Novel anti-Gremlin 1 antibody

Abstract

The disclosure herein provides anti-Gremlin 1 antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding same, pharmaceutical compositions comprising same, and uses thereof.

Description

[Technical field]

[0001] The present disclosure relates generally to novel anti-Gremlin 1 (GREM1) antibodies that specifically bind to human Gremlin 1. [Background technology]

[0002] Gremlin 1 (GREM1) is a highly conserved secreted protein with a cysteine-rich region and a cysteine ​​knot (Wordinger et al., Exp Eye Res. 2008 Aug;87(2):78-79). GREM1 is a member of the family of differential screening-selected gene aberrative in neuroblastoma (DAN), which functions as an antagonist of bone morphogenetic proteins (BMPs) (Wordinger et al., Exp Eye Res. 2008 Aug;87(2):78-79). GREM1 can physically bind to BMP-2, BMP-4, or BMP-7 to form heterodimers, preventing BMP ligands from interacting with their corresponding BMP receptors, and then inhibiting the activation of the BMP signaling pathway.

[0003] GREM1 is closely associated with fibrotic lesions in the kidney, lung, liver and retina, as well as several tumor types, including pancreatic, colon, lung, glioma, gastric and prostate cancer (Sneddon et al., PNAS 2006 October;103(40):14842-14847). For example, aberrant Gremlin 1 upregulation confers tumorigenic potential to colon cells outside the stem cell niche. Tumor stem cells have also been found to highly express and secrete Gremlin 1 to maintain their stemness in gliomas (Yan, K. et al., Genes Dev 28,1085-1100 (2014)). Thus, Gremlin 1 has been used as a therapeutic target in treating Gremlin-related diseases.

[0004]

[0004] However, since GREM1 is also expressed in other normal tissues, there is currently no effective treatment for GREM1-associated diseases due to side effects, including toxicity to normal tissues. Therefore, there is a need for a novel anti-Gremlin 1 antibody with reduced side effects. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] 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.

[0006]

[0006] The present disclosure provides, inter alia, novel monoclonal anti-Gremlin 1 (GREM1) antibodies, nucleotide sequences encoding the antibodies, and uses thereof. The anti-GREM1 antibodies provided herein bind to different regions of GREM1 and have differential effects on modulating the activity of GREM1 on bone morphogenetic protein (BMP) binding when compared with existing anti-GREM1 antibodies. In particular, the anti-GREM1 antibodies provided herein can selectively reduce GREM1-mediated inhibition of BMP signaling in cancer cells compared with non-cancer cells. This is unexpected and solves the long-standing problem of side effects caused by anti-GREM1 antibodies due to the ubiquitous expression of Gremlin in cancer cells as well as non-cancer cells. [Means for solving the problem]

[0007] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof against human Gremlin 1 (hGREM1), comprising the following characteristics: a) reducing hGREM1-mediated inhibition of BMP signaling selectively in cancer cells compared to non-cancer cells; b) non-cancer cells exhibit a 50% or less reduction in hGREM1-mediated inhibition of BMP signaling; c) capable of binding to a chimeric hGREM1 comprising the amino acid sequence of SEQ ID NO:68; d) capable of binding to hGREM1 but unable to specifically bind to mouse Gremlin 1; e) binds to hGREM1 at an epitope that includes residues Gln27 and / or Asn33, where the residue numbering is according to SEQ ID NO:69, or binds to a fragment of hGREM1 that includes residues Gln27 and / or Asn33, optionally the hGREM1 fragment has a length of at least 3 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acid residues; f) K<1nM as measured by Fortebio D can bind to hGREM1; h) capable of blocking the binding of hGREM1 to BMP7 with a maximum blocking percentage of greater than 50% as measured by ELISA; i) capable of blocking the interaction of GREM1 with FGFR; and / or j) capable of binding to both hGREM1 and DAN; The present invention provides an isolated antibody or antigen-binding fragment thereof, comprising at least one of:

[0008]

[0008] In certain embodiments, the epitope is a linear epitope or a conformational epitope.

[0009] In another aspect, the disclosure provides an isolated 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 a sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31, 114, 119 and 123; b) HCDR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22, 32 and 115, and c) HCDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33, 116, 120 and 124 and / or The light chain variable region is: d) LCDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 24, 34, 117, 121, 122 and 125; 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, 36 and 118; The present invention provides an isolated antibody or antigen-binding fragment thereof comprising:

[0010] In certain embodiments, the heavy chain variable region of an antibody or antigen-binding fragment thereof against hGREM1 provided herein comprises: a) a heavy chain variable region comprising 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; b) a heavy chain variable region comprising 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; c) a heavy chain variable region comprising 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; d) a heavy chain variable region comprising 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; e) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 114, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 116; f) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; and g) a heavy chain variable region comprising HCDR1 comprising the sequence of SEQ ID NO: 123, HCDR2 comprising the sequence of SEQ ID NO: 115, and HCDR3 comprising the sequence of SEQ ID NO: 124 is selected from the group consisting of:

[0011] In certain embodiments, the light chain variable region of an antibody or antigen-binding fragment thereof against hGREM1 provided herein comprises: 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; 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; e) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 117, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; f) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 121, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; g) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 122, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; and h) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 125, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118. may be selected from the group consisting of:

[0012] In certain embodiments, in the antibody or antigen-binding fragment thereof provided herein: 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; 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; e) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 114, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 116; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 117, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; f) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 121, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; g) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 122, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; or h) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 123, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 124; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 125, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118.

[0013]

[0013] In certain embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof against hGREM1 provided herein 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, SEQ ID NO:57, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134, as well as sequences thereof having at least 80% sequence identity thereto while still retaining specific binding specificity or affinity to hGREM1.

[0014]

[0014] In certain embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof against hGREM1 provided herein 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, SEQ ID NO:61, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137, as well as sequences having at least 80% sequence identity thereto while still retaining specific binding specificity or affinity to hGREM1.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein 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 the sequence of SEQ ID NO: 126 and a light chain variable region comprising the sequence of SEQ ID NO: 127; or f) a heavy chain variable region comprising the sequence of SEQ ID NO: 128 and a light chain variable region comprising the sequence of SEQ ID NO: 129; or g) a heavy chain variable region comprising the sequence of SEQ ID NO: 128 and a light chain variable region comprising the sequence of SEQ ID NO: 130; or h) 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 i) a pair of heavy 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 j) 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 k) a pair of heavy 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; or l) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 135, SEQ ID NO: 136 and SEQ ID NO: 137; or m) a pair of heavy chain variable region sequences and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 131 / 135, 131 / 136, 131 / 137, 132 / 135, 132 / 136, 132 / 137, 133 / 135, 133 / 136, 133 / 137, 134 / 135, 134 / 136, and 134 / 137; Includes.

[0016]

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises a substitution or modification of one or more amino acid residues that still retains specific binding specificity or affinity for hGREM1.

[0017]

[0017] In certain embodiments, at least one of the substitutions or modifications is in one or more CDR sequences and / or in one or more non-CDR regions of the VH or VL sequence.

[0018]

[0018] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region.

[0019]

[0019] In certain embodiments, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0020]

[0020] In certain embodiments, the constant region comprises a heavy chain constant region comprising the sequence of SEQ ID NO:138 and / or a light chain constant region comprising the sequence of SEQ ID NO:139.

[0021]

[0021] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are humanized.

[0022]

[0022] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are diabodies, Fab, Fab', F(ab')2, Fd, 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.

[0023]

[0023] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are bispecific.

[0024]

[0024] In certain embodiments, the antibody or antigen-binding fragment provided herein is capable of specifically binding to a first and a second epitope of hGREM1, or is capable of specifically binding to both hGREM1 and a second antigen.

[0025]

[0025] In certain embodiments, the second antigen provided herein is an immune-related target.

[0026]

[0026] 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.

[0027] In certain embodiments, the second antigen comprises a tumor antigen.

[0028]

[0028] In certain embodiments, the tumor antigen comprises a tumor-specific antigen or a tumor-associated antigen.

[0029] In certain embodiments, the tumor antigen is prostate specific antigen (PSA), CA-125, gangliosides 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., VEGFR3), estrogen, or IL-16. gen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2, GPC-3, nectin-4, ROR1, methemoglobin, 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.

[0030]

[0030] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are not cross-reactive to mouse gremlin 1.

[0031]

[0031] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are cross-reactive to mouse gremlin 1.

[0032]

[0032] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are linked to one or more conjugate moieties.

[0033]

[0033] In certain embodiments, the conjugate moiety comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anticancer drug such as an androgen receptor inhibitor.

[0034]

[0034] In another aspect, the disclosure provides an antibody or antigen-binding fragment thereof that competes with an antibody or antigen-binding fragment thereof provided herein for binding to hGREM1.

[0035]

[0035] In another aspect, the present disclosure provides a pharmaceutical composition or kit comprising an antibody or antigen-binding fragment thereof provided herein and a pharma- ceutically acceptable carrier.

[0036]

[0036] In certain embodiments, the pharmaceutical composition or kit further comprises a second therapeutic agent.

[0037]

[0037] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein.

[0038]

[0038] In another aspect, the present disclosure provides a vector comprising an isolated polynucleotide provided herein.

[0039]

[0039] In another aspect, the present disclosure provides a host cell comprising the vector provided herein.

[0040]

[0040] In another aspect, the present disclosure provides a method for expressing an antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which a vector provided herein can be expressed.

[0041]

[0041] In another aspect, the present disclosure provides a method for treating a GREM1-related disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein.

[0042]

[0042] In another aspect, the disclosure provides a method of treating a GREM1-associated disease or condition in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of an anti-human GREM1 antibody, or antigen-binding fragment thereof, wherein the anti-human GREM1 antibody, or antigen-binding fragment thereof: a) capable of binding to hGREM1 at an epitope comprising residue Gln27 and / or residue Asn33, where the residue numbers are according to SEQ ID NO: 69; and / or b) capable of binding to a hGREM1 fragment that includes 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; and / or c) reducing hGREM1-mediated inhibition of BMP signaling selectively in cancer cells relative to non-cancer cells; and / or d) non-cancer cells exhibit less than 50% reduction in hGREM1-mediated inhibition of BMP signaling; and / or e) capable of binding to a chimeric hGREM1 comprising the amino acid sequence of SEQ ID NO: 68; and / or f) K<1nM as measured by Fortebio D and / or capable of binding to hGREM1 at h) capable of blocking the binding of hGREM1 to BMP7 with a maximum blocking percentage of greater than 50% as measured by ELISA; and / or i) It is possible to block the interaction between GREM1 and FGFR.

[0043]

[0043] In certain embodiments, the GREM1-related disease or condition is selected from the group consisting of cancer, fibrotic disease, angiogenesis, glaucoma or retinal disease, kidney disease, pulmonary arterial hypertension, or osteoarthritis (OA).

[0044]

[0044] In certain embodiments, the cancer is a GREM1-expressing cancer. In certain embodiments, the GREM1-expressing cancer is also PD-L1-expressing. In certain embodiments, the GREM1-expressing cancer is not a PD-L1-expressing cancer. In certain embodiments, the GREM1-expressing cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor.

[0045]

[0045] In certain embodiments, the subject is identified as having GREM1-expressing cancer cells or as having a GREM1-expressing cancer microenvironment.

[0046]

[0046] In certain embodiments, the cancer is a solid tumor or a hematological cancer.

[0047] In certain embodiments, the solid tumor is adrenocortical carcinoma, anal carcinoma, astrocytoma, pediatric cerebellar or cerebral, 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 ... cell carcinoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin's lymphoma, islet cell carcinoma (pancreatic endocrine), 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, throat cancer, thyroid cancer, or vaginal cancer.

[0048] In certain embodiments, the hematological cancer is a leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML)), a lymphoma (e.g., Hodgkin's lymphoma, or a non-Hodgkin's lymphoma (e.g., Waldenstrom's macroglobulinemia (WM)), or a myeloma (e.g., multiple myeloma (MM)).

[0049]

[0049] 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, liver and 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, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, or adenocarcinoma.

[0050] 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.

[0051]

[0051] In certain embodiments, the cancer is prostate cancer.

[0052] In certain embodiments, the fibrotic disease is a fibrotic disease of the lung, liver, kidney, eye, skin, heart, intestine, or muscle.

[0053] In certain embodiments, the subject is a human.

[0053]

[0054] In certain embodiments, administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0054]

[0055] In certain embodiments, the methods provided herein further comprise administering a therapeutically effective amount of a second therapeutic agent.

[0055]

[0056] In certain embodiments, the second therapeutic agent comprises an anti-cancer therapy, and optionally the anti-cancer therapy is selected from the group consisting of chemotherapeutic agents (e.g., cisplatin), anti-cancer drugs, radiation therapy, immunotherapy agents (e.g., immune checkpoint modulators, such as PD-1 / PD-L1 axis inhibitors, and TGF-beta inhibitors), anti-angiogenic 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 ( For example, tumor resection), one or more antiemetics, treatment of complications resulting from chemotherapy, nutritional supplements for cancer patients (e.g., indole-3-carbinol), agents that modulate the tumor microenvironment (e.g., bifunctional molecules comprising a PD-L1 binding moiety and an extracellular domain of a TGF-beta receptor), or anti-fibrotic therapies (e.g., BMP7 treatment, ACE inhibitors (or ARBs), anti-MASP2 antibodies, endothelin receptor antagonists, NRF2 inhibitors steroids, CTLA4-IgG, or TNF inhibitors). In certain embodiments, the second therapeutic agent comprises cisplatin. In certain embodiments, the second therapeutic agent comprises a PD-1 / PD-L1 axis inhibitor.

[0056]

[0057] In certain embodiments, the anti-cancer therapy comprises an anti-prostate cancer drug.

[0057]

[0058] In certain embodiments, the anti-prostate cancer drug comprises an androgen axis inhibitor; an androgen synthesis inhibitor; a poly ADP-ribose polymerase (PARP) inhibitor; or a combination thereof.

[0058]

[0059] 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.

[0059]

[0060] In certain embodiments, the androgen axis inhibitor is degarelix, bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide or abiraterone.In certain embodiments, the androgen synthesis inhibitor is abiraterone acetate or ketoconazole.In certain embodiments, the PARP inhibitor is olaparib or rucaparib.

[0060]

[0061] 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), Firmagone (degarelix), flutamide, goserelin acetate, Jevtana (cabazitaxel), leuprolide acetate, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lympa The agent is selected from the group consisting of: ixazomib (olaparib), mitoxantrone hydrochloride, nilandrone (nilutamide), nilutamide, nubequo (darolutamide), olaparib, provenzi (sipuleucel-T), radium-223 dichloride, rubraca (rucaparib camsylate), rucaparib camsylate, sipuleucel-T, taxotere (docetaxel), zofigo (radium-223 dichloride), xtandi (enzalutamide), zoladex (goserelin acetate) and zytiga (abiraterone acetate).

[0061]

[0062] In certain embodiments, the second therapeutic agent comprises indole-3-carbinol.

[0062]

[0063] In another aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment provided herein.

[0063]

[0064] In another aspect, the disclosure provides a method for detecting the presence or amount of GREM1 in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of GREM1 in the sample.

[0064]

[0065] In another aspect, the disclosure provides use of an antibody, or antigen-binding fragment thereof, provided herein in the manufacture of a medicament for treating a GREM1-related disease or condition in a subject.

[0065]

[0066] In certain embodiments, the GREM1-associated disease or condition is cancer.

[0066]

[0067] In certain embodiments, the GREM1-associated disease or condition is a fibrotic disease, angiogenesis, glaucoma, a retinal disease, a kidney disease, pulmonary arterial hypertension, or osteoarthritis (OA). [Brief description of the drawings]

[0067] [Figure 1-1]

[0068] Figure 1 shows the binding of anti-gremlin 1 antibodies 69H5, 56C11, 22F1 and 14E3 to human gremlin 1 (hGREM1) (Figures 1A, 1C) and mouse gremlin 1 (Figures 1B, 1C), and the binding of anti-gremlin 1 antibodies 42B9, 36F5, and 67G11 to human gremlin 1 (hGREM1) (Figures 1D, 1F) and mouse gremlin 1 (Figures 1E, 1F), respectively, as measured by ELISA. [Figure 1-2]

[0068] Figure 1 shows the binding of anti-gremlin 1 antibodies 69H5, 56C11, 22F1 and 14E3 to human gremlin 1 (hGREM1) (Figures 1A, 1C) and mouse gremlin 1 (Figures 1B, 1C), and the binding of anti-gremlin 1 antibodies 42B9, 36F5, and 67G11 to human gremlin 1 (hGREM1) (Figures 1D, 1F) and mouse gremlin 1 (Figures 1E, 1F), respectively, as measured by ELISA. [Figure 1-3]

[0068] Figure 1 shows the binding of anti-gremlin 1 antibodies 69H5, 56C11, 22F1 and 14E3 to human gremlin 1 (hGREM1) (Figures 1A, 1C) and mouse gremlin 1 (Figures 1B, 1C), and the binding of anti-gremlin 1 antibodies 42B9, 36F5, and 67G11 to human gremlin 1 (hGREM1) (Figures 1D, 1F) and mouse gremlin 1 (Figures 1E, 1F), respectively, as measured by ELISA. [Diagram 2]

[0069] FIG. 2 shows the binding specificity of anti-Gremlin 1 antibody 14E3 to Gremlin-1 compared to Gremlin-2, COCO, and DAN-hFc as measured by ELISA. [Figure 3-1]

[0070] FIG. 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (FIGS. 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (FIG. 3B), to BMP4 (FIG. 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (FIGS. 3E, 3H), to BMP4 (FIGS. 3F, 3H), or to BMP7 (FIGS. 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Figure 3-2]

[0070] Figure 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (Figures 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (Figure 3B), BMP4 (Figure 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (Figures 3E, 3H), BMP4 (Figures 3F, 3H), or BMP7 (Figures 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Figure 3-3]

[0070] Figure 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (Figures 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (Figure 3B), BMP4 (Figure 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (Figures 3E, 3H), BMP4 (Figures 3F, 3H), or BMP7 (Figures 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Diagram 3-4]

[0070] Figure 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (Figures 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (Figure 3B), BMP4 (Figure 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (Figures 3E, 3H), BMP4 (Figures 3F, 3H), or BMP7 (Figures 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Figure 3-5]

[0070] Figure 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (Figures 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (Figure 3B), BMP4 (Figure 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (Figures 3E, 3H), BMP4 (Figures 3F, 3H), or BMP7 (Figures 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Diagram 3-6]

[0070] Figure 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (Figures 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (Figure 3B), BMP4 (Figure 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (Figures 3E, 3H), BMP4 (Figures 3F, 3H), or BMP7 (Figures 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Diagram 3-7]

[0070] Figure 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (Figures 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (Figure 3B), BMP4 (Figure 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (Figures 3E, 3H), BMP4 (Figures 3F, 3H), or BMP7 (Figures 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Diagram 3-8]

[0070] Figure 3 shows the binding affinity of Gremlin 1 or XM5 (Gremlin 1-DAN fusion protein) to BMP2 / 4 / 7 (Figures 3A, 3D) as measured by ELISA, the blocking activity of Gremlin 1 binding to BMP2 (Figure 3B), BMP4 (Figure 3C) by anti-Gremlin 1 antibodies 69H5, 56C11, 14E3 or benchmark antibody 6245P, and the blocking activity of Gremlin 1 binding to BMP2 (Figures 3E, 3H), BMP4 (Figures 3F, 3H), or BMP7 (Figures 3G, 3H) by anti-Gremlin 1 antibodies 42B9, 36F5, 67G11, and 14E3 HaLa or benchmark antibody 6245P. [Figure 4]

[0071] FIG. 4 shows blockade of Gremlin-mediated inhibition of BMP4 signaling by anti-Gremlin 1 antibodies 69H5, 56C11, 22F1, 14E3, and the benchmark antibody 6245P, as measured by a BMP-inducible reporter assay. [Figure 5-1]

[0072] Figure 5 shows blockade of Gremlin-mediated inhibition of BMP4 signaling by anti-Gremlin 1 antibodies 14E3 (Figure 5A), 22F1 (Figure 5B), 56C11 (Figure 5C), and 69H5 (Figure 5D) compared to the benchmark antibody 6245P, as measured by BMP4-induced ATDC-5 cell differentiation. [Figure 5-2]

[0072] Figure 5 shows blockade of Gremlin-mediated inhibition of BMP4 signaling by anti-Gremlin 1 antibodies 14E3 (Figure 5A), 22F1 (Figure 5B), 56C11 (Figure 5C), and 69H5 (Figure 5D) compared to benchmark antibody 6245P, as measured by BMP4-induced ATDC-5 cell differentiation. [Figure 5-3]

[0072] Figure 5 shows blockade of Gremlin-mediated inhibition of BMP4 signaling by anti-Gremlin 1 antibodies 14E3 (Figure 5A), 22F1 (Figure 5B), 56C11 (Figure 5C), and 69H5 (Figure 5D) compared to benchmark antibody 6245P, as measured by BMP4-induced ATDC-5 cell differentiation. [Figure 5-4]

[0072] Figure 5 shows blockade of Gremlin-mediated inhibition of BMP4 signaling by anti-Gremlin 1 antibodies 14E3 (Figure 5A), 22F1 (Figure 5B), 56C11 (Figure 5C), and 69H5 (Figure 5D) compared to benchmark antibody 6245P, as measured by BMP4-induced ATDC-5 cell differentiation. [Figure 6-1]

[0073] Figure 6 shows that Gremlin 1 reduces the level of BMP4-induced smad phosphorylation in prostate cancer cells (PC-3 cells) as measured by Western blot (Figure 6A), but this level is restored by anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 in prostate cancer cells (Figure 6B). [Figure 6-2]

[0073] Figure 6 shows that Gremlin 1 reduces the level of BMP4-induced smad phosphorylation in prostate cancer cells (PC-3 cells) as measured by Western blot (Figure 6A), but this level is restored by anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 in prostate cancer cells (Figure 6B). [Figure 7]

[0074] FIG. 7 shows blockade of Gremlin-mediated inhibition of BMP4 signaling in cancer and non-cancer cells by anti-Gremlin 1 antibody 14E3. [Figure 8]

[0075] FIG. 8 shows the binding affinity of chimeric anti-Gremlin 1 antibodies 56C11-C and 14E3-C to hGREM1 as measured by ELISA. [Figure 9]

[0076] FIG. 9 shows the binding kinetics of chimeric anti-Gremlin 1 antibodies 14E3-C and 22F1-C to hGREM1 as measured by Biacore. [Figure 10-1]

[0077] FIG. 10 shows results from an epitope study, with FIGS. 10A-C showing the results of epitope binning of anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assay (FIG. 10A), the results of a cross-competition assay of antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (FIG. 10B), and the binding of antibody 14E3-C and benchmark 6245P to the gremlin-DAN fusion protein XM5 as measured by ELISA (FIG. 10C), FIG. 10D showing the epitope mapping of 14E3 as measured by a biolayer interference (BLI) assay, and FIGS. 10E and 10F showing the binding of antibodies 42B9, 36F5, and 67G11 to human gremlin or the gremlin-DAN fusion protein XM5 as measured by ELISA. [Figure 10-2]

[0077] Figure 10 shows results from epitope studies, with Figures 10A-C showing epitope binning results for anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assay (Figure 10A), cross-competition assay results for antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show binding of antibodies 42B9, 36F5, and 67G11 to human gremlin or gremlin-DAN fusion protein XM5 as measured by ELISA. [Figure 10-3]

[0077] Figure 10 shows results from epitope studies, with Figures 10A-C showing epitope binning results for anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assay (Figure 10A), cross-competition assay results for antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show binding of antibodies 42B9, 36F5, and 67G11 to human gremlin or gremlin-DAN fusion protein XM5 as measured by ELISA. [Figure 10-4]

[0077] Figure 10 shows results from epitope studies, with Figures 10A-C showing epitope binning results for anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assay (Figure 10A), cross-competition assay results for antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show binding of antibodies 42B9, 36F5, and 67G11 to human gremlin or gremlin-DAN fusion protein XM5 as measured by ELISA. [Figure 10-5]

[0077] Figure 10 shows results from epitope studies, with Figures 10A-C showing epitope binning results for anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assay (Figure 10A), cross-competition assay results for antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show binding of antibodies 42B9, 36F5, and 67G11 to human gremlin or gremlin-DAN fusion protein XM5 as measured by ELISA. [Figure 10-6]

[0077] Figure 10 shows results from epitope studies, with Figures 10A-C showing epitope binning results for anti-Gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assay (Figure 10A), cross-competition assay results for antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show binding of antibodies 42B9, 36F5, and 67G11 to human gremlin or gremlin-DAN fusion protein XM5 as measured by ELISA. [Figure 11-1]

[0078] Figures 11A and 11B show the epitope analysis results of the anti-Gremlin 1 antibodies 14E3, 56C11, 22F1, 69H5 and benchmark antibody 6245P provided herein as measured by Fortebio. The results showed that 14E3 has an epitope that does not overlap at all with that of 6245P, while 56C11 shares a similar epitope with that of 6245P. [Figure 11-2]

[0078] Figures 11A and 11B show the epitope analysis results of the anti-Gremlin 1 antibodies 14E3, 56C11, 22F1, 69H5 and the benchmark antibody 6245P provided herein as measured by Fortebio. [Figure 12-1]

[0079] FIG. 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1 as measured by ELISA (FIGS. 12A-C) or by Fortebio (FIG. 12D) in comparison to the benchmark antibody 6245P. [Figure 12-2]FIG. 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1 as measured by ELISA (FIGS. 12A-C) or by Fortebio (FIG. 12D) in comparison to the benchmark antibody 6245P. [Figure 12-3] FIG. 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1 as measured by ELISA (FIGS. 12A-C) or by Fortebio (FIG. 12D) in comparison to the benchmark antibody 6245P. [Figure 12-4] FIG. 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1 as measured by ELISA (FIGS. 12A-C) or by Fortebio (FIG. 12D) in comparison to the benchmark antibody 6245P. [Figure 13]

[0080] FIG. 13 shows that anti-GREM1 antibody 14E3 reduced prostate cancer tumor volume (FIG. 13A) and tumor weight (FIG. 13B) in a PC3 xenograft model. [Figure 14]

[0081] FIG. 14 shows the anti-tumor effect of anti-GREM1 antibody 56C11 on the CT-26 colon cancer model. [Figure 15-1]

[0082] Figures 15A and 15B show the synergistic anti-tumor effect of combination therapy with anti-mGREM1 antibody and immune checkpoint inhibitor (e.g., MPDL-3280A) on the CT-26 model. [Figure 15-2]

[0082] Figures 15A and 15B show the synergistic anti-tumor effect of combination therapy with anti-mGREM1 antibody and immune checkpoint inhibitor (e.g., MPDL-3280A) on the CT-26 model. [Figure 16]

[0083] Figure 16 shows IHC staining of GREM1 or PD-L1 in E7PDX tumor samples with either anti-GREM1 antibody (14E3) or anti-PD-L1 antibody (22C3). [Figure 17-1]

[0084] Figures 17A and 17B show that humanized 14E3 (hzd 14E3), alone or in combination with cisplatin, inhibited tumor growth in a gremlin-positive esophageal PDX model. Humanized 14E3 alone achieved about 43% tumor growth inhibition (TGI). Cisplatin alone achieved about 60% TGI. The combination of humanized 14E3 and cisplatin achieved about 64% TGI. [Figure 17-2]

[0084] Figures 17A and 17B show that humanized 14E3 (hzd 14E3), alone or in combination with cisplatin, inhibited tumor growth in a gremlin-positive esophageal PDX model. [Figure 18-1]

[0085] Figure 18 shows the binding of gremlin to FGFR1 as measured by ELISA (Figure 18A) and the blocking activity of huIgG1, hIgG4, anti-gremlin 1 antibodies 42B9, 36F5, 67G11, 69H5-chi, 36F5-chi, 22F1chi, 56C11-chi and 14E3 HaLa, and the benchmark antibody 6245P, respectively, of gremlin binding to FGFR1 (Figures 18B, 18C). [Figure 18-2]

[0085] Figure 18 shows the binding of gremlin to FGFR1 as measured by ELISA (Figure 18A), and the blocking activity of huIgG1, hIgG4, anti-gremlin 1 antibodies 42B9, 36F5, 67G11, 69H5-chi, 36F5-chi, 22F1chi, 56C11-chi and 14E3 HaLa, and the benchmark antibody 6245P, respectively, in blocking gremlin binding to FGFR1 (Figures 18B, 18C). [Figure 18-3]

[0085] Figure 18 shows the binding of gremlin to FGFR1 as measured by ELISA (Figure 18A), and the blocking activity of huIgG1, hIgG4, anti-gremlin 1 antibodies 42B9, 36F5, 67G11, 69H5-chi, 36F5-chi, 22F1chi, 56C11-chi and 14E3 HaLa, and the benchmark antibody 6245P, respectively, in blocking gremlin binding to FGFR1 (Figures 18B, 18C). [Figure 18-4]

[0085] Figure 18 shows the binding of gremlin to FGFR1 as measured by ELISA (Figure 18A), and the blocking activity of huIgG1, hIgG4, anti-gremlin 1 antibodies 42B9, 36F5, 67G11, 69H5-chi, 36F5-chi, 22F1chi, 56C11-chi and 14E3 HaLa, and the benchmark antibody 6245P, respectively, in blocking gremlin binding to FGFR1 (Figures 18B, 18C). [Figure 19-1]

[0086] FIG. 19 shows the binding activity of hybridoma 36F5 (FIG. 19A) and chimera 36F5 (36F5-chi) (FIG. 19B) to Gremlin-his and DAN-his in ELISA. [Figure 19-2]

[0086] Figure 19 shows the binding activity of hybridoma 36F5 (Figure 19A) and chimera 36F5 (36F5-chi) (Figure 19B) to Gremlin-his and DAN-his in ELISA. [Figure 20-1]

[0087] FIG. 20 shows that chimeric 36F5 (36F5-chi) blocks BMP4 binding to DAN proteins (FIG. 20A) and BMP2 binding to DAN proteins (FIG. 20B). [Figure 20-2] FIG. 20 shows that chimeric 36F5 (36F5-chi) blocks BMP4 binding to DAN protein (FIG. 20A) and BMP2 binding to DAN protein (FIG. 20B). [Figure 21]

[0088] Figure 21 shows the anti-tumor activity of hybridoma 36F5 against the EMT6 / hPD-L1 tumor model. Figure 21A shows that the EMT6 / hPD-L1 tumor model has low responsiveness to anti-PD-L1 antibody AM4B6. Figure 21B shows that hybridoma 36F5 exhibits anti-tumor activity against the EMT6 / hPD-L1 tumor model. [Figure 22-1]

[0089] Figure 22 shows the anti-tumor activity of hybridoma 14E3 or 36F5 against E7 tumor model. Figure 22A shows that E7 tumor model has low responsiveness to anti-PD-1 antibody nivolumab, and hybridoma 14E3 shows anti-tumor activity against E7 tumor model. Figure 22B shows that E7 tumor model has low responsiveness to anti-PD-1 antibody nivolumab, and hybridoma 36F5 shows anti-tumor activity against E7 tumor model. [Figure 22-2] FIG. 22 shows the antitumor activity of hybridomas 14E3 or 36F5 against the E7 tumor model. [Figure 23]

[0090] Figure 23 shows the anti-tumor activity of 56C11 combination therapy with anti-PDL1 antibodies against the MC38 / hPD-L1 tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068]

[0091] The following description of the present disclosure is intended to merely illustrate various embodiments of the present disclosure. As such, the specific modifications discussed should not be interpreted as limitations on the scope of the present disclosure. It is clear to those skilled in the art that various equivalents, variations and modifications can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification. All references cited in this specification, including publications, patents and patent applications, are incorporated herein by reference in their entirety.

[0069] definition

[0092] 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 construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0070]

[0093] 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 naturally occurring 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 λ or κ, and each light chain is composed of a variable region (V L) and constant regions. Antibodies have the shape of a "Y" with the stem of the Y being composed of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable region 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 including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3). The boundaries of the CDRs of the antibodies and antigen binding domains disclosed herein may be determined according to the Kabat, IMGT, AbM, Chothia, or Al-Lazikani convention (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. Engineering, vol. 13(12), pp. 819-824 (2000); Chothia, C. et al., Nature. Dec. 21-28; 342(6252):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 (2nd ed.), Chapter 26, pp. 481-514, (2015)). The three CDRs are interposed 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, but 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 main 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 main 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 include any antigen-binding fragment thereof.

[0071]

[0094] As used herein, the term "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, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fd, 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-chain domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments can bind to the same antigen that the parent antibody binds. In certain embodiments, antigen-binding fragments can include one or more CDRs from a particular parent antibody.

[0072]

[0095] "Fab" with respect to an antibody refers to a monovalent antigen-binding fragment of an antibody that is composed 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. Fabs can be obtained by papain digestion of an antibody at residues proximal to the N-terminus of the disulfide bond between the heavy chains in the hinge region.

[0073]

[0096] "Fab'" refers to a Fab fragment containing a portion of the hinge region, which may be obtained by pepsin digestion of an antibody at the residues proximal to the C-terminus of the inter-heavy chain disulfide bond in the hinge region, and which therefore differs from Fab in a small number of residues in the hinge region, including one or more cysteines.

[0074]

[0097] "F(ab')2" refers to a dimer of Fab' containing two light chains and part of two heavy chains.

[0075]

[0098] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that retains a complete antigen-binding site. An Fv fragment is composed 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 link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.

[0076]

[0099] "Single chain Fv antibody" or "scFv" refers to an engineered antibody composed 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)). "scFv dimer" refers to 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 bivalent diabody or a bivalent ScFv (BsFv), which is a V H A part of V L and to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). H -V L The V dimerized with the moiety H -V L (linked by a peptide linker). In another embodiment, an "scFv dimer" is a bispecific diabody, which comprises H1 and V L1 VH2 and V L2 V is arranged so that they cooperate and each cooperated pair has a different antigen specificity. L1 -V H2 (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker).

[0077]

[0100] "Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody composed of an scFv attached to the Fc region of an antibody.

[0078]

[0101] "Camelized single domain antibodies", "heavy chain antibodies", "nanobodies" or "HCAbs" are antibodies that consist of two V HIt refers to antibodies that contain heavy chain domains and do not contain 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 obtained from the Camelidae family (camels, dromedaries and llamas). Although lacking light chains, camelized antibodies have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3;363(6428):446-8 (1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", Immunogenetics. April;54(1):39-47 (2002); Nguyen VK. et al. Immunology. May;109(1):93-101 (2003)). The variable domains of heavy-chain antibodies (VHH domains) represent the smallest known antigen-binding units generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November;21(13):3490-8. Epub 2007 June 15 (2007)). "Diabodies" include small antibody fragments with two antigen-binding sites, where the fragments are arranged in a single polypeptide chain. L V connected to the domain H Domain (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. July 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 they are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The two antigen-binding sites can target the same or different antigens (or epitopes).

[0079]

[0102] A "domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or only 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 can target the same or different antigens.

[0080]

[0103] In certain embodiments, a "(dsFv)2" is a Fv comprising three peptide chains: a Fv comprising two V L Two V's joined together H Includes parts.

[0081]

[0104] In certain embodiments, a "bispecific ds diabody" is H1 and V L1 Through a disulfide bridge between V L1 -V H2 V bound to (linked by a peptide linker) H1 -V L2 (also linked by a peptide linker) Includes.

[0082]

[0105] In certain embodiments, a "bispecific dsFv" or "dsFv-dsFv'" comprises three peptide chains: H1 -V H2The two heavy chains are linked by a peptide linker (e.g., a long flexible linker) and are connected via disulfide bridges to each other via V L1 Part and V L2 Partially paired, V H1 -V H2 Each disulfide-paired heavy and light chain has a different antigen specificity.

[0083]

[0106] 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, if applicable, a constant region 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.

[0084]

[0107] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of the heavy and / or light chain derived from one species and the remaining portion of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody can include a constant region derived from a human and a variable region derived from a non-human species, such as a mouse.

[0085]

[0108] The term "germline sequence" refers to a nucleic acid sequence encoding a variable region amino acid sequence or subsequence that shares the highest determined amino acid sequence identity with a reference variable region amino acid sequence or subsequence in comparison with 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 in comparison with all other evaluated variable region amino acid sequences. A germline sequence may 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 by aligning the two sequences using the methods described herein, for example, 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.

[0086]

[0109] As used herein, "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 uses.

[0087]

[0110] 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.

[0088]

[0111] As used herein, the terms "specific binding" or "specifically bind" refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein have a specific 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 to 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 using 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 ​​may 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 may be used to determine specific immunoreactivity). Usually, a specific or selective binding reaction will produce a signal that is at least 2-fold over background, more usually at least 10-100-fold over background.

[0089]

[0112] As used herein, the term "amino acid" refers to an organic compound that contains an amine (-NH2) functional group and a carboxyl (-COOH) functional group along with a side chain characteristic of each amino acid. In this disclosure, the names of amino acids are also represented as standard one-letter or three-letter codes, which are summarized below.

[0090] [Table 1]

[0091]

[0113] "Conservative substitution" in the context of amino acid sequence refers to replacing an amino acid residue with a different amino acid residue that has 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 acids 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 substitution usually does not cause significant changes in the conformational structure of protein, and therefore may retain the biological activity of protein.

[0092]

[0114] "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 obtain maximum correspondence. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be performed using publicly available tools such as, for example, BLASTN, BLASTp (available at 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 at 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, such as by selecting an appropriate algorithm, as necessary for alignment. 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, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference.

[0093]

[0115] 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, optionally when aligned.

[0094]

[0116] An "isolated" material is one that has been altered by the hand of man from its natural state. An "isolated" composition or material, when present in nature, has been changed 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 is "isolated" when the same polynucleotide or polypeptide is sufficiently separated from the coexisting materials of its natural state so that it exists in a substantially pure state. Isolated "nucleic acid" or "polynucleotide" are used interchangeably and refer to the sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment having 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 electrophoretic (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic (e.g., ion exchange chromatography or reverse phase HPLC) methods.

[0095]

[0117] 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" and "subject" are used interchangeably herein.

[0096]

[0118] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the rate of onset or occurrence of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0097]

[0119] The term "vector" as used herein refers to a vehicle into which a genetic element is operably inserted to cause expression of the genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element carried by the vector in the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector can contain an origin of replication. A vector can also contain substances that aid in the entry of the vector into a cell, including, but not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0098]

[0120] As used herein, a "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0099]

[0121] The terms "gremlin 1" or "GREM1" refer to variant 1 of gremlin and encompass gremlin 1 in various species, such as 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 accessible through the GenBank database under accession number NP-037504 or through 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.

[0100]

[0122] 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 related to 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).

[0101]

[0123] As used herein, "cancer" refers to any medical condition characterized by malignant cell proliferation or neoplasia, abnormal growth, invasion or metastasis, and can be either benign or malignant, and includes both solid tumors and non-solid cancers such as leukemia (e.g., hematological malignancies). As used herein, a "solid tumor" refers to a solid mass of neoplastic and / or malignant cells.

[0102]

[0124] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation and physiologically compatible with the recipient thereof.

[0103]

[0125] Reference herein to a value or parameter of "about" includes (and describes) an embodiment that is directed to the value or parameter itself. For example, a reference to "about X" includes the reference to "X". A numerical range includes the numbers defining the range. Generally speaking, the term "about" refers to the stated value of the variable and all values ​​of the variable that are within experimental error of the stated value (e.g., within a 95% confidence interval for the mean) or within 10 percent of the stated value, whichever is greater. When the term "about" is used within the context of a period of time (years, months, weeks, days, etc.), the term "about" means the period plus or minus a certain amount of the next subperiod (e.g., about 1 year means 11-13 months; about 6 months means 6 months ± 1 week; about 1 week means 6-8 days; etc.), or within 10 percent of the stated value, whichever is longer.

[0104]

[0126] Anti-human Gremlin 1 antibody

[0127] The present disclosure provides anti-human Gremlin 1 (hGREM1) antibodies and antigen-binding fragments thereof. The anti-hGREM1 antibodies and antigen-binding fragments provided herein are uniquely distinguished from existing anti-hGREM1 antibodies in that: a) they can selectively reduce hGREM1-mediated inhibition of BMP signaling in cancer cells compared to non-cancer cells; b) they exhibit less than 50% reduction in hGREM1-mediated inhibition of BMP signaling in non-cancer cells; c) they can bind to chimeric hGREM1 comprising the amino acid sequence of SEQ ID NO: 68; d) they can bind to hGREM1 but cannot specifically bind to mouse Gremlin 1; e) they bind to hGREM1 at an epitope comprising residue Gln27 and / or residue Asn33, where the residue numbering is according to SEQ ID NO: 69, or they bind to an hGREM1 fragment comprising residue Gln27 and / or residue Asn33. and optionally, the hGREM1 fragment has a length of at least 3 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acid residues; f) is capable of reducing hGREM1-mediated activation of MAPK signaling; g) is capable of binding to hGREM1 with a Kd of 1 nM or less as measured by Fortebio; h) is capable of blocking the binding of hGREM1 to BMP7 with a maximum blocking percentage of at least 50% as measured by ELISA; i) is capable of blocking the interaction of GREM1 (e.g., hGREM1 or mGREM1) with FGFR (e.g., FGFR1, preferably human FGFR1 (hFGFR1) or mouse FGFR1 (mFGFR1)); and / or j) is capable of binding to both hGREM1 and DAN.

[0105]

[0128] Bone morphogenetic proteins (BMPs), such as BMP2, BMP4 and BMP7, are known as glycosylated extracellular matrix (ECM)-associated members of the transforming growth factor beta (TGF-beta) superfamily and play key roles in the processes of morphogenesis, general organogenesis, cartilage and limb formation, cell proliferation, differentiation and apoptosis. BMP signaling is activated by the binding of BMP ligands (e.g., BMP2, BMP4 and BMP7) to the BMP receptor, which activates the phosphorylation of the receptor, resulting in phosphorylation of R-Smads (e.g., Smad1 / 5 / 9) and complex formation with co-Smad4. The formed Smad complex then translocates to the nucleus to regulate the expression of BMP target genes. Thus, the activation of BMP signaling can be assayed, for example, by measuring the phosphorylation level of smads and / or the expression level of BMP target genes. The activation of BMP signaling can also be assayed by measuring the expression level of differentiation marker genes, for example, alkaline phosphatase, an early marker of osteoblast differentiation.

[0106] It is known that BMP signaling is inhibited by BMP antagonists such as noggin, chordin, gremlin 1 and twisted gastrulation 1. GREM1 can physically bind to BMP ligands such as BMP-2, BMP-4, or BMP-7 to form heterodimers and prevent these BMP ligands from interacting with their corresponding BMP receptors, thereby inhibiting the activation of the BMP signaling pathway. Therefore, it is expected that eliminating GREM1 and / or reducing the activity of GREM1 will reverse or reduce the inhibition of BMP signaling.

[0107]

[0129] i.Binding affinity

[0130] In certain embodiments, the anti-GREM1 antibodies provided herein can bind to hGREM1 with a Kd of 1 nM or less as measured by Fortebio. The binding affinity of the anti-GREM1 antibodies and antigen-binding fragments provided herein is D This can be expressed by the ratio of the dissociation rate to the association rate (k off / k on ) represents the antigen binding affinity (e.g., K D ) may be suitably determined using any suitable method known in the art, including, for example, Kinetic Exclusion Assay (KinExA), Biacore, Fortebio, or flow cytometry.

[0108]

[0131] In certain embodiments, the "K D " or "K DThe "value" is measured in one embodiment by a Biacore or Fortebio assay, which is performed with an anti-GREM1 antibody and GREM1 as described by the following assay that measures the solution binding affinity of an anti-GREM1 antibody. In general, Biacore works by equilibrating a fixed amount of one binding partner (CBP) with various concentrations of the other binding partner (titrant), and then capturing a portion of the free CBP with a fluorescently labeled secondary antibody within a contact time that is shorter than the time required for dissociation of the previously formed CBP-titrant complex. The fluorescent signal generated from the captured CBP is directly proportional to the concentration of free CBP in the equilibrated sample and is used to generate a binding curve (percent free CBP vs. total titrant concentration) when measured in series. Further details are available from Schreiber, G., Fersht, AR Nature Structural Biology. 1996, 3(5), pp. 427-431. If an anti-GREM1 antibody is used as the CBP in a fixed amount, then the GREM1 protein can be used as the titrant, or vice versa. Fortebio generally works in a similar manner to Biacore by equilibrating a fixed amount of CBP (e.g., GREM1 protein) with various concentrations of the titrant (e.g., anti-GREM1 antibody). The binding kinetics (k on and k off Information about the interferometry of the fragments can be obtained from changes in the interference patterns generated by the biosensors used by Fortebio. Further details are available in Charles Wartchow, Frank Podlaski, Shirley Li, Karen Rowan, Xiaolei Zhang, David Mark, Kuo-Sen Huang, "Biosensor-based Small Molecule Fragment Screening with Biolayer Interferometry": J Comput Aided Mol Des 2011, 25(7), 669-76.

[0109]

[0132] In certain embodiments, the K D is determined according to the method described in Example 14 of the present disclosure.

[0110]

[0133] K D Other methods suitable for measuring may also be used where applicable, such as radiolabeled antigen binding assays (see, e.g., Chen, et al. (1999) J. Mol Biol 293:865-881) or surface plasmon resonance assays other than BIAcore.

[0111]

[0134] In certain embodiments, the anti-GREM1 antibodies and antigen-binding fragments thereof provided herein have a K of 100 nM or less (or 90, 80, 70, 60, 50, 40, 30, 29, 27, 20, 19, or 18 nM or less) as measured by a Biacore assay. D In certain embodiments, the anti-GREM1 antibodies and antigen-binding fragments thereof provided herein specifically bind to human GREM1 with a K of 10 nM or less (or 9, 8, 7, 6, 5, 4, 3, 2, or 1 nM or less) as measured by the Fortebio assay. D It specifically binds to human GREM1 at a value of 0.01.

[0112]

[0135] Alternatively, the binding affinity of the anti-GREM1 antibodies and antigen-binding fragments provided herein to human GREM1 may also be expressed as the "half maximal effective concentration" (EC 50 ) value, which refers to the concentration of antibody at which 50% of its maximal effect (e.g., binding) is observed. 50Values ​​may be measured by methods known in the art, for example, sandwich assays such as ELISA, Western blots, flow cytometry assays, and other binding assays. In certain embodiments, the anti-GREM1 antibodies and fragments thereof provided herein have an EC50 of 120 ng / ml or less (or 100, 90, 80, 60, 40, 30, 20, 14.5, 14, 13.5, 13, 12.5, 12, 11.5, 11, 10.5, 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3, 2, or 1 ng / ml or less) as measured by ELISA. 50 The antibody specifically binds to human GREM1 (eg, cells expressing human GREM1) at a concentration of 0.1 mM.

[0113]

[0136] In certain embodiments, some of the antibodies and antigen-binding fragments thereof provided herein have an EC of 20 ng / ml or less as measured by ELISA. 50 In certain embodiments, the antibodies and antigen-binding fragments thereof can specifically bind to mouse GREM1 with an EC value of 4 ng / ml to 20 ng / ml (e.g., 4 ng / ml to 9 ng / ml, 5 ng / ml to 8 ng / ml, 6 ng / ml to 7 ng / ml, 6 ng / ml to 14 ng / ml, 6 ng / ml to 12 ng / ml, 4.564 ng / ml, 7.713 ng / ml, 8.512 ng / ml, or 17.2 ng / ml) as measured by ELISA. 50 It binds to mouse GREM1 at 100 ng / s.

[0114]

[0137] In certain embodiments, some of the antibodies and antigen-binding fragments thereof provided herein do not specifically bind to mouse GREM1.

[0115]

[0138] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein do not specifically bind to GREM2.

[0116]

[0139] Antibody sequence

[0140] In one aspect, the disclosure provides an anti-GREM1 antibody or antigen-binding fragment thereof provided herein, wherein the heavy chain variable region comprises: a) HCDR1 comprising a sequence selected from SEQ ID NOs: 1, 11, 21, 31, 114, 119 and 123; b) HCDR2 comprising a sequence selected from SEQ ID NOs: 2, 12, 22, 32 and 115, and c) HCDR3 comprising a sequence selected from SEQ ID NOs: 3, 13, 23, 33, 116, 120 and 124 and / or The light chain variable region is: d) LCDR1 comprising the sequences of SEQ ID NOs: 4, 14, 24, 34, 117, 121, 122 and 125; e) LCDR2 comprising the sequences of SEQ ID NOs: 5, 15, 25 and 35, and f) LCDR3 comprising a sequence selected from SEQ ID NOs: 6, 16, 26, 36 and 118; Includes.

[0117]

[0141] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising: a) a heavy chain variable region comprising 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; b) a heavy chain variable region comprising 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; c) a heavy chain variable region comprising 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; d) a heavy chain variable region comprising 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; e) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 114, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 116; f) a heavy chain variable region comprising an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; and g) a heavy chain variable region comprising HCDR1 comprising the sequence of SEQ ID NO: 123, HCDR2 comprising the sequence of SEQ ID NO: 115, and HCDR3 comprising the sequence of SEQ ID NO: 124 An antibody or antigen-binding fragment thereof selected from the group consisting of:

[0118]

[0142] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a light chain variable region comprising: 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; 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; e) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 117, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; f) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 121, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; g) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 122, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; and h) a light chain variable region comprising an LCDR1 comprising the sequence of SEQ ID NO: 125, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118. An antibody or antigen-binding fragment thereof selected from the group consisting of:

[0119]

[0143] In certain embodiments, an antibody or antigen-binding fragment thereof provided herein comprises: 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; 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; e) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 114, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 116; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 117, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; f) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 121, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; g) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 122, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; h) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 123, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 124; the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 125, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; An antibody or an antigen-binding fragment thereof.

[0120]

[0144] In certain embodiments, the antibodies provided herein comprise the CDR sequences of one or more (e.g., one, two, three, four, five, or six) of the anti-hGREM1 antibodies 14E3, 69H5, 22F1, 56C11, 36F5, 42B9, and 67G11.

[0121]

[0145] 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.

[0122]

[0146] 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.

[0123]

[0147] 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.

[0124]

[0148] 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.

[0125]

[0149] As used herein, "36F5" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:126 and a light chain variable region of SEQ ID NO:127.

[0126]

[0150] As used herein, "42B9" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:128 and a light chain variable region of SEQ ID NO:129.

[0127]

[0151] As used herein, "67G11" refers to a murine antibody having a heavy chain variable region of SEQ ID NO:128 and a light chain variable region of SEQ ID NO:130.

[0128]

[0152] Table 1 shows the CDR sequences of these anti-hGREM1 antibodies. The CDRs are determined according to the Kabat numbering. Those skilled in the art will understand that other known methods for CDR determination can also be applied to the antibodies provided herein, and their CDR sequences are also encompassed within the present disclosure. The heavy and light chain variable region sequences are also provided below in Table 2.

[0129]

[0153]

[0130] [Table 2]

[0131] Where X 32 is S or Y;X 27 is A or G;X 28 is H or N; X 29 is H or N; X 30 is R or I;X 31 is L or V.

[0132]

[0154]

[0133] [Table 3]

[0134]

[0155] 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. A recombinant antibody is one that is prepared in vitro using recombinant methods rather than in an animal.

[0135]

[0156] CDRs are known to be responsible for antigen binding, however, it has been found that not all six CDRs are necessarily essential or invariant. In other words, it is possible to replace, alter or modify one, two or three CDRs in anti-hGREM1 antibodies 14E3, 69H5, 22F1, 56C11, 36F5, 42B9 or 67G11 (corresponding to any one of SEQ ID NOs: 1-36 and 114-125) and still substantially retain specific binding affinity to hGREM1.

[0136]

[0157] In certain embodiments, the anti-hGREM1 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence of one of the anti-hGREM1 antibodies 14E3, 69H5, 22F1, 56C11, 36F5, 42B9 or 67G11. In certain embodiments, the anti-hGREM1 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence of SEQ ID NOs: 3, 13, 23, 33, 116, 120 and 124. The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore believed to make the most contact with the antigen and provide the most free energy for the affinity of the antibody to the antigen. The heavy chain CDR3 is also believed to be by far the most diverse CDR for the antigen-binding site in terms of length, amino acid composition and conformation due to multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81). Diversity in the heavy chain CDR3 is sufficient to generate most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) as well as desirable antigen binding affinities (Schier R, et al. J Mol Biol. 263:551-67).

[0137]

[0158] In some embodiments, the anti-hGREM1 antibodies and antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-hGREM1 antibodies and antigen-binding fragments provided herein are single-domain antibodies consisting of all or a portion of the heavy chain variable domain provided herein. Further information regarding such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0138]

[0159] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of specifically binding to GREM1. The CDR sequences provided in Table 1 are taken from mouse antibodies, but can be grafted to any suitable FR sequences of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0139]

[0160] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are chimeric: VH / VL are grafted onto human IgG1 sequences and human kappa sequences.

[0140]

[0161] In certain embodiments, the antibodies and antigen-binding fragments provided herein are humanized. Humanized antibodies or antigen-binding fragments are desirable in terms of their reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can essentially be performed by substituting corresponding human CDR genes in human immunoglobulin genes with non-human (e.g., mouse) CDR genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536). Simulation of the three-dimensional structure of the variable region or domain of the parent non-human antibody can be performed before or after such humanization.

[0141]

[0162] Suitable human heavy and light chain variable domains can be selected using methods known in the art to perform CDR grafting. In an illustrative example, a "best-fit" approach can be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain germline sequences (e.g., Protein Data Bank, http: / / www.rcsb.org / ), and the human sequence that is closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequence (see, e.g., Sims et al., (1993) J.Immunol. 151:2296; Chothia et al., (1987) J.Mot.Biol. 196:901). Alternatively, frameworks derived from the consensus sequence of all human antibodies can be used for grafting of non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0142]

[0163] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR, and, if present, the constant region, are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences may be derived from different human immunoglobulin genes, e.g., the FR sequences may be derived from one human antibody and the constant region may be derived from another human antibody. In some embodiments, the humanized antibodies or antigen-binding fragments comprise human heavy / light chain FR1-4.

[0143]

[0164]

[0144] [Table 4-1]

[0145] [Table 4-2]

[0146]

[0165]

[0147] [Table 5]

[0148] where X1 is V, I or A; X2 is A or S; X3 is V or L; X4 is Y or S; X5 is T or S; X6 is V, L or M; X7 is R or K; X8 is Q or K; X9 is E or T; 10 is M or I;X 11 is V or A;X 12 is M or L;X 13 is R or V; X 14 is T or K;X 15 is S or R; X 16 is V or A;X 17 is M or L;X 18 is R or V; X 19is T or K;X 20 is T, M or L; X 21 is L, F or Y; X 22 is R or L; X 23 is Y or S;X 24 is V or F;X 25 is G or Q;X 26 is V or L.

[0149]

[0166] In some embodiments, the FR region derived from a human may contain the same amino acid sequence as the human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are replaced with the corresponding residues from the parent non-human antibody. This may be desirable in certain embodiments to make the humanized antibody or fragment thereof more similar to the non-human parent antibody structure, thereby reducing or avoiding immunogenicity, and / or improving or retaining binding activity or binding affinity.

[0150]

[0167] In certain embodiments, the humanized antibody or antigen-binding fragment provided herein comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in each human FR sequence, or no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue substitutions in all FRs of the heavy or light chain variable domain. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR region, only in the light chain FR region, or in both chains. In certain embodiments, one or more amino acid residues are mutated, e.g., back-mutated to the corresponding residue found in the non-human parent antibody from which the CDR sequence is derived (e.g., in the mouse framework region). The positions suitable for mutation can be selected by the skilled artisan according to principles known in the art. For example, the position of the mutation may be selected at the following locations: 1) where the residue in the framework of the human Gremlin sequence is rarely present (e.g., in less than 20% or less than 10% of the human variable region sequences); 2) where the location is directly adjacent to one or more of the three CDRs in the primary sequence of the human germline chain, because it is more likely to interact with the residues in the CDRs; or 3) where the location is close to the CDRs in the three-dimensional model, and therefore may have a good possibility of interacting with the amino acids in the CDRs. The residue at the selected location may be backmutated to the corresponding residue in the parent antibody, or to a residue that is neither the corresponding residue in the human germline sequence nor the corresponding residue in the parent antibody, but is a common residue in human sequences, i.e., occurs more frequently at that position in known human sequences that belong to the same subgroup as the human germline sequence (see U.S. Patent No. 5,693,762).In certain embodiments, in the case of hu14E3, the heavy chain variants Ha, Hb and Hc were obtained by directly grafting the three CDRs into their germline sequences, which have back mutations of V37M for the heavy chain variant Ha, V37M, V68A, V2I for the heavy chain variant Hb, and V37M, V68A, V2I, Y27S, R38K, E46T for the heavy chain variant Hc, respectively, all based on the Kabat numbering; the light chain variants were obtained by directly grafting the three CDRs into their germline sequences, which have back mutations of F36L for the light chain variant La, and F36L, Y49S for the light chain variant Lb, respectively, all based on the Kabat numbering. In certain embodiments, for hu22F1, the heavy chain variants Ha, Hb, Hc and Hd have the following back mutations, respectively, for heavy chain variant Ha: no back mutations, for heavy chain variant Hb: R72V, T74K, T28S, R98S, and for heavy chain variant Hc: R71V, T73K, T28S, R94S, S82, all based on Kabat numbering. A The heavy chain variant Hd had R71V, T73K, T28S, R94S, and S82 AThe light chain variants were obtained by directly grafting the three CDRs onto their germline sequences, which have the following back mutations: R, M69L, V2A, V67A, M48I, V37L; the light chain variants were obtained by directly grafting the three CDRs onto their germline sequences, which have the following back mutations: F36L for light chain variant La, and F36L, V58F for light chain variant Lb, all based on Kabat numbering. In certain embodiments, for hu56C11, heavy chain (HC) variants 1, 2, 3, and 4 were obtained by directly grafting the three CDRs onto their germline sequences, with no back mutations for heavy chain variant H0, R71V, T73K back mutations for heavy chain variant Ha, R71V, T73K, M69L, M48I back mutations for heavy chain variant Hb, and R71V, T73K, M69L, M48I, Q43K, R38K back mutations for heavy chain variant Hc, all based on Kabat numbering; light chain (LC) variants 1, 2, and 3 were obtained by directly grafting the three CDRs onto their germline sequences, with no back mutations for light chain variant L0, R46L back mutations for light chain variant La, and F36Y, R46L back mutations for light chain variant Lb, all based on Kabat numbering.

[0151]

[0168] In certain embodiments, the humanized light chains and humanized heavy chains of the present disclosure are substantially non-immunogenic in humans and retain substantially the same or even higher affinity to hGREM1 than the parental antibodies. Specifically, the humanized antibodies provided herein (e.g., Hu22F1 and Hu56C11) and humanized anti-hGREM1 antibodies derived from 69H5, 36F5, 42B9 and 67G11 are expected to exhibit similar properties (e.g., high binding affinity to human gremlin 1 and / or mouse gremlin 1, high blocking effect on gremlin-mediated inhibition of BMP4 signaling, high blocking activity of gremlin 1 binding to FGFR1, and / or high anti-tumor effect) or even better than their parental antibodies.

[0152]

[0169] In certain embodiments, the humanized antibodies and antigen-binding fragments thereof provided herein comprise one or more light chain FR sequences of the human germline framework sequence IGKV / 2-30, with or without back mutations, and / or one or more heavy chain FR sequences of the human germline framework sequence IGHV / 7-4 or IGHV / 1-46 or IGHV1-2. Optionally, back mutations may be introduced into the human germline framework sequences.

[0153]

[0170] In certain embodiments, the humanized antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1.

[0154]

[0171] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein further comprises a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1.

[0155]

[0172] In certain embodiments, the humanized antibody or antigen-binding fragment provided herein comprises 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 homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:49, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1.

[0156]

[0173] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein further comprises 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 a pair of sequences having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto while still retaining specific binding specificity or affinity to hGREM1.

[0157]

[0174] In certain embodiments, the humanized antibody or antigen-binding fragment provided herein comprises 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 homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:59 and SEQ ID NO:61, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1.

[0158]

[0175] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein further comprises 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; or a pair of sequences having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto while still retaining specific binding specificity or affinity to hGREM1.

[0159]

[0176] In certain embodiments, the humanized antibody or antigen-binding fragment provided herein comprises a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137, and homologous sequences thereof having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity while still retaining specific binding specificity or affinity to hGREM1.

[0160]

[0177] In certain embodiments, a humanized antibody or antigen-binding fragment thereof provided herein further comprises a pair of heavy chain and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 131 / 135, 131 / 136, 131 / 137, 132 / 135, 132 / 136, 132 / 137, 133 / 135, 133 / 136, 133 / 137, 134 / 135, 134 / 136, and 134 / 137, or a pair of sequences having at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto while still retaining specific binding specificity or affinity to hGREM1.

[0161]

[0178]

[0162] [Table 6-1]

[0163] [Table 6-2]

[0164] [Table 6-3]

[0165]

[0179] The humanized anti-hGREM1 antibodies provided herein retain specific binding affinity to hGREM1, and in these respects are at least comparable to or even better than the parent antibody. In certain embodiments, the anti-hGREM1 antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ.

[0166]

[0180] In certain embodiments, the anti-hGREM1 antibodies and fragments thereof provided herein further comprise a human IgG1, IgG2, IgG3, or IgG4 constant region. In certain embodiments, the anti-hGREM1 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of an IgG1 isotype. In certain embodiments, the anti-hGREM1 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of an IgG2b isotype.

[0167]

[0181] In certain embodiments, the humanized antibodies provided herein can comprise a heavy chain variable region fused to a constant region of a human IgG1 isotype and a light chain variable region fused to a constant region of a human kappa chain.

[0168]

[0182] Antibody variants

[0183] The anti-hGREM1 antibodies and antigen-binding fragments thereof provided herein also encompass various types of variants of the antibody sequences provided herein.

[0169]

[0184] In certain embodiments, the variants contain one or more modifications or substitutions in one, two, or three CDR sequences provided in Table 1, in one or more FR sequences, in the variable region sequences of the heavy or light chains provided herein, and / or in the constant region (e.g., Fc region). Such antibody variants retain the specific binding affinity of their parent antibody to hGREM1, but have one or more desirable properties imparted by the modifications or substitutions. For example, the antibody variants may have improved antigen binding affinity, improved glycosylation patterns, reduced risk of glycosylation, reduced deamination, reduced or improved effector functions, improved FcRn receptor binding, extended pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues), to name a few.

[0170]

[0185] The parent antibody sequence can be screened to identify suitable or preferred residues for modification or substitution using methods known in the art, such as "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are generated and screened for the desired properties. If substitution at a particular amino acid position manifests a desired functional change, that position can be identified as a potential residue for modification or substitution. Potential residues can be further evaluated by substituting with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.). i. Affinity variants

[0186] Affinity variants retain the specific binding affinity of parent antibody to hGREM1 or even have improved specific binding affinity to hGREM1 over parent antibody. Various methods known in the art can be used to achieve this goal. For example, a library of antibody variants (e.g., Fab variants or scFv variants) can be generated and expressed using phage display technology and then screened for binding affinity to human GREM1. For another example, computer software can be used to virtually simulate the binding of an antibody to human GREM1 and identify the amino acid residues on the antibody that form the binding interface. Such residues can be avoided in substitutions to prevent a decrease in binding affinity, or targeted for substitutions that result in stronger binding.

[0171]

[0187] In certain embodiments, at least one (or all) of the substitutions in the CDR sequence, FR sequence, or variable region sequence comprises a conservative substitution.

[0172]

[0188] In certain embodiments, the anti-hGREM1 antibodies or antigen-binding fragments provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variants comprise a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in one or more CDR sequences and / or FR sequences.

[0173]

[0189] In certain embodiments, anti-hGREM1 antibodies and antigen-binding fragments thereof comprise one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the CDR sequence(s) listed in Table 1, while retaining binding affinity to hGREM1 at a similar or even higher level than its parent antibody. ii. Glycosylation variants

[0190] The anti-hGREM1 antibodies and antigen-binding fragments provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.As used herein, the term "glycosylation" refers to the enzymatic process of attaching glycans, such as fucose, xylose, mannose, or GlcNAc phosphoserine glycans, to proteins, lipids, or other organic molecules.Depending on the carbon that is linked to the glycan, glycosylation can be divided into five classes, including N-linked glycosylation, O-linked glycosylation, phosphate glycosylation, C-linked glycosylation, and glypiation.

[0174]

[0191] Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence, such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine.

[0175]

[0192] In certain embodiments, antibody glycosylation variants may be obtained by removal of a native glycosylation site (e.g., by N297A substitution), such that, for example, the tripeptide sequence of the N-linked glycosylation site, or the serine or threonine residue of the O-linked glycosylation site, is no longer present in the antibody or in the Fc sequence. Alternatively, in certain embodiments, antibody glycosylation variants may be obtained by producing the antibody in a host cell line that is not capable of adding the selected sugar group to the mature core carbohydrate structure of the antibody. iii. Cysteine ​​engineered variants

[0193] The anti-hGREM1 antibodies and antigen-binding fragments provided herein also include cysteine ​​engineered variants, which contain one or more introduced free cysteine ​​amino acid residues.

[0176]

[0194] A free cysteine ​​residue is a cysteine ​​residue that is not part of a disulfide bridge.Cysteine ​​engineered variants are useful for, for example, conjugation with cytotoxic compounds and / or imaging compounds, labels, or radioisotopes, among others, at the site of engineered cysteine, for example, through maleimide or haloacetyl.Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0177]

[0195] antigen binding fragment

[0196] Also provided herein are anti-hGREM1 antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-hGREM1 antibodies provided herein, including, for example, the exemplary antibodies whose CDR sequences are shown in Table 1 and various variants thereof (e.g., affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0178]

[0197] In certain embodiments, the anti-hGREM1 antigen-binding fragment provided herein 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, or bivalent domain antibody.

[0179]

[0198] A variety of techniques can be used to generate such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression by host cells such as E. coli (for example, for Fab, Fv, and ScFv antibody fragments), screening from a phage display library as discussed above (for example, for ScFv), and chemical coupling of two Fab'-SH fragments to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for generating antibody fragments will be apparent to those skilled in the art.

[0180]

[0199] In certain embodiments, the antigen-binding fragment is scFv. The generation of scFv is described, for example, in WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. scFv can be fused to effector protein at either amino or carboxyl terminus to produce fusion protein (see, for example, Antibody Engineering, edited by Borrebaeck).

[0181]

[0200] In certain embodiments, the anti-hGREM1 antibody and its antigen-binding fragments provided herein are bivalent, tetravalent, hexavalent, or multivalent.The term "valency" as used herein refers to the presence of a specified number of antigen-binding sites in a given molecule.Thus, the terms "bivalent", "tetravalent" and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule.Any molecule that is more than bivalent is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0182]

[0201] A bivalent molecule can be monospecific when both binding sites are specific for binding to the same antigen or epitope. This allows for stronger binding to antigen or epitope than its monovalent counterpart in certain embodiments. Similarly, a multivalent molecule can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have different sequences, despite having the same specificity).

[0183]

[0202] Bivalent can also be bispecific, when two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific, when two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0184]

[0203] Epitope

[0204] In another aspect, the disclosure provides an antibody that binds to the same epitope as the antibody or antigen-binding fragment thereof provided herein. In another aspect, the disclosure provides an antibody that competes with the antibody or antigen-binding fragment thereof provided herein for binding to hGREM1.

[0185]

[0205] The term "epitope" as used herein refers to a specific group of atoms or amino acids of an antigen to which an antibody binds. An epitope can include specific amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups that directly contact the antibody. Those skilled in the art will understand that it is possible to determine, without undue experimentation, whether an antibody binds to the same epitope as an antibody of the present disclosure (e.g., any of the hybridoma / chimeric or humanized antibodies 14E3, 69H5, 22F1, 56C11, 36F5, 42B9, and 67G11 provided herein, and their chimeric and humanized variants) by determining whether the two antibodies compete for binding to a GREM1 antigen polypeptide.

[0186]

[0206] The term "compete for binding" as used herein with respect to two antigen-binding proteins (e.g., antibodies) means that one antigen-binding protein blocks or reduces the binding of the other antigen-binding protein to an antigen (e.g., human / mouse GREM1) as determined by competitive binding assays. Competitive binding assays are well known in the art and include, for example, direct or indirect radioimmunoassays (RIA), direct or indirect enzyme immunoassays (EIA), Fortebio, competitive ELISA assays, and sandwich competition assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253). Typically, such assays involve the use of purified antigen or antigen-bearing cells bound to a solid surface, an unlabeled test antibody, and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Typically, the test antibody is present in excess. When two antibodies compete for binding to hGREM1, the two antibodies bind to the same or overlapping epitopes, or to adjacent epitopes close enough to sterically interfere with the epitope bound by the other antibody. Typically, a competing antibody, when present in excess, will inhibit (e.g., reduce) specific binding of a test antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90% or more.

[0187]

[0207] Groups of antibodies classified by biological properties

[0208] The antibodies provided herein have certain unique biological properties. Thus, antibodies sharing certain unique biological properties can be classified into multiple groups.

[0188]

[0209] i) Antibodies with reduced cancer cell selectivity of hGREM1-mediated inhibition of BMP signaling

[0210] In certain embodiments, the antibody provided herein can selectively reduce hGREM1-mediated inhibition of BMP signal transduction in cancer cells compared to non-cancer cells, and comprises the heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and the light chain CDR1 (LCDR1), LCDR2 and LCDR3 of an antibody selected from the group consisting of 14E3, 22F1, 56C11, 69H5, 42B9, 36F5 and 67G11.These antibodies are particularly useful in the method of treating cancer.

[0189]

[0211] The present disclosure has unexpectedly found that neutralization of GREM1 using certain anti-GREM1 antibodies provided herein selectively inhibits GREM1-mediated inhibition of BMP signaling in cancer cells, but shows no or only very limited inhibition in non-cancer cells.

[0190]

[0212] Eliminating GREM1 and / or reducing the activity of GREM1 in cancer cells is preferable because it has an inhibitory effect on cancer cell proliferation and sphere formation and also induces apoptosis of cancer cells. However, traditional knockout of GREM1 in mice causes abnormal intestinal development and impaired hematopoietic system, so inhibition of GREM1 may be generally undesirable (Rowan, SC et al. Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure. J Pathol 251, 117-122). This suggests that traditional elimination of GREM1 and / or reducing its activity will inevitably cause harmful effects on other normal tissues. In this regard, the unexpected cancer-specific regulation of GREM1-mediated inhibition of BMP signaling by anti-GREM1 antibodies provided herein is beneficial by avoiding unwanted side effects on normal tissues.

[0191]

[0213] In certain embodiments, the anti-GREM1 antibodies provided herein exhibit a reduction in GREM1-mediated inhibition of BMP signaling in non-cancer cells of less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%.

[0192]

[0214] As used herein, the phrase "BMP signaling" refers to the signaling of one or more BMP ligands that can be inhibited by GREM1. In certain embodiments, the BMP signaling is BMP-2 signaling and / or BMP-4 signaling.

[0193]

[0215] As used herein, the phrase "non-cancerous cells" refers to cells that are not cancer cells. Non-cancerous cells may be cell lines, primary cells isolated from a subject.

[0194]

[0216] GREM1-mediated inhibition of BMP signaling can be determined by measuring the BMP signaling of BMP ligand in the presence and absence of GREM1, respectively, where the difference indicates GREM1-mediated inhibition. However, anti-GREM1 antibody can reduce GREM1-mediated inhibition of BMP signaling, or in other words, can restore BMP signaling. The reduction or restoration of GREM1-mediated inhibition of BMP signaling can be calculated as the increase of BMP signaling in the presence of anti-GREM1 antibody compared to BMP signaling in the absence of anti-GREM1 antibody. The percentage of such reduction or such restoration can be calculated as the ratio of the reduction of GREM1-mediated inhibition to the total GREM1-mediated inhibition. A 100% reduction of GREM1-mediated inhibition of BMP signaling would mean that BMP signaling is restored to substantially the same level as in the absence of GREM1, and a 0% reduction would mean that BMP signaling is not restored.

[0195]

[0217] ii) Antibodies that bind to chimeric hGREM1 (i.e., XM5)

[0218] In certain embodiments, certain anti-GREM1 antibodies provided herein bind to GREM1 at an epitope outside the BMP binding loop. In certain embodiments, the BMP binding loop comprises the amino acid sequence of SEQ ID NO:63.

[0196]

[0219] In certain embodiments, the anti-GREM1 antibodies provided herein can bind to chimeric GREM1 (also referred to herein as "XM5") comprising the amino acid sequence of SEQ ID NO: 68. Chimeric GREM1 XM5 comprises a mutant version of hGREM1 in which the BMP binding loop (i.e., amino acid residues 123-143 of hGREM1 (NSFYIPRHIRKEEGSFQSCSF, SEQ ID NO: 63)) is replaced by amino acids 63-83 of DNA (FSYSVPNTFPQSTESLVHCDS, SEQ ID NO: 64), which does not bind to BMP. Thus, this chimeric hGREM1 does not bind to BMP. Certain existing anti-GREM1 antibodies cannot bind to this chimeric hGREM1, suggesting that their binding to hGREM1 requires the BMP binding loop in hGREM1. In contrast, the anti-GREM1 antibodies provided herein are able to bind to this chimeric hGREM1, indicating that the anti-GREM1 antibodies bind to hGREM1 at an epitope outside of this BMP binding loop.

[0197]

[0220] In certain embodiments, the antibodies provided herein are capable of binding to chimeric hGREM1 comprising the amino acid sequence of SEQ ID NO:68, and include heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and light chain CDR1 (LCDR1), LCDR2, and LCDR3 of an antibody selected from the group consisting of 14E3, 42B9, 67G11, 36F5, 56C11, 22F1 and 69H5.

[0198]

[0221] The benchmark antibody 6245P is unable to bind to X5.

[0199]

[0222] iii) Antibodies that are cross-reactive or not cross-reactive with mouse GREM1

[0223] In certain embodiments, some of the anti-GREM1 antibodies provided herein are capable of binding to hGREM1, but are unable to specifically bind to mouse GREM1.

[0200]

[0224] hGREM1 and mouse GREM1 share 98% sequence identity, with the differing amino acid residues found only in the N-terminal portion of hGREM1, including Gln27 and Asn33 of hGREM1, where the residue numbering is according to SEQ ID NO: 69. Thus, for an anti-GREM1 antibody that does not cross-react with mouse GREM1, it is expected that the antibody may bind to hGREM1 at an epitope that includes Gln27 and / or Asn33 of hGREM1, where the residue numbering is according to SEQ ID NO: 69, or that the antibody may bind to a hGREM1 fragment that includes residues Gln27 and / or Asn33, optionally with the hGREM1 fragment having a length of at least 3 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acid residues. The epitope may be a conformational or linear epitope.

[0201]

[0225] In certain embodiments, the antibodies provided herein are capable of binding to hGREM1 but cannot specifically bind to mouse Gremlin 1, and comprise the heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and the light chain CDR1 (LCDR1), LCDR2 and LCDR3 of an antibody selected from the group consisting of 69H5, 22F1, and 14E3.

[0202]

[0226] In certain embodiments, the anti-hGREM1 antibody provided herein is cross-reactive to mouse GREM1 and comprises the heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and the light chain CDR1 (LCDR1), LCDR2, and LCDR3 of an antibody selected from the group consisting of 56C11, 42B9, 36F5, and 67G11.

[0203]

[0227] The benchmark antibody 6245P is cross-reactive to mouse GREM1.

[0204]

[0228] iv) Antibodies that block the binding of hGREM1 to BMP7

[0229] In certain embodiments, the antibodies provided herein are capable of blocking the binding of hGREM1 to BMP7 with a maximal blocking percentage of at least 50% as measured by ELISA.

[0205]

[0230] As used herein, the term "blocking percentage" refers to the percentage of reduced interaction between two proteins (e.g., reduced binding of human Gremlin 1 to BMP7) in the presence of a blocking agent (e.g., an anti-Gremlin 1 antibody) compared to the interaction between the two proteins in the absence of the blocking agent.

[0206]

[0231] As used herein, the term "maximum blocking percentage" refers to the highest blocking percentage (i.e., the plateau of blocking percentage) that can be achieved by a blocking agent (e.g., anti-gremlin 1 antibody) to block the interaction between two proteins (e.g., human gremlin 1 and BMP7). Generally, the percentage of blocking increases with increasing concentration of the blocking agent (e.g., anti-gremlin 1 antibody), but may reach a plateau where no further blocking can be achieved despite further increasing concentration of the blocking agent (e.g., anti-gremlin 1 antibody). The higher the maximum blocking percentage, the more effective the blocking. The maximum blocking percentage may vary to some extent depending on various assays, such as competitive ELISA assay and competitive FACS assay.

[0207]

[0232] In certain embodiments, certain anti-gremlin 1 antibodies provided herein have a maximum blocking percentage of at least 50% for hGREM1-BMP7 interaction as measured by competitive ELISA assay. The assay conditions can be similar to those provided in Example 6 of the present disclosure (human gremlin 1 concentration is 1 μg / ml, and BMP7 concentration is 0.5 μg / ml). Exemplary anti-gremlin 1 antibodies having the above-mentioned blocking activity for hGREM1-BMP7 interaction include 14E3 (e.g., 14E3HaLa), 42B9, 36F5 and 67G11.

[0208]

[0233] In certain embodiments, certain anti-gremlin 1 antibodies provided herein have a maximum blocking percentage of at least 60%, at least 70%, or at least 75% for hGREM1-BMP7 interaction as measured by competitive ELISA assay. The assay conditions can be similar to those provided in Example 6 of the present disclosure (human gremlin 1 concentration is 1 μg / ml, and BMP7 concentration is 0.5 μg / ml). Exemplary anti-gremlin 1 antibodies having the above-mentioned blocking activity for hGREM1-BMP7 interaction include 42B9, 36F5, and 67G11.

[0209]

[0234] BMP-7 is a homodimeric protein with a cysteine ​​knot that is selectively expressed in several adult organs, including the kidney (Rui et al., Role of bone morphogenetic protein-7 in renal fibrosis, Front. Physiol., April 23, 2015).BMP-7 expression in normal kidney is highest in adult organs and is downregulated in kidneys of patients with ischemia-reperfusion injury, diabetic nephropathy, and hypertensive nephrosclerosis (Dudley et al., A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev. 9, pp. 2795-2807, 1995; Luo et al., BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev. 9, pp. 2808-2820, 1995; Simon et al., Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney. Am. J. Physiol. 276, pp. F382-F389, 1999; Wang et al., Loss of tubular bone morphogenetic protein-7 in diabetic kidney. nephropathy.J.Am.Soc.Nephrol.12, pp. 2392-2399, 2001;Bramlage et al., Bone morphogenetic protein(BMP)-7 expression is decreased in human hypertensive nephrosclerosis.BMC Nephrol.11:31., 2010;Vukicevic et al., Osteogenic protein-1(bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat.J.Clin.Invest.102, pp.202-214., 1998;Simon et al., Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney.Am.J.Physiol.276, pp.F382-F389., 1999).

[0210]

[0235] As one of the key cytokines in the TGFβ superfamily, BMP7 plays an anti-fibrotic role in chronic kidney disease by balancing the TGF-beta signaling pathway, which mediates renal fibrogenesis by increasing the production of extracellular matrix (ECM) and decreasing its degradation (Rui et al., Role of bone morphogenetic protein-7 in renal fibrosis, Front. Physiol., April 23, 2015). BMP7 treatment has been reported to reverse renal fibrosis and restore renal function in several animal models of renal injury (Hruska et al., Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction. Am. J. Physiol. Renal Physiol. 279, F130-F143. (2000); Jeremiah et al., Bone morphogenetic protein-7 improves renal fibrosis and accelerates the return of renal function, J Am Soc Nephrol. January 2002;).

[0211]

[0236] However, the activity of BMP7 in the kidney is determined not only by the availability of BMP7 itself, but also by the balance of agonists and antagonists (e.g., Gremlin). When BMP7 is used to treat renal fibrosis and other kidney diseases (e.g., acute and chronic kidney injury), the presence of BMP7 antagonists (e.g., Gremlin) needs to be considered in terms of its treatment efficacy (Michael et al., Reversal of experimental renal fibrosis by BMP7 provides insights into novel therapeutic strategies for chronic kidney disease, Pediatr Nephrol. 2008 September;23(9):1395-8).

[0212]

[0237] The present disclosure shows that Gremlin 1 binds to BMP7, and the anti-Gremlin 1 antibodies provided herein (e.g., 42B9, 36F5, 67G11 and 14E3HaLa) have a stronger blocking activity of Gremlin 1 binding to BMP7 compared to benchmark antibodies. As used herein, the term "benchmark antibody" refers to any existing anti-GREM1 antibody, such as 6245P, which is made according to the sequence of H4H6245P disclosed in WO2014159010, the entire disclosure of which is incorporated by reference. In other words, the anti-Gremlin 1 antibodies provided herein (e.g., 42B9, 36F5, 67G11 and 14E3HaLa) can restore BMP7 activity in the function of BMP7-expressing organs (e.g., kidneys). Thus, it can be reasonably expected that the anti-Gremlin 1 antibodies provided herein (e.g., 42B9, 36F5, 67G11 and 14E3HaLa) can improve the therapeutic efficacy of treatments for fibrotic and renal diseases (e.g., renal fibrosis).

[0213]

[0238] In certain embodiments, the antibodies provided herein can block hGREM1 binding to BMP7 with a maximum blocking percentage of at least 50% as measured by ELISA, and include the heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, and the light chain CDR1 (LCDR1), LCDR2, and LCDR3 of an antibody selected from the group consisting of 42B9, 36F5, and 67G11. In certain embodiments, the antibodies provided herein can block hGREM1 binding to BMP7 with a maximum blocking percentage of 30% to 50% as measured by ELISA, and include the heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, and the light chain CDR1 (LCDR1), LCDR2, and LCDR3 of the antibody 14E3.

[0214]

[0239] v) Antibodies that block FGFR binding to GREM1

[0240] In certain embodiments, the antibodies provided herein can block the binding of GREM1 (e.g., hGREM1 or mGREM1) and FGFR (e.g., FGFR1, preferably human FGFR1 (hFGFR1) or mouse FGFR1 (mFGFR1)) blocking interaction, and comprise the heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and the light chain CDR1 (LCDR1), LCDR2 and LCDR3 of an antibody selected from the group consisting of 14E3, 42B9, 67G11 and 36F5. These antibodies are particularly useful in methods of treating conditions or diseases associated with GREM1 binding to FGFR or FGFR activity.

[0215]

[0241] In certain embodiments, the antibodies provided herein do not block binding of the blocking interaction between GREM1 (e.g., hGREM1 or mGREM1) and FGFR (e.g., FGFR1, preferably human FGFR1 (hFGFR1) or mouse FGFR1 (mFGFR1)), and comprise the heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and the light chain CDR1 (LCDR1), LCDR2, and LCDR3 of an antibody selected from the group consisting of 56C11.

[0216]

[0242] In certain embodiments, the antibodies provided herein partially block binding of the blocking interaction between GREM1 (e.g., hGREM1 or mGREM1) and FGFR (e.g., FGFR1, preferably human FGFR1 (hFGFR1) or mouse FGFR1 (mFGFR1)) (with an IC50 of at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, or at least 7 nM) and comprise the heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, and the light chain CDR1 (LCDR1), LCDR2, and LCDR3 of antibody 22F1 or 69H5.

[0217]

[0243] vi) Antibodies that reduce GREM1-mediated activation of MAPK signaling

[0244] In certain embodiments, the anti-GREM1 antibody provided herein can reduce GREM1-mediated activation of MAPK signaling. It is well known in the art that MAPK signaling is a crucial signal pathway for maintaining tumor cell proliferation, migration, angiogenesis and epithelial-mesenchymal transition (EMT). It is known in the art that MAPK signaling can be activated either through epidermal growth factor (EGF) receptor by its ligand called EGF, or through fibroblast growth factor (FGF) receptor by its ligand called FGF. The present disclosure surprisingly found that GREM1 appears to play a role in activating MAPK signaling, possibly functioning as a novel ligand for FGFR. The present disclosure further found that the anti-GREM1 antibody provided herein can reduce GREM1-mediated activation of MAPK signaling, and can, in particular, block the interaction between GREM1 and FGFR.

[0218]

[0245] vii) Antibody that binds to both hGREM1 and DAN

[0246] The antibody provided in the present disclosure can specifically bind to one or more (e.g., 1, 2, 3 or more) DAN family members, including GREM1. In certain embodiments, the antibody provided herein can bind to both hGREM1 and DAN. As used herein, the term "DAN" refers to the founding member of the DAN family (also called NbI1 and DAND1), which is a moderate antagonist for modulating BMP signaling. DAN initially acted as a tumor suppressor gene in neuroblastoma. Misregulation of the balance between BMP signaling and DAN inhibition can lead to many disease conditions, including cancer, nephropathy and pulmonary arterial hypertension. Gremlin acts as a strong antagonist, while DAN functions as a moderate antagonist. Both of them can antagonize BMP2, BMP4 and BMP7, but only share approximately 20% identity.

[0219]

[0247] In certain embodiments, the anti-hGREM1 antibodies provided herein can bind to both hGREM1 and DAN and comprise the heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and the light chain CDR1 (LCDR1), LCDR2 and LCDR3 of an antibody selected from the group consisting of 36F5, 42B9 and 67G11. These antibodies are particularly useful in methods of treating conditions or diseases associated with both GREM1 and DAN.

[0220]

[0248] In certain embodiments, the anti-hGREM1 antibodies provided herein are capable of binding to hGREM1 but are unable to bind to DAN and comprise the heavy chain CDR1 (HCDR1), HCDR2 and HCDR3, and the light chain CDR1 (LCDR1), LCDR2, and LCDR3 of antibodies 14E3, 22F1, 56C11, or 69H5.

[0221]

[0249] bispecific antibody

[0250] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein are bispecific. The term "bispecific" as used herein encompasses molecules with three or more specificities, and molecules with three or more specificities, i.e., multispecific. In certain embodiments, the bispecific antibodies and antigen-binding fragments 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 different from each other or do not overlap. In certain embodiments, the bispecific antibodies and antigen-binding fragments thereof can simultaneously bind to both the first epitope and the second epitope. In certain embodiments, the second antigen is different from hGREM1.

[0222]

[0251] In certain embodiments, the second antigen is an immune-related target.As used herein, immune-related target includes the biological molecule that is involved in immune response, optionally in the generation, inhibition or modulation of cellular immune response.Examples of immune-related target include immune checkpoint molecules.

[0223]

[0252] Immune checkpoint molecules can mediate costimulatory signals to amplify immune responses, or mediate costenulatory signals to suppress immune responses. Examples of immune checkpoint molecules include, for example, PD-L1, PD-L2, PD-1, CLTA-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 second antigen comprises PD-1, PD-L1, CTLA-4, or LAG-3.

[0224]

[0253] In certain embodiments, the second antigen comprises a tumor antigen. As used herein, "tumor antigen" refers to tumor-specific antigens (e.g., antigens that are unique to tumor cells and are not commonly found in non-tumor cells) and tumor-associated antigens (e.g., found in both tumor cells and non-tumor cells, but differentially expressed in tumor cells or found in the tumor microenvironment). Tumor-specific antigens can also comprise tumor neo-antigens (e.g., expressed in cancer cells because somatic mutations change protein sequence or create fusion proteins between two unrelated sequences).

[0225]

[0254] Examples of tumor antigens include, but are not limited to, prostate specific antigen (PSA), CA-125, gangliosides 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., VEGFR3), estrogen receptor agonists, and the like. Examples of such proteins include IL-1, IL-2R, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-2A, IL-2B, IL-2C, IL-2D, IL-2E, IL-2F, IL-2H, IL-2H-IL-3, IL-2F, IL-2H-IL-4, IL-2F, IL-2H-IL-5, IL-2F, IL-2H-IL-6, IL-2F, IL-2H-IL-7, IL-2F, IL-2H-IL-8, IL-2F, IL-2H-IL-9 ...

[0226]

[0255] In certain embodiments, 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., VEGFR3), estrogen receptor. receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2, GPC-3, nectin-4, ROR1, methemoglobin, 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.

[0227]

[0256] The bispecific antibodies and antigen-binding fragments thereof provided herein may be in any suitable format known in the art. For example, exemplary bispecific formats include bispecific diabodies, scFv-based bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), BiTEs, CrossMab, CrossFab, Duobody, SEEDbody, leucine zipper, dual acting Fab (DAF)-IgG, and Mab. 2 It may be in a bispecific format (see, for example, Brinkmann et al. 2017, Mabs, 9(2):182-212). Bispecific molecules may be of symmetric or asymmetric structure.

[0228]

[0257] The bispecific antibodies and antigen-binding fragments provided herein can be made using any suitable method known in the art.

[0229]

[0258] In one embodiment, two immunoglobulin heavy chain-light chain pairs with different antigen specificities are co-expressed in a host cell, resulting in the recombinant production of a bispecific antibody (see, e.g., Milstein and Cuello, Nature, 305:537 (1983)), which is subsequently purified by affinity chromatography.

[0230]

[0259] In another embodiment, sequences encoding antibody heavy chain variable domains for the two specificities are each fused to an immunoglobulin constant domain sequence and subsequently inserted into one or more expression vectors which are co-transfected with an expression vector for the light chain sequences into a host cell suitable for recombinant expression of the bispecific antibody (see, e.g., WO 94 / 04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be constructed and expressed recombinantly from a host cell (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)).

[0231]

[0260] In another method, the leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. The linked antibodies are reduced at the hinge region into four half antibodies (i.e., monomers) and then reoxidized to form heterodimers (Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)).

[0232]

[0261] Two antigen-binding domains can also be conjugated or cross-linked to form bispecific antibodies or antigen-binding fragments. For example, one antibody can be coupled with biotin, while the other antibody can be coupled with avidin, and the strong association between biotin and avidin will combine the two antibodies together to form a bispecific antibody (see, for example, U.S. Pat. No. 4,676,980, WO91 / 00360, WO92 / 00373, and EP03089). In another example, two antibodies or antigen-binding fragments can be cross-linked by conventional methods known in the art, for example, as disclosed in U.S. Pat. No. 4,676,980.

[0233]

[0262] Bispecific antigen-binding fragments can be produced from bispecific antibodies, for example, by proteolytic cleavage or by chemical cross-linking. For example, an antigen-binding fragment of an antibody (e.g., Fab') can be prepared and converted to a Fab'-thiol derivative and then mixed and reacted with another converted Fab' derivative having a different antigen specificity to form a bispecific antigen-binding fragment (see, for example, Brennan et al., Science, 229:81 (1985)).

[0234]

[0263] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein can be engineered at the interface such that knob-into-hole associations can form to promote heterodimerization of two distinct antigen-binding sites. Such can maximize the percentage of heterodimers recovered from recombinant cell culture. As used herein, "knobs-into-holes" refers to the interaction between two polypeptides (e.g., Fc) where one polypeptide has a protrusion (i.e., "knobs") due to the presence of an amino acid residue with a bulky side chain (e.g., tyrosine or tryptophan) and the other polypeptide has a recess (i.e., "hole") with a small side chain amino acid residue (e.g., alanine or threonine), but the protrusion can be positioned in the recess to promote the interaction of the two polypeptides, thereby forming a heterodimer or complex. Methods for generating polypeptides with knobs-into-holes are known in the art, for example, as described in U.S. Pat. No. 5,731,168.

[0235]

[0264] Conjugates

[0265] In some embodiments, the anti-hGREM1 antibody and its antigen-binding fragment are linked to one or more conjugate moieties. A conjugate is a moiety that can be attached to an antibody or its antigen-binding fragment. It is contemplated that various conjugates can be linked to the antibody or antigen-binding fragment provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, 1989). These conjugates can be linked to the antibody or antigen-binding fragment by covalent bonding, affinity bonding, intercalation, coordinate bonding, complex formation, association, blending, or addition, among other methods. In certain embodiments, the antibody or its antigen-binding fragment is linked to one or more conjugates via a linker. In certain embodiments, the linker is a hydrazone linker, a disulfide linker, a bifunctional linker, a dipeptide linker, a glucuronide linker, a thioether linker.

[0236]

[0266] In certain embodiments, the anti-hGREM1 antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites outside the epitope-binding moiety that can be utilized for binding to one or more conjugates. For example, such sites can include one or more reactive amino acid residues, such as, for example, cysteine ​​or histidine residues, to facilitate covalent linkage to a conjugate.

[0237]

[0267] The conjugate may be a clearance modifier, a therapeutic agent (e.g., a chemotherapeutic agent), a toxin, a radioisotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, or an enzyme substrate label), a pharmacokinetic-modifying moiety, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent, or other anti-cancer drug, such as an androgen receptor inhibitor.

[0238]

[0268] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, sugar oxidase, or β-D-galactosidase), radioisotopes, other lanthanides, luminescent labels, chromophore moieties, digoxigenin, biotin / avidin, detectable DNA molecules, or gold.

[0239]

[0269] Examples of radioisotopes include: 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 Examples include P. Radioisotope-labeled antibodies are useful in receptor-targeted imaging experiments.

[0240]

[0270] In certain embodiments, the conjugate may be a pharmacokinetic-modifying moiety, such as PEG, which serves to extend the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like.

[0241]

[0271] In certain embodiments, the conjugate can be a purification moiety, such as a magnetic bead or nanoparticle.

[0242]

[0272] Pharmaceutical Compositions

[0273] The present disclosure provides pharmaceutical compositions comprising an anti-hGREM1 antibody or antigen-binding fragment thereof and one or more pharma- ceutically acceptable carriers.

[0243]

[0274] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary agents, other ingredients known in the art, or various combinations thereof.

[0244]

[0275] Suitable ingredients can include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorings, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants can include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments and conjugates provided herein reduces the oxidation of the antibodies or antigen-binding fragments. Such reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided that include one or more antibodies or antigen-binding fragments disclosed herein and one or more antioxidants, such as methionine. Additionally, methods are provided to prevent oxidation of the antibodies or antigen-binding fragments provided herein, extending their shelf life, and / or improving their effectiveness by combining the antibodies or antigen-binding fragments provided herein with one or more antioxidants, such as methionine.

[0245]

[0276] To further illustrate, pharma- ceutically acceptable carriers include, for example, aqueous vehicles such as Sodium Chloride Injection, Ringer's Solution, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics. The carrier may include drugs such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN®-80), sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multi-dose containers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0246]

[0277] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained release formulations, or powders. Oral formulations can include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharine, cellulose, magnesium carbonate, and the like.

[0247]

[0278] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition.The injectable pharmaceutical composition can be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid form suitable for producing liquid solution, suspension or emulsion.The preparation for injection can include sterile and / or non-pyrogenic solution ready for injection, sterile dry soluble, such as lyophilized powder, ready to be combined with solvent immediately before use, including subcutaneous tablets, sterile suspension ready for injection, sterile dry insoluble, ready to be combined with vehicle immediately before use, and sterile and / or non-pyrogenic emulsion.The solution can be either aqueous or non-aqueous.

[0248]

[0279] In certain embodiments, unit dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. As is known and practiced in the art, all preparations for parenteral administration are required to be sterile and non-pyrogenic.

[0249]

[0280] In certain embodiments, a sterile, freeze-dried powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients to improve stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffer known to those skilled in the art, in one embodiment, at about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-hGREM1 antibody or antigen-binding fragment thereof or composition thereof. Overfilling the vial by a small amount (e.g., about 10%) beyond that needed for a dose or series of doses is permissible to facilitate accurate sampling and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0250]

[0281] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution, sterile water and / or non-pyrogenic water, or other liquid suitable carrier, is added to the lyophilized powder. The exact amount depends on the selected therapy being given, but may be empirically determined.

[0251]

[0282] In certain embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0252]

[0283] In certain embodiments, the second therapeutic agent can be an agent for treating cancer, such as a chemotherapeutic agent, an anti-cancer drug, radiation therapy, immunotherapy, an anti-angiogenic agent (e.g., an antagonist of VEGFR such as VEGFR-1, VEGFR-2, and VEGFR-3), targeted therapy, cell therapy, gene therapy agent, hormonal therapy agent, cytokine, palliative care, surgery for the treatment of cancer (e.g., tumor removal), or one or more antiemetic agents or other treatments for complications resulting from chemotherapy.

[0253]

[0284] In certain embodiments, the second therapeutic agent comprises an anti-angiogenic agent, such as a VEGFR or VEGF antagonist. In certain embodiments, the second therapeutic agent comprises an anti-VEGFR antibody or an anti-VEGF antibody. In certain embodiments, the second therapeutic agent comprises an anti-VEGFR-2 antibody.

[0254]

[0285] In certain embodiments, the second therapeutic agent can be an agent for treating a fibrotic disease.

[0255]

[0286] In certain embodiments, the second therapeutic agent addresses or treats at least one complication associated with fibrosis or cancer.

[0256]

[0287] Polynucleotides and Recombinant Methods

[0288] The present disclosure provides isolated polynucleotides encoding anti-hGREM1 antibodies and their antigen-binding fragments.As used herein, each term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers, either in single-stranded or double-stranded form.Unless otherwise specified, a specific polynucleotide sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be made by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell Probes 8:91-98 (1994)).

[0257]

[0289] In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences set forth in SEQ ID NOs: 9, 10, 19, 20, 29, 30, 39, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, and 142-147, and / or a sequence having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto, and / or a variant thereof having only degenerate substitutions, wherein the isolated polynucleotide encodes the variable region of an exemplary antibody provided herein.

[0258]

[0290] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0259]

[0291] The present disclosure provides a vector (e.g., an expression vector) comprising the isolated polynucleotide provided herein. In certain embodiments, the expression vector provided herein comprises a polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the polynucleotide sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET , pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, p LexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

[0260]

[0292] A vector containing a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in the vectors herein are the above-mentioned prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive bacteria, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0261]

[0293] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for vectors encoding anti-hGREM1 antibodies. Saccharomyces cerevisiae (or common baker's yeast) is the most commonly used of the lower eukaryotic host microorganisms. However, several other genera, species, and strains are commonly available and useful herein, e.g., Schizosaccharomyces pombe; Kluyveromyces hosts, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. wartsii (ATCC 56,500), K. drosophila, K. spp. (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia spp. (EP 402,226); Pichia pastoris (EP 183,070); Candida spp.; Trichoderma lesia (EP 244,234); Neurospora crassa; Schwanniomyces spp., such as Schwanniomyces oxidentalis; and filamentous fungi, such as Neurospora spp., Penicillium spp., Tolypocladium spp., and Aspergillus hosts, such as A. nidulans and A. niger.

[0262]

[0294] Suitable host cells for the expression of glycosylated antibodies or antigen fragments provided herein are derived from multicellular organisms, such as invertebrate cells, e.g., plant cells and insect cells. Numerous baculovirus strains and variants from hosts such as Fall Armyworm (caterpillar), Aedes aegypti (mosquito), Aedes japonica (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified, and corresponding insect permissive host cells. Various virus strains for transfection, such as the L-1 variant of Pseudomonas nigroma NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used as viruses herein according to the present invention, particularly for the transfection of Fall Armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

[0263]

[0295] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the host cells are mammalian cultured cell lines, such as CHO, BHK, NS0, 293, and derivatives thereof.

[0264]

[0296] Host cells are transformed with the above-described expression or cloning vectors for producing anti-hGREM1 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In another embodiment, antibodies may be produced by homologous recombination, as known in the art.

[0265]

[0297] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Pat. No. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium chloride, magnesium chloride, and phosphates), such as (HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source, as appropriate. Any other necessary supplements may also be included at appropriate concentrations that would be known to one of skill in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression and will be apparent to one of skill in the art.

[0266]

[0298] When using recombinant techniques, antibodies may be produced intracellularly, in the periplasmic space, or may be secreted directly into the medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris may be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is usually first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0267]

[0299] Anti-hGREM1 antibodies and antigen-binding fragments thereof prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0268]

[0300] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and antigen-binding fragments thereof. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond is often used. ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0269]

[0301] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, preferably performed at a low salt concentration (e.g., about 0-0.25 M salt) and using an elution buffer at a pH between about 2.5-4.5.

[0270]

[0302] How to use

[0303] In one aspect, the disclosure provides therapeutic uses of the antibodies provided herein.

[0271]

[0304] In certain embodiments, the present disclosure provides a method for treating or preventing a GREM1-related disease or condition in a subject in need of such treatment or prevention, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein and / or a pharmaceutical composition provided herein, thereby treating or preventing the GREM1-related disease or condition.

[0272]

[0305] In another aspect, the disclosure provides a method of treating a GREM1-associated disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an anti-human GREM1 antibody or antigen-binding fragment thereof, wherein the anti-human GREM1 antibody or antigen-binding fragment thereof: a) capable of binding to hGREM1 at an epitope comprising residue Gln27 and / or residue Asn33, where the residue numbers are according to SEQ ID NO: 69; and / or b) capable of binding to a hGREM1 fragment that includes 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; and / or c) reducing hGREM1-mediated inhibition of BMP signaling selectively in cancer cells relative to non-cancer cells; and / or d) non-cancer cells exhibit less than 50% reduction in hGREM1-mediated inhibition of BMP signaling; and / or e) capable of binding to a chimeric hGREM1 comprising the amino acid sequence of SEQ ID NO: 68; and / or g) K<1nM as measured by Fortebio D and / or capable of binding to hGREM1 at h) capable of blocking the binding of hGREM1 to BMP7 with a maximum blocking percentage of greater than 50% as measured by ELISA; and / or i) It is possible to block the interaction between GREM1 (eg, hFGFR1 or mGREM1) and FGFR (eg, FGFR1, preferably human FGFR1 (hFGFR1) or mouse FGFR1 (mFGFR1)).

[0273]

[0306] In another aspect, the disclosure provides a method of inhibiting FGFR1 activity in a subject in need of inhibition of FGFR1 activity, or a method of treating a disease or condition associated with GREM1-mediated FGFR1 activation, comprising administering to the subject a therapeutically effective amount of an anti-human GREM1 antibody, or antigen-binding fragment thereof, wherein the anti-human GREM1 antibody, or antigen-binding fragment thereof, comprises: 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; 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) 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; 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) 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; 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; d) HCDR1 comprising the sequence of SEQ ID NO: 31, HCDR2 comprising the sequence of SEQ ID NO: 32, HCDR3 comprising the sequence of SEQ ID NO: 33; 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; e) HCDR1 comprising the sequence of SEQ ID NO: 114, HCDR2 comprising the sequence of SEQ ID NO: 115, and HCDR3 comprising the sequence of SEQ ID NO: 116; LCDR1 comprising the sequence of SEQ ID NO: 117, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 118; f) HCDR1 comprising the sequence of SEQ ID NO: 119, HCDR2 comprising the sequence of SEQ ID NO: 115, and HCDR3 comprising the sequence of SEQ ID NO: 120; LCDR1 comprising the sequence of SEQ ID NO: 121, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 118; or g) HCDR1 comprising the sequence of SEQ ID NO: 119, HCDR2 comprising the sequence of SEQ ID NO: 115, and HCDR3 comprising the sequence of SEQ ID NO: 120; LCDR1 comprising the sequence of SEQ ID NO: 122, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 118.

[0274]

[0307] In another aspect, the disclosure provides a method of inhibiting FGFR1 activity in a subject in need of inhibition of FGFR1 activity, or a method of treating a disease or condition associated with GREM1-mediated FGFR1 activation, comprising administering to the subject a therapeutically effective amount of an anti-human GREM1 antibody, or antigen-binding fragment thereof, wherein the anti-human GREM1 antibody, or antigen-binding fragment thereof, comprises: a) HCDR1 comprising the sequence of SEQ ID NO: 123, HCDR2 comprising the sequence of SEQ ID NO: 115, HCDR3 comprising the sequence of SEQ ID NO: 124; LCDR1 comprising the sequence of SEQ ID NO: 125, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 118; b) HCDR1 comprising the sequence of SEQ ID NO: 114, HCDR2 comprising the sequence of SEQ ID NO: 115, HCDR3 comprising the sequence of SEQ ID NO: 116; LCDR1 comprising the sequence of SEQ ID NO: 117, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 118; c) HCDR1 comprising the sequence of SEQ ID NO: 119, HCDR2 comprising the sequence of SEQ ID NO: 115, HCDR3 comprising the sequence of SEQ ID NO: 120; LCDR1 comprising the sequence of SEQ ID NO: 121, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 118; or d) HCDR1 comprising the sequence of SEQ ID NO: 119, HCDR2 comprising the sequence of SEQ ID NO: 115, HCDR3 comprising the sequence of SEQ ID NO: 120; LCDR1 comprising the sequence of SEQ ID NO: 122, LCDR2 comprising the sequence of SEQ ID NO: 35, and LCDR3 comprising the sequence of SEQ ID NO: 118.

[0275]

[0308] A GREM1-associated disease or condition can be a disease or condition that would benefit from modulation of GREM1 activity (e.g., decreasing GREM1 activity). In some embodiments, a GREM1-associated disease or condition is characterized by GREM1 expression or overexpression.

[0276]

[0309] The term "overexpression" with respect to GREM1 as used herein refers to an increased expression level compared to a reference level. The reference level may be the level of GREM1 expression found in normal cells of the same tissue type, and may optionally be normalized to the expression level of another gene (e.g., a housekeeping gene). Alternatively, the reference level may be the level of GREM1 expression found in a healthy subject. In some embodiments, the GREM1-expressing cancer has a GREM1 expression level that is at least 10% higher (e.g., at least 15%, 20%, 30%, 35%, 40%, 50%, or 1-fold, 2-fold, 3-fold or even higher) than the reference level.

[0277]

[0310] The expression of GREM1 can be determined based on nucleic acid level or protein level. The expression level of GREM1 can be measured at the nucleic acid level by any method known in the art, for example, but not limited to, amplification assay (e.g., polymerase chain reaction, quantitative real-time PCR, rolling circle replication, isothermal amplification, etc.), hybridization assay (e.g., Northern blotting, microarray, fluorescence in situ hybridization (FISH), etc.), or sequencing assay (e.g., RNA sequencing). Alternatively, the expression level of GREM1 can be measured at the protein level by any method known in the art, for example, but not limited to, immunoassay (e.g., 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)).

[0278]

[0311] In certain embodiments, the subject is a human. In certain embodiments, the subject is identified as having expression or overexpression of GREM1, optionally in a biological sample obtained from the subject.

[0279]

[0312] In some embodiments, the GREM1-related disease or condition is selected from the group consisting of cancer, fibrotic disease, angiogenesis, glaucoma or retinal disease, kidney disease, pulmonary arterial hypertension, and osteoarthritis (OA). Elevated levels of GREM1 have been associated with many of these diseases and conditions, for example, scleroderma, diabetic nephropathy, glioma, head and neck cancer, prostate cancer, and colorectal cancer.

[0280]

[0313] i. Cancer Treatment

[0314] In certain embodiments, the present disclosure provides methods of treating or preventing cancer using the antibodies provided herein.

[0281]

[0315] In some embodiments, the cancer is GREM1-expressing cancer.As used herein, the phrase "GREM1-expressing cancer" refers to cancer characterized by having GREM1-expressing cancer cells and / or having GREM1 expression in cancer microenvironment.In some embodiments, GREM1-expressing cancer has GREM1 overexpression in cancer cells and / or in cancer microenvironment.

[0282]

[0316] GREM1 acts through an autocrine manner to promote the proliferation of tumor cells that express it, and it may also be secreted by non-cancer cells present within or surrounding the tumor microenvironment to create a niche suitable for cancer cell proliferation or survival, even though the cancer cells themselves may not need to express GREM1.

[0283]

[0317] As used herein, the cancer microenvironment refers to the tissues, cells and environment surrounding cancer cells. The cancer microenvironment can include stromal cells, such as fibroblasts, pericytes, endothelial cells, adipocytes, and bone marrow mesenchymal stromal cells (MSCs). The cancer microenvironment can also include extracellular matrix associated with cancer cells or associated with stromal cells surrounding cancer cells. The extracellular matrix is ​​primarily composed of ground substance - a porous hydrated gel made primarily of proteoglycan aggregates - and connective tissue fibers. Expression of GREM1 in the cancer microenvironment can be observed, for example, in stromal cells or extracellular matrix. In certain embodiments, GREM1-expressing cancers have expression or overexpression of GREM1 in stroma (e.g., desmoplastic stroma) or stromal cells.

[0284]

[0318] In certain embodiments, the subject is identified as having GREM1-expressing cancer cells or GREM1 expression in the cancer microenvironment. The presence and / or expression level of GREM1 on cancer cells or in the cancer microenvironment can be determined by various methods known in the art or provided herein using a biological sample obtained from the subject. A biological sample containing or suspected to contain cancer cells, or a biological sample from the cancer microenvironment can be obtained or derived from a subject, for example, 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 separated 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.

[0285]

[0319] GREM1 expressing cancer can be any type of cancer.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, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin's lymphoma, islet cell carcinoma (pancreatic endocrine), 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, throat cancer, thyroid cancer, or vaginal cancer.

[0286]

[0320] In certain embodiments, the GREM1-expressing cancer is also PD-L1-expressing. In certain embodiments, the GREM1-expressing cancer is not a PD-L1-expressing cancer. As used herein, the term "PD-L1-expressing" with respect to cancer refers to a cancer that is positive for PD-L1 expression using any detection method known in the art, such as immunohistochemistry (IHC), flow cytometry (e.g., FACS), etc. For example, a PD-L1-expressing cancer can refer to a cancer that is positive for PD-L1 expression using a simple positive / negative binary representation approach based on IHC data, where a positive result is defined as the percentage of cancer cells in tumor tissue sections that exhibit cell surface membrane staining for PD-L1 being at least 1%, 2%, 3%, 4%, or 5% of the total cancer cells. Detailed descriptions can be found in Thompson, RH, et al., PNAS 101(49); pp. 17174-17179 (2004); Thompson, RH, et al., Cancer Res. 66: pp. 3381-3385 ​​(2006); Gadiot, J., et al., Cancer 117: pp. 2192-2201 (2011); Taube, JM, et al., Sci Transl Med 4, 127ra37 (2012); and Toplian, SL, et al., New Eng. J Med. 366(26): pp. 2443-2454 (2012). PD-L1 expressing cancers may refer to cancers positive for PD-L1 expression using the scoring process described in WO2014165422A1. In certain embodiments, the GREM1-expressing cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor.

[0287]

[0321] In certain embodiments, the hematological tumor is a leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML)), a lymphoma (e.g., Hodgkin's lymphoma, or a non-Hodgkin's lymphoma (e.g., Waldenstrom's macroglobulinemia (WM)), or a myeloma (e.g., multiple myeloma (MM)).

[0288]

[0322] 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, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and 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, vaginal cancer, thyroid cancer, glial cancer, blastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, adenocarcinoma, leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML)), lymphoma (e.g., Hodgkin's lymphoma, or non-Hodgkin's lymphoma (e.g., Waldenstrom's macroglobulinemia (WM)), or myeloma (e.g., multiple myeloma (MM)).

[0289]

[0323] 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.

[0290]

[0324] 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.

[0291]

[0325] In certain embodiments, the cancer is prostate cancer, breast cancer, or liver cancer.

[0292]

[0326] In certain embodiments, breast cancer is triple-negative breast cancer.The terms "triple-negative breast cancer" or "TNBC" refer to breast cancer that is negative for estrogen receptor, progesterone receptor, and excess HER2 protein.TNBC may be non-responsive to hormone therapy or drugs that target HER2.

[0293]

[0327] In certain embodiments, the cancer is multiple myeloma (MM). GREM1 has been found to be secreted in large amounts by a subset of bone marrow (BM) mesenchymal stromal cells and is believed to play a pivotal 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 controls. Anti-GREM1 antibodies have been shown to reduce MM tumor burden in mice (K. Clark et al., Cancers 2020, 12, 2149).

[0294]

[0328] ii. Treatment of fibrotic diseases

[0329] In some embodiments, GREM1-related disease or condition is fibrotic disease.Fibrotic disease is a disease or condition that involves fibrosis.Fibrosis is a scarring process that is a common feature of chronic organ injury, for example, in lung, liver, kidney, skin, heart, intestine or muscle.Fibrosis is characterized by the increased activity of transforming growth factor beta (TGF-beta), which leads to increased deposition and alteration of extracellular matrix and other fibrosis-related 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).

[0295]

[0330] Fibrotic diseases can include fibrotic diseases in the lungs, liver, kidneys, eyes, skin, heart, intestines or muscles. Examples of fibrotic diseases in the lungs include pulmonary fibrosis, cystic fibrosis, pulmonary hypertension, progressive massive fibrosis, bronchiolitis obliterans, chronic asthma or idiopathic lung-related airway remodeling. Examples of fibrotic diseases in the liver include cirrhosis or non-alcoholic steatohepatitis. Examples of fibrotic diseases in the kidneys include renal fibrosis, ischemic renal injury, tubulointerstitial fibrosis, diabetic nephropathy, nephrosclerosis or nephrotoxicity. Examples of fibrotic diseases in the eyes include corneal fibrosis, subretinal fibrosis, etc. Examples of fibrotic diseases in the skin include nephrogenic systemic fibrosis, keloid or scleroderma, etc. Examples of fibrotic diseases in the heart include myocardial fibrosis or old myocardial infarction.

[0296]

[0331] In another aspect, the present disclosure also provides a method of improving the efficacy of BMP7 treatment in treating a fibrotic disease (e.g., renal fibrosis) in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an anti-GREM1 antibody provided herein. In certain embodiments, the subject has been treated with BMP7 treatment. In another aspect, the present disclosure also provides a method of treating a fibrotic disease (e.g., renal fibrosis) in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of an anti-Gremlin 1 antibody provided herein in combination with BMP7 treatment. The term "BMP7 treatment" as used herein may be any treatment involving increasing the level of BMP7 in a subject in need of such treatment. For example, the BMP7 treatment may be administration of recombinant BMP7 or administration of a peptide mimetic of BMP7. BMP7 treatment may also include restoring endogenous BMP7, such as by decreasing the level and / or activity of a BMP7 antagonist (e.g., Noggin, or Uterine Sensitization Associated Gene-1 (USAG-1)) or by increasing the level and / or activity of a BMP7 agonist (e.g., Kirin / Chordin-like protein (KCP) or BMP receptor). (Michael et al., Reversal of experimental renal fibrosis by BMP7 provides insights into novel therapeutic strategies for chronic kidney disease, Pediatr Nephrol. 2008 Sep;23(9):1395-8). In certain embodiments,

[0332] In another aspect, the present disclosure also provides a method for treating a disease that can benefit from increasing BMP7 activity or decreasing Gremlin-mediated inhibition of BMP7 activity, comprising administering to a subject a therapeutically effective amount of an anti-Gremlin 1 antibody provided herein. In certain embodiments, the disease is a fibrotic disease and / or a kidney disease. In certain embodiments, the disease is selected from the group consisting of ischemia-reperfusion injury, ischemic acute kidney failure, diabetic nephropathy and hypertensive nephrosclerosis, renal fibrosis, chronic kidney disease, acute kidney disease, and other autoimmune diseases such as hypertensive nephrosclerosis, immunoglobulin A nephropathy (IgAN) and lupus nephritis or systemic lupus erythematosus (SLE).

[0297]

[0333] iii. Treatment of other diseases

[0334] 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 persistently elevated pulmonary arterial pressure.GREM1 has been found to be elevated in the wall of small blood vessels in the lungs of mice during hypoxia. Anti-GREM1 antibodies have been found to reduce or ameliorate one or more symptoms associated with PAH, for example, by inhibiting pulmonary artery thickening, increasing stroke volume and / or stroke volume to end-systolic volume ratio ("SV / ESV"), increasing right ventricular cardiac output and / or cardiac index (CI), and improving other hemodynamic measures in subjects with PAH, such as, for example, right atrial pressure, pulmonary artery pressure, pulmonary capillary wedge pressure in the presence of end-expiratory pressure, systemic arterial pressure, heart rate, pulmonary vascular resistance, and / or systemic vascular resistance (see U.S. Patent Application No. 20180057580 A1 for details).

[0298]

[0335] 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 tension or hydrostatic 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, leading to the subsequent induction of catabolic enzymes. Intra-articular administration of GREM1 antibodies or chondrocyte-specific deletion of GREM1 in mice was reported to slow down the development of osteoarthritis (see SHChang et al., Nature Communications, (2019)10:1442).

[0299]

[0336] In some embodiments, the GREM1-associated disease or condition is angiogenesis. GREM1 is an agonist of the major pro-angiogenic receptor vascular endothelial growth factor receptor-2 (VEGFR-2). 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 Application No. 20200157194 for details).

[0300]

[0337] In some embodiments, GREM1-related disease or condition is glaucoma.Glaucoma may be caused by the expression change of one or more BMP family genes in eye, which causes increased intraocular pressure and / or glaucomatous optic neuropathy.GREM1 has been found to have increased expression in glaucomatous trabecular meshwork cells.GREM1 antagonists have been found to reduce or improve one or more symptoms associated with neovascularization or glaucoma (see US Patent No. 7,744,873 for details).

[0301]

[0338] 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.

[0302]

[0339] Route of Administration and Dosage

[0340] The antibody or antigen-binding fragment provided herein can be administered in a therapeutically effective dose.The therapeutically effective amount of the antibody or antigen-binding fragment provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current prescription drugs, subject's health condition and the possibility of cross-reaction, allergy, sensitivity and adverse side effects, as well as route of administration and the degree of disease development.The dosage may be proportionally increased or decreased by a person skilled in the art (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0303]

[0341] In certain embodiments, the antibodies or antigen-binding fragments provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose may vary over the course of treatment. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0304]

[0342] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0305]

[0343] The antibodies and antigen-binding fragments disclosed herein can be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0306]

[0344] Combination therapy

[0345] In some embodiments, the antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic procedures or agents, which may be selected based on the disease or condition being treated.

[0307]

[0346] In some embodiments, the antibodies or antigen-binding fragments disclosed herein may be administered to treat cancer in combination with a second anti-cancer drug, e.g., a chemotherapeutic agent, an anti-cancer drug, radiation therapy, immunotherapy, an anti-angiogenic agent, targeted therapy, cell therapy, gene therapy agent, hormonal therapy agent, cytokines, palliative care, surgery for the treatment of cancer (e.g., tumor removal), one or more antiemetic agents, treatment of complications resulting from chemotherapy, or nutritional supplements for cancer patients, or agents that modulate the tumor microenvironment.

[0308]

[0347] The term "chemotherapeutic agent" refers to a biological (polymer) or chemical (small molecule) compound that can be used to treat cancer. Types of chemotherapeutic agents include, but are not limited to, histone deacetylase inhibitors (HDACIs), alkylating agents, antimetabolites, alkaloids, cytotoxic / anticancer antibiotics, topoisomerase inhibitors, tubulin inhibitors, proteins, antibodies, kinase inhibitors, and the like. Examples of chemotherapeutic agents include erlotinib, afatinib, docetaxel, adriamycin, 5-FU (5-fluorouracil), panobinostat, gemcitabine, cisplatin, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, metformin, temozolomide, tamoxifen, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, and trametinib. In certain embodiments, the chemotherapeutic agent is cisplatin.

[0309]

[0348] The term "immunotherapy" as used herein refers to a type of stimulating the immune system to fight against disease such as cancer, or generally boosting the immune system. Immunotherapy includes passive immunotherapy (e.g., antibody therapy or CAR-T cell therapy) by delivering agents (e.g., effector cells) with established tumor immune reactivity that can directly or indirectly mediate antitumor effects and do not necessarily rely on intact host immune system. Immunotherapy can also include active immunotherapy, in which case treatment utilizes in vivo stimulation of the endogenous host immune system to react against diseased cells by administering immune response modifiers.

[0310]

[0349] Examples of immunotherapeutics include, but are not limited to, checkpoint modulators, adoptive cell transfer, cytokines, oncolytic viruses and therapeutic vaccines.

[0311]

[0350] Checkpoint modulators can interfere with the ability of cancer cells to evade immune system attack and help the immune system to mount a stronger response against tumors. Immune checkpoint molecules can either mediate costimulatory signals to increase immune responses or mediate costimulatory signals to suppress immune responses. Examples of checkpoint modulators include, but are not limited to, 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, or CD83. In certain embodiments, the immune checkpoint modulator comprises a PD-1 / PD-L1 axis inhibitor.

[0312]

[0351] Adoptive cell transfer, 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, expanded, and activated in vitro. In certain embodiments, the T cells are engineered in vitro into CAR-T cells. The T cells or CAR-T cells that are most active against cancer are cultured in large batches in vitro for 2-8 weeks. During this time, the patient will undergo 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 will be returned to the patient. In certain embodiments, the immunotherapy is CAR-T therapy.

[0313]

[0352] Cytokine therapy can also be used to enhance tumor antigen presentation to the immune system. The two main types of cytokines used to treat cancer are interferon and interleukin. Examples of cytokine therapy include, but are not limited to, interferon, 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), interleukin, 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.

[0314]

[0353] Oncolytic viruses are genetically modified viruses that can kill cancer cells. Oncolytic viruses can specifically infect tumor cells, leading to tumor cell lysis and subsequent release of large amounts of tumor antigens, which trigger the immune system to target and eliminate the cancer cells that carry such tumor antigens. Examples of oncolytic viruses include, but are not limited to, talimogene laherparepvec.

[0315]

[0354] Therapeutic vaccines work against cancer by boosting the immune system's response to cancer cells. Therapeutic vaccines can contain a non-pathogenic microorganism (e.g., Mycobacterium bovis Calmette-Guerin, BCG) with a genetically modified virus that targets tumor cells or one or more immunogenic components. For example, BCG can be inserted directly into the bladder using a catheter to trigger an immune response against bladder cancer cells.

[0316]

[0355] 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.

[0317]

[0356] "Targeted therapy" is a type of therapy that acts on specific molecules related to cancer, such as specific proteins that are present in cancer cells but not in normal cells or that are more abundant in cancer cells, or target molecules in the cancer microenvironment that contribute to the growth and survival of cancer.Targeted therapy directs therapeutic agents to target tumors, thereby sparing normal tissues from the effects of therapeutic agents.

[0318]

[0357] 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), estrogen receptor (ER), nuclear receptor (NR), and PR.

[0319]

[0358] Targeted therapies can target molecules in the tyrosine kinase or nuclear receptor signaling cascade, for example, 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, Box03A, MDM2, IAPs, NF-kB, Myc, P13K, Ra, FLIP, heregulin (HRG) (also known as gp30), Bcl-2, Bcl-xL, Bax, Bak, Bad, Bok, Bik, Blk, Hrk, BNIP3, BimL, Bid, and EGL-1.

[0320]

[0359] Targeted therapy can also target tumor-associated ligands, such as estrogen, estradiol (E2), progesterone, estrogen, androgen, glucocorticoids, prolactin, thyroid hormone, 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.

[0321]

[0360] In certain embodiments, the second therapeutic agent modulates the tumor microenvironment. In certain embodiments, the second therapeutic agent is a bifunctional molecule comprising a PD-L1 binding portion and the extracellular domain of a TGF-beta receptor.

[0322]

[0361] In some embodiments, the antibody or antigen-binding fragment disclosed herein can be administered in combination with a second anticancer drug to treat prostate cancer.In certain embodiments, the anticancer drug comprises an antiprostate cancer drug.In some embodiments, the antiprostate cancer drug comprises an androgen axis inhibitor; an androgen synthesis inhibitor; an ADP-ribose polymerase (PARP) inhibitor; or a combination thereof.

[0323]

[0362] 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.

[0324]

[0363] In certain embodiments, the androgen axis inhibitor is degarelix, bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, or abiraterone.

[0325]

[0364] In certain embodiments, the androgen synthesis inhibitor is abiraterone acetate or ketoconazole.

[0326]

[0365] In certain embodiments, the PARP inhibitor is olaparib or rucaparib.

[0327]

[0366] 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), Firmagone (degarelix), flutamide, goserelin acetate, Jevtana (cabazitaxel), leuprolide acetate, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lympa The agent is selected from the group consisting of: ixazomib (olaparib), mitoxantrone hydrochloride, nilandrone (nilutamide), nilutamide, nubequo (darolutamide), olaparib, provenzi (sipuleucel-T), radium-223 dichloride, rubraca (rucaparib camsylate), rucaparib camsylate, sipuleucel-T, taxotere (docetaxel), zofigo (radium-223 dichloride), xtandi (enzalutamide), zoladex (goserelin acetate) and zytiga (abiraterone acetate).

[0328]

[0367] In certain embodiments, the nutritional supplement for cancer patients can be a suitable supplement that has protective effect against cancer.In certain embodiments, the nutritional supplement comprises indole-3-carbinol or its derivative that produces indole-3-carbinol after ingestion.Indole-3-carbinol is believed to have protective effect against cancer and may be preventive against precancerous conditions.

[0329]

[0368] In certain embodiments, the antibody or antigen-binding fragment disclosed herein can be administered in combination with indole-3-carbinol or its derivative that produces indole-3-carbinol after ingestion.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.

[0330]

[0369] In some embodiments, the antibody or antigen-binding fragment disclosed herein may be administered in combination with a second therapeutic agent, such as a second antifibrotic agent (e.g., recombinant BMP7 or a peptide mimetic of BMP7) to treat fibrotic diseases. In certain embodiments, the second antifibrotic agent is an ACE inhibitor (or ARB), an anti-MASP2 antibody, an endothelin receptor antagonist, an NRF2 inhibitor steroid, a CTLA4-IgG, or a TNF inhibitor.

[0331]

[0370] In another embodiment, the second therapeutic agent is selected from the group consisting of anti-fibrotic agents such as pirfenidone, anti-inflammatory drugs, NSAIDs, corticosteroids such as prednisone, nutritional supplements, vascular endothelial growth factor (VEGF) antagonists (e.g., "VEGF traps", such as aflibercept or other VEGF inhibitory fusion proteins described in U.S. Pat. No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab or ranibizumab)), antibodies against cytokines such as IL-1, IL-6, IL-13, IL-4, IL-17, IL-25, IL-33 or TGF-β, negative regulators of the TGF-β / Smad signaling pathway (recombinant BMP7 or peptide mimics of BMP7), and any other palliative therapy 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.

[0332]

[0371] In some embodiments, the second therapeutic agent may be administered to manage or treat at least one complication associated with fibrosis or cancer.

[0333]

[0372] In certain of these embodiments, the antibodies or antigen-binding fragments disclosed herein that are administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibodies or antigen-binding fragments and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment that is administered "in combination" with another therapeutic agent may not be administered simultaneously with the agent or in the same composition as the agent. An antibody or antigen-binding fragment that is administered before or after another agent is considered to be administered "in combination" with the agent, as the phrase "in combination" is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment 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, Vol. 57; Medical Economics Company; ISBN: 1563634457; Vol. 57 (November 2002)), or according to protocols well known in the art.

[0334]

[0373] In another aspect, the disclosure provides a kit or pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent, which may be formulated in one composition or in different compositions. Instructions or labels for use may further be included to provide information regarding how the combination therapy is to be performed.

[0335]

[0374] Methods of Detection and / or Diagnosis

[0375] In some embodiments, the disclosure provides a method for detecting the presence or amount of GREM1 in a sample derived from a subject, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof, and determining the presence or amount of GREM1 in the sample.

[0336]

[0376] The presence or amount of GREM1 in a sample can also be detected by measuring the mRNA levels of GREM1 using techniques including, but not limited to, RNA sequencing (RNA-seq) and RNAscope (Wang, Z., Gerstein, M., & Snyder, M. (2009). RNA-seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics, 10(1), pp. 57-63; Wang et al., RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues, J Mol Diagn. January 2012; 14(1): pp. 22-9.). Briefly, RNA-seq involves reverse transcribing target mRNA into cDNA, fragmenting and sequencing the cDNA, and analyzing the sequence data for mRNA quantification; RNAscope involves in situ hybridization of target mRNA with one or more oligonucleotides conjugated with a fluorescent probe and detecting the levels of mRNA by measuring the fluorescence intensity.

[0337]

[0377] In certain embodiments, the biological sample comprises cancer cells, or a sample from the tumor microenvironment (e.g., stromal cells or stroma).

[0338]

[0378] In some embodiments, the present disclosure provides a method for detecting the presence or amount of GREM1 in a sample or diagnosing a GREM1-associated disease or condition in a subject, the method comprising: a) contacting a sample obtained from a subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of GREM1 in the sample; and optionally c) correlating the presence or amount of GREM1 with the presence or status of a GREM1-associated disease or condition in the subject. In certain embodiments, the biological sample comprises cancer cells, stromal cells, stroma or fibrotic cells.

[0339]

[0379] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety. The kit may be useful in detecting the presence or amount of GREM1 in a biological sample, or may be useful in the diagnostic methods provided herein.

[0340]

[0380] In some embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent. The kit may be useful in treating, preventing, and / or ameliorating a GREM1-associated disease.

[0341]

[0381] In some embodiments, the disclosure also provides the use of an antibody, or antigen-binding fragment thereof, provided herein in the manufacture of a medicament for treating or diagnosing a GREM1-related disease or condition in a subject. EXAMPLES

[0342]

[0382] While the present disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.

[0343] Example 1: Antigen Preparation

[0383] HGREM1-His (R&D): Recombinant HGREM1 protein (accession number O60565) was expressed in NS-0 cells. Briefly, the coding region of hGREM1 gene from Lys25 to Asp184 with a 10x his tag at the C-terminus was used for transfection. The supernatant was purified using a His-tag affinity column. The resulting purified protein was characterized using SDS PAGE gel. The protein was purchased from R&D Systems (catalog number 5190-GR).

[0344]

[0384] Mouse Gremlin-His (R&D): Recombinant mouse Gremlin (Accession No. O70326) Lys25-Asp184 was fused to a 10xHis tag at the C-terminus and produced in NS-0 cells. Transfection supernatant was purified using a His-tag affinity column. The resulting purified protein was characterized using SDS PAGE gels. Protein was purchased from R&D Systems (Cat. No. 956-GR).

[0345]

[0385] HGREM1-His (ACRO): Recombinant hGREM1 protein Lys25-Asp184 (accession no. NP_037504) was fused with a polyhistidine tag at the C-terminus and produced in human 293 cells (HEK293). Transfection supernatants from HEK293 cells were purified using a His-tag affinity column. The resulting purified protein was characterized using SDS PAGE gels. The protein was purchased from ACRO Biosystems (catalog no. GR1-H52H3).

[0346]

[0386] HGREM1-Fc (ACRO): Recombinant hGREM1 protein Lys25-Asp184 (accession no. NP_037504) was fused to a hIgG1 Fc tag at the C-terminus and produced in human 293 cells (HEK293). The resulting purified protein was characterized using SDS PAGE gels. The protein was purchased from ACRO Biosystems (catalog no. GR1-H5254).

[0347]

[0387] The above Gremlin protein was used in the following experiments.

[0348] Example 2: Antibody Generation

[0388] 1. Antigen Conjugation and Immunization

[0389] For immunization, recombinant hGremlin-His protein was conjugated with various MabSpace immune enhancing peptides. Briefly, a 2-8 fold molar excess of peptide was mixed with Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate, Pierce, Cat. No. 22322) activated hGremlin protein and incubated for 1 hour at room temperature. The reaction was stopped and the conjugated protein was analyzed using SDS-PAGE gels and QC was performed.

[0349]

[0390] The above conjugated hGremlin-His proteins were emulsified with complete Freund's adjuvant (Pierce, Cat. No. 77140) at a 1:1 ratio, and then immunized subcutaneously and intraperitoneally into C57B / L6 mice. Booster immunizations were performed with CpG and alum to preserve the native conformation of the protein. Immunizations were performed at least every 2 weeks, and antisera from mice were collected after the first immunization for anti-hGremlin titer analysis by ELISA assay.

[0350]

[0391] For serum titer determination, 20 μl of mouse serum was prepared from each immunized mouse. High-binding clear polystyrene 96-well plates (Nunc) were coated with 100 μl / well of a 1 μg / ml solution composed of hGREM1-His in high pH coating buffer (0.16% Na2CO3, 0.3% NaHCO3, pH 9.8). Plates were incubated overnight at 4° C. and then washed once in an automated plate washer using wash buffer PBS+0.1% Tween® 20 (Sigma). 200 μl of blocking buffer (PBS+1% BSA+1% goat serum+0.05% Tween® 20) was added to each well and incubated for 2 hours at room temperature. The blocking buffer was then aspirated and 100 μl of serum serially diluted in dilution buffer (PBS+1% BSA+1% goat serum+0.01% Tween®20) was transferred to each well of the ELISA plate and incubated for 60 min at room temperature. The plate was then washed three times using the method described above. 100 μl / well of a solution of HRP-conjugated goat anti-mouse Fc antibody (Abcam, Cat. No. Ab98808) diluted in dilution buffer was then added to each well of the plate. The ELISA plate was then incubated for 60 min at RT and the plate was washed three times with 250 μl / well of wash buffer. Finally, 100 μl / well of TMB was added to each well and the reaction was terminated using 0.64 M H2SO4. The plate was read at 450 nM on a Thermo Multiscan FC.

[0351]

[0392] 2.Fusion

[0393] Four days prior to fusion, each mouse was boosted intraperitoneally with unconjugated hGremlin-His protein in PBS. On the day of fusion, the spleen was aseptically removed and the organ processed into a single cell suspension. Red blood cells were lysed and splenocytes were washed with DMEM (Gibco). Live logarithmic growth phase myeloma cells (SP2 / 0) were mixed with mouse splenocytes at a 1:4 ratio. The cells were then washed twice prior to fusion with PEG. The fused cells were washed with DMEM and suspended in cell growth medium supplemented with 10% FBS+HFCS+OPI+1xHAT. 200 μl per well of this cell suspension was plated into a 96-well cell culture plate and incubated overnight in a humidified 10% CO2 incubator at 37°C. The cultures were incubated for 7 days, then the growth medium was aspirated from the wells and replaced with fresh growth medium. Screening of hybridoma supernatants was initiated 2-3 days after medium change.

[0352]

[0394] 3. Antibody Screening by ELISA Assay

[0395] The same protocol as for serum titration above was used. Briefly, hGremlin-His 1 μg / ml was coated overnight at 4°C. After washing, 100 μl of hybridoma supernatant was added and allowed to bind completely. HRP-conjugated goat anti-mouse Fc antibody was then added to detect bound Gremlin antibodies. Finally, after TMB reaction and H2SO4 termination, the plates were read at 450 nM on a Thermo Multiscan FC. Cells from ELISA-positive hybridoma wells were then expanded in cell culture for further characterization studies.

[0353] Example 3: Subcloning of positive hybridoma clones and small-scale antibody production

[0396] 1. Subcloning of positive hybridoma clones

[0397] Cells from ELISA positive hybridoma wells with desired binding profile and blocking activity were selected and plated in 96-well plate respectively using limiting dilution. These cells were grown for 7 days. When appropriate cell amount was reached, the supernatant from each well was collected and rescreened for antigen binding ability (see screening in Example 2).

[0354]

[0398] From each 96-well plate, the clone with the highest antigen binding activity was identified and expanded by further limiting dilution into a 96-well plate containing 200 μl of hybridoma growth medium per well. After 7 days, cells from the 96-well plate were tested for antigen binding. Subcloning was performed three or more times. When more than 90 wells showed positive binding signals, two clones with the highest antigen binding activity were identified and transferred to a 24-well plate containing medium and expanded for another 2 days. When the 24-well plate became confluent, the cells were transferred to a 6-well plate. After 5 days of incubation, a portion of the cells was frozen. The rest of the cells were transferred to a flask and expanded. When the flask became confluent, half of the cells were frozen (3 vials per clone) for further backup. The remaining half was further expanded in a flask containing medium for antibody production. The isotype was determined using standard methods.

[0355]

[0399] 2. Small-scale Antibody Production

[0400] Hybridoma cells were inoculated into roller bottles and cultured in 200-300 ml of hybridoma culture medium (Invitrogen) for 14 days. Gremlin monoclonal antibodies (mAbs) were purified from hybridoma cell cultures as follows. All purification processes were carried out at room temperature. One purification scheme was used to purify the various mAbs, using affinity chromatography.

[0356]

[0401] The host cell culture fluid (CCF) was centrifuged to remove cell debris, and the CCF supernatant was then filtered, diluted, and then loaded onto a protein G chromatography medium in the form of a column, Protein G High Performance (Bio-Rad), and allowed to equilibrate.

[0357]

[0402] After loading, the Protein G column was washed until the absorbance at 280 nm of the flow-through returned to baseline. The Gremlin mAb was then eluted from the column using glycine, pH 2.5, and immediately neutralized by adding 50 μL of a stock solution of 1 M Tris base per mL of elution volume. The absorbance at 280 nm of the eluate was monitored, and fractions containing protein were collected to create the Protein A pool.

[0358]

[0403] Following purification, the Gremlin mAb was formulated in PBS by dialysis using a 10,000 MWCO membrane (Pierce Slide-A-Lyzer or dialysis tubing). Following formulation, the Gremlin mAb was filtered.

[0359] Example 4: Binding analysis of purified hybridoma anti-Gremlin 1 antibodies to captured human and mouse gremlin by ELISA

[0404] Clear polystyrene plates (BEAVER) were coated with 100 μl / well of hGREM1 (ACRO) and mouse Gremlin (R&D) 0.5 μg / ml in high pH coating buffer overnight at 4° C. The plates were then washed once in an automated plate washer using PBS+0.1% Tween® 20 (Sigma). 100 μl of blocking solution consisting of PBS+1% BSA+1% normal goat serum+0.5% Tween® 20 (Sigma) was added to each well and incubated for 2 hours at room temperature. 100 μl of antibody in antibody dilution buffer containing PBS+1% BSA+1% normal goat serum+0.01% Tween® 20, starting at 2 μg / ml, followed by serial dilutions, was then added to each well of the plate and incubated for 1 hour at room temperature. Afterwards, the plates were washed 3 times with 200 μl of PBS+0.1% Tween® 20, followed by the addition of 100 μl / well of 1:10000 goat anti-mouse IgG-HRP (abcam) and incubation at room temperature for 1 hour. They were then washed 3× with PBS+0.1% Tween® 20. Finally, 100 μl / well of TMB (Pierce) was added to each well, and after a few minutes, 50 μl of stop solution was added to each well. The plates were read at 450 nM on a Multiscan FC microplate reader (Thermo Scientific). As shown in FIG. 1, 56C11, 42B9, 36F5, and 67G11 showed high binding affinity to both hGREM1 and mouse gremlin (EC50 values ​​of 13.42ng / ml and 17.2ng / ml for 56C11, 8.058ng / ml and 8.512ng / ml for 42B9, 5.869ng / ml and 4.564ng / ml for 36F5, and 7.841ng / ml and 7.713ng / ml for 67G11), while 69H5, 22F1, and 14E3 showed selective binding affinity to hGREM1 compared to mouse gremlin (EC50 values ​​of 105.9ng / ml, 14.13ng / ml, and 13.6ng / ml, respectively).

[0360]

[0405] The humanized antibodies provided herein (eg, Hu14E3, Hu22F1 and Hu56C11) exhibited similar high binding affinity to hGREM1 and / or mGREM1.

[0361] Example 5: Characterization of the binding specificity of hybridoma antibodies binding to captured hGREM1 or related family proteins by ELISA

[0406] In addition, the specificity of binding of 14E3 to the benchmark antibodies was evaluated by ELISA. Briefly, clear polystyrene plates (BEAVER) were coated overnight at 4° C. with 100 μl / well of hGREM1 (ACRO), or human Gremlin-2 (R&D), human COCO (R&D) and human DAN protein (R&D) at 0.5 μg / ml in high pH coating buffer. Plates were then washed once in an automated plate washer using PBS+0.1% Tween® 20 (Sigma). 100 μl of blocking solution consisting of PBS+1% BSA+1% normal goat serum+0.5% Tween® 20 (Sigma) was added to each well and incubated for 2 hours at room temperature. Then, 100ul of the antibody in antibody dilution buffer containing PBS+1%BSA+1% normal goat serum+0.01%Tween®20, starting at 2μg / ml, followed by serial dilutions of 5-fold dilutions, were added to each well of the plate and incubated at room temperature for 1 hour. Afterwards, the plate was washed 3 times with 200ul of PBS+0.1%Tween®20, followed by the addition of 100μl / well of 1:10000 goat anti-mouse IgG-HRP (Abcam) and incubated at room temperature for 1 hour. They were then washed 3× with PBS+0.1%Tween®20. Finally, 100μl / well of TMB (Pierce) was added to each well, and 50ul of stop solution was added to each well after 5 minutes. The plate was read at 450nM on a Multiscan FC microplate reader (Thermo Scientific). The results showed that 14E3 specifically bound to hGREM1 but not to human Gremlin-2, COCO, and DAN proteins, with which it shares high structural homology (Fig. 2 ).

[0362] Example 6: Characterization of antibody activity in blocking gremlin binding to captured BMP2 / 4 / 7

[0407] Gremlin is known to be capable of binding to BMP proteins, and is confirmed by this disclosure. Briefly, Gremlin Fc was tested for its binding ability to BMP2 / 4 / 7 immobilized on a plate. Briefly, a plate was coated with recombinant human BMP2, BMP4 (hBMP4, Peprotech) or human BMP7 (R&D) 0.5 μg / ml overnight, wild-type Gremlin-1 was added to the coated plate, and incubated at RT for an additional hour. The plate was then washed, and plate-bound biotin-hGremlin was detected using neutravidin conjugated with HRP (thermo). The plate was then developed with TMB solution and stopped by adding stop solution. The plate was read at 450 nm on a plate reader. Figures 3A and 3D show that wild-type gremlin was capable of binding to BMP2, BMP4 and BMP7, and that its binding affinity to BMP2 and 4 was stronger than its binding affinity to BMP7.

[0363]

[0408] A mutant version of Gremlin XM5 was also tested for its binding ability to BMP2 / 4 / 7 directly with Gremlin. XM5 was constructed and expressed by Mabspace, in this case, the 123-143 amino acids of Gremlin (NSFYIPRHIRKEEGSFQSCSF, SEQ ID NO: 63), known as the binding loop of BMP, were replaced by the 63-83 amino acids of DNA (FSYSVPNTFPQSTESLVHCDS, SEQ ID NO: 64), which does not bind to BMP. A His-tag or Fc-tag was constructed at the C-terminus of the protein. For the adhesion of XM5 to Gremlin, unpurified supernatant was used for this assay. Briefly, anti-human Fc antibody (1 μg / ml) was coated and XM5-Fc or Gremlin-Fc supernatant (1:32 dilution) was added. After incubation, serially diluted BMPs carrying a his-tag (BMP2 / 4 / 7) were added and then a secondary antibody anti-his-HRP was used for detection of binding.

[0364]

[0409] As shown in Figure 3A, in contrast to Gremlin, BMPs did not bind to XM5 at all, which was expected, confirming that at least one amino acid in the binding loop of BMPs, including SEQ ID NO: 63 on Gremlin, is essential for its binding to BMPs. The ability of antibodies to block Gremlin binding to human bone morphogenetic proteins 2 and 4 (hBMP2, hBMP4) was examined via ELISA. Plates were coated overnight with recombinant human BMP2 and BMP4 (0.25 μg / ml) (hBMP2, hBMP4, Peprotech), and then serial dilutions of antibodies were incubated with 0.1 μg / ml of hGREM1 modified with a biotin tag (Peprotech) for 1 hour at RT, after which the complex was added to the coated plate and incubated for another hour at RT. Plates were then washed, and plate-bound biotin-hGremlin was detected using neutravidin (thermo) conjugated with HRP. The plate was then developed with TMB solution and stopped by adding stop solution. The plate was read at 450 nm on a plate reader. The results are shown in Figure 3B and Figure 3C, which show that the anti-Gremlin 1 antibodies provided herein (e.g., 14E3, 56C11 and 69H5, as well as benchmark antibody 6245P, which was produced according to the sequence of H4H6245P disclosed in WO2014159010, the disclosure of which is incorporated by reference in its entirety) can inhibit the binding of Gremlin to BMP2 and BMP4 in a dose-dependent manner, albeit to different degrees.

[0365]

[0410] In addition, the ability of the antibody dilutions provided herein to block the binding of gremlin to human BMP2 / 4 / 7 was also tested via ELISA. Plates were coated overnight with recombinant human BMP2 / 4 / 7 (0.5 μg / ml), and then the antibody dilutions were incubated with modified human gremlin-his 1 μg / ml at RT for 1 hour, after which the complex was added to the coated plate and incubated for another hour at RT. Plates were then washed, and anti-hisHRP (GenScript) was added to the plate. Plates were then developed with TMB solution and stopped by adding stop solution. Plates were read at 450 nm on a plate reader. The results are shown in Figures 3E-H, which show that the anti-gremlin 1 antibodies provided herein (e.g., 42B9, 36F5, 67G11, 14E3 HaLa, chimeric antibody 69H5 (69H5-chi), chimeric antibody 56C11 (56C11-chi) and chimeric antibody 22F1 (22F1-chi)), as well as the benchmark antibody 6245P, can inhibit binding of gremlin to BMP2 (see Figure 3E), BMP4 (see Figure 3F) and BMP7 (see Figure 3G). The chimeric anti-gremlin 1 antibody (69H5-chi) and the chimeric anti-gremlin 1 antibody (22F1-chi) can also inhibit binding of gremlin to BMP2 and BMP4 (see Figures 3E and 3F). A chimeric anti-gremlin 1 antibody (56C11-chi) can inhibit the binding of gremlin to BMP2 (see FIG. 3E).

[0366]

[0411] In particular, as shown in Figure 3G, the anti-Gremlin 1 antibodies provided herein (42B9, 36F5, 67G11 and 14E3 HaLa) were able to significantly block the binding of Gremlin to BMP7, exhibiting a maximum blocking percentage of at least 50% and even at least 70%. In contrast, the benchmark antibody 6245P exhibited a maximum blocking of less than 30% against the binding of human Gremlin to BMP7, suggesting that 6245P was much less effective at blocking BMP7.

[0367] Example 7: Characterization of antibody activity in blocking Gremlin-mediated BMP signaling using a BMP-responsive reporter assay

[0412] These purified hybridoma antibodies were also tested for their ability to reduce Gremlin-mediated inhibition of BMP signaling using a BMP4-inducible luciferase reporter assay in BRITER (BMP-responsive osteoblast reporter cell line, Abmgood, T3105). Briefly, BRITER cells were plated at 10,000 cells / well in 96-well plates and incubated overnight at 37°C and 5% CO2. The next day, cells were stimulated with either control medium or medium containing BMP4 (BMP4 30) 30ng / ml or BMP4 30ng / ml + Gremlin 200ng / ml (B 30+Gremlin 200); or BMP4 30ng / ml + Gremlin 200ng / ml with various concentrations of the antibodies provided herein (B+G+0.096 / 0.048 / 0.24 / 1.2 / 6 / 30μg / ml), as indicated on the x-axis of Figure 4. Luciferase activity of cells in each well was measured after 3 hours of incubation at 37°C and 5% CO2 using a plate reader (Thermo Scientific Varioskan Flash).

[0368]

[0413] As shown in FIG. 4, in contrast to the benchmark antibodies, the antibodies did not show any activity in reducing Gremlin-mediated inhibition of BMP signaling in reporter cells that were not of cancer cell origin, whereas the benchmark antibodies can reduce or reverse Gremlin-mediated inhibition of BMP signaling.

[0369] Example 8: Characterization of antibody activity in reducing Gremlin-mediated inhibition of BMP signaling and cell differentiation

[0414] The purified hybridoma antibodies were also tested for their ability to reduce Gremlin-mediated inhibition of BMP signaling using BMP4-induced ATDC-5 cell differentiation. ATDC-5 is a chondrogenic cell line and can differentiate in response to BMP4 signaling. Differentiation of the chondrogenic cell line can be blocked by Gremlin, a known BMP inhibitor. In this assay, blocking Gremlin leads to reversal of BMP4 inhibition. Differentiation can be measured colorimetrically by using a substrate that detects the endogenous expression of alkaline phosphatase (ALP), an early marker of osteoblast differentiation. The differentiation level can positively reflect the activity of BMP4 signaling.

[0370]

[0415] ATDC-5 cells were plated at 3000 cells / well in 96-well plates, grown in DMEM / F12, 10% FBS + 1% PS in a volume of 100ul / well, and incubated overnight at 37°C and 5% CO2. The next day, hGREM1 was mixed with serially diluted antibodies in serum-free medium and incubated for 30 minutes at 37°C. Human BMP4 (Peprotech) diluted in serum-free medium was added to the Gremlin / antibody mixture, which was then incubated for an additional 30 minutes at 37°C. After incubation, 100ul of the mixture was added to ATDC-5 cells plated in 100μl of complete medium. The final concentrations of hBMP4 and hGremlin on the cells in each well were 100ng / ml and 400ng / ml, respectively. After 3 days of growth at 37°C and 5% CO2, the medium was aspirated and washed twice with cold PBS. Cells were lysed with M-PER buffer (Thermo) plus protein inhibitor (Roche). ALP was measured using p-nitrophenyl phosphate (PNPP) (Sigma). OD405 was measured with a Multiscan FC microplate reader (Thermo Scientific).

[0371]

[0416] As shown in Figure 5, the benchmark antibody 6245P was able to reduce the Gremlin-mediated inhibition of BMP signaling in cell differentiation, whereas the antibodies 14E3 (Figure 5A), 22F1 (Figure 5B), 56C11 (Figure 5C), and 69H5 (Figure 5D) did not show any such effect. In other words, the antibodies provided herein were unable to restore the Gremlin inhibition of BMP signaling involved in the differentiation of cells not of cancer origin.

[0372] Example 9: Characterization of Gremlin-mediated inhibition of BMP signaling in PC3 prostate cancer cells and evaluation of antibodies provided herein in reversing Gremlin-mediated inhibition of BMP signaling in PC3 cells

[0417] PC3 prostate cancer cells were plated in 12-well plates at 100,000 cells / well in DMEM / 10% FBS, 1% PS (complete medium) and grown to 90% confluence at 37°C and 5% CO2. After overnight starvation in serum-free medium, PC3 cells were stimulated with BMP4 with or without Gremlin for 30 min. For Western blot analysis, cells were lysed in RIPA buffer (CST). Cell lysates were resolved using 4%-12% SDS-PAGE (Genscript) and transferred to PVDF membranes (Millipore). The membranes were incubated overnight at 4°C with antibodies specific for Smad phosphorylation (p-Smad1 / 5 / 9) (1:1000, CST), which positively reflects the activity of BMP signaling, and with an antibody specific for β-actin as a control (1:5000, Abbkine), respectively, followed by incubation with the corresponding secondary antibodies. Color development was performed using Pierce ECL Western Blotting Substrate (Thermo Scientific) and visualized using a Cheniluminescent Imager (MiniChemi, Sagecreation).

[0373]

[0418] The results showed that gremlin dose-dependently reduced the levels of BMP-induced p-smad1 / 5 / 9, suggesting that gremlin could indeed inhibit BMP signaling (Figure 6A).

[0374]

[0419] The ability of various antibodies provided herein to block Gremlin-mediated inhibition of BMP-induced p-smad1 / 5 / 9 was further evaluated. Gremlin was incubated with various concentrations of the antibodies provided herein at 37° C. for 30 minutes, and the mixture of Gremlin and antibodies was incubated with BMP4 at 37° C. for 30 minutes, and then added to plated PC3 cells. After 30 minutes, cells were lysed in RIPA buffer (CST) for Western blot analysis using the protocol described above.

[0375]

[0420] As shown in Figure 6B, the intensity of p-smad1 / 5 / 9 or pSMAD1 / 5 / 9 recovery from GREM1-mediated inhibition increased with increasing concentrations of anti-hGREM1 antibodies provided herein (e.g., 14E3, 22F1, 56C11, 69H5). This experiment demonstrated that these antibodies provided herein can dose-dependently modulate (e.g., reduce) Gremlin-mediated inhibition of BMP signaling.

[0376] Example 10: Characterization of differential reversal of Gremlin-mediated inhibition of BMP signaling by the anti-Gremlin 1 antibodies provided herein in various cell types.

[0377]

[0421] Since Gremlin is expressed in multiple physiological tissues and has effects on multiple cell types, we evaluated whether the anti-Gremlin 1 antibody provided herein has similar effects on various cell types that are responsive to Gremlin-mediated inhibition of BMP signaling.In brief, multiple cell types of different origins, including osteoblast ATDC-5, kidney fibroblast NRK49F, kidney epithelial HK2 cells and tumor PC3 cells, were stimulated with BMP4 or BPM4 together with Gremlin.Then, the cells stimulated with both BMP4 and Gremlin were supplemented with Gremlin antibody or control IgG 1 or 10 μg / ml.The antibodies tested here include 14E3 and benchmark antibody 6245P. As shown in FIG. 7, Gremlin can potently inhibit BMP4-induced pSMAD1 / 5 / 9, and the benchmark antibody 6245P can reverse Gremlin-mediated inhibition of BMP4-induced pSMAD1 / 5 / 9 in all cell types tested, whereas 14E3 only reverses Gremlin-mediated inhibition of BMP4 signaling in tumor cells such as PC3 cells, but not in other cell types (e.g., ATDC-5 osteoblasts, NRK-49F renal fibroblasts, HK-2 renal epithelial cells). This unexpected result suggests that the anti-Gremlin 1 antibodies provided herein (14E3, 22F1, 56C11, 42B9, 36F5, 67G11 and 69H5, Hu14E3, Hu22F1 and Hu56C11) are quite different in their biological activity from the benchmark antibodies, which is consistent with their differential activity using the ALP assay in ATDC-5 cells, as described in Example 8. Such selectivity towards reverting BMP signaling in cancer cells compared to non-cancer cells indicates that the anti-hGREM1 antibodies provided herein (e.g., 14E3, 22F1, 56C11, 42B9, 36F5, 67G11 and 69H5, Hu14E3, Hu22F1 and Hu56C11) can selectively affect cancer cells while having little toxicity to non-cancer cells.

[0378] Example 11: Cloning and sequencing of hybridoma antibodies

[0422] Four of the lead antibodies that exhibited desirable profiles were selected for gene cloning. The sequences of the variable regions of mouse anti-hGREM1 light and heavy chains were obtained by a polymerase chain reaction (PCR) amplification technique known as 5'RACE (rapid amplification of cDNA ends). Total RNA from gremlin antibody-producing hybridoma cells was isolated using Trizol (Invitrogen), and cDNA was synthesized using the Superscript first-strand synthesis system (Invitrogen) with Oligo(dT)12-18 primer. The variable regions of mouse IgG genes were cloned by PCR using MuIgG VH3'-2 and MuIg-5' leader primers for the heavy chain variable region and MuIgK VL3'-1 and MuIg-5" leader primers for the light chain variable region (NOVAGEN). The resulting bands were cloned into TOPO TA cloning vector and DNA from more than 10 clones was subjected to sequencing and determined using an ABI DNA sequencer (Perkin Elmer). The consensus sequence was determined using Vector NTI Advance 10 software (Invitrogen). After sequence analysis and confirmation, the variable regions of the gremlin genes were cloned into recombinant expression vectors (VL into pCP-mCK; VH into pCP-mCg2a) for antibody production and purification. The sequences of these hybridoma antibodies are shown in Table 10. Table 10 shows all sequences used in this application.

[0379]

[0423] The antibody was isotyped as mouse IgG2b. To facilitate secretion of the antibody, a signal peptide (MGWSCIILFLVATGVHS (SEQ ID NO: 65)) was fused at the N-terminus of the antibody.

[0380] Example 12: Expression and purification of recombinant antibody proteins in 293E6 cells.

[0381]

[0424] Expression and purification of recombinant antibody proteins were carried out as follows: 1 × 10 in Freestyle 293 Expression Medium containing 10% Pluronic F-68. 6 HEK293E cells cultured at 1000 cells / ml were transfected with equal amounts of heavy and light chain vector DNA at a final concentration of 0.5 μg / ml and PEI (polyethyleneimine-linear, Polyscience) at 1.0 μg / ml. The ratio of DNA to PEI was 1:2. The formation time of DNA and PEI complexes with Optimal MEM should be 15 min at room temperature. The transfected cells were cultured in flasks at 5% CO2, 37°C and a shaking speed of 125 rpm. 1% peptone medium was added 22-26 hours after transfection. The conditioned medium was harvested on day 6 and the supernatant was centrifuged at 3000 rpm for 30 min. The clarified conditioned medium was then loaded onto a nProteinA column (GE Healthcare), washed with PBS+0.1% Triton-X100, and finally the bound IgG was eluted with a 0.1 M glycine-containing solution at pH 3.5. The eluted antibody protein was dialyzed into PBS and stored at −80° C. To remove endotoxin, the purified protein was further processed by passing it through a Hitrap DEAE Sepharose FF column, and the resulting antibody was analyzed to determine the purity level using size exclusion chromatography (Superdex 200 5 / 150 GL, GE Healthcare).

[0382]

[0425] Binding analysis of recombinant chimeric anti-Gremlin 1 antibodies prepared according to the methods described above is shown in Figure 8, where antibodies 56C11-C (i.e., a chimeric antibody of 56C11) and 14E3-C (i.e., a chimeric antibody of 14E3) showed significantly higher binding affinity with lower EC50 values ​​(5.240 ng / ml for 14E3-C and 4.887 ng / ml for 56C11-C) compared to the benchmark antibody 6245P (115.2 ng / ml).

[0383] Example 13: Large-scale production of selected antibodies for in vivo studies

[0426] Each of these cloned antibodies was scaled up to produce large amounts of antibody for in vivo testing in renal failure models.

[0384]

[0427] The hybridoma cells are cultured in roller bottles containing in vitro production medium, and then the monoclonal antibodies produced in the conditioned medium will be processed and purified by Protein A affinity column with low endotoxin procedure.

[0385]

[0428] Briefly, hybridoma cells were harvested and expanded, the cells were adapted to grow in hybridoma production medium (DMEM + 2% Low IgG FBS) and inoculated into roller bottles containing 300 ml of culture medium each. The cells were then cultured in roller bottles for 2-3 weeks and the culture medium was harvested and clarified before purification. The produced mAb from the culture medium was then purified by Protein A affinity column, dialyzed against PBS (pH 7.4) and concentrated to 1.0 mg / ml or higher as required. The following parameters were measured for quality control: purity of the antibody product, endotoxin level, aggregation level as well as binding to the target antigen.

[0386]

[0429] Expected Product Specifications: a. Buffer solution: Phosphate buffered saline (PBS), pH 7.2-7.4, sterile filtered and preservative free.

[0387] b.Concentration: 1.0mg / ml or more c. Purity: 90% or more by SDS-PAGE and HPLC d. Aggregation rate: less than 10% by HPLC e. Endotoxin: 3EU / mg or less Example 14: Characterization of binding affinity measurements of selected Gremlin antibodies to captured hGREM1

[0430] The affinity of the antibodies was measured by Biacore T200 at 25°C. HGREM1 (Peprotech) was immobilized on a Biacore sensor chip. Kinetic experiments were performed using HBS-EP+ as both running and sample buffer. Antibody-antigen association rates were measured by injecting various concentrations of antibody (ranging from 12.5 to 400 nM, 2-fold dilutions) over the captured hGREM1 surface. Antibody-antigen association was monitored for 180 seconds, while dissociation in buffer was monitored for 360 seconds. Kinetic analysis was performed to determine Ka and Kd values ​​using Biacore T200 evaluation software. KD was calculated from the experimentally determined Ka and Kd values ​​as KD=Kd / Ka. As shown in FIG. 9, the recombinant chimeric anti-Gremlin 1 antibody 14E3 (14E3-C) has a KD value of 17.68 nM, and the chimeric anti-Gremlin 1 antibody 22F1 (22F1-C) has a KD value of 27.28 nM.

[0388] Example 15: Epitope studies

[0431] Epitope binning analysis by competitive ELISA assay

[0432] Epitope binning of anti-Gremlin hybridoma antibodies was performed using a competitive ELISA assay. Clear polystyrene plates (BEAVER) were coated with 100 μl / well of hGREM1 (ACRO) 0.5 μg / ml in high pH coating buffer overnight at 4° C. The plates were then washed once in an automated plate washer using PBS+0.1% Tween® 20 (Sigma). 100 μl of blocking solution consisting of PBS+1% BSA+1% normal goat serum+0.05% Tween® 20 (Sigma) was added to each well and incubated for 2 hours at room temperature. 50 μl / well of saturating antibody (hybridoma antibody) 20 μg / ml in antibody dilution buffer (PBS+1% BSA+1% normal goat serum+0.01% Tween® 20) was then added to each well of the plate and incubated for 1 hour. Then, 50 μl / well of 40ng / ml of competing antibody (chimeric antibody, e.g., 14E3-C, 22F1-C, 56C11-C, and 69H5-C) is added to each well of the plate and incubated for another hour at room temperature. They are then washed 3× with PBS+0.1% Tween® 20, followed by the addition of 100 μl of 1:10000 goat anti-human IgG-HRP (abcam) and incubation for 1 hour at room temperature. Finally, 100 μl / well of TMB (Pierce) is added to each well, and 5 minutes later, 50 μl of stop solution is added to each well. The plate is read at 450 nm on a plate reader. No binding of the second antibody in the presence of a saturating amount of the first antibody indicates that the two antibodies were in the same epitope bin; successful binding of the second antibody in the presence of a saturating amount of the first antibody indicates that the two antibodies were in different epitope bins. Based on the complete data set, there are multiple antibody bins, and an example of the data is shown in Figure 10A.

[0389]

[0433] As shown in Figure 10A, 14E3 and 22F1 competed with each other and were also able to block 69H5-C binding, indicating that these three antibodies were in one epitope bin. On the other hand, the binding of 56C11 to Gremlin was not competed by 14E3, 22F1 or 69H5, indicating that 56C11 was in an epitope bin different from those of 14E3, 22F1 and 69H5. This result reiterates the data shown in Figure 1, where 56C11 was able to bind mouse Gremlin, whereas 14E3, 22F1 and 69H5 were unable to bind.

[0390]

[0434] Cross-competition experiments were also performed to analyze whether 14E3 / 22F1 binds to an epitope different from that of the benchmark antibody 6245P. Briefly, a fixed amount of either the chimeric antibody 14E3-C / 22F1-C or 6245P was added, followed by increasing amounts of the hybridoma antibodies 14E3 and 22F1. The amount of chimeric antibody bound to Gremlin was determined. As shown in Figure 10B, 14E3 and 22F1 competed with each other but could not compete with 6245P binding even at 100-fold excess, indicating that 14E3-C and 22F1-C bind to epitopes different from that of 6245P.

[0391]

[0435] Epitope mapping of 14E3

[0436] The sequences of human and mouse gremlin were aligned, and only two different amino acids were observed: Q27 (human)-P27 (mouse) and N33 (human)-T33 (mouse), where the numbering is according to SEQ ID NO: 69 and SEQ ID NO: 70, respectively. 14E3 binds to human gremlin but not to mouse gremlin, and therefore we speculate that these two different amino acids may be key amino acids that affect antibody binding. To verify the two key residues for 14E3 binding, four variants with signal peptide (SEQ ID NO: 71) and C-terminal His-tag, including human gremlin_WT, human gremlin_Q27P, human gremlinN33T and human gremlin_Q27P / N33T, were cloned into pcDNA3.1(+) vector, and all of the constructs were confirmed by DNA sequencing. The plasmids of the four constructs purified by using QIAGEN Plasmid Midi Kit were transfected into ExpiCHO cells at 3ml scale by using ExpiCHO transfection kit. The transfected cells were cultured in shake flasks at 125 rpm in an incubator at 8% CO2 and 37°C. The cell cultures were harvested on day 4 and centrifuged at 8000 rpm for 30 min, then the supernatants were used for antibody binding assays.

[0392]

[0437] Human (Huam) Gremlin 1 sequence without the signal peptide: KKKGSQGAIPPPDKAQHNDSEQTQSP Q QPGSR N RGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD (SEQ ID NO: 69)

[0438] Mouse Gremlin 1 sequence without the signal peptide: KKKGSQGAIPPPDKAQHNDSEQTQSP PQPGSR T RGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD (SEQ ID NO: 70)

[0439] Note: The two amino acids that differ between human and mouse gremlin are in bold and underlined. The signal peptide sequence of human gremlin 1 is set forth in SEQ ID NO:71, and the signal peptide sequence of mouse gremlin 1 is set forth in SEQ ID NO:72.

[0393]

[0440] Biolayer Interference (BLI) Assay

[0441] Human Gremlin Abs, 14E3 and 6245p, were diluted in kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.2% Tween®-20) to obtain a concentration of 100 nM at 100 ul per well in the Loading Column of a 96-well half-area Microplate (Greiner Bio-one). Supernatants of Gremlin WT and mutants to be tested were added to the Association Column of the plate at 100 ul per well. Media or KD buffer was used as a reference control. The AHC sensor was placed in the first Baseline Column for 60 seconds to obtain a first baseline, then in the Loading Column for 300 seconds to capture the Gremlin antibody. The sensor was then placed in the second Baseline Column for 60 seconds to obtain a second baseline. The sensor was then placed in the Association Column for 300 seconds to allow complete association of Gremlin / Gremlin Ab, and the sensor was placed in the Dissociation Column for 300 seconds. Analyze group data using ForteBio (Octet96).

[0394]

[0442] Figure 10D shows that 14E3 can still bind to human gremlin 1 variants with Asn33 substituted by Thr (i.e., N33T), but the binding was partially reduced. However, the substitution of Gln27 by Pro (i.e., Q27P) in human gremlin 1 significantly reduces 14E3 binding, but this reduction was also observed in human gremlin 1 variants with both N33T and Q27P substitutions. In contrast, neither the single mutation of N33T nor the single mutation of Q27P in human gremlin 1 significantly reduced the binding of 6245P, indicating that 6245P binds to an epitope that does not contain N33 or Q27 in human gremlin 1. This was consistent with the binding results observed in human gremlin 1 variants with both N33T and Q27P mutations. These data suggest that 14E3 binds to an epitope that includes Q27 of SEQ ID NO:69. The epitope bound by 14E3 may include, to a lesser extent, N33 of SEQ ID NO:69.

[0395] Example 16: Binding to Gremlin-DAN fusion protein XM5

[0443] To further demonstrate that 14E3 binds to an epitope distinct from that of 6245P, we assessed the binding of these antibodies to either wild-type Gremlin protein or XM5, as described in Example 6.

[0396]

[0444] Using a similar ELISA protocol described above, we tested the binding of chimeric antibody 14E3 to XM5 or Gremlin. Briefly, 14E3-C or 6245P were coated (1 μg / ml) and serial dilutions of XM5-his supernatant or Gremlin-his (2 μg / ml to 0.49 μg / ml) were added for binding. Secondary antibody anti-his-HRP was used for detection. As shown in Figure 10C, both 14E3-C and 6245P were able to bind to Gremlin. However, only 14E3-C could bind to XM5, whereas 6245P could not. This indicates that 14E3-C and 6245P bind to different epitopes, which is consistent with the epitope binning results described above.

[0397]

[0445] Binding affinities to hGREM1 and XM5 were also compared. Briefly, XM5-Fc or Gremlin-his (1 μg / ml) were coated. 100 μl of blocking solution consisting of PBS+1% BSA+1% normal goat serum+0.5% Tween® 20 (Sigma) was added to each well and incubated for 2 hours at room temperature. 4-fold serial antibody dilutions from 2 μg / ml were added. Afterwards, the plates were washed 3 times with 200 ul of PBS+0.1% Tween® 20, followed by the addition of 100 μl / well of 1:10000 goat anti-mouse IgG-HRP (abcam) and incubation for 1 hour at room temperature. They were then washed 3× with PBS+0.1% Tween® 20. Finally, 100 μl / well of TMB (InnoReagents) was added to each well, and 50 ul of stop solution was added to each well after 2 minutes. Plates were read at 450 nM on a Multiscan FC microplate reader (Thermo Scientific). EC50 values ​​are tabulated below.

[0398]

[0446] As shown in Figures 10E and 10F, 42B9, 36F5 and 67G11 bind to hGREM1 with EC50 values ​​ranging from 0.012 ng / ml to 0.015 ng / ml and bind to XM5 with EC50 values ​​ranging from 0.006 ng / ml to 0.008 ng / ml.

[0399] Example 17: Epitope analysis by Fortebio

[0447] The epitope analysis of the anti-gremlin 1 antibody provided herein was further carried out by Fortebio. Briefly, the first anti-gremlin 1 antibody (first Ab) was diluted with kinetic buffer (PBS) at 250 μl / well in the Loading Column of a Microplate (Greiner Bio-one). hGremlin-his was in kinetic buffer at 250 μl / well in the Association Column of the plate; the AHC sensor was placed in the first Baseline Column for 60 seconds to obtain a first baseline, then in the Loading Column for 300 seconds to capture the first anti-gremlin 1 antibody. The sensor was then placed in the second Baseline Column for 180 seconds to obtain a second baseline, then in the Association Column for 300 seconds to allow gremlin to fully associate with the first anti-gremlin 1 antibody. The sensor was placed in a column of a second anti-Gremlin 1 antibody (second Ab) for 300 seconds to allow the second anti-Gremlin 1 antibody to compete or not compete with the first Ab. Data were analyzed by ForteBio (Octet96).

[0400]

[0448] If the second anti-gremlin 1 antibody cannot bind to gremlin, this indicates that the second anti-gremlin 1 antibody binds to the same epitope as the first anti-gremlin 1 antibody; if the second anti-gremlin 1 antibody can bind to gremlin without being affected by the first anti-gremlin 1 antibody, this indicates that the epitopes of the antibodies are separate. As shown in Figure 11A, 6245P as the second anti-gremlin 1 antibody was still able to bind to gremlin, indicating that 6245P could not compete with 14E3 but binds to a different epitope; on the other hand, 22F1 / 69H5 could not bind to gremlin in the presence of 14E3, indicating that 22F1 / 69H5 and 14E3 may bind to the same epitope site. Conversely, 6245P, as the first anti-Gremlin 1 antibody, was unable to block 14E3 / 22F1 binding to the antigen, but completely blocked 56C11 / 69H5 binding (Figure 11B). Thus, depending on the type of epitope, there are likely three groups: one group includes 14E3 and 22F1, one group includes 56C11 and 69H5, and these epitopes may overlap with those of 6245P (Table 6).

[0401]

[0449]

[0402] [Table 7]

[0403] Example 18: Humanization of anti-GREM1 antibodies

[0450] Humanized 14E3

[0451] A humanized antibody of 14E3 was designed using three-dimensional structure simulation and humanization by CDR grafting with the following protocol.

[0404]

[0452] The first step in antibody humanization is the simulation of the three-dimensional structure of the variable domain of 14E3. The sequences of each variable domain (Vk and Vh) of the mouse antibody were BLAST searched against the PDB database (Protein Data Bank, http: / / www.rcsb.org / ) to identify the most homologous antibody sequence with known high-resolution structure. The structural template selected to model 14E3 had the highest similarity to the target antibody. We manually modified each residue of the structure to fit the target sequence. Certain side chain conformations were adjusted while the main chain conformation was maintained. At positions where the parent and simulated structures have the same residues, the side chain conformation remains unchanged; if the residues differ between the template and modeled structures at some positions, the side chain conformation is mutated and refined according to the template structure and packaging considerations.

[0405]

[0453] We also simulated the structure of CDR-grafted 14E3 to guide the design of back mutations and the evaluation of the developability and stability of the humanized antibody. Structural simulations were also performed in a similar manner.

[0406]

[0454] Humanization was performed by CDR grafting. After BLAST searching the sequence of mouse 14E3 in the IMGT human immunoglobulin gene database, human germline framework sequences IGHV / 7-4 for the heavy chain and IGKV / 2-30 for the light chain, respectively, were used for CDR grafting to obtain humanized 14E3 without back mutations. To further maintain the activity of humanized 14E3, we aligned the framework sequence of the humanized antibody with that of its corresponding mouse antibody. Various residues were double-checked with the mouse antibody structure model: if any of them was present in a position that could interact with and affect the CDR residues, it should be back mutated to the mouse residue. The present disclosure obtained three humanized heavy chain variable regions with different reverse mutations, labeled Hu14E3_Ha VH, Hu14E3_Hb VH and Hu14E3_Hc VH, and two humanized light chain variable regions with different reverse mutations, labeled Hu14E3-La V and Hu14E3-Lb VL, respectively (see Table 5). The cDNAs of these humanized heavy and light chain variable regions were fused to the constant regions of hIgG1 and hKappa and inserted into a mammalian vector. Each humanized heavy chain was co-expressed with a humanized light chain to obtain six versions of humanized antibodies, namely Hu14E3_HaLa, Hu14E3_HaLb, Hu14E3_HbLa, Hu14E3_HbLb, Hu14E3_HcLa and Hu14E3_HcLb. The expression and purification procedures are similar to those of chimeric antibodies.

[0407]

[0455] Humanized 22F1

[0456] The humanized antibody of 22F1 was designed by a similar protocol. Briefly, human germline framework sequences IGHV / 1-46 for heavy chain and IGKV / 2-30 for light chain were used for CDR grafting, respectively, and then computer modeling was used to design humanized variants with CDR grafting and back mutations. The present disclosure obtained four humanized heavy chain variable regions with back mutations, namely Hu22F1_Ha VH, Hu22F1_Hb VH, Hu22F1_Hc VH and Hu22F1_Hd VH, and two light chain variable regions with different back mutations, namely Hu22F1-La VL and Hu22F1-Lb VL. (See Table 5). Each humanized heavy chain was co-expressed with the humanized light chain to obtain eight versions of humanized antibodies for 22F1, namely Hu22F1_HaLa, Hu22F1_HaLb, Hu22F1_HbLa, Hu22F1_HbLb, Hu22F1_HcLa, Hu22F1_HcLb, Hu22F1_HdLa and Hu22F1_HdLb. The expression and purification procedures are similar to those of the chimeric antibody.

[0408]

[0457] Expression and purification of recombinant antibody protein was carried out by the following steps: ExpiCHO cells were cultured at 5-6 × 10 6The cells were seeded in ExpiCHO Expression Medium at 1.0 cells / ml. ExpiCHO cells were then transfected with equal amounts of heavy and light chain vector DNA at a final concentration of 1.0 μg / ml using the ExpiCHO transfection kit. The transfected cells were cultured in shake flasks at 125 rpm in a 37°C incubator supplemented with 8% CO2. ExpiCHO feed was added 18-22 hours after transfection. The cell culture was harvested on day 10. Harvested cell culture fluid (HCCF) was obtained by centrifugation. The HCCF was then loaded onto an rProteinA column (GE Healthcare) and washed with PBS. The final IgG antibody was eluted using a solution containing 20 mM citric acid at pH 3.2. Finally, the eluted antibody protein was neutralized and stored at -80°C for long-term use. The resulting antibodies were analyzed to determine purity levels using SDS-PAGE and size exclusion chromatography (TSKgel G3000SWXL, TOSOH).

[0409]

[0458] Humanized 56C11

[0459] The humanized antibody of 56C11 was designed with a similar protocol. Briefly, human germline framework sequences IGHV1-2*02 for the heavy chain and IGKV2-30*02 for the light chain, respectively, were used for CDR grafting.

[0410]

[0460] Heavy chain (HC) variants 1, 2, 3, and 4 were obtained by directly grafting three CDRs into the germline sequence. The combination of the heavy and light chain variable regions described above produces the following humanized 56C11 antibodies: 56C11-H0L0, 56C11-HaL0, 56C11-HbL0, 56C11-HcL0, 56C11-H0La, 56C11-HaLa, 56C11-HbLa, 56C11-HcLa, 56C11-H0Lb, 56C11-HaLb, 56C11-HbLb, 56C11-HcLb.

[0411]

[0461] The humanized variants of the heavy and light chains of 56C11 are linked to the human IgG1 heavy chain constant region and the kappa light chain constant region as shown below:

[0462] Human IgG1 heavy chain constant region (SEQ ID NO: 138):

[0463] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0464] Human Kappa Light Chain Constant Region (SEQ ID NO:139):

[0465] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0466] The variable regions of the heavy and light chain cDNAs mentioned above were synthesized and fused with the constant regions of human IgG1 and human kappa. The heavy and light chains of the selected antibody genes were cloned into expression vectors, and large-scale DNA was prepared using the Plasmid Maxiprep System from Qiagen. Transfection was performed using ExpiFectamine™ CHO Reagent from Invitrogen according to the manufacturer's protocol. The supernatant was harvested when the cell viability was approximately 60%. The cell culture supernatant was filtered through a 0.22 μm filtration capsule to remove cell debris. The supernatant was loaded onto a pre-equilibrated Protein A affinity column. The Protein A resin inside the column was then washed with equilibration buffer (PBS), and the antibody was eluted using 25 mM citrate (pH 3.5). The pH was adjusted to approximately 6.0-7.0 using 1 M Tris base (pH 9.0). Endotoxin was controlled to less than 1 EU / mg. The purified antibodies were then characterized by SDS-PAGE and SEC-HPLC.

[0412] Example 19: Binding of humanized antibodies to hGremlin in ELISA and Fortebio

[0467] Same protocol as in Example 4. As shown in Figure 12A, the humanized variants of 14E3 retained similar binding activity as chimeric 14E3 (e.g., 14E3 hIgG1 or 14E3-C), indicating that biological activity was probably not affected by humanization. However, most of the humanized variants of 22F1 lost binding significantly, and only 22F1-HdLa and 22F1-HdLb still had good affinity (Figures 12B and C).

[0413]

[0468] The affinity of the humanized antibodies was also measured by Fortebio. Human Gremlin protein was diluted in kinetic buffer to obtain a concentration of 2 μg / ml. 0 nM was used as a reference control. Antibodies to be tested were diluted to concentrations of 100 nM, 50 nM, and 25 nM in ForteBio kinetic buffer (PBS pH 7.4, 0.1% BSA + 0.002% Tween®-20). Human Gremlin-his was immobilized onto an NTA biosensor. The baseline was detected for 60 seconds, and then the association of the anti-Gremlin antibody was detected for 120 seconds to obtain the K on This was followed by 90 s dissociation in kinetic buffer to obtain K off Factor data was obtained. As shown in Figure 12D, the humanized anti-Gremlin 1 antibody 14E3 has a KD value of less than 1 nM, much lower than the KD values ​​of the benchmark antibodies.

[0414] Example 20: Tumor growth inhibitory activity of humanized antibody 14E3 in PC-3 xenograft tumor model

[0469] Briefly, human prostate cancer PC3 cells were maintained in vitro as monolayer cultures in RPMI1640 medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), penicillin 100U / ml, streptomycin (Hyclone) 100ug / ml, and puromycin (Gibco) 1ug / mL at 37°C in an atmosphere of 5% CO2 in air. Tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment (Hyclone). Cells growing in logarithmic growth phase were harvested and counted for tumor inoculation. SPF grade male nude mice were subcutaneously inoculated with 1*10^6 mixed PC3 cells containing 50% Matrigel. After 10 days, inoculated mice were castrated by orchiectomy. When tumor size reached approximately 200mm^3, tumor-bearing mice were selected and randomized into two groups (n=8). Animals were injected intraperitoneally with hIgG1 control 10mg / kg and Hu14E3_HaLa 10mg / kg every 4 days for 3 weeks. Tumor size was measured every 4 days in two dimensions using calipers (INSIZE) and the volume was expressed in mm^3 using the formula: V=0.5a*b^2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between the two groups were performed by T-test, with p < 0.05. * <0.05 and ** Differences were considered significant if <0.01. As a result, Hu14E3_HaLa was able to effectively inhibit tumor growth by either volumetric or weight assessment (FIGS. 13A and 13B).

[0415] Example 21: Efficacy of 56C11 against the CT-26 tumor model in BALB / c (MSB-Pharm2018025)

[0470] Since Gremlin 1 can be expressed in both tumor cells and stromal fibroblasts, we evaluated the contribution of Gremlin 1 in modulating the tumor microenvironment and its potential in modulating tumor growth either alone or in combination with checkpoint inhibitors. Therefore, we evaluated the antitumor activity of 56C11 against the CT-26 model, a syngeneic tumor model. Briefly, 24 female Balb / c mice aged 5-6 weeks were inoculated with 2 × 10 6 CT26 tumor cells were inoculated into the tumor. The tumor volume was approximately 100 mm 3 When the tumor reached 100 mg / kg, the animals were randomized and grouped as follows: 1) isotype control at 20 mg / kg, and 2) 56C11 at 20 mg / kg. Antibodies were injected intraperitoneally (IP) twice a week for 2 weeks. The results shown in FIG. 14 demonstrated that the anti-GREM1 antibody 56C11 (which is cross-reactive with mouse GREM1) alone can have significant anti-tumor activity without significant effect on total body weight. The humanized anti-GREM1 antibodies provided herein (e.g., Hu14E3, Hu14E3, Hu22F1 and Hu56C11) are expected to show similar technical efficacy in humans as their chimeric counterparts in exhibiting significant anti-tumor activity without significant effect on total body weight.

[0416] Example 22: Efficacy of combination therapy of MPDL-3820A and anti-GREM1 antibody against CT26 tumor model (MSB-Pharm2018004)

[0471] To further test the antitumor efficacy of the combination of anti-GREM1 antibodies with immune checkpoint inhibitors, we evaluated the antitumor activity of combining MPDL-3280A with a surrogate anti-mouse GREM1 antibody (anti-mGREM1 antibody) in the CT26 tumor model. Briefly, 5-6 week-old female Balb / c mice were inoculated with 5 × 10 CT26 cells. 5 Animals were randomized. Tumor volumes were approximately 100 mm 3When the total number of tumor cells reached 100, the mice were divided into 4 groups treated with 1) control IgG1 alone at 10mpk, 2) MPDL3280A alone at 3mpk, 3) anti-mGREM1 antibody alone at 10mpk, or 4) a combination of MPDL3280a at 3mpk and anti-mGREM1 antibody at 10mpk by IP twice a week for 2 weeks. As shown in Figure 15A and Figure 15B, the combination of anti-mGREM1 antibody with immune checkpoint inhibitor (e.g., antibody against PD-L1) resulted in significantly higher anti-tumor activity (in terms of either tumor volume or tumor weight) compared with anti-mGREM1 antibody alone or immune checkpoint inhibitor alone. This suggests that anti-mGREM1 antibody can enhance the anti-tumor activity of immune checkpoint inhibitor.

[0417] Example 23: Efficacy of humanized 14E3 in combination with cisplatin on esophageal cancer PDX models

[0472] Human Gremlin IHC specific positive esophageal tumor tissue (E7) was obtained from the Beijing Cancer Hospital's passage and established PDX bank of NOD / SCID mice. We tested GREM1 expression and PD-L1 expression in the E7 esophageal PDX model by immunohistochemistry using either anti-GREM1 antibody (14E3) or anti-PD-L1 antibody (22C3). Figure 16 shows that the esophageal cancer PDX model E7 was positive for GREM1 expression but did not have PD-L1 expression.

[0418]

[0473] Each mouse was subcutaneously inoculated with a small tumor tissue mass approximately 3 mm in diameter that was sheared from an integral tumor skinned wound from a tumor-bearing mouse. 18 days after inoculation, approximately 70 mm 3Animals with tumor sizes of 0.01 mm were selected and randomly divided into 4 groups, each consisting of 8 mice. Mice were then treated with isotype control + PBS, humanized 14E3 (hzd14E3) at a dose of 20 mg / kg, cisplatin at a dose of 3 mg / kg, and a combination of hzd14E3 and cisplatin. Isotype control and hzd14E3 were administered by ip injection and PBS twice a week for 4 weeks, while cisplatin was administered by iv injection once a week for 4 weeks. Animals were sacrificed by CO2 inhalation at the end of the study. Tumor size was measured in two dimensions using a caliper (INSIZE) two or three times a week, and the volume was expressed in mm^3 using the formula: V=0.5a*b2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparison between the two groups was performed using the T-test, with p<0.05. * <0.05 and ** Differences are considered significant if <0.01.

[0419]

[0474] Figure 17A and Figure 17B show the significantly enhanced tumor growth inhibition when humanized 14E3 alone is used in this experiment compared to the isotype control with a TGI of 42.92%. The combination of humanized 14E3 and cisplatin further inhibited tumor growth when compared to either humanized 14E3 alone (63.97% TGI vs. 42.92% TGI) or cisplatin alone (63.97% TGI vs. 59.79% TGI), suggesting the synergistic effect of the combination treatment of humanized 14E3 and cisplatin on esophageal cancer.

[0420]

[0475] To date, first-line therapy for esophageal cancer generally includes esophagectomy, chemotherapy, targeted therapy, immunotherapy (e.g., targeting PD-1 or PD-L1), and / or combinations thereof. Second-line and subsequent therapy for esophageal cancer may involve targeted therapy, such as ramucirumab targeting vascular endothelial growth factor (VEGF) receptors or trastuzumab for metastatic adenocarcinomas overexpressing HER2 (NCCN Clinical Practice Guidelines in Oncology. Esophageal and Esophagogastric Junction Cancers. National Comprehensive Cancer Network. V1.2020). The inventors' data above indicates that the anti-GREM1 antibody provided herein can effectively treat tumors that do not express PD-L1, such as esophageal cancers that do not overexpress PD-L1, and can further achieve synergistic effects when combined with chemotherapy, such as cisplatin. This suggests that the anti-GREM1 antibody provided herein can serve as a new option for either first-line or second-line therapy for esophageal cancer.

[0421] Example 24: Efficacy of hzd14E3 (in combination with DC101) and 6245P on esophageal cancer PDX models

[0476] Human gremlin IHC-specific positive esophageal tumor tissues (E7) were obtained from the passaged and established PDX bank of the Beijing Cancer Hospital in NOD / SCID mice. Each mouse was subcutaneously inoculated with a small tumor tissue mass of about 3 mm in diameter that was sheared from an integral tumor-denuded wound from a tumor-bearing mouse. 18 days after inoculation, tumors of about 70 mm in diameter were observed. 3Animals with tumor sizes of 0.01 to 0.01 mm were selected and randomly divided into 4 groups, each consisting of 8 mice. Mice were then treated with isotype control, hzd14E3 and 6245P at a dose of 20 mg / kg, DC101 at a dose of 10 mg / kg, and a combination of hzd14E3 and DC101. DC101 is a monoclonal antibody that reacts with mouse VEGFR-2 and is commercially available (e.g., under catalog number BE0060 from BioXell). Controls and test substances were administered by ip injection twice a week for 4 weeks. Animals were sacrificed by CO2 inhalation at the end of the study. Tumor size was measured twice or thrice a week in two dimensions using a caliper (INSIZE) and calculated according to the formula: V=0.5a*b 2 Volumes were expressed in mm^3 using the formula: (where a and b are the long and short diameters of the tumor, respectively). Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test, with p < 0.05. * <0.05 and ** The difference is considered significant if it is <0.01. Other humanized anti-GREM1 antibodies provided herein (e.g., Hu22F1 and Hu56C11) are expected to show similar technical effects.

[0422] Example 25: Characterization of antibody activity in blocking Gremlin binding to captured FGFR1

[0477] Gremlin-his was tested for its binding ability to FGFR1 immobilized on a plate. Briefly, a plate was coated with recombinant human FGFR1-Fc (Sino-Biological) at 2 μg / ml overnight, and then two-fold serial dilutions of Gremlin-his (ACRO) from 2 μg / ml were added to the coated plate and incubated at RT (room temperature) for 1 hour. The plate was then washed, and plate-bound Gremlin-his was detected using anti-his HRP (GenScript). The plate was then developed with TMB solution and stopped by adding stop solution. The plate was read at 450 nm in a plate reader. The incubation time was about 20 minutes. As shown in FIG. 18A, hGREM1 can bind to FGFR1.

[0423]

[0478] Gremlin 0.25μg / ml was then selected to test blocking activity. The ability of the antibodies to block gremlin binding to human FGFR1-Fc was examined via ELISA. Plates were coated overnight with recombinant FGFR1-Fc (2μg / ml), and then serial dilutions of the antibodies were incubated with modified human gremlin-his 0.25μg / ml for 1 hour at RT, after which the complex was added to the coated plate and incubated for an additional hour at RT. The plate was then washed and anti-hisHRP (GenScript) was added to the plate. The plate was then developed with TMB solution and stopped by adding stop solution. The plate was read at 450nm on a plate reader. As shown in Figures 18B and 18C, the anti-Gremlin 1 antibodies provided herein (e.g., 42B9, 36F5, 67G11 and 14E3 HaLa, chimeric antibody 69H5 (69H5-chi), chimeric antibody 36F5 (36F5-chi), chimeric antibody 22F1 (22F1-chi)) can inhibit or block the binding of hGREM1 to FGFR1, whereas the benchmark antibody 6245P does not block the binding of hGREM1 to FGFR1. 36F5-chi was able to block the binding of hGREM1 to FGFR1 with an IC50 of 1.368 nM. 69H5-chi and 22F1-chi had partial blocking activity, where 69H5-chi was able to block hGREM1 binding to FGFR1 with an IC50 of 7.138 nM, and 22F1-chi was able to block hGREM1 binding to FGFR1 with an IC50 of 5.117 nM. The humanized anti-GREM1 antibodies provided herein (e.g., Hu14E3, Hu22F1) are expected to show similar technical efficacy as their chimeric counterparts in blocking hGREM1 binding to FGFR1.

[0424] Example 26: Binding analysis of purified hybridoma or chimeric anti-gremlin antibodies to captured human gremlin and DAN proteins by ELISA

[0479] Clear polystyrene plates (BEAVER) were coated with 100 μl / well of human Gremlin (ACRO) and DAN (Sinobiological) 0.5 μg / ml in high pH coating buffer overnight at 4° C. Plates were then washed once in an automated plate washer using PBS+0.1% Tween® 20 (Sigma). 100 μl of blocking solution consisting of PBS+1% BSA+1% normal goat serum+0.5% Tween® 20 (Sigma) was added to each well and incubated for 2 hours at room temperature. Then, 100ul of antibodies (hybridoma or chimeric antibody 36F5, chimeric antibody 67G11, chimeric antibody 42B9) in antibody dilution buffer containing PBS+1% BSA+1% normal goat serum+0.01% Tween® 20, starting at 2μg / ml (13.33nM), followed by 4-fold dilutions, were added to each well of the plate and incubated at room temperature for 1 hour. Afterwards, the plate was washed 3 times with 200ul of PBS+0.1% Tween® 20, followed by the addition of 100μl / well of 1:10000 goat anti-mouse IgG-HRP or mouse anti-human IgG-HRP (abcam) and incubated at room temperature for 1 hour. Then, they were washed 3× with PBS+0.1% Tween® 20. Finally, 100μl / well of TMB (InnoReagents) was added to each well, and 50ul of stop solution was added to each well after 2 minutes. Plates were read at 450 nM on a Multiscan FC microplate reader (Thermo Scientific). EC50 values ​​are also shown below in Figures 19A, 19B, and 19C. As a result, hybridoma or chimeric antibody 36F5, chimeric antibody 67G11, and chimeric antibody 42B9 had similar binding activity of Gremlin with DAN protein.

[0425] Example 27: Characterization of antibody activity in blocking DNA protein binding to captured BMP2 / 4

[0480] Plates were coated overnight with recombinant human BMP2 / 4 (0.5 μg / ml). Plates were then washed once with an automatic plate washer using PBS+0.1% Tween® 20 (Sigma). 100 μl of blocking solution consisting of PBS+1% BSA+1% normal goat serum+0.5% Tween® 20 (Sigma) was added to each well and incubated for 2 hours at room temperature. Plates were then washed 3 times. 55 ul of serial dilutions of chimeric antibody 36F5 were then mixed separately with 55 ul of dilution buffer containing 0.5 μg / ml of human DAN-his in PBS+1% BSA+1% normal goat serum+0.01% Tween® 20 and incubated for 1 hour at RT, after which 100 ul of this complex was added to the coated plate and incubated for another hour at room temperature. The plate was then washed three times and 100ul of anti-hisHRP (GenScript) dilution buffer was added to the plate. The plate was then developed with TMB solution and stopped by adding stop solution. After washing three times with wash buffer, the plate was read at 450nm in a plate reader. Also, consistent with previous results, 36F5 could block BMP2 / 4 binding to DAN protein in addition to blocking Gremlin activity. (Figures 20A and 20B).

[0426] Example 28: Efficacy of hybridoma 36F5 against EMT6 / hPD-L1 tumor model

[0481] The mouse breast cancer cell line EMT6, transfected with human PD-L1 gene and screened for stable expression of human PD-L1, is named EMT6 / hPD-L1. EMT6 / hPD-L1 cells were maintained in vitro as monolayer cultures at 37°C in an atmosphere of 5% CO2 in air in DMEM medium (Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100U / ml penicillin, and 100ug / ml streptomycin (Hyclone). Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment (Hyclone). Cells growing in logarithmic growth phase were harvested and counted for tumor inoculation. SPF grade male BABL / c mice were inoculated with mixed 2*10^6 EMT6 / hPD-L1 cells containing 50% Matrigel. In the first study, tumor-bearing mice were selected when tumor size was approximately 80mm^3 and randomized into two groups (n=10). Animals were treated with hIgG1 control 24.9mg / kg and AM4B6 24.9mg / kg by ip injection twice a week for 4 weeks. In the second study, tumor-bearing mice were selected when tumor size was approximately 70mm^3 and randomized into two groups (n=8). Animals were treated with hIgG1 control 10mg / kg and 36F5 10mg / kg by ip injection twice a week for 3 weeks. Tumor size was measured twice a week in two dimensions using a caliper (INSIZE) and volume was expressed in mm^3 using the formula: V=0.5a*b^2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparison between the two groups was performed using the T-test, with p<0.05. * <0.05 and **Differences are considered significant when <0.01. Table 7 and Figure 21A were the results of the first study. The results show that anti-PD-L1 antibody (AM4B6) had no anti-tumor activity against the EMT6 / hPD-L1 tumor model. The EMT6 / hPD-L1 tumor model exhibits poor response to PD-L1 antibody. Table 8 and Figure 21B were the results of the second study. The results show that anti-Gemlin1 antibody (36F5) has promising anti-tumor activity against the EMT6 / hPD-L1 tumor model exhibits poor response to PD-L1 antibody.

[0427]

[0482]

[0428] [Table 8]

[0429]

[0483]

[0430] [Table 9]

[0431] Example 29: Efficacy of hybridoma 14E3, hybridoma 36F5 or nivolumab against the E7 tumor model in PBMC-humanized mice

[0484] E7 is a human gremlin-high expression esophageal cancer PDX obtained from the passaged and established PDX bank of Beijing Cancer Hospital in NOD / SCID mice. NOG mice were severely immunodeficient purchased from Vital River. Each mouse was subcutaneously inoculated with a small tumor tissue mass of about 3 mm in diameter sheared from an integral tumor-denuded wound from a tumor-bearing mouse (moue). 27 days after inoculation, animals with tumor sizes of about 50 mm^3 were selected and intravenously injected with human PBMCs 5x10^6 / mouse. One week later, animals were screened for remodeling profile and randomized into 6 groups, each consisting of 8 mice. Animals were treated with isotype control 30mg / kg, 14E3 30mg / kg, 36F5 30mg / kg, and nivolumab 10mg / kg by ip injection twice a week for 5 weeks. Tumor size was measured twice weekly in two dimensions using calipers (INSIZE) and the volume was expressed in mm^3 using the formula: V=0.5a*b^2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test, with p < 0.05. * <0.05 and ** Difference is considered significant when it is <0.01. Table 9, Figure 22A and Figure 22B were the results of the study. The results show that anti-PD-1 antibody (Nivolumab) did not have anti-tumor activity against E7 tumor model. Anti-Gemlin1 antibody 36F5 and 14E3 have promising anti-tumor activity against E7 tumor model, which shows poor response to PD-1 antibody.

[0432]

[0485]

[0433] [Table 10]

[0434] Example 30: Efficacy of 56C11 combination therapy with anti-PDL1 antibody on MC38 / hPD-L1 tumor model

[0486] The mouse colon cancer cell line MC38, transfected with human PD-L1 gene and screened for stable expression of human PD-L1, is named MC38 / hPD-L1. MC38 / hPD-L1 cells were maintained in vitro as monolayer cultures at 37℃ in an atmosphere of 5% CO2 in air in 1640 medium (Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100U / ml penicillin, and 100ug / ml streptomycin (Hyclone). Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment (Hyclone). Cells growing in logarithmic growth phase were harvested and counted for tumor inoculation. SPF grade female C57BL / 6 mice were inoculated with mixed 2*10^6 MC38 / hPD-L1 cells containing 50% Matrigel. Tumor-bearing mice were selected when tumor size was approximately 120 mm^3 and randomized into 4 groups (n=8). Animals were treated with hIgG1 control 3 mg / kg, 56C11 20 mg / kg, 23F11 (anti-PDL1 antibody) 3 mg / kg, and a combination of 56C11 20 mg / kg and 23F11 3 mg / kg by ip injection twice a week for 3 weeks. Tumor size was measured twice a week in two dimensions using calipers (INSIZE) and the volume was expressed in mm^3 using the formula: V=0.5a*b^2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparisons between two groups were performed by T-test, with p < 0.05. * <0.05 and ** Differences are considered significant if <0.01. Combination of 56C11 with anti-PDL1 antibodies enhances anti-tumor activity against the MC38 / hPD-L1 tumor model (Table 10 and Figure 23).

[0435]

[0487]

[0436] [Table 11]

[0437]

[0488]

[0438]

Table 12-1

[0439]

Table 12-2

[0440]

Table 12-3

[0441]

Table 12-4

[0442]

Table 12-5

[0443]

Table 12-6

[0444]

Table 12-7

[0445]

Table 12-8

[0446]

Table 12-9

[0447]

Table 12-10

[0448]

Table 12-11

[0449]

Table 12-12

[0450]

Table 12-13

[0451]

Table 12-14

[0452]

Table 12-15

[0453]

Table 12-16

Claims

1. An isolated 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: 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; 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; e) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 114, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 116; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 117, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; f) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 121, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; g) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 122, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; h) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 123, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 124; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 125, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; i) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 114, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 116; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 117, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; j) the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 121, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO: 118; or k) an antibody or antigen-binding fragment thereof, wherein the heavy chain variable region comprises an HCDR1 comprising the sequence of SEQ ID NO: 119, an HCDR2 comprising the sequence of SEQ ID NO: 115, and an HCDR3 comprising the sequence of SEQ ID NO: 120; and the light chain variable region comprises an LCDR1 comprising the sequence of SEQ ID NO: 122, an LCDR2 comprising the sequence of SEQ ID NO: 35, and an LCDR3 comprising the sequence of SEQ ID NO:

118.

2. 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 the sequence of SEQ ID NO: 126 and a light chain variable region comprising the sequence of SEQ ID NO: 127; or f) a heavy chain variable region comprising the sequence of SEQ ID NO: 128 and a light chain variable region comprising the sequence of SEQ ID NO: 129; or g) a heavy chain variable region comprising the sequence of SEQ ID NO: 128 and a light chain variable region comprising the sequence of SEQ ID NO: 130; or h) 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 i) a pair of heavy 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 j) 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 k) a pair of heavy 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; or l) a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133 and SEQ ID NO: 134, and a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 135, SEQ ID NO: 136 and SEQ ID NO: 137; or m) a pair of heavy chain variable region sequences and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 131 / 135, 131 / 136, 131 / 137, 132 / 135, 132 / 136, 132 / 137, 133 / 135, 133 / 136, 133 / 137, 134 / 135, 134 / 136, and 134 / 137; The antibody or antigen-binding fragment thereof of claim 1 .

3. The antibody or antigen-binding fragment thereof of claim 1, further comprising an immunoglobulin constant region or further comprising a constant region of a human IgG, optionally wherein the constant region comprises a heavy chain constant region comprising the sequence of SEQ ID NO: 138 and / or a light chain constant region comprising the sequence of SEQ ID NO:

139.

4. The antibody or antigen-binding fragment thereof of claim 1 , which is humanized or chimeric.

5. 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.

6. The antibody or antigen-binding fragment thereof described in claim 1, which is bispecific, 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.

7. The second antigen 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, B 7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16, CD83, prostate-specific antigen (PSA), CA-125, gangliosides 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, transferrin receptor, claudin The antibody or antigen-binding fragment thereof of claim 6, which is selected from the group consisting of 18.2, GPC-3, nectin-4, ROR1, methocelin, 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.

8. The antibody or antigen-binding fragment thereof of claim 1 , linked to one or more conjugate moieties.

9. A pharmaceutical composition or kit comprising the antibody or antigen-binding fragment thereof described in claim 1 and a pharma- ceutically acceptable carrier.

10. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1.

11. A vector comprising the isolated polynucleotide of claim 10.

12. A host cell comprising the vector of claim 11.

13. A method for expressing the antibody or antigen-binding fragment thereof described in claim 1, comprising the step of culturing a host cell containing a vector comprising an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof described in claim 1 under conditions in which the vector is expressed.

14. A pharmaceutical composition for treating a GREM1-related disease or condition in a subject, inhibiting FGFR1 activation in a subject in need of inhibition of FGFR1 activation, or treating a disease or condition associated with FGFR1 activation mediated by GREM1, comprising an antibody or antigen-binding fragment thereof described in claim 1.

15. The pharmaceutical composition of claim 14, wherein the GREM1-related disease or condition is selected from the group consisting of cancer, fibrotic disease, angiogenesis, glaucoma or retinal disease, kidney disease, pulmonary arterial hypertension, or osteoarthritis (OA), or the GREM1-related disease or condition is associated with elevated levels of GREM1 selected from the group consisting of scleroderma, idiopathic pulmonary fibrosis, diabetic nephropathy, IgAN, lupus nephritis, Alport syndrome, glioma, head and neck cancer, prostate cancer, lung cancer, gastric cancer, pancreatic cancer, esophageal cancer, bladder cancer, breast cancer and colorectal cancer.

16. The pharmaceutical composition of claim 15, wherein the cancer is a GREM1-expressing cancer, optionally a PD-L1-expressing cancer or a non-PD-L1-expressing cancer, and optionally is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor.

17. The pharmaceutical composition of claim 14, wherein the subject has been identified as having GREM1-expressing cancer cells or as having GREM1 expression in the cancer microenvironment.

18. The cancer may be prostate cancer, gastroesophageal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, pancreatic cancer, breast cancer, bronchial cancer, bone cancer, liver and 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, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, bone marrow cancer, 16. The pharmaceutical composition of claim 15, wherein the tumor is a dysplastic syndrome, a sarcoma, a teratoma, a glioma, an adenocarcinoma, a leukemia (e.g., acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML)), a lymphoma (e.g., Hodgkin's lymphoma, or a non-Hodgkin's lymphoma (e.g., Waldenstrom's macroglobulinemia (WM))), or a myeloma (e.g., multiple myeloma (MM)).

19. The pharmaceutical composition of claim 15, wherein the breast cancer is triple-negative breast cancer.

20. 16. The pharmaceutical composition of claim 15, wherein the cancer is esophageal cancer, optionally resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and optionally resistant or refractory to treatment with an anti-PD-1 antibody (e.g., nivolumab).

21. 16. The pharmaceutical composition of claim 15, wherein the fibrotic disease is a fibrotic disease of the lung, liver, kidney, eye, skin, heart, intestine or muscle.

22. The pharmaceutical composition of claim 14, wherein the subject is a human.

23. and is administered in combination with a second therapeutic agent, said second therapeutic agent comprising an anti-cancer therapy, optionally said anti-cancer therapy being selected from the group consisting of chemotherapeutic agents (e.g., cisplatin), radiation therapy, immunotherapy agents (e.g., immune checkpoint modulators, e.g., PD-1 / PD-L1 axis inhibitors, TGF-beta inhibitors), anti-angiogenic agents (e.g., antagonists of VEGFR such as VEGFR-1, VEGFR-2, and VEGFR-3), targeted therapy agents, cell therapy agents, gene therapy agents, hormone therapy agents, cytokines, palliative care, surgery for the treatment of cancer (e.g., tumor 15. The pharmaceutical composition of claim 14, wherein the therapeutic agent is selected from the group consisting of tumor resection, one or more antiemetics, treatment of complications resulting from chemotherapy, nutritional supplements for cancer patients (e.g., indole-3-carbinol), agents modulating the tumor microenvironment (e.g., a bifunctional molecule comprising a PD-L1 binding moiety and the extracellular domain of the TGF-beta receptor), or antifibrotic therapies (e.g., BMP7 treatment, ACE inhibitors (or ARBs), anti-MASP2 antibodies, endothelin receptor antagonists, NRF2 inhibitors steroids, CTLA4-IgG, or TNF inhibitors).

24. The anti-cancer therapy comprises an androgen axis inhibitor; an androgen synthesis inhibitor; a PARP inhibitor; or a combination thereof for treating prostate cancer, or abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), darolutamide, degarelix, docetaxel, Eligard (leuprolide acetate), enzalutamide, Erleada (apalutamide), Firmagone (degarelix), flutamide, goserelin acetate, Jevtana (cabazitaxel), leuprolide acetate, Lupron (leuprolide acetate), 24. The pharmaceutical composition of claim 23, comprising abiraterone acetate, abiraterone HCl, nilandrone (nilutamide), nilutamide, nubequo (darolutamide), olaparib, provenzi (sipuleucel-T), radium 223 dichloride, rubraca (rucaparib camsylate), rucaparib camsylate, sipuleucel-T, taxotere (docetaxel), zofigo (radium 223 dichloride), xtandi (enzalutamide), zoladex (goserelin acetate) or zytiga (abiraterone acetate).

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

26. A method for detecting the presence or amount of Gremlin in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in claim 1 and determining the presence or amount of Gremlin in the sample.

27. A pharmaceutical composition for treating a disease that could benefit from increasing BMP7 activity or decreasing gremlin-mediated inhibition of BMP7 activity, or for improving the effectiveness of BMP7 treatment in a subject in need thereof, comprising an antibody or antigen-binding fragment thereof described in claim 1.

Images