the protein level can be measured by any method known in the art, including, but not limited to, immunoassays.

[0323]

[0441] The activity level of a biomarker can be measured by any suitable functional assay known in the art.

[0324]

[0442] These methods are well known in the art and are described in detail below as exemplary examples.

[0325]

[0443] i. Amplification assay

[0444] Nucleic acid amplification assays involve copying a target nucleic acid (e.g., DNA or RNA), thereby increasing the number of copies of the amplified nucleic acid sequence. Amplification can be exponential or linear. Exemplary nucleic acid amplification methods include polymerase chain reaction ("PCR", see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide To Methods And Applications (Innis et al., eds., 1990)), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR); quantitative PCR such as TaqMan®, nested PCR, ligase chain reaction (see Abravaya, K. et al., Nucleic Acids Research, 23:675-682, (1995)), branched DNA signal amplification (Urdea, M.S. et al., AIDS, 7(suppl 2:S11-S14, (1993)), amplifiable RNA reporters, Q-beta replication (see Lizardi et al., Biotechnology (1988) 6:1197), transcription-based amplification (see Kwoh et al., Proc. Natl. Acad. Sci. USA (1989) 86:1173-1177), boomerang DNA amplification, strand displacement activation, cycling probe technology, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87:1874-1878), rolling circle replication (U.S. Patent No. 5,854,033), isothermal nucleic acid sequence-based amplification (NASBA), and amplification using sequential analysis of gene expression (SAGE).

[0326]

[0445] Hybridization assay

[0446] Nucleic acid hybridization assays use probes that hybridize to target nucleic acids, thereby allowing detection of the target nucleic acids. Non-limiting examples of hybridization assays include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplex hybridization-based assays.

[0327]

[0447] In certain embodiments, the probe for hybridization assay is detectably labeled. In certain embodiments, the nucleic acid-based probe for hybridization assay is unlabeled. Such unlabeled probe can be immobilized on a solid support such as a microarray and can hybridize to a target nucleic acid molecule that is detectably labeled.

[0328]

[0448] In some embodiments, the hybridization assay can be performed on a microarray.

[0329]

[0449] Sequencing Methods

[0450] Sequencing method can determine the nucleic acid sequence of target nucleic acid, and can also count the sequenced target nucleic acid, thereby measuring the level of target nucleic acid.Examples of sequencing method include, but are not limited to, RNA sequencing, pyrosequencing, and high-throughput sequencing.

[0330]

[0451] High-throughput sequencing includes sequencing-by-synthesis, sequencing-by-ligation, and ultra-deep sequencing (such as described in Marguiles et al., Nature 437(7057):376-80 (2005)). Sequencing-by-synthesis can be performed on a solid surface (or microarray or chip) using fold-back PCR and anchored primers. Target nucleic acid fragments can be attached to the solid surface by hybridization to anchored primers and amplified bridges. This technology is used, for example, in the Illumina® sequencing platform.

[0331]

[0452] In certain embodiments, the detection of the mutation and / or wild-type status of the biomarker of interest described herein and the measurement of its level are by whole transcriptome sequencing, or RNA sequencing (e.g., RNA-Seq). Briefly, RNA-Seq includes reverse transcribing target mRNA into cDNA, fragmenting and sequencing the cDNA, and analyzing sequence data for mRNA quantification; RNA scope includes in situ hybridization of target mRNA with one or more oligonucleotides conjugated with fluorescent probes and detecting the level of mRNA by measuring fluorescence intensity.

[0332]

[0453] Immunoassays

[0454] Immunoassays typically involve detecting or measuring the presence or level of a target polypeptide or protein using an antibody that specifically binds to a biomarker polypeptide or protein (e.g., ATM, ATR, MDM2, and / or p53 protein as provided herein). Such antibodies can be obtained using methods known in the art (see, e.g., Huse et al., Science (1989) 246:1275-1281; Ward et al., Nature (1989) 341:544-546) or from commercial sources. Examples of immunoassays include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), sandwich assay, competitive assay, immunofluorescence staining and imaging, immunohistochemistry (IHC), and fluorescence-activated cell sorting (FACS). For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites & Terr, eds., 7th ed., 1991). Furthermore, immunoassays can be performed in any of a number of configurations, which are extensively reviewed in Enzyme Immunoassays (Maggio, ed., 1980); and Harlow & Lane (see above). For reviews of general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, vol. 37 (Asai, ed., 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed., 1991).

[0333]

[0455] In certain embodiments, the method of the present disclosure comprises measuring the expression level or gene copy of AR, PTEN, p53 and / or GREM1.The activity of p53 can be measured by detecting the phosphorylation of the 15th amino acid residue of p53 or by detecting the change in the expression level of downstream target genes of p53.Due to the ability of a protein to exert multiple biological activities, several acceptable bioassays may exist for a particular protein. Exemplary functional assays for measuring the activity of AR, PTEN, p53 and / or GREM1 can be found in Lee JH et al., J Biol Chem, 288:12840-12851 (2013); Loughery J, et al., Nucleic Acids Research, 42:7666-7680 (2014); Thompson T, et al., Journal Biological Chemistry, 279:53015-53022 (2004); Wienken, M. et al., J. Mol. Cell Biol. 2017;9(1):74-80.

[0334]

[0456] In certain embodiments, a decrease in expression levels of AR, PTEN and / or p53 gene products (e.g., at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% decrease) compared to a reference level of the AR, PTEN and / or p53 gene product, respectively, indicates a deficiency in AR, PTEN and / or p53 activity or levels in the biological sample.

[0335]

[0457] In certain embodiments, the expression level of AR, PTEN and / or p53 can be normalized to an internal control value or to a standard curve.For example, the respective levels of AR, PTEN and / or p53 described herein can be normalized to the standard level for standard marker.The standard level of standard marker can be determined in advance, at the same time, or after the sample is obtained from the subject.The standard marker can be performed in the same assay or can be a known standard marker from a previous assay.When the level of PTEN and / or p53 is determined by sequencing assay (such as RNA sequencing), the level of biomarker can be normalized to the total reading data of sequencing.

[0336]

[0458] In certain embodiments, if the RNA or DNA level of AR, PTEN and / or p53 is to be measured, the method further comprises isolating nucleic acid from the sample.Various extraction methods are suitable for isolating DNA or RNA from cells or tissues, such as phenol and chloroform extraction, and various other methods described, for example, in Ausubel et al., Current Protocols of Molecular Biology (1997) John Wiley & Sons, and in Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition (2001).

[0337]

[0459] Commercially available kits can also be used to isolate RNA, including NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France), QIAamp™ mini blood kit, Agencourt Genfind™, Rneasy® mini columns (Qiagen), PureLink® RNA mini kit (Thermo Fisher Scientific), and Eppendorf Phase Lock Gels™. A person skilled in the art can easily extract or isolate RNA or DNA following the manufacturer's protocol.

[0338]

[0460] kit

[0461] In another aspect, the present disclosure further provides kits for use in the methods described herein.

[0339]

[0462] In one embodiment, the kit comprises a first reagent, or a first set of reagents, for detecting the presence or absence of one or more inactivating mutations in PTEN / p53; or one or more reagents for measuring the expression level of PTEN / p53. In one embodiment, the kit further comprises a second reagent for detecting the presence or absence or expression level of GREM1.

[0340]

[0463] In one embodiment, the kit comprises a first reagent for measuring the expression level or the presence or absence of an inactivating mutation of AR. In one embodiment, the kit further comprises a second reagent for detecting the presence or absence or expression level of GREM1.

[0341]

[0464] In certain embodiments, the first reagent comprises one or more primers, one or more probes, and / or one or more antibodies against PTEN or p53 or AR. In certain embodiments, the second reagent comprises one or more primers, one or more probes, and / or one or more antibodies against GREM1. The primers, probes, and / or antibodies may or may not be detectably labeled.

[0342]

[0465] In certain embodiments, the kit may further comprise other reagents for carrying out the methods described herein. In such applications, the kit may comprise any or all of the following: suitable buffers, reagents for isolating nucleic acids, reagents for amplifying nucleic acids (e.g., polymerase, dNTP mixture), reagents for hybridizing nucleic acids, reagents for sequencing nucleic acids, reagents for quantifying nucleic acids (e.g., intercalating agents, detection probes), reagents for isolating proteins, and reagents for detecting proteins (e.g., secondary antibodies). Typically, the reagents useful in any of the methods provided herein are contained in a carrier or multi-chambered container. The carrier can be, for example, a container or carrier in the form of a bag, box, tube, rack, and is optionally partitioned.

[0343]

[0466] In certain embodiments, the present disclosure provides for the use of a first reagent provided herein, optionally together with a second reagent, in the manufacture of a diagnostic reagent for use in the diagnostic methods provided herein.

[0344]

[0467] In some embodiments, the present disclosure also provides the use of a GREM1 antagonist (e.g., an antibody or antigen-binding fragment thereof provided herein) in the manufacture of a pharmaceutical agent for treating or diagnosing a GREM1-expressing cancer in a subject, where the GREM1-associated disease or condition has been determined to be a deficiency of PTEN and / or p53.

[0345]

[0468] In some embodiments, the present disclosure also provides the use of a GREM1 antagonist (e.g., an antibody or antigen-binding fragment thereof provided herein) in the manufacture of a pharmaceutical for treating or diagnosing a GREM1-related disease or condition in a subject, where the GREM1-related disease or condition has been determined to be a deficiency of PTEN and / or p53. EXAMPLES

[0346]

[0001] Although the present disclosure has been particularly shown and described with reference to certain embodiments, some of which are preferred embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure disclosed herein.

[0347] Example 1: Upregulation of Gremlin 1 in prostate cancer (PCa) strongly correlates with the development of castration resistance and poor disease outcome

[0002] Secreted proteins are an important group of potential therapeutic targets for anti-cancer drug development. To screen for secreted proteins specifically upregulated in castration-resistant prostate cancer (CRPC), we performed data mining on publicly available RNA sequencing datasets. Gremlin 1 expression was ranked among the top differentially expressed genes encoding secreted proteins in hormone-resistant PCa (Best, CJ et al., Molecular alterations in primary prostate cancer after androgen ablation therapy. Clin Cancer Res 11, 6823-6834 (2005)). Additional analyses on other PCa datasets suggested that Gremlin 1 expression levels were significantly increased in advanced metastatic CRPC compared to primary PCa, or in hormone-refractory PCa compared to hormone-naive PCa (based on sequencing data from Yu, YP et al., Gene expression alterations in prostate cancer predicting tumor aggression and preceding development of malignancy. J Clin Oncol 22, pp. 2790-2799 (2004); Best, CJ et al., Molecular alterations in primary prostate cancer after androgen ablation therapy. Clin Cancer Res 11, pp. 6823-6834 (2005)). Importantly, GREM1 amplification was associated with shortened disease-free / progression-free survival based on data analysis on prostate adenocarcinoma (TCGA, Firehose Legacy). Next, we performed Gremlin 1 immunohistochemistry (IHC) staining in a large cohort of 139 human PCa patients at the Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Of the 139 patient specimens, 60 samples were obtained from patients with castration-resistant prostate tumors.Quantitative study of IHC results showed significantly enhanced staining intensity of Gremlin 1 in CRPC samples than in hormone-sensitive PCa (HSPC) (Figure 1A, 1B). Patients with higher Gremlin 1 expression in PCa had significantly shorter overall survival (Figure 1H). Based on sequencing data from the SU2C 2019 PCa dataset (A Robinson et al., Cell 161(5), pp. 1215-1228 (2015)), it was also revealed that patients with elevated GREM1 transcription exhibited shorter overall survival. Taken together, Gremlin 1 is upregulated in CRPC and strongly correlates with poor disease outcome.

[0348] Example 2: Transcription of GREM1 is repressed by AR and increases after ADT

[0003] AR plays a central role in PCa. To evaluate the relationship between Gremlin 1 and AR signaling, we performed further IHC staining for Gremlin 1 and PSA, a classical downstream target of AR, on sections of CRPC specimens. Statistical analysis revealed that Gremlin 1 expression was obviously upregulated in CRPC with low PSA staining intensity (Fig. 1C). Furthermore, we detected a significant increase in GREM1 in castration-resistant LNCaP cell lines (referred to as LNCaP R) derived from xenograft LNCaP tumors implanted in castrated mice compared with control LNCaP cell lines arising from xenografts in intact mice (Fig. 2A). Next, we investigated whether the expression of GREM1 is regulated by AR signaling. AR-expressing lentivirus or CRISPR / Cas9 methods were utilized to achieve AR upregulation (Fig. 2B) or knockout in LNCaP PCa cell lines (Fig. 2D). Combined immunoblotting and q-PCR experiments demonstrated that AR represses GREM1 transcription (Figures 2B-2E), a conclusion supported by AR agonist R1881 and antagonist enzalutamide treatment experiments (Figures 1D and 1E).

[0349]

[0004] Furthermore, we performed a luciferase reporter assay. GREM1 promoter-driven luciferase activity was significantly inhibited by treatment with R1881 and enhanced by the addition of enzalutamide (Fig. 1F). ChIP experimental results further suggested that AR bound to the promoter region of GREM1 (Fig. 1G). Together, these results demonstrated that GREM1 was elevated in CRPC and negatively regulated by AR.

[0350] Example 3: GREM1 promotes PCa cell proliferation and tumor growth upon androgen deprivation

[0005] Metastatic prostate cancer is a devastating disease, and most cancers progress with successive treatments with androgen receptor antagonists or chemotherapy. One important cell type that is resistant to these treatments is cells with stem cell-like properties that have the ability to form tumor spheres in suspension culture. To investigate the role of GREM1 in CRPC progression, we utilized the AR-independent CRPC cell line PC3, and the AR-dependent PCa cell lines LNCaP and LAPC4. Cell sublines that lost or gained GREM1 expression were generated (Figures 3A, 4A, and 5A). GREM1 knockdown in PC3 suppressed sphere-forming ability, cell growth, and survival, while GREM1 overexpression or addition of 100 ng / ml GREM1 protein in the culture medium significantly increased sphere formation and cell proliferation over the corresponding control sublines (Figures 3B, 3C, 3D, and 4B). Furthermore, we found that knockdown of GREM1 significantly suppressed PC3 tumor growth in vivo, and overexpression of GREM1 enhanced tumor growth and the incidence of tumor formation in limiting dilution assays (Figures 3E, 3F).

[0351]

[0006] Furthermore, exogenous expression of GREM1 in PCa organoids generated from Hi-Myc mice, a genetically engineered mouse model (GEMM) for PCa, promoted organoid growth under androgen deprivation conditions (Fig. 3G and 3H). GREM1 knockdown greatly enhanced the inhibitory effect of enzalutamide on AR-dependent LNCaP and LAPC4 cells, and GREM1 overexpression or addition of GREM1 protein impaired the response to enzalutamide treatment in LNCaP and LAPC4 cells (Fig. 4A-4D and 5A-5D). Together, these data suggested a tumor-promoting role of GREM1 in the development of PCa and castration resistance.

[0352] Example 4: The oncogenic effect of GREM1 in PCa is dependent on activation of the FGFR1 / MEK / ERK signaling pathway

[0007] To address the mechanism underlying the oncogenic effect of GREM1, we performed RNA sequencing to compare the transcriptional differences between GREM1-overexpressing LNCaP sublines and their control cells. Figure 6A lists the most significantly differentially expressed gene sets. FGFR and MAPK signaling were the top hits in upregulated signaling pathways. Further gene set enrichment analysis (GSEA) showed enrichment of signaling by FGFR1 and activation of MARK activity in LNCaP cells transfected with GREM1-expressing lentivirus (Figure 6B). Furthermore, in addition to the upregulation of GREM1 (Figure 2A), we found that both MARK and FGFR1 were activated in castration-resistant LNCaP cells compared to hormone-naive LNCaP cells (Figure 7A). This is particularly relevant in light of recent findings that FGF signal activation is required for AR-independent growth of CRPC.

[0353] To examine whether Gremlin 1 promoted FGFR-MAPK signaling, we first performed an analysis of the expression levels of four FGFRs in CRPC patients. Based on the sequencing data of the SU2C CRPC cohort (Robinson et al., Cell 161(5), pp. 1215-1228 (2015)), FGFR1 was the most abundantly expressed FGFR in CRPC. Therefore, we primarily examined FGFR1 activation following GREM1 treatment. We treated LNCaP and PC3 with GREM1 at different concentrations (1 ng / ml, 10 ng / ml, 100 ng / ml) and examined the phosphorylation levels of FGFR1, MEK, and ERK. FGF1, a known FGFR1 ligand, was used as a positive control. As shown in Figure 6C, GREM1 treatment increased p-FGFR1, p-MEK1 / 2, and p-ERK1 / 2 in a dose-dependent manner. The addition of BMP4 did not alter the phosphorylation levels of FGFR1, MEK1 / 2, and ERK1 / 2 following GREM1 stimulation, further revealing that the activation of the FGFR1-MAPK axis by GREM1 is BMP-independent (Figure 6D). Interestingly, we found that GREM1 induced a more prolonged activation of MARK signaling than FGF1. MARK activation in response to GREM1 treatment was maintained at a high level for up to 1 h after GREM1 addition, and MARK signaling was most highly activated at 10 min after FGF1 stimulation and then rapidly decreased (Figure 6E, 6F). Furthermore, exogenous expression of GREM1 activated the MAPK / FGFR1 signaling axis in PCa organoids derived from the Hi-myc mouse PCa model (Figure 7B).

[0354]

[0009] MAPK signaling can be activated through many membrane receptors other than FGFR. To investigate whether GREM1 activates the MAPK pathway through FGFR, we constructed FGFR1 knockout LNCaP sublines by CRISPR / Cas9 method. As shown in Figure 6G, phosphorylation of ERK1 / 2 and MEK1 / 2 by GREM1 treatment can be abrogated by FGFR1 knockout. Furthermore, the GREM1-mediated promoting effect on PCa cell growth and sphere formation can be abolished by knockout of FGFR1 (Figures 8A-8E). To further investigate whether the tumor-promoting effect of GREM1 is through FGFR, we utilized small molecule inhibitors of FGFR or EGFR. Activation of the MAPK / FGFR1 signaling axis by GREM1 could be attenuated by the FGFR1 inhibitor BGJ398, but not by the EGFR inhibitor erlotinib (Figures 6H, 6I). Furthermore, as shown in Figures 8A, B, the PCa proliferation and sphere formation-promoting role of GREM1 was significantly impaired by treatment with the FGFR inhibitor BGJ398 or the MEK inhibitor trametinib, but was not affected by the addition of BMP4. These results indicated that the oncogenic effect of Gremlin1 could be attributed to the activation of the FGFR1 / MEK / ERK signaling pathway.

[0355] Example 5: GREM1 is a novel FGFR1 ligand in PCa

[0010] We next investigated the mechanism that leads to the activation of the FGFR1 / MEK / ERK signaling pathway by GREM1. We performed surface plasmon resonance analysis (Fortebio) to first evaluate whether GREM1 can bind to FGFR1. As shown in Figure 9A, FGFR1 bound with high affinity to GREM1 immobilized on a ForteBio sensor chip (KD=1.06E-08M). Computer simulation mimicked that GREM1 bound to the extracellular domain of FGFR1 as a dimer (Figure 9B). The binding between FGFR1 and GREM1 was further demonstrated by co-immunoprecipitation assay using exogenously expressed Flag-tagged GREM1 and HA-tagged FGFR1 in both 293T and LNCaP cells (Figure 9C), or exogenous proteins in LNCaP cells (Figure 9D). The direct association between FGFR1 and GREM1 was supported by pull-down experiments shown in Figure 9F. Figure 9E further shows that Gremlin 1, but not Gremlin 2 or other members of the DNA protein family, binds to FGFR1 as measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, activation of GREM1 on the FGFR1 / MEK / ERK signaling pathway could be attenuated by adding excess amounts of soluble FGFR1 (Figure 9G).

[0356]

[0011] Bimolecular fluorescence complementation (BiFC) assay was performed to examine the interaction between GREM1 and FGFR1. GREM1 and FGFR1 cDNAs fused with a fragment of the coding sequence of yellow fluorescent protein (YFP) were transfected individually or simultaneously into 293T cells. As shown in Figure 9H, YFP signal could be detected only when GREM1 and FGFR1 plasmids were co-transfected. Consistent with that, confocal microscopic imaging of immunofluorescence staining showed the co-localization of Gremlin 1 and FGFR1 on the membrane of LNCaP-R cells (Figure 9I). Furthermore, soluble FGFR1 could compete for the binding between Gremlin 1 and FGFR1, as revealed by competitive ELISA experiments (Figure 9G). The activation of FGFR1 / MEK / ERK signaling pathway caused by Gremlin 1 could be attenuated by applying an excess amount of soluble FGFR1 (Figure 9G). These data provide strong supporting evidence for specific binding between Gremlin and FGFR1 and support the idea that this binding is required for activation of FGFR1 and its downstream MAPK signaling (FIG. 9G).

[0357] To further delineate the mode of Gremlin1 / FGFR1 interaction, we performed co-immunoprecipitation between truncated FGFR1 (Fig. 9J-9K) and either Gremlin1 or the classical FGFR1 ligand, FGF1. It has been clearly defined that the extracellular region of FGFR1 contains domain 1 (D1), domain 2 (D2) and domain 3 (D3). Consistent with previous reports that the linker between D2 and D3 is the critical binding region between FGF1 and FGFR1, we found that loss of D2 or D3 abolished the co-immunoprecipitation of FGF1 and FGFR1. In contrast, the mere absence of D2 abrogated the association between Gremlin1 and FGFR1, suggesting that Gremlin1 binds to FGFR1 at D2 (Fig. 9J-9K). Furthermore, we mutated previously identified critical amino acid residues (C176 and R248) of FGFR1 in the binding pocket for FGF1 (Fig. 9P). As expected, FGFR1-C176G or FGFR1-R248Q indeed disrupted the co-immunoprecipitation of FGF1 and FGFR1, and these two mutations did not affect the interaction between Gremlin 1 and FGFR1 (Figures ​(Figures9P-Q). 9P-Q). These data indicated that FGFR1 binds to Gremlin 1 in a manner that is distinct from that of its classical ligand, FGF1. Consistent with this, the Fortebio assay, co-immunofluorescence staining, and co-immunoprecipitation experiments together demonstrated that the addition of Gremlin 1 did not compete with the association of FGF1 and FGFR1, and vice versa (Figures ​(Figures9R-U). 9R-U). Thus, Gremlin 1 and FGF1 likely bind to different sites on FGFR1. We next assessed the expression levels of Gremlin 1 and various FGFs in CRPC patient samples from published RNA-seq datasets (Robinson et al., Cell 161(5), pp. 1215-1228 (2015)). We observed that GREM1 transcripts were higher than FGFs. Taken together, these data suggested that Gremlin 1 serves as the primary ligand for FGFR1 in CRPC.

[0358]

[0013] The next problem was to decipher the structural basis of Gremlin1 / FGFR1 interaction. We used the HDOCK platform (http: / / hdock.phys.hust.edu.cn / ) to perform docking of the previously characterized protein structure of Gremlin1 (PDB:5AEJ) (Kisonaite et al., Structure of Gremlin-1 and analysis of its interaction with BMP-2. Biochem J 473, 1593-1604 (2016)) with the extracellular domain of FGFR1 (PDB:3OJV) (Beenken et al., Plasticity in interactions of fibroblast growth factor 1 (FGF1)N terminus with FGF receptors underlies promiscuity of FGF1. J Biol Chem 287, 3067-3078 (2012)). As shown in FIG. 9B, two positively charged clusters of amino acid residues in Gremlin1 (Lys66-Arg67; R92-Lys123-Lys124-Arg148-Lys150-Arg153 relative to SEQ ID NO: 69) and extracellular domain 2 of FGFR1 were predicted to be essential binding regions between Gremlin1 and FGFR1. Next, mutagenesis described in FIG. 9L and 9N was performed to test protein binding simulations. Mutations of Lys123Lys124 to Ala123Ala124 on Gremlin1 (numbering relative to SEQ ID NO: 69) or E160A mutant of FGFR1 significantly impaired co-immunoprecipitation between Gremlin1 and FGFR1 (FIG. 9M and 9O). The docking module highlights the important amino acid residues in the binding pocket between Gremlin1 and FGFR1 (FIG. 9V). Thus, Lys123-Lys124 of Gremlin 1 (numbering relative to SEQ ID NO: 69) and the corresponding Glu160 of FGFR1 were important amino acids for the formation of the Gremlin 1 / FGFR1 protein complex.

[0359] Example 6: Anti-GREM1 antibody is Pbsn-Cre4;Pten fl / fl;Trp53 fl / fl Greatly inhibits the development of castration-resistant PCa in GEMM

[0014] The upregulation and oncogenic effects of GREM1 in CRPC make it a promising therapeutic target. fl / fl ;Trp53 fl / fl A schematic diagram illustrating treatment with GEMM is shown. Mice castrated at 2 months received anti-mouse gremlin 1 antibody (anti-mGREM1 antibody) (intraperitoneally, 10 mg / kg) or IgG as indicated, 3 times a week for 2 months. To target GREM1, a monoclonal antibody against mouse GREM1 was developed with high affinity, which is also referred to as a surrogate antibody in this disclosure. This surrogate antibody did not bind to gremlin 2 or other BMP antagonists such as COCO and DNA (Figure 10A). To examine the effect of anti-mGREM1 antibody on PCa, we used Pbsn-Cre4;PTEN mice that developed spontaneous invasive PCa in an average of 3 months. fl / fl ;Trp53 fl / fl GEMM was used. Pbsn-Cre4;PTEN fl / fl ;Trp53 fl / fl It has been previously demonstrated that PCa is castration-resistant from the outset. We demonstrated by immunohistochemistry that intact or castrated Pbsn-Cre4;PTEN fl / fl ;Trp53 fl / fl The expression levels of GREM1 in PCa and wild-type prostate samples were quantified. As shown in FIG. 10B, GREM1 was significantly increased in castrated Pbsn-Cre4;PTEN compared to wild-type prostate. fl / fl ;Trp53 fl / fl Tumors followed by intact castration Pbsn-Cre4;PTEN fl / fl ;Trp53 fl / fl Highly upregulated in tumors and castrated Pbsn-Cre4;Pten fl / fl ;Trp53 fl / fl It was further enriched in tumors.

[0360] Co-immunostaining of E-cadherin and GREM1 revealed that Gremlin is primarily involved in the castration of Pbsn-Cre4;PTEN fl / fl ;Trp53fl / fl It was suggested that it was expressed by tumor-like epithelial cells in the tumor (Figure 11A). fl / fl ;Trp53 fl / fl Quantitative PCR analysis of Grem1 in several major organs in mice showed that Grem1 expression was highest in prostate cancer tissues (Figure 1I), making Gremlin 1 a compelling therapeutic target for PCa.

[0361]

[0016] The present inventors have demonstrated that castrated Pbsn-Cre4;PTEN fl / fl ;Trp53 fl / fl Mice were treated with anti-mGREM1 antibody and its in vivo effect on CRPC development was evaluated. Δ / Δ Mice were castrated and subjected to anti-GREM1 antibody or control IgG2a treatment at 10 mg / kg three times a week for 8 weeks (Figure 10C). Two months after castration, animals were sacrificed for analysis. As shown in Figures 10C, 10D, all control IgG2a-treated mice developed invasive CRPC. Anti-mGREM1 antibody exerted a profound inhibitory effect on PCa growth, as evidenced by the marked inhibition of macroscopic tumor appearance, tumor weight, and a significant reduction in proliferating PCNA-positive cells (Figure 10E). Anti-GREM1-treated Pbsn-Cre4;PTEN fl / fl ;Trp53 fl / fl H&E staining of prostate sections from mice showed mainly intraductal hyperplasia with intact basement membrane, which was in stark contrast to the invasive PCa phenotype in IgG2a-injected mice (Figure 10F). At a dosage of 10 mg / kg, no obvious differentiation was observed in major organs including intestine, lung, liver, spleen, bone marrow and kidney, nor alterations in peripheral blood cell counts between the two experimental groups (Figures 11B, 11C), demonstrating the significant antitumor effect of anti-mGREM1 antibody without obvious side effects.

[0362] To understand the mechanism of the potent inhibitory effect of anti-mGREM1 antibody on CRPC in mice, we performed RNA sequencing on prostate samples from IgG2a- or anti-mGREM1 antibody-treated mice. KEGG and GESA analysis demonstrated that FGFR and MAPK signaling were the most significantly altered signaling pathways in the anti-mGREM1 antibody-treated group (Fig. 10G, 10H). Further immunostaining and immunoblotting experiments demonstrated that administration of anti-mGREM1 antibody significantly reduced FGFR1, MEK, and ERK phosphorylation (Fig. 10I, 10J). Taken together, these results suggest that anti-mGREM1 antibody inhibits Pbsn-Cre4;PTEN phosphorylation through inhibition of the FGFR1 / MAPK signaling pathway. fl / fl ;Trp53 fl / fl This suggested that it suppresses the onset of CRPC in mice.

[0363] Example 7: Antibodies targeting GREM1 exert potent antitumor effects in human PCa cell lines

[0018] 14E3 targeting GREM1 was tested in human PCa. The affinity and specificity of this antibody for hGREM1 was confirmed by ELISA (Figure 12A). Anti-human GREM1 antibody 14E3 exerted an inhibitory effect on sphere formation, proliferation, and induced apoptosis of LNCaP and PC3 cells. This antibody enhanced the antitumor effect of enzalutamide in AR-dependent LNCap cells (Figures 12B, 12C, 10K, 10L). Biochemical analysis showed dose-dependent inhibition of FGFR1 / MEK / ERK signaling pathway by anti-GREM1 antibody treatment in both PC3 and LNCap cells (Figures 12D, 10M).

[0364]

[0019] To examine whether BMP signaling is involved in the anti-tumor effect of 14E3, BMPRII was knocked out by CRISP / Cas9, but this did not abrogate the inhibitory effect of 14E3 on PCa cells, indicating that BMP signaling is an independent role of anti-GREM1 antibody (Figures 17A, 17B, and 17C).

[0365] To investigate the effect of 14E3 in vivo in CRPC, nude mice bearing CRPC cell line PC3 xenografts were intraperitoneally injected with anti-GREM1 antibody or IgG2a (10 mg / kg body weight) three times a week for 2 weeks. 14E3 significantly inhibited PC3 xenograft tumor growth. The tumor inhibition caused by 14E3 became more evident during secondary tumor passage (Figures 12E, 12F).

[0366] The tumor growth inhibitory activity of 14E3 in the castrated PC3 CRPC model was investigated. Briefly, PC3 cells were cultured at 1×10 per mouse. 6 The cells were subcutaneously implanted in Balb / c nude mice, and the mice were then castrated to generate a CRPC (castration-resistant prostate cancer) model. 3 At 37 days (day 20), mice were treated with isotype control mouse IgG2a or 14E3 hybridoma antibody (mIgG2a). Each group had eight mice and the antibodies were given intraperitoneally (ip) at 10 mg / kg twice weekly. Tumor volumes were measured twice weekly in two dimensions using calipers (INSIZE). Figure 13 shows that the Gremlin antibody 14E3 reduced PC3 tumor growth and extended survival of tumor-bearing mice.

[0367] Example 8: Antibodies against Gremlin 1 exert potent antitumor effects in LNCaP PCa cells 14E3 targeting GREM1 was tested in LNCaP PCa cells. In Figure 14, 14E3 was shown to exert an inhibitory effect on sphere formation and proliferation in AR-dependent LNCaP cells, enhancing the antitumor effect of enzalutamide (Figure 14A, B). Biochemical analysis showed dose-dependent inhibition of FGFR1 / MEK / ERK signaling pathway by 14E3 treatment in LNCaP cells (Figure 14C). Figures 14D and 10N showed the synergistic effect of combination therapy of anti-GREM1 antibody (e.g., 14E3 or anti-mGREM1 antibody) and enzalutamide on tumor inhibition of CRPC. These data demonstrated that antibodies targeting GREM1 can serve as a promising therapeutic approach for human CRPC patients.

[0368] Example 9: 14E3 inhibited GREM1-mediated tumor cell migration

[0023] PC-3 cells in logarithmic growth phase were harvested and resuspended in cell culture medium (DMEM medium supplemented with 10% FBS). The cells were either untreated or treated with 1 μg / ml of Gremlin, 10 μg / ml of 14E3, or 10 μg / ml of control mIgG2a for 3 days. The cells were then plated in 10 wells of a 6-well cell culture plate. 5 Cells were plated at 100x the cell count / well. After the cells reached 100% coverage of the bottom of the well, the medium was changed to serum-free medium. Each well was scratched with a single stroke using a 200 μl-tip. The migration rate of the cells was analyzed by calculating the area of ​​cell growth on the scratch using Image J software.

[0369]

[0024] As shown in FIG. 15, addition of GREM1 accelerated cell migration of prostate cancer cells, and 14E3 reduced such acceleration, ie, inhibited GREM1-mediated tumor cell migration.

[0370] Example 10: 14E3 inhibited GREM1-induced EMT / stem cell formation in LNCaP and PC3 suspension cultures To evaluate the role of Gremlin 1 in regulating tumor prostate cell growth, we tested the antibodies provided herein in an assay involving prostate cancer cells LNCaP. This cell line was transfected with a PSA promoter-driven GFP-expressing lentiviral plasmid. PSA is known as a differentiation marker for prostate cells, and prostate cancer cells with low levels of PSA represent poorly differentiated or undifferentiated prostate cancer cells. These cells usually have stem cell-like characteristics and have more invasive growth characteristics. The LNCaP reporter cell assay is briefly described below.

[0371]

[0026] LNCaP-PSA cells were plated in 24-well plates at 10000 / well in RPMI 1640 / 10% FBS (GIBCO), 1% P / S (complete medium) and incubated overnight at 37°C and 5% CO2. The next day, the medium was removed and 1 μg / ml human gremlin (ACRO) or human gremlin with serially diluted antibodies was added to the cells. The medium was changed every 3 days. On the 7th day, the medium was removed from the wells, washed twice with PBS, and run a flow cytometer (Beckman) using the FITC channel. As shown in Figure 16A, the percentage of PSA-low LNCaP population in the control wells (blank) was about 6%, and the percentage increased in a dose-dependent manner as GREM1 was added, suggesting that GREM1 promotes the invasiveness of prostate cancer cells. The hybridoma antibody provided herein, 14E3, dose-dependently neutralized Gremlin-mediated expansion of the PSA-low LNCaP population, as shown in FIG. 16B, indicating that 14E3 can reverse GREM1-promoted invasiveness.

[0372] FIG. 16C shows that replacing the BMP-binding loop with a non-BMP-binding loop did not inhibit PSA in prostate cancer cells (LNCaP). - / lo These results showed that GREM1 did not affect the GREM1-mediated increase in the percentage of the cell population, suggesting that GREM1-promoted cancer cell invasiveness is independent of the BMP-binding loop on GREM1.

[0373] Example 11: Effect of anti-GREM1 antibodies in suppressing the growth of organoids derived from human CRPC patients Next, we investigated whether anti-GREM1 antibody exhibited an inhibitory effect on patient-derived organoids (PDO). PDO were freshly collected from PCa patients at Renji Hospital. In brief, tumor tissues from nine CRPC patients were collected from surgery and were divided into 1-5 mm 3 After cutting into pieces and washing once with HBSS, the tissue was then digested to a single cell suspension using collagenase II + 10 μM Y-27632 for 4 h at 37 °C, followed by neutralization of the digestion using basal culture medium. Afterwards, the tumor tissue was further digested using 1 ml of TrypLE + 10 μM Y-27632 for 15 min at 37 °C, followed by neutralization using medium with 10% FBS. The cells thus obtained were resuspended in 50% Matrigel + 50% medium, and 50 μl of the cell suspension was distributed into each well of a 96-well plate. Afterwards, pre-warmed PDO medium (B27, N-acetylcysteine, EGF, Noggin, R-spondin 1, A83-1, FGF10, FGF2, prostaglandin E2, nicotinamide, SB202190, DHT and Y27632) was added to the cultures, and fresh medium was added every 2-3 days. Figures 18A, 18B, 18C and 18D show that 14E3 reduced organoid growth to various degrees in seven of nine PDOs, demonstrating a potential mechanism for 14E3-mediated tumor growth inhibition and pointing to the therapeutic potential of Gremlin-based inhibition.

[0374] Example 12: Observations

[0029] The widespread application of second-generation ADT drugs for PCa significantly increased the number of AR-independent CRPC. In this study, we found that the expression of GREM1 in CRPC was abnormally increased compared to hormone-naive or newly diagnosed PCa. GREM1 promotes prostate cancer progression and resistance to androgen ablation. Their effects are achieved through direct GREM1-FGFR binding, which activates the FGFR / MAPK signaling pathway. GREM1-blocking antibodies can effectively inhibit castration-resistant development of PCa in GEMM mouse PCa models, human PCa cell lines, patient-derived organoids and xenografts. Together, these results strongly support that the GREM1 / FGFR1 / MAPK signaling axis promotes PCa progression, pointing to Gremlin as an important and promising therapeutic target.

[0375]

[0030] Second generation antiandrogens have been shown to trigger the upregulation of key drivers for AR-independent CRPC. However, the mechanism by which these drivers are regulated by AR signaling remains incompletely understood. We find that GREM1 is negatively correlated with the AR signaling pathway in CRPC patient samples. AR activation or overexpression leads to a strong decrease in GREM1 expression in PCa cells. Conversely, GREM1 transcription is significantly increased when AR is knocked out or inhibited by enzalutamide. Together, CHIP and luciferase reporter assay data strongly support that the suppression of GREM1 is achieved through the binding of AR at the GREM1 promoter region for transcriptional repression. These results suggest that the gene expression of GREM1 is transcriptionally inhibited by AR as a strong driver of CRPC. The mechanisms of castration resistance in PCa can be summarized into two broad categories: 1) reactivation of the AR signaling pathway through AR amplification, mutation or alternative splicing, or upregulation of the glucocorticoid receptor (GR); 2) activation of alternative signaling pathways, such as FGF, PRC1, and BCL2, to avoid AR-independent tumor growth and cell death. We found that the FGFR / MAPK signaling pathway was aberrantly activated in prostate cancer cell lines with GREM1 overexpression. This is especially relevant in light of recent findings that FGF signal activation is an essential molecular signature of AR-independent CRPC and is required for AR-independent growth of CRPC. The association of the FGF-FGFR1 signal axis in bone metastasis of prostate cancer has also been reported. In this study, we found that GREM1 caused FGFR1 phosphorylation and activation in a concentration-dependent manner. Gremlin-induced FGFR1 phosphorylation is more persistent than FGF1 stimulation. Furthermore, sequencing data from the SU2C PCa cohort showed that the RNA transcript abundance of GREM1 was higher than that of other FGFs in CRPC. Therefore, we propose that GREM1 is at least one of the major contributors to the aberrant activation of FGFR1 / MAPK signaling in CRPC.Critically, using co-immunostaining, bi-FC, fortebio, co-IP, pull-down and computer simulation approaches, we provide compelling evidence that GREM1 can directly bind to FGFR1. Thus, GREM1-induced FGFR1 activation results from direct ligand-receptor binding. GREM1 functions as a novel ligand for FGFR1 in PCa.

[0376] GREM1 was previously regarded as a classical antagonist of BMP. It was reported that the BMP signaling pathway and downstream target genes have significant effects on the progression and metastasis of PCa based on findings from conditional knockout mouse models. However, we found that BMP4 has no significant effect on the activation of FGFR / MAPK and the tumor-promoting effect of GREM1 on PCa. Furthermore, the inhibitory effect of GREM1-blocking antibody on PCa cells cannot be abolished by BMPRII knockout. However, the positive role of GREM1 on PCa can be largely abolished by FGFR1 knockout. Taken together, the activation of FGFR / MAPK and the CRPC-promoting effect of GREM1 are independent of the BMP signaling pathway. Our present study identifies an entirely new function of GREM1 and adds a new member to the FGFR ligands. More broadly, the FGFR signaling pathway is also a central oncogenic driver in other tumors, such as bladder cancer, gastric cancer, lung cancer and breast cancer. Further studies are needed to understand whether the GREM1 / FGFR / MAPK axis is involved in tumor initiation and progression in other cancer types.

[0377]

[0032] We found by immunostaining that GREM1 is expressed by tumor epithelial cells in both GEMM and human PCa samples. Consistent with this, other independent laboratories have reported that tumor cells or tumor stem cells highly express GREM1 in colon cancer and glioma. However, Julie B. Sneddon et al. analyzed the expression of GREM1 RNA in 774 different tumor cases and found that more than 50% of tumor stromal cells from colon, lung, pancreatic and breast cancer were positive for GREM1. Michael Quante et al. showed that Gremlin was significantly increased in tumor-associated fibroblasts (CAFs) in a gastric cancer model. The promoting effect of GREM1 on CRPC may act not only in an autocrine manner on tumor cells but also possibly through regulating the tumor microenvironment to create a microenvironment suitable for PCa cell growth and evasion of cell death in harsh conditions such as androgen deprivation.

[0378] Secreted proteins are an important category of drug targets. In this study, we develop a monoclonal antibody against GREM1. Experiments in human PCa cell lines, PDO and PDX, and Pbsn-Cre4;PTEN fl / fl ;Trp53 fl / fl Based on in vivo studies in mouse PCa models, we demonstrate the outstanding antitumor effect of anti-GREM1 antibodies. Anti-GREM1 antibodies show strong synergistic effects with ADT against PCa. However, it must be taken into account that GREM1 is also expressed in other tissues. Studies have revealed that conventional knockout of GREM1 in mice causes abnormal development of the intestine and impaired hematopoietic system. In our study, we carefully investigate the major organs, including the intestine, of mice after anti-GREM1 antibody treatment. At a dose of 10 mg / kg through intraperitoneal injections three times a week, no obvious toxic effects or significant damage to major organs or peripheral blood cell counts are observed. These findings suggest that undesirable side effects can be avoided with an appropriate administration window.

[0379] The majority of the 24 antibody drugs approved by the FDA for cancer therapy target immune checkpoint proteins or cell surface proteins in hematopoietic malignancies, while a few others block HER2, EGFR, VEGF, or VEGFR in the treatment of a few solid tumors. "Cold tumors," including PCa, have low CD8+ cytotoxic T cell infiltration but respond poorly to immune checkpoint therapy. Therefore, it becomes clinically crucial to identify novel targets for new antibody-based drugs against "cold tumors." Our discovery that the GREM1 / FGFR1 / MAPK axis is a critical driver for CRPC not only provides insights for understanding the molecular mechanisms of castration resistance expression, but also demonstrates the direct therapeutic relevance of GREM1 as a novel drug target. Anti-GREM1 monoclonal antibodies hold great therapeutic promise for the treatment of CRPC.

[0380] Example 13: 14E3 could potently reduce the formation of PCa metastases in the lungs To evaluate the effect of Gremlin 1 antibodies on prostate cancer metastasis in the lung, PC3 cells (ATCC) were transfected with a plasmid constitutively expressing luciferase. PC3-luc cells were harvested from logarithmic growth phase and cultured in 80 ml of basal medium (DMEM) at 1 × 10 6The cells were suspended in 14E3. The cell suspension was then intracardially injected into the ventricle of BALB / C nude mice (Shanghai SLAC Laboratory Animal). Gremlin 1 hybridoma antibody 14E3 or isotype control was given intraperitoneally at a dose of 10 mg / kg twice a week for 3 weeks. Body weight was measured every 2 days. Mice were anesthetized and administered D-luciferin (ThermoFisher, L2916) at 15 mg / kg for 5 minutes. Images were taken by an in vivo imaging system (Caliper IVIS bioluminescence system, Caliper LifeScience.USA). As a result of imaging in Figures 19A, 19B and 19C, 14E3 shows obvious inhibition on the mean radiance intensity without affecting the body weight, indicating that antibodies against Gremlin 1 (e.g., 14E3) significantly reduced PCa metastasis in mouse models receiving intracardiac injection. According to the micrometastasis statistics in Figures 19D and 19E, 14E3 was able to potently reduce the formation of PCa metastases in the lung.

[0381]

[0036]

[0382] [Table 5-1]

[0383] [Table 5-2]

[0384] [Table 5-3]

[0385] [Table 5-4]

[0386] [Table 5-5]

[0387] [Table 5-6]

[0388]

Table 5-7

[0389]

Table 5-8

[0390]

Table 5-9

[0391]

Table 5-10

Claims

1. A pharmaceutical composition comprising a GREM1 antagonist for use in a method of treating a GREM1-expressing cancer in a subject in need of such treatment, wherein the cancer is characterized by reduced androgen receptor (AR) signaling.

2. Cancer, a) the cancer is AR-expressing or AR-negative, b) selected from the group consisting of prostate cancer, breast cancer, lung cancer, head and neck cancer, testicular cancer, endometrial cancer, ovarian cancer, and skin cancer; c) is metastatic, optionally wherein the cancer is metastatic prostate cancer, and further optionally wherein the cancer is lung metastasis of prostate cancer; and / or d) characterized by GREM1 overexpression; The pharmaceutical composition of claim 1.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the subject is undergoing or has undergone androgen deprivation therapy, or is resistant to androgen deprivation therapy.

4. A pharmaceutical composition comprising a GREM1 antagonist for use in a method for increasing the sensitivity of AR-expressing cancer to androgen deprivation therapy in a subject.

5. A pharmaceutical composition comprising a GREM1 antagonist for use in a method for treating a GREM1-related disease or condition characterized by PTEN and / or p53 deficiency in a subject in need of such treatment, or for inhibiting FGFR1 activation in a subject in need of such inhibition, or for inhibiting MAPK signaling in a subject in need of such inhibition.

6. Deficiency of PTEN and / or p53 a) characterized by the absence of functional PTEN and / or p53; b) characterized by the absence of PTEN and / or p53 expression; and / or c) characterized by the presence of inactivating mutations in PTEN and / or p53; The pharmaceutical composition according to claim 5.

7. The pharmaceutical composition of claim 5 or 6, wherein the GREM1-associated disease or condition is characterized by GREM1 expression or overexpression.

8. The pharmaceutical composition of any one of claims 5 to 7, wherein the GREM1-related disease or condition is selected from the group consisting of cancer, fibrotic diseases, angiogenesis, glaucoma or retinal diseases, renal diseases, pulmonary arterial hypertension, and osteoarthritis (OA).

9. 9. The pharmaceutical composition of claim 8, wherein the cancer is prostate cancer, breast cancer, glioma, liposarcoma, hepatocellular carcinoma, lung cancer, cervical cancer, endometrial cancer, uterine leiomyosarcoma, squamous cell carcinoma of the head and neck, thyroid cancer, liver cancer, pancreatic cancer, bladder cancer, colon cancer, esophageal cancer, bile duct cancer, osteosarcoma, glioblastoma, ovarian cancer, gastric cancer, triple-negative breast cancer (TNBC), small cell lung cancer or melanoma.

10. Prostate cancer, a) negative for androgen receptor (AR) expression; b) both androgen receptor (AR) expression and neuroendocrine (NE) differentiation are negative; c) resistant to androgen deprivation therapy, optionally castration-resistant; d) exhibiting a level of prostate specific antigen (PSA) lower than a reference level; or e) Any combination of a) to d). The pharmaceutical composition according to claim 9.

11. The pharmaceutical composition of claim 9, wherein the breast cancer is triple-negative breast cancer.

12. 9. The pharmaceutical composition of claim 8, wherein the fibrotic disease is pulmonary fibrosis, skin fibrosis, diabetic nephropathy, or ischemic kidney injury.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the GREM1 antagonist comprises an anti-GREM1 antibody or an antigen-binding fragment thereof, an inhibitory GREM1 mimetic peptide, an inhibitory nucleic acid targeting GREM1 RNA or DNA, a polynucleotide encoding the inhibitory nucleic acid, a compound that inhibits the interaction between Gremlin and BMP, or a compound that inhibits GREM1 activity.

14. The pharmaceutical composition of claim 13, wherein the GREM1 antagonist comprises a GREM1-FGFR1 axis inhibitor.

15. The pharmaceutical composition of claim 14, wherein the GREM1-FGFR1 axis inhibitor comprises an FGFR1 binding inhibitor.

16. The pharmaceutical composition of claim 14, wherein the GREM1 antagonist or GREM1-FGFR1 axis inhibitor comprises an antibody against hGREM1 or an antigen-binding fragment thereof.

17. The antibody or antigen-binding fragment thereof against hGREM1, comprising a heavy chain variable region and a light chain variable region, a) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:1, an HCDR2 comprising the sequence of SEQ ID NO:2, and an HCDR3 comprising the sequence of SEQ ID NO:3; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:4, an LCDR2 comprising the sequence of SEQ ID NO:5, and an LCDR3 comprising the sequence of SEQ ID NO:6; b) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:11, an HCDR2 comprising the sequence of SEQ ID NO:12, and an HCDR3 comprising the sequence of SEQ ID NO:13; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:15, and an LCDR3 comprising the sequence of SEQ ID NO:16; c) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO:21, an HCDR2 comprising the sequence of SEQ ID NO:22, and an HCDR3 comprising the sequence of SEQ ID NO:23; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO:24, an LCDR2 comprising the sequence of SEQ ID NO:25, and an LCDR3 comprising the sequence of SEQ ID NO:26; or d) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 31, an HCDR2 comprising the sequence of SEQ ID NO: 32, and an HCDR3 comprising the sequence of SEQ ID NO: 33; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 34, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO:

36.

18. The antibody or antigen-binding fragment thereof against hGREM1 is a) a heavy chain variable region comprising the sequence of SEQ ID NO:7 and a light chain variable region comprising the sequence of SEQ ID NO:8; or b) a heavy chain variable region comprising the sequence of SEQ ID NO: 17 and a light chain variable region comprising the sequence of SEQ ID NO: 18; or c) a heavy chain variable region comprising the sequence of SEQ ID NO:27 and a light chain variable region comprising the sequence of SEQ ID NO:28; or d) a heavy chain variable region comprising the sequence of SEQ ID NO: 37 and a light chain variable region comprising the sequence of SEQ ID NO: 38; or e) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO: 45, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 47 and SEQ ID NO: 49; or f) a pair of heavy chain and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 41 / 47, 41 / 49, 43 / 47, 43 / 49, 45 / 47, and 45 / 49; or g) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55 and SEQ ID NO:57, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:59 and SEQ ID NO:61; or h) The pharmaceutical composition of claim 17, comprising a pair of heavy chain and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 51 / 59, 51 / 61, 53 / 59, 53 / 61, 55 / 59, 55 / 61, 57 / 59, and 57 / 61.

19. The pharmaceutical composition of any one of claims 16 to 18, wherein the antibody or antigen-binding fragment thereof against hGREM1 further comprises an immunoglobulin constant region, optionally a constant region of human IgG, or optionally a constant region of human IgG1, IgG2, IgG3, or IgG4.

20. The pharmaceutical composition of any one of claims 16 to 18, wherein the antibody or antigen-binding fragment thereof against hGREM1 is humanized and / or bispecific.

21. A pharmaceutical composition according to any one of claims 16 to 18, wherein the antibody or antigen-binding fragment thereof against hGREM1 is capable of specifically binding to a first and a second epitope of Gremlin, or is capable of specifically binding to both hGREM1 and a second antigen.

22. The second antigen comprises an immune-related target or a tumor antigen, and the immune-related target is PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD47, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7. , NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16 and CD83, and the tumor antigen is selected from the group consisting of prostate specific antigen (PSA), CA-125, ganglioside G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep -CAM, CEA, bombesin-like peptide, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, Ki-67, cancer-associated mucins, VEGF, VEGFR (e.g., VEGFR-1, VEGFR-2, VEGFR-3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, trastuzumab, erythrocyte serine / erythrocyte sarcoma, ... The pharmaceutical composition of claim 21, wherein the receptor is selected from the group consisting of sarcolemmal receptor, claudin 18.2, GPC-3, nectin-4, ROR1, mesothelin, PCMA, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5, BCR-ABL, E2APRL, H4-RET, IGH-IGK, MYL-RAR, IL-2R, CO17-1A, TROP2, and LIV-1.

23. The pharmaceutical composition of any one of claims 1 to 22, wherein the method further comprises the step of administering a therapeutically effective amount of a second therapeutic agent, the second therapeutic agent comprising an anti-cancer therapeutic agent, optionally selected from a chemotherapy agent, a radiation therapy, an immunotherapy agent, an anti-angiogenesis agent (e.g., an antagonist of VEGFR such as VEGFR-1, VEGFR-2, and VEGFR-3), a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, a cytokine, palliative care, surgery for the treatment of cancer (e.g., tumor removal), one or more antiemetic agents, a treatment for complications resulting from chemotherapy, or a dietary supplement for cancer patients (e.g., indole-3-carbinol).

24. Anticancer drugs include androgen deprivation therapy drugs, androgen axis inhibitors, androgen synthesis inhibitors, PARP inhibitors, abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), darolutamide, degarelix, docetaxel, Eligard (leuprolide acetate), enzalutamide, Erleada (apalutamide), Filmagone (degarelix), flutamide, goserelin acetate, histrelin (Bantas), Jevtana (cabazitaxel), leuprolide acetate, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lynparza (olaparib), keto 24. The pharmaceutical composition of claim 23, comprising an anti-prostate cancer drug selected from the group consisting of conazole (Nizoral), mitoxantrone hydrochloride, nilandrone (nilutamide), nilutamide, Nubequo (darolutamide), olaparib, Provenzi (sipuleucel-T), radium-223 dichloride, relugolix (Orgovix), Rubraca (rucaparib cansylate), rucaparib cansylate, sipuleucel-T, Taxotere (docetaxel), triptorelin (Trelstar), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Zoladex (goserelin acetate) and Zytiga (abiraterone acetate) or any combination thereof.

25. 25. The pharmaceutical composition of claim 24, wherein the androgen axis inhibitor is degarelix, bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, or abiraterone.

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