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]

[0001] This disclosure relates to a novel anti-gremlin 1 (GREM1) antibody that specifically binds to human gremlin 1 in general. [Background technology]

[0002]

[0002] Gremlin 1 (GREM1) is a highly conserved secreted protein having a cysteine-rich region and a cysteine ​​knot (Wordinger et al., Exp Eye Res. August 2008; 87(2): pp. 78-79). GREM1 is a member of the family of differential screening-selected gene aberrative in neuroblastoma (DANs), and it functions as an antagonist of bone morphogenetic proteins (BMPs) (Wordinger et al., Exp Eye Res. August 2008; 87(2): pp. 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 subsequently inhibiting the activation of the BMP signaling pathway.

[0003]

[0003] GREM1 is closely associated with fibrotic lesions of the kidneys, lungs, liver, and retina, as well as several tumor types, including pancreatic cancer, colon cancer, lung cancer, glioma, gastric cancer, and prostate cancer (Sneddon et al., PNAS October 2006; 103(40): pp. 14842-14847). For example, abnormal upregulation of gremlin 1 confers tumorigenic ability to colon cells outside the stem cell niche. It has also been found that tumor stem cells highly express and secrete gremlin 1 to maintain their stem cell nature in gliomas (Yan, K. et al., Genes Dev 28, pp. 1085-1100 (2014)). Therefore, 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 are currently no effective treatments for GREM1-related diseases due to side effects such as toxicity to normal tissues. Therefore, there is a need for novel anti-gremlin 1 antibodies with reduced side effects. [Overview of the project] [Problems that the invention aims to solve]

[0005]

[0005] Throughout this disclosure, the articles “a,” “an,” and “the” are used herein to refer to one or more grammatical objects of the article (i.e., at least one). For example, “an antibody” means one antibody or two or more antibodies.

[0006]

[0006] This disclosure provides, in particular, novel monoclonal anti-gremlin 1 (GREM1) antibodies, nucleotide sequences encoding these antibodies, and their uses. The anti-GREM1 antibodies provided herein have differential effects on binding to different regions of GREM1 and modulating the activity of GREM1 to bone morphogenetic protein (BMP) binding 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 to non-cancer cells. This is unexpected and addresses long-term issues related to side effects caused by anti-GREM1 antibodies due to the universal expression of gremlin in cancer and non-cancer cells. [Means for solving the problem]

[0007]

[0007] In one embodiment, the present disclosure relates to an isolated antibody or antigen-binding fragment against human gremlin 1 (hGREM1), characterized by the following: a) It can selectively reduce hGREM1-mediated inhibition of BMP signaling in cancer cells compared to non-cancer cells; b) Non-cancer cells exhibit a reduction of less than 50% in hGREM1-mediated inhibition of BMP signaling; c) Can bind to chimeric hGREM1 containing the amino acid sequence of SEQ ID NO: 68; d) It can bind to hGREM1, but cannot specifically bind to mouse gremlin 1; e) Binding to hGREM1 with an epitope containing residue Gln27 and / or residue Asn33, where residue numbering follows Sequence ID No. 69, or binding to an hGREM1 fragment containing residue Gln27 and / or residue Asn33, where 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) When measured with Fortebio, K is less than 1 nM. D It can then bind to hGREM1; h) When measured by ELISA, the binding of hGREM1 to BMP7 can be blocked at a maximum blockage percentage exceeding 50%; i) The interaction between GREM1 and FGFR can be blocked; and / or j) It can be coupled to both GREM1 and DAN. The present invention provides an isolated antibody or its antigen-binding fragment having at least one of the following:

[0008]

[0008] In certain embodiments, the epitope is a linear epitope or a three-dimensional epitope.

[0009]

[0009] In another embodiment, the present disclosure relates to an isolated antibody or antigen-binding fragment 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 containing a sequence selected from the group consisting of sequence numbers 1, 11, 21, 31, 114, 119 and 123, b) HCDR2 containing sequences selected from the group consisting of sequence numbers 2, 12, 22, 32 and 115, and c) HCDR3 containing a sequence selected from the group consisting of sequence numbers 3, 13, 23, 33, 116, 120, and 124. including and / or The light chain variable region is: d) LCDR1 containing a sequence selected from the group consisting of sequence numbers 4, 14, 24, 34, 117, 121, 122 and 125, e) LCDR2 containing a sequence selected from the group consisting of sequence numbers 5, 15, 25, and 35, and f) LCDR3 containing a sequence selected from the group consisting of sequence numbers 6, 16, 26, 36 and 118, The present invention provides an isolated antibody or its antigen-binding fragment, which includes [the specified substance].

[0010]

[0010] In a particular embodiment, the heavy chain variable region of the antibody against hGREM1 or its antigen-binding fragment provided herein is: a) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 1, HCDR2 containing the sequence of sequence number 2, and HCDR3 containing the sequence of sequence number 3; b) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 11, HCDR2 containing the sequence of sequence number 12, and HCDR3 containing the sequence of sequence number 13; c) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 21, HCDR2 containing the sequence of sequence number 22, and HCDR3 containing the sequence of sequence number 23; d) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 31, HCDR2 containing the sequence of sequence number 32, and HCDR3 containing the sequence of sequence number 33; e) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 114, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 116; f) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 119, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 120; and g) Heavy chain variable region including HCDR1 containing the sequence of SEQ ID NO: 123, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 124. It is selected from the group consisting of the following.

[0011]

[0011] In a particular embodiment, the light chain variable region of the antibody against hGREM1 or its antigen-binding fragment provided herein is: a) Light chain variable region including LCDR1 containing the sequence of sequence number 4, LCDR2 containing the sequence of sequence number 5, and LCDR3 containing the sequence of sequence number 6; b) Light chain variable regions including LCDR1 containing the sequence of sequence number 14, LCDR2 containing the sequence of sequence number 15, and LCDR3 containing the sequence of sequence number 16; c) Light chain variable region containing LCDR1 containing the sequence of sequence number 24, LCDR2 containing the sequence of sequence number 25, and LCDR3 containing the sequence of sequence number 26; d) Light chain variable regions including LCDR1 containing the sequence of sequence number 34, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 36; e) Light chain variable region containing LCDR1 containing the sequence of sequence number 117, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; f) Light chain variable region containing LCDR1 containing the sequence of sequence number 121, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; g) Light chain variable regions including LCDR1 containing the sequence of sequence number 122, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; and h) Light chain variable region including LCDR1 containing the sequence of sequence number 125, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118. A selection can be made from the group consisting of the following:

[0012]

[0012] In a particular embodiment, in an antibody or antigen-binding fragment provided herein: a) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 1, HCDR2 containing the sequence of SEQ ID NO: 2, and HCDR3 containing the sequence of SEQ ID NO: 3; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 4, LCDR2 containing the sequence of SEQ ID NO: 5, and LCDR3 containing the sequence of SEQ ID NO: 6; b) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 11, HCDR2 containing the sequence of SEQ ID NO: 12, and HCDR3 containing the sequence of SEQ ID NO: 13; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 14, LCDR2 containing the sequence of SEQ ID NO: 15, and LCDR3 containing the sequence of SEQ ID NO: 16; c) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 21, HCDR2 containing the sequence of SEQ ID NO: 22, and HCDR3 containing the sequence of SEQ ID NO: 23; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 24, LCDR2 containing the sequence of SEQ ID NO: 25, and LCDR3 containing the sequence of SEQ ID NO: 26; d) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 31, HCDR2 containing the sequence of SEQ ID NO: 32, and HCDR3 containing the sequence of SEQ ID NO: 33; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 34, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 36; e) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 114, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 116; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 117, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118; f) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 119, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 120; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 121, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118; g) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 119, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 120; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 122, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118; or h) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 123, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 124; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 125, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118.

[0013]

[0013] In a particular embodiment, the heavy chain variable region of an antibody against hGREM1 or its antigen-binding fragment provided herein includes a sequence selected from the group consisting of SEQ ID NOs: 7, SEQ ID NOs: 17, SEQ ID NOs: 27, SEQ ID NOs: 37, SEQ ID NOs: 41, SEQ ID NOs: 43, SEQ ID NOs: 45, SEQ ID NOs: 51, SEQ ID NOs: 53, SEQ ID NOs: 55, SEQ ID NOs: 57, SEQ ID NOs: 126, SEQ ID NOs: 128, SEQ ID NOs: 131, SEQ ID NOs: 132, SEQ ID NOs: 133, and SEQ ID NOs: 134, and sequences having at least 80% sequence identity while still retaining specific binding specificity or affinity to hGREM1.

[0014]

[0014] In a particular embodiment, the light chain variable region of an antibody against hGREM1 or its antigen-binding fragment provided herein includes a sequence selected from the group consisting of SEQ ID NOs: 8, 18, 28, 38, 47, 49, 59, 61, 127, 129, 130, 135, 136, and 137, as well as sequences having at least 80% sequence identity while still retaining specific binding specificity or affinity to hGREM1.

[0015]

[0015] In a particular embodiment, the antibody or its antigen-binding fragment provided herein is: a) Heavy chain variable region containing the sequence of Sequence ID 7 and light chain variable region containing the sequence of Sequence ID 8; or b) A heavy chain variable region containing the sequence of Sequence ID No. 17 and a light chain variable region containing the sequence of Sequence ID No. 18; or c) Heavy chain variable region containing the sequence of Sequence ID No. 27 and light chain variable region containing the sequence of Sequence ID No. 28; or d) A heavy chain variable region containing the sequence of sequence number 37 and a light chain variable region containing the sequence of sequence number 38; or e) A heavy chain variable region containing the sequence of sequence number 126 and a light chain variable region containing the sequence of sequence number 127; or f) A heavy chain variable region containing the sequence of sequence number 128 and a light chain variable region containing the sequence of sequence number 129; or g) A heavy chain variable region containing the sequence of sequence number 128 and a light chain variable region containing the sequence of sequence number 130; or h) A heavy chain variable region containing a sequence selected from the group consisting of SEQ ID NOs. 41, 43, and 45, and a light chain variable region containing a sequence selected from the group consisting of SEQ ID NOs. 47 and 49; or i) A pair of heavy chain variable region sequences 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 containing a sequence selected from the group consisting of SEQ ID NOs. 51, SEQ ID NOs. 53, SEQ ID NOs. 55, and SEQ ID NOs. 57, and a light chain variable region containing a sequence selected from the group consisting of SEQ ID NOs. 59 and SEQ ID NOs. 61; or k) A pair of heavy chain variable region sequences and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 51 / 59, 51 / 61, 53 / 59, 53 / 61, 55 / 59, 55 / 61, 57 / 59, and 57 / 61; or l) A heavy chain variable region containing a sequence selected from the group consisting of SEQ ID NOs: 131, 132, 133, and 134, and a light chain variable region containing a sequence selected from the group consisting of SEQ ID NOs: 135, 136, and 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 provided herein further comprises substitutions or modifications of one or more amino acid residues that still retain specific binding specificity or affinity to hGREM1.

[0017]

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

[0018]

[0018] In certain embodiments, the antibody or antigen-binding fragment provided herein further comprises an immunoglobulin constant region, optionally a human Ig constant region, or optionally a human IgG constant region.

[0019]

[0019] In a particular embodiment, the steady-state region includes the steady-state region of human IgG1, IgG2, IgG3, or IgG4.

[0020]

[0020] In a particular embodiment, the steady region includes a heavy chain steady region containing the sequence of sequence number 138 and / or a light chain steady region containing the sequence of sequence number 139.

[0021]

[0021] In certain embodiments, the antibody or its antigen-binding fragment provided herein is humanized.

[0022]

[0022] In certain embodiments, the antibodies or antigen-binding fragments 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 (dsdiabodies), 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 provided herein are bispecific.

[0024]

[0024] In certain embodiments, the antibody or antigen-binding fragment provided herein can specifically bind to the first and second epitopes of hGREM1, or can specifically bind to both hGREM1 and the 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 includes 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]

[0027] In a particular embodiment, the second antigen includes a tumor antigen.

[0028]

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

[0029]

[0029] In certain embodiments, tumor antigens include 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-related mucin, VEGF, VEGFR (e.g., VEGFR3), estrogen Contains Gen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2, GPC-3, nectin-4, ROR1, metoserin, 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 antibody or its antigen-binding fragment provided herein is not cross-reactive to mouse gremlin 1.

[0031]

[0031] In certain embodiments, the antibody or antigen-binding fragment provided herein is cross-reactive to mouse gremlin 1.

[0032]

[0032] In certain embodiments, the antibody or antigen-binding fragment provided herein is linked to one or more conjugate portions.

[0033]

[0033] In certain embodiments, the conjugate portion includes clearance modifiers, chemotherapeutic agents, toxins, radioisotopes, lantanides, luminescence labels, fluorescent labels, enzyme substrate labels, DNA alkylating agents, topoisomerase inhibitors, tubulin binders, or other anticancer drugs such as androgen receptor inhibitors.

[0034]

[0034] In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof that competes with the antibody or antigen-binding fragment 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 pharmaceutically acceptable carrier.

[0036]

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

[0037]

[0037] In another aspect, the present disclosure provides isolated polynucleotides encoding antibodies or antigen-binding fragments thereof, as provided herein.

[0038]

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

[0039]

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

[0040]

[0040] In another aspect, the present disclosure provides a method for expressing an antibody or an antigen-binding fragment provided herein, comprising the step of 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, comprising the step of administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein or a pharmaceutical composition provided herein to the subject.

[0042]

[0042] In another aspect, the present disclosure provides a method for treating a GREM1-related disease or condition in a subject requiring treatment, comprising the step of administering a therapeutically effective amount of an anti-human GREM1 antibody or its antigen-binding fragment to the subject, wherein the anti-human GREM1 antibody or its antigen-binding fragment is: a) An epitope containing residue Gln27 and / or residue Asn33 that can bind to hGREM1, where the residue numbers follow SEQ ID NO: 69, and / or b) It can bind to an hGREM1 fragment containing 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; and / or c) It can selectively reduce hGREM1-mediated inhibition of BMP signaling in cancer cells compared to non-cancer cells; and / or d) Non-cancer cells exhibit a reduction of 50% or less in hGREM1-mediated inhibition of BMP signaling; and / or e) Can bind to a chimeric hGREM1 containing the amino acid sequence of SEQ ID NO: 68; and / or f) When measured with Fortebio, K is less than 1 nM. D It can be bound to hGREM1; and / or h) When measured by ELISA, the binding of hGREM1 to BMP7 can be blocked at a maximum blockage percentage of more than 50%; and / or i) The interaction between GREM1 and FGFR can be blocked.

[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 to or unresponsive to treatment with PD-1 / PD-L1 axis inhibitors.

[0045]

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

[0046]

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

[0047]

[0047] In certain embodiments, solid tumors include adrenocortical carcinoma, anal cancer, astrocytoma, pediatric cerebellar or cerebral cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectoderm tumor, visual tract and hypothalamic glioma, breast cancer, Burkitt lymphoma, cervical cancer, colon cancer, emphysema, endometrial cancer, esophageal cancer, Ewing's sarcoma, and retinoblastoma. These include cell tumors, gastric cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (pancreatic endocrine cancer), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, liver cancer, lung cancer, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, Ewing family tumors, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, or vaginal cancer.

[0048]

[0048] In certain embodiments, blood cancers include leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin lymphoma, or non-Hodgkin lymphoma (e.g., Waldenström macroglobulinemia (WM))), or myeloma (e.g., multiple myeloma (MM)).

[0049]

[0049] In certain embodiments, 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, stomach cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, glioma, or adenocarcinoma.

[0050]

[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 a particular embodiment, the cancer is prostate cancer.

[0052]

[0052] In certain embodiments, fibrotic disease is a fibrotic disease of the lungs, liver, kidneys, eyes, skin, heart, intestines, or muscles.

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

[0053]

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

[0054]

[0055] In certain embodiments, the method provided herein further includes the step of administering a therapeutically effective dose of a second therapeutic agent.

[0055]

[0056] In certain embodiments, the second therapeutic agent includes, optionally, anticancer therapy, chemotherapeutic agents (e.g., cisplatin), anticancer drugs, radiotherapy, immunotherapy agents (e.g., immune checkpoint modulators, e.g., PD-1 / PD-L1 axis inhibitors, and TGF-beta inhibitors), anti-angiogenic agents (e.g., VEGFR antagonists 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 cancer treatment. For example, the second therapeutic agent is selected from (for example, tumor resection), one or more antiemetics, treatment of complications arising from chemotherapy, nutritional supplements for cancer patients (e.g., indole-3-carbinol), agents that modulate the tumor microenvironment (e.g., a bifunctional molecule containing a PD-L1 binding moiety and the extracellular domain of a TGF-beta receptor), or antifibrotic therapies (e.g., BMP7 treatment, ACE inhibitors (or ARBs), anti-MASP2 antibodies, endothelin receptor antagonists, NRF2 inhibitory 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, anticancer therapy includes anti-prostate cancer drugs.

[0057]

[0058] In certain embodiments, the anti-prostate cancer drug includes androgen axis inhibitors; androgen synthesis inhibitors; poly-ADP-ribose polymerase (PARP) inhibitors; or combinations thereof.

[0058]

[0059] In certain embodiments, the androgen axis inhibitor is selected from the group consisting of luteinizing hormone-releasing hormone (LHRH) agonists, LHRH antagonists, and androgen receptor antagonists.

[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, Elleada (apalutamide), filmagon (degarelix), flutamide, goserelin acetate, Jevtana (cabazitaxel), leuprolide acetate, Leupron (leuprolide acetate), Leupron Depot (leuprolide acetate), Lympa The following are selected from the group consisting of -za (olaparib), mitoxantrone hydrochloride, nilandrone (nilutamide), nilutamide, Nubequa (darolutamide), olaparib, Provenzi (cypuroicell-T), radium-223 chloride, Rubraca (lucaparib cansylate), lucaparib cansylate, cypuroicell-T, Taxotere (docetaxel), Zofigo (radium-223 chloride), Extanzi (enzalutamide), Zoladex (goserelin acetate), and Zytiga (abiraterone acetate).

[0061]

[0062] In a particular embodiment, the second therapeutic agent comprises indole-3-carbinol.

[0062]

[0063] In another embodiment, 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, comprising the steps of contacting the sample with an antibody or antigen-binding fragment provided herein, and determining the presence or amount of GREM1 in the sample.

[0064]

[0065] In another aspect, the disclosure provides the use of antibodies or antigen-binding fragments provided herein in the manufacture of a pharmaceutical product for treating a GREM1-related disease or condition in a subject.

[0065]

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

[0066]

[0067] In certain embodiments, the GREM1-related disease or condition is a fibrotic disease, neovascularization, glaucoma, retinal disease, kidney disease, pulmonary arterial hypertension, or osteoarthritis (OA). [Brief explanation of the drawing]

[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) (Figure 1A, 1C) and mouse gremlin 1 (Figure 1B, 1C), respectively, as measured by ELISA, as well as the binding of anti-gremlin 1 antibodies 42B9, 36F5, and 67G11 to human gremlin 1 (hGREM1) (Figure 1D, 1F) and mouse gremlin 1 (Figure 1E, 1F). [Figure 1-2]

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

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

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

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

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

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

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

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

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

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

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

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

[0072] Figure 5 shows the 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 the 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 5-3]

[0072] Figure 5 shows the 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 5-4]

[0072] Figure 5 shows the 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 6-1]

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

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

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

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

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

[0077] Figure 10 shows the results from epitope studies, with Figures 10A-C showing the results of epitope binning of anti-gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assays (Figure 10A), the results of cross-competitive assays of antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and the binding of antibody 14E3-C and benchmark 6245P to the gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C). Figure 10D shows the epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show 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 the results from epitope studies, where Figures 10A-C show the results of epitope binning of anti-gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assays (Figure 10A), the results of cross-competitive assays of antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and the binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows the epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show the 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 the results from epitope studies, where Figures 10A-C show the results of epitope binning of anti-gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assays (Figure 10A), the results of cross-competitive assays of antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and the binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows the epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show the 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 the results from epitope studies, where Figures 10A-C show the results of epitope binning of anti-gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assays (Figure 10A), the results of cross-competitive assays of antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and the binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows the epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show the 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 the results from epitope studies, where Figures 10A-C show the results of epitope binning of anti-gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assays (Figure 10A), the results of cross-competitive assays of antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and the binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows the epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show the 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 the results from epitope studies, where Figures 10A-C show the results of epitope binning of anti-gremlin 1 antibodies 14E3, 22F1, 56C11, and 69H5 as measured by competitive ELISA assays (Figure 10A), the results of cross-competitive assays of antibodies 14E3-C, 22F1-C, and benchmark antibody 6245P (Figure 10B), and the binding of antibody 14E3-C and benchmark 6245P to gremlin-DAN fusion protein XM5 as measured by ELISA (Figure 10C), Figure 10D shows the epitope mapping of 14E3 as measured by biolayer interference (BLI) assay, and Figures 10E and 10F show the 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 results of epitope analysis 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 the epitope of 6245P, while 56C11 shares an epitope similar to that of 6245P. [Figure 11-2]

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

[0079] Figure 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1, as measured by ELISA (Figures 12A-C) or Fortebio (Figure 12D), compared to the benchmark antibody 6245P. [Figure 12-2]

[0079] Figure 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1, as measured by ELISA (Figures 12A-C) or Fortebio (Figure 12D), compared to benchmark antibody 6245P. [Figure 12-3]

[0079] Figure 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1, as measured by ELISA (Figures 12A-C) or Fortebio (Figure 12D), compared to benchmark antibody 6245P. [Figure 12-4]

[0079] Figure 12 shows the binding affinity of humanized anti-hGREM1 antibodies 14E3 and 22F1 to hGREM1, as measured by ELISA (Figures 12A-C) or Fortebio (Figure 12D), compared to benchmark antibody 6245P. [Figure 13]

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

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

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

[0082] Figures 15A and 15B show the synergistic antitumor effect of combination therapy with an anti-mGREM1 antibody and an 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 using 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), either alone or in combination with cisplatin, inhibited tumor growth in a gremlin-positive esophageal PDX model. Humanized 14E3 alone achieved approximately 43% tumor growth inhibition (TGI). Cisplatin alone achieved approximately 60% TGI. The combination of humanized 14E3 and cisplatin achieved approximately 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, as well as the benchmark antibody 6245P, to the binding of gremlin 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 benchmark antibody 6245P, respectively, to the binding of gremlin 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 benchmark antibody 6245P, respectively, to the binding of gremlin 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 benchmark antibody 6245P, respectively, to the binding of gremlin to FGFR1 (Figures 18B, 18C). [Figure 19-1]

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

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

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

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

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

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

[0089] Figure 22 shows the antitumor activity of hybridoma 14E3 or 36F5 against the E7 tumor model. [Figure 23]

[0090] Figure 23 shows the antitumor activity of 56C11 combination therapy with an anti-PDL1 antibody against an MC38 / hPD-L1 tumor model. [Modes for carrying out the invention]

[0068]

[0091] The following descriptions of this disclosure are intended merely to illustrate various embodiments of this disclosure. Certain modifications discussed in this manner should not be construed as limitations on the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, modifications, and alterations can be made without departing from the scope of this disclosure, and it will be understood that such equivalent embodiments should be included herein. All references cited herein, 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 used in light of the present invention (especially in light of the claims) should be construed to cover both singular and plural forms unless otherwise specified herein or unless otherwise clearly contradicted by the context.

[0070]

[0093] As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen. Naturally intact antibodies contain 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 has a variable region (V H ) and the first, second, and third steady-state regions (each C H1 , C H2 , C H3 ) is composed of; mammalian light chains are classified as λ or κ, and each light chain has a variable region (V LAntibodies consist of a light chain variable region and a constant region. Antibodies have a "Y" shape, and the main stem of the Y consists of a second and third constant region of two heavy chains linked to each other via disulfide bonds. Each arm of the Y contains a variable region of a single light chain and a variable region of a single heavy chain bound to the constant region, as well as a first constant region. 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, and heavy chain CDRs including HCDR1, HCDR2, and HCDR3). The boundaries of the CDRs of the antibody and antigen-binding domains disclosed herein are conventional to Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J.Mol.Biol., 273(4), p. 927 (1997); Chothia, C. et al., J Mol Biol. December 5; 186(3): pp. 651-63 (1985); Chothia, C. and Lesk, AM, J.Mol.Biol., 196, p. 901 (1987); NR. Whitelegg et al., Protein It may be defined or identified by: Engineering, Vol. 13(12), pp. 819-824 (2000); Chothia, C. et al., Nature, December 21-28; 342(6252): pp. 877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: pp. 55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd edition), Chapter 26, pp. 481-514, (2015). The three CDRs are inserted between adjacent stretches known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loop.The constant regions of the heavy and light chains do not participate 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 chain. 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 mono-heavy chain), IgG2 (gamma double-heavy chain), IgG3 (gamma triple-heavy chain), IgG4 (gamma quadruple-heavy chain), IgA1 (alpha mono-heavy chain), or IgA2 (alpha double-heavy chain). In certain embodiments, the antibodies provided herein encompass any of their antigen-binding fragments.

[0071]

[0094] As used herein, the term “antigen-binding fragment” refers to an antibody fragment formed from an antibody fragment containing one or more CDRs, or any other antibody moiety 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 (dsdiabodies), 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 to which a parent antibody binds. In certain embodiments, an antigen-binding fragment may contain one or more CDRs derived from a particular parent antibody.

[0072]

[0095] In relation to antibodies, "Fab" refers to a monovalent antigen-binding fragment of an antibody, consisting of a single light chain (both variable and constant regions) attached by a disulfide bond to the variable region and primary constant region of a single heavy chain. Fab can be obtained by papain digestion of the antibody at the hinge region, at a residue proximal to the N-terminus of the disulfide bond between the heavy chains.

[0073]

[0096] "Fab'" refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of an antibody at the residues proximal to the C-terminus of the disulfide bond between the heavy chains in the hinge region, and thus it differs from Fab at a small number of residues (including one or more cysteines) in the hinge region.

[0074]

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

[0075]

[0098] "Fv" with respect to an antibody refers to the smallest fragment of an antibody that possesses a complete antigen-binding site. The 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, where the linkage 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 that are connected directly to each other 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 that includes two heavy chain variable regions and two light chain variable regions together with a linker. In certain embodiments, "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv), which forms two binding sites such that a portion of V H cooperates with another portion of V L to target the same antigen (or epitope) or different antigens (or epitopes) and is dimerized with another V H -V L portion and includes V H -V L (linked by a peptide linker). In other embodiments, "scFv dimer" is a bispecific diabody, which means that V H1 and V L1 cooperate and VH2 and V L2 V L1 -V H2 V associated with (linked by a peptide linker) H1 -V L2 (Also includes those linked by peptide linkers)

[0077]

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

[0078]

[0101] "Camelized single-domain antibody," "heavy chain antibody," "nanobody," or "HCAb" refers to two V HThis refers to antibodies that contain a domain but do not contain a light chain (Riechmann L. and Muyldermans S., J Immunol Methods. December 10; 231(1-2): pp. 25-38 (1999); Muyldermans S., J Biotechnol. June; 74(4): pp. 277-302 (2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally obtained from the camelid family (Camelidae) (camels, dromedaries, and llamas). Although lacking a light chain, camelized antibodies possess a reliable antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428): pp. 446-448 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. April; 54(1): pp. 39-47 (2002); Nguyen VK. et al., Immunology. May; 109(1): pp. 93-101 (2003)). The variable domain (VHH domain) of heavy-chain antibodies becomes the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. November; 21(13): pp. 3490-3498. Epub June 15, 2007 (2007)). A "diabody" contains a small antibody fragment having two antigen-binding sites, in this case, this fragment is V in a single polypeptide chain. L V connected to the domain H Domain (V H -V L or V L -V H) include (see, for example, Holliger P. et al., Proc Natl Acad Sci USA. July 15; 90(14): pp. 6444-648 (1993); EP404097; WO93 / 11161). Two domains on the same strand cannot pair because the linker is too short; therefore, these domains are forced to pair with complementary domains on another strand, thereby creating two antigen-binding sites. These 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 the heavy chain or only the variable region of the light chain. In certain embodiments, two or more V H The domains covalently combine with the peptide linker to form a bivalent or polyvalent domain antibody. The two Vs of a bivalent domain antibody H Domains can target the same or different antigens.

[0080]

[0103] In a particular embodiment, "(dsFv)2" consists of three peptide chains linked by a peptide linker and two V cross-links by disulfide cross-links. L Two V's joined together in the part H Includes a portion.

[0081]

[0104] In a particular embodiment, the "dual specificity ds diabody" is V H1 and V L1 V L1 -V H2 V (linked by a peptide linker) H1 -V L2 (Also linked by a peptide linker) Includes.

[0082]

[0105] In certain embodiments, "bispecific dsFv" or "dsFv-dsFv'" refers to three peptide chains: V H1 -V H2In this portion, these two heavy chains are linked by a peptide linker (e.g., a long, flexible linker) and are connected via disulfide bridges, respectively. L1 Part and V L2 V is opposed to the part. H1 -V H2 It includes a portion. The heavy and light chains paired with each disulfide have different antigen specificities.

[0083]

[0106] As used herein, the term “humanized” means that an antibody or antigen-binding fragment includes a CDR derived from a non-human animal, an FR region derived from a human, and, where applicable, a constant region derived from a human. In certain embodiments, amino acid residues of the variable region framework of a humanized gremlin antibody are substituted for sequence optimization. In certain embodiments, the variable region framework sequence of a humanized gremlin antibody chain is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to the corresponding human variable region framework sequence.

[0084]

[0107] As used herein, the term "chimera" refers to an antibody or antigen-binding fragment having heavy chain and / or light chain portions derived from one species, as well as the remaining heavy chain and / or light chain portions derived from a different species. In exemplary cases, a chimeric antibody may include a constant region derived from humans and a variable region derived from a non-human species, such as 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 may 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 a framework region only, a complementarity-determining region only, a framework and complementarity-determining region, a variable segment (as defined above), or any other combination of a sequence or subsequence containing a variable region. Sequence identity may be determined by aligning two sequences using the methods described herein, for example, BLAST, ALIGN, or another alignment algorithm known in the art. The germline nucleic acid sequence or amino acid sequence may have at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the nucleic acid sequence or amino acid sequence of the reference variable region. The germline sequence may 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 against human gremlin 1” are interchangeable and refer to antibodies that can specifically bind to human gremlin 1 with sufficient specificity and / or affinity to be provided, for example, for therapeutic purposes.

[0087]

[0110] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0088]

[0111] As used herein, the terms “specific binding” or “specifically binding” refer to a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibody or antigen binding fragments provided herein are ≤10 -6 M (for example, ≤ 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) of M D ) specifically binds to human and / or non-human gremlin 1. D This refers to the ratio (k) between the dissociation rate and the association rate. off / k on ) refers to the ratio which can 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 a particular embodiment, K D The values ​​can be appropriately determined using flow cytometry. Various immunoassay formats can be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that are specifically immunoreactive with a given protein (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, specific or selective binding reactions will produce a signal at least twice as high as the background signal, and more commonly at least 10 to 100 times higher than the background signal.

[0089]

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

[0090] [Table 1]

[0091]

[0113] In relation to amino acid sequences, a "conservative substitution" refers to the exchange of an amino acid residue with a different amino acid residue that has a side chain with similar physiological and chemical properties. For example, conservative substitutions can occur 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 substitutions usually do not cause significant changes in the conformational structure of a protein and therefore can preserve the biological activity of the protein.

[0092]

[0114] With respect to amino acid sequences (or nucleic acid sequences), "sequence identity percentage (%)" is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in the reference sequence after aligning the sequences, introducing gaps as necessary, and obtaining maximum match. Alignment for the purpose of determining the percentage of amino acid (or nucleic acid) sequence identity can be performed using publicly available tools such as BLASTN, BLASTp (available on the US National Center for Biotechnology Information (NCBI) website; see also Altschul SF et al., J.Mol.Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the European Bioinformatics Institute website; see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or customize the parameters as needed for alignment, for example, by selecting an appropriate algorithm. In certain embodiments, the positions of non-identical residues may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution in which one 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). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted up to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art.For example, see Pearson (1994) Methods Mol. Biol. 24: pp. 307–331, which is incorporated herein by reference.

[0093]

[0115] As used herein, “homologous sequence” means a polynucleotide sequence (or its complementary chain) or amino acid sequence that, when arbitrarily aligned, has at least 80% sequence identity (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with another sequence.

[0094]

[0116] An “isolated” substance is one that has been modified by human hands from its natural state. An “isolated” composition or substance, if it exists naturally, has been altered or extracted from its original environment, or both. For example, a polynucleotide or polypeptide that exists naturally in a living animal is not “isolated,” but it is “isolated” if the same polynucleotide or polypeptide is sufficiently separated from the coexisting substances in its natural state to exist in a substantially pure state. The terms “isolated nucleic acid” and “polynucleotide” are used interchangeably and refer to a sequence of isolated nucleic acid molecules. In certain embodiments, “isolated antibody or antigen-binding fragment” 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%, or 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatography (e.g., ion-exchange chromatography or reverse-phase HPLC).

[0095]

[0117] The term “subject” includes humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise noted, the terms “patient” and “subject” are used interchangeably herein.

[0096]

[0118] As used herein, “to treat” or “to treat” a condition includes preventing or mitigating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the onset of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial regression of a condition, curing a condition, or any combination thereof.

[0097]

[0119] As used herein, the term “vector” means a vehicle into which a genetic factor is operably inserted so as to cause the expression of that genetic factor, for example, to produce the protein, RNA, or DNA encoded by that genetic factor, or to replicate that genetic factor. A vector may be used to transform, transduce, or transfect a host cell so as to cause the expression of the genetic factor carried by the vector within 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 phages or M13 phages, and animal viruses. A vector may contain various elements for controlling expression, including promoter sequences, transcription start sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector may contain an origin of replication. A vector may also contain substances that assist in the entry of the vector into the cell, including, but are not limited to, viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. This disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid sequence provided herein encoding an antibody or an antigen-binding fragment, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0098]

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

[0099]

[0121] The terms “Gremlin 1” and “GREM1” refer to variant 1 of gremlin, encompassing gremlin 1 in various species such as humans, mice, and monkeys. GREM1 is evolutionarily conserved, and the human gremlin 1 gene (hGREM1) has been mapped to chromosomes 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 via the GenBank database under accession number NP-037504 or via the Uniprot Database under accession number O60565, and is provided herein as Sequence ID No. 66. The terms “human gremlin 1” and “hGREM1” are used interchangeably in this disclosure.

[0100]

[0122] As used herein, “gremlin 1-related” or “GREM1-related” disease or condition means any disease or condition caused by, aggravated by, or otherwise related to increased expression or activity of GREM1. In some embodiments, GREM1-related conditions are, 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 neoplasm, abnormal growth, invasion or metastasis, which may be benign or malignant, and includes both solid tumors and non-solid cancers such as leukemia (e.g., hematological malignancies). As used herein, “solid tumor” refers to a solid mass of neoplasm and / or malignant cells.

[0102]

[0124] The term "pharmaceutically acceptable" means that a specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its recipient.

[0103]

[0125] References to values ​​or parameters "about" in this specification include (and describe) embodiments that apply to the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X". A numerical range includes the number that defines the range. Generally speaking, the term "about" refers to all values ​​of a variable that are greater than the stated value of the variable and either within the experimental error of the stated value (e.g., within the 95% confidence interval for the mean) or within 10 percent of the stated value. When the term "about" is used in the context of a period (year, month, week, day, etc.), the term "about" means the period ± a certain amount of the next subperiod (e.g., about 1 year means 11 to 13 months; about 6 months means 6 months ± 1 week; about 1 week means 6 to 8 days; etc.) or within 10 percent of the stated value, whichever is longer.

[0104]

[0126] Anti-human gremlin 1 antibody

[0127] This disclosure provides an anti-human gremlin 1 (hGREM1) antibody and its antigen-binding fragment. The anti-hGREM1 antibody and antigen-binding fragment provided herein are uniquely distinguished from existing anti-hGREM1 antibodies in the following respects: a) they can selectively reduce hGREM1-mediated inhibition of BMP signaling in cancer cells compared to non-cancer cells; b) they exhibit a reduction of less than 50% in hGREM1-mediated inhibition of BMP signaling in non-cancer cells; c) they can bind to chimeric hGREM1 containing 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 in an epitope containing residues Gln27 and / or Asn33, where the residue numbering follows SEQ ID NO: 69, or they bind to an hGREM1 fragment containing residues Gln27 and / or Asn33. In addition, 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) it can reduce hGREM1-mediated activation of MAPK signaling; g) it can bind to hGREM1 with a Kd of 1 nM or less as measured by Fortebio; h) it can block the binding of hGREM1 to BMP7 with a maximum blockage percentage of at least 50% as measured by ELISA; i) it can block the interaction between GREM1 (e.g., hGREM1 or mGREM1) and FGFR (e.g., FGFR1, preferably human FGFR1 (hFGFR1) or mouse FGFR1 (mFGFR1)); and / or j) it can bind to both hGREM1 and DAN.

[0105]

[0128] Bone morphogenetic proteins (BMPs), such as BMP2, BMP4, and BMP7, are known as glycosylated extracellular matrix (ECM)-related members of the transforming growth factor beta (TGF-beta) superfamily and play key roles in 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 BMP receptors, activating receptor phosphorylation, resulting in phosphorylation of R-Smad (e.g., Smad1 / 5 / 9) and complex formation with co-Smad4. The formed Smad complex then translocates to the nucleus and regulates the expression of BMP target genes. Therefore, activation of BMP signaling can be assayed, for example, by measuring the phosphorylation level of smad and / or the expression level of BMP target genes. 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] BMP signaling is known to be 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, preventing 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 its activity will reverse or reduce the inhibition of BMP signaling.

[0107]

[0129] i.Binding affinity

[0130] In certain embodiments, the anti-GREM1 antibody 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 antibody and antigen-binding fragment provided herein is K D This can be expressed by a value, which is the ratio (k) of the dissociation rate to the association rate at the point when the binding between the antigen and the antigen-binding molecule reaches equilibrium. off / k on ) represents antigen binding affinity (e.g., K D ) can be appropriately determined using any suitable method known in the art, including, for example, Kinetic Exclusion Assay (KinExA), Biacore, Fortebio, or flow cytometry.

[0108]

[0131] In a particular embodiment, "K" according to the disclosure D " or "K DThe value is measured in one embodiment by the Biacore assay or the Fortebio assay, which is performed using an anti-GREM1 antibody and GREM1, as described by the assay below which measures the solution binding affinity of the anti-GREM1 antibody. Generally, Biacore works by equilibrating a fixed amount of one binding partner (CBP) with various concentrations of the other binding partner (titrate), and then capturing a portion of the free CBP with a fluorescently labeled secondary antibody within a contact time shorter than the time required for the dissociation of the CBP-titrate complex formed up to that point. The fluorescence signal generated from the captured CBP is directly proportional to the concentration of free CBP in the equilibrium sample and, when measured in series, is used to create a binding curve (percentage of free CBP versus total titrate concentration). Further details are available in Schreiber, G., Fersht, ARNature Structural Biology. 1996, 3(5), pp. 427-431. When anti-GREM1 antibody is used as CBP in a fixed amount, then GREM1 protein can be used as a titrant, and vice versa. Fortebio generally works in a similar manner to Biacore by equilibrating a fixed amount of CBP (e.g., GREM1 protein) with titrants of various concentrations (e.g., anti-GREM1 antibody). The binding kinetics (k) between CBP and titrant are also important. on and k off Information regarding this can be obtained from changes in interference patterns generated from the biosensors used by Fortebio. Further details are available in Charles Wartchow, Frank Podlaski, Shirley Li, Karen Rowan, Xiaolei Zhang, David Mark, and Kuo-Sen Huang, "Biosensor-based Small Molecule Fragment Screening with Biolayer Interferometry": J Comput Aided Mol Des 2011, 25(7), pp. 669–676.

[0109]

[0132] In certain embodiments, the K of the anti-GREM1 antibody provided herein D This is determined according to the method described in Example 14 of this disclosure.

[0110]

[0133] K D Other methods suitable for the measurement may also be used under applicable circumstances, for example, 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 antibody and its antigen-binding fragment provided herein have a K content 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 the Biacore assay. D It specifically binds to human GREM1 at a certain value. In certain embodiments, the anti-GREM1 antibody and its antigen-binding fragment provided herein have a K value of ≤10 nM (or ≤9, 8, 7, 6, 5, 4, 3, 2, or 1 nM) as measured by the Fortebio assay. D It specifically binds to human GREM1 based on its value.

[0112]

[0135] Alternatively, the binding affinity of the anti-GREM1 antibodies and antigen-binding fragments provided herein to human GREM1 is also "median effective concentration" (EC2). 50 This can be expressed as an EC value, which refers to the antibody concentration at which 50% of its maximum effect (e.g., binding) is observed. 50The values ​​can be measured by methods known in the art, such as sandwich assays including ELISA, Western blotting, flow cytometry assays, and other binding assays. In certain embodiments, the anti-GREM1 antibody and its fragments provided herein, when measured by ELISA, have an EC 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) 50 It specifically binds to human GREM1 (for example, cells that express human GREM1).

[0113]

[0136] In certain embodiments, some of the antibodies and antigen-binding fragments provided herein have an EC of 20 ng / ml or less when measured by ELISA. 50 The antibody can specifically bind to mouse GREM1 at a certain value. In certain embodiments, the antibody and its antigen-binding fragment, when measured by ELISA, have an EC 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). 50 The value binds to mouse GREM1.

[0114]

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

[0115]

[0138] In certain embodiments, the antibody and its antigen-binding fragment provided herein do not specifically bind to GREM2.

[0116]

[0139] antibody sequence

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

[0117]

[0141] In a particular embodiment, an antibody or antigen-binding fragment provided herein, wherein the heavy chain variable region is: a) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 1, HCDR2 containing the sequence of sequence number 2, and HCDR3 containing the sequence of sequence number 3; b) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 11, HCDR2 containing the sequence of sequence number 12, and HCDR3 containing the sequence of sequence number 13; c) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 21, HCDR2 containing the sequence of sequence number 22, and HCDR3 containing the sequence of sequence number 23; d) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 31, HCDR2 containing the sequence of sequence number 32, and HCDR3 containing the sequence of sequence number 33; e) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 114, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 116; f) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 119, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 120; and g) Heavy chain variable region including HCDR1 containing the sequence of SEQ ID NO: 123, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 124. An antibody or its antigen-binding fragment, selected from the group consisting of the following.

[0118]

[0142] In a particular embodiment, an antibody or antigen-binding fragment provided herein, wherein the light chain variable region is: a) Light chain variable region including LCDR1 containing the sequence of sequence number 4, LCDR2 containing the sequence of sequence number 5, and LCDR3 containing the sequence of sequence number 6; b) Light chain variable regions including LCDR1 containing the sequence of sequence number 14, LCDR2 containing the sequence of sequence number 15, and LCDR3 containing the sequence of sequence number 16; c) Light chain variable region containing LCDR1 containing the sequence of sequence number 24, LCDR2 containing the sequence of sequence number 25, and LCDR3 containing the sequence of sequence number 26; d) Light chain variable regions including LCDR1 containing the sequence of sequence number 34, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 36; e) Light chain variable region containing LCDR1 containing the sequence of sequence number 117, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; f) Light chain variable region containing LCDR1 containing the sequence of sequence number 121, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; g) Light chain variable regions including LCDR1 containing the sequence of sequence number 122, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; and h) Light chain variable region including LCDR1 containing the sequence of sequence number 125, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118. An antibody or its antigen-binding fragment, selected from the group consisting of the following.

[0119]

[0143] In certain embodiments, an antibody or its antigen-binding fragment provided herein: a) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 1, HCDR2 containing the sequence of SEQ ID NO: 2, and HCDR3 containing the sequence of SEQ ID NO: 3; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 4, LCDR2 containing the sequence of SEQ ID NO: 5, and LCDR3 containing the sequence of SEQ ID NO: 6; b) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 11, HCDR2 containing the sequence of SEQ ID NO: 12, and HCDR3 containing the sequence of SEQ ID NO: 13; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 14, LCDR2 containing the sequence of SEQ ID NO: 15, and LCDR3 containing the sequence of SEQ ID NO: 16; c) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 21, HCDR2 containing the sequence of SEQ ID NO: 22, and HCDR3 containing the sequence of SEQ ID NO: 23; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 24, LCDR2 containing the sequence of SEQ ID NO: 25, and LCDR3 containing the sequence of SEQ ID NO: 26; d) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 31, HCDR2 containing the sequence of SEQ ID NO: 32, and HCDR3 containing the sequence of SEQ ID NO: 33; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 34, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 36; e) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 114, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 116; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 117, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118; f) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 119, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 120; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 121, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118; g) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 119, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 120; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 122, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118; h) The heavy chain variable region includes HCDR1 containing the sequence of SEQ ID NO: 123, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 124; the light chain variable region includes LCDR1 containing the sequence of SEQ ID NO: 125, LCDR2 containing the sequence of SEQ ID NO: 35, and LCDR3 containing the sequence of SEQ ID NO: 118; An antibody or its antigen-binding fragment.

[0120]

[0144] In certain embodiments, the antibody provided herein comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences from among anti-hGREM1 antibodies 14E3, 69H5, 22F1, 56C11, 36F5, 42B9, and 67G11.

[0121]

[0145] As used herein, "14E3" refers to a mouse antibody having the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8.

[0122]

[0146] As used herein, "69H5" refers to a mouse antibody having the heavy chain variable region of SEQ ID NO: 27 and the light chain variable region of SEQ ID NO: 28.

[0123]

[0147] As used herein, "22F1" refers to a mouse antibody having the heavy chain variable region of SEQ ID NO: 17 and the light chain variable region of SEQ ID NO: 18.

[0124]

[0148] As used herein, "56C11" refers to a mouse antibody having the heavy chain variable region of SEQ ID NO: 37 and the light chain variable region of SEQ ID NO: 38.

[0125]

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

[0126]

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

[0127]

[0151] As used herein, "67G11" refers to a mouse antibody having the heavy chain variable region of SEQ ID NO: 128 and the 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 Kabat numbering. Those skilled in the art will understand that other known methods for CDR determination may also be applied to the antibodies provided herein, and that their CDR sequences are also included in this disclosure. The variable region sequences of the heavy and light chains are also provided below in Table 2.

[0129]

[0153]

[0130] [Table 2]

[0131] Here, 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 It is L or V.

[0132]

[0154]

[0133] [Table 3]

[0134]

[0155] The anti-hGREM1 antibodies or their antigen-binding fragments provided herein may be monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, bispecific antibodies, labeled antibodies, bivalent antibodies, or anti-idiotype antibodies. Recombinant antibodies are antibodies prepared in vitro using recombinant methods rather than in animals.

[0135]

[0156] While CDRs are known to be responsible for antigen binding, it has been found that not all six CDRs are necessarily essential or immutable. In other words, it is possible to replace, change, 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) while still substantially maintaining specific binding affinity to hGREM1.

[0136]

[0157] In certain embodiments, the anti-hGREM1 antibody and antigen-binding fragment provided herein includes one of the following heavy chain CDR3 sequences: 14E3, 69H5, 22F1, 56C11, 36F5, 42B9, or 67G11. In certain embodiments, the anti-hGREM1 antibody and antigen-binding fragment provided herein includes the heavy chain CDR3 sequences of SEQ ID NOs: 3, 13, 23, 33, 116, 120, and 124. The heavy chain CDR3 region is thought to be centrally located at the antigen-binding site and therefore has the most contact with the antigen, supplying the most free energy to the antibody's affinity for the antigen. Furthermore, due to multiple diversification mechanisms, the heavy chain CDR3 is considered to be by far the most diverse CDR in terms of length, amino acid composition, and three-dimensional structure with respect to the antigen-binding site (Tonegawa S. Nature. 302: pp. 575-581). The diversity of heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM.Immunity.13:pp. 37-45) and desirable antigen-binding affinities (Schier R, et al. J Mol Biol.263:pp. 551-67).

[0137]

[0158] In some embodiments, the anti-hGREM1 antibodies and antigen-binding fragments provided herein include all or part of the heavy chain variable domain and / or all or part of the light chain variable domain. In one embodiment, the anti-hGREM1 antibodies and antigen-binding fragments provided herein are single-domain antibodies comprising all or part 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 provided herein include a suitable framework region (FR) sequence, insofar as the antibody and antigen-binding fragment can specifically bind to GREM1. The CDR sequences provided in Table 1 are obtained from mouse antibodies but can be transplanted into any suitable FR sequence of any suitable species, such as mouse, human, rat, or rabbit, using suitable methods known in the art, such as recombinant techniques.

[0139]

[0160] In certain embodiments, the antibody and its antigen-binding fragment provided herein are chimeric. VH / VL are transplanted into a human IgG1 sequence and a human kappa sequence.

[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 region, as a non-human CDR sequence is transplanted into a human or substantially human FR sequence. Humanization of antibodies or antigen-binding fragments can be essentially carried out by replacing the corresponding human CDR gene in a human immunoglobulin gene with a non-human (e.g., mouse) CDR gene (see, e.g., 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 parental non-human antibody can be performed before or after such humanization.

[0141]

[0162] Appropriate human heavy and light chain variable domains can be selected using methods known in the art for CDR transplantation. In an exemplary example, a “best-fit” approach may be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened against a database of known human variable domain germline sequences (e.g., Protein Data Bank, http: / / www.rcsb.org / ) or BLASTed (BLASTed), and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for transplanting 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, a framework derived from the consensus sequences of all human antibodies can be used for the transplantation of non-human CDRs (see, for example, 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 substantially entirely of human sequences, except for the non-human CDR sequences. In some embodiments, the variable region FRs, and where present, the constant regions are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and 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 antibody or antigen-binding fragment comprises human heavy / light chain FR1-4.

[0143]

[0164]

[0144]

Table 4-1

[0145]

Table 4-2

[0146]

[0165]

[0147]

Table 5

[0148] Here, 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; X 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 comprise 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 parental non-human antibody. This may be desirable in certain embodiments in order to approximate the humanized antibody or fragment thereof to the non-human parental antibody structure to reduce or avoid immunogenicity and / or to improve or maintain binding activity or binding affinity.

[0150]

[0167] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein contain 10, 9, 8, 7, 6, 5, 4, 3, 2, or one or fewer amino acid residue substitutions in each human FR sequence, or contain 10, 9, 8, 7, 6, 5, 4, 3, 2, or one or fewer amino acid residue substitutions in all FRs of the variable domains of the heavy chain or light chain. 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, for example, by backmutation to corresponding residues found in the non-human parental antibody from which the CDR sequence is derived (e.g., in the mouse framework region). Suitable sites for mutation can be selected by those skilled in the art according to principles known in the art. For example, the site of mutation may be selected from the following locations: 1) a location in the human gremlin sequence framework where there are few residues (e.g., less than 20% or less than 10% of the human variable region sequence); 2) a location where the site is directly adjacent to one or more of the three CDRs in the primary sequence of the human germline chain, because it is likely to interact with residues in the CDRs; or 3) a location where the site is close to the CDRs in the three-dimensional model and therefore has a good chance of interacting with amino acids in the CDRs. The residue at the selected site may be reverse-mutated 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 residue that is normal in the human sequence, i.e., a residue that is more frequently present at that location in known human sequences belonging to the same subgroup as the human germline sequence (see U.S. Patent No. 5,693,762).In a particular embodiment, for hu14E3, the heavy chain variants Ha, Hb, and Hc were obtained by directly transplanting three CDRs into their germline sequences, each having, based on Kabat numbering, a V37M revertant mutation for heavy chain variant Ha, V37M, V68A, and V2I revertant mutations for heavy chain variant Hb, and V37M, V68A, V2I, Y27S, R38K, and E46T revertant mutations for heavy chain variant Hc; the light chain variants were obtained by directly transplanting three CDRs into their germline sequences, each having, based on Kabat numbering, a F36L revertant mutation for light chain variant La, and F36L and Y49S revertant mutations for light chain variant Lb. In a particular embodiment, for hu22F1, heavy chain variants Ha, Hb, Hc, and Hd are all assigned Kabat numbering as follows: heavy chain variant Ha has no revert mutations; heavy chain variant Hb has the revert mutations R72V, T74K, T28S, and R98S; and heavy chain variant Hc has the revert mutations R71V, T73K, T28S, R94S, and S82. A It has revertant mutations R, M69L, and V2A, and for the heavy chain variant Hd, R71V, T73K, T28S, R94S, and S82. AThe germline sequences containing the R, M69L, V2A, V67A, M48I, and V37L reverse mutations were obtained by directly transplanting three CDRs; the light chain variants were all obtained based on Kabat numbering, by directly transplanting three CDRs into the germline sequences containing the F36L reverse mutation for light chain variant La, and the F36L and V58F reverse mutations for light chain variant Lb, respectively. In a particular embodiment, for hu56C11, heavy chain (HC) variants 1, 2, 3, and 4 were all obtained by directly transplanting three CDRs into the germline sequence having, based on Kabat numbering, no revert mutations for heavy chain variant H0, R71V and T73K revert mutations for heavy chain variant Ha, R71V, T73K, M69L and M48I revert mutations for heavy chain variant Hb, and R71V, T73K, M69L, M48I, Q43K and R38K revert mutations for heavy chain variant Hc; light chain (LC) variants 1, 2, and 3 were all obtained by directly transplanting three CDRs into the germline sequence having, based on Kabat numbering, no revert mutations for light chain variant L0, R46L revert mutations for light chain variant La, and F36Y and R46L revert mutations for light chain variant Lb.

[0151]

[0168] In certain embodiments, the humanized light and heavy chains of this disclosure are substantially non-immunogenic in humans and retain substantially the same or even higher affinity to hGREM1 as the parental antibodies. Specifically, the humanized antibodies provided herein (e.g., Hu22F1 and Hu56C11) and the 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 against gremlin-mediated inhibition of BMP4 signaling, high blocking activity of gremlin 1 binding to FGFR1, and / or high antitumor effect), or even better than their parental antibodies.

[0152]

[0169] In certain embodiments, the humanized antibody and its antigen-binding fragment provided herein comprises one or more light chain FR sequences of the human germline framework sequence IGKV / 2-30, with or without reverse mutations, and / or one or more heavy chain FR sequences of the human germline framework sequence IGHV / 7-4, IGHV / 1-46, or IGHV1-2. Reverse mutations may be introduced into the human germline framework sequence as needed.

[0153]

[0170] In certain embodiments, the humanized antibody and its antigen-binding fragment provided herein include a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 41, 43, 45, 51, 53, 55, 57, 131, 132, 133, and 134, as well as homologous sequences 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 its antigen-binding fragment provided herein further comprises a light chain variable region including a sequence selected from the group consisting of SEQ ID NOs: 47, 49, 59, 61, 135, 136, and 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 includes a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 41, 43, and 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 NOs: 47 and 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 its antigen-binding fragment provided herein comprises a pair of heavy chain variable region sequences 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 while still retaining specific binding specificity or affinity to hGREM1.

[0157]

[0174] In certain embodiments, the humanized antibody or antigen-binding fragment provided herein includes a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs. 51, SEQ ID NOs. 53, SEQ ID NOs. 55, and SEQ ID NOs. 57, and a homologous sequence 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 NOs. 59 and SEQ ID NOs. 61, and a homologous sequence 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 its antigen-binding fragment provided herein comprises a pair of heavy chain variable region sequences and light chain variable region sequences selected from the group consisting of SEQ ID NOs: 51 / 59, 51 / 61, 53 / 59, 53 / 61, 55 / 59, 55 / 61, 57 / 59, and 57 / 61; or a pair of sequences 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.

[0159]

[0176] In certain embodiments, the humanized antibody or antigen-binding fragment provided herein includes a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 131, 132, 133, and 134, and a homologous sequence 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 NOs: 135, 136, and 137, and a homologous sequence 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, the humanized antibodies or antigen-binding fragments thereof provided herein further comprise 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, 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 retained specific binding affinity to hGREM1 and in this regard were at least comparable or even better than the parental antibody. In certain embodiments, the anti-hGREM1 antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG. In some embodiments, the immunoglobulin constant region comprises a constant region of the heavy chain and / or light chain. The heavy chain constant region comprises CH1, hinge, and / or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises the Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.

[0166]

[0180] In certain embodiments, the anti-hGREM1 antibody and its fragment provided herein further comprises a constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the anti-hGREM1 antibody and its antigen-binding fragment provided herein comprises a constant region of an IgG1 isotype. In certain embodiments, the anti-hGREM1 antibody and its antigen-binding fragment provided herein comprises a constant region of an IgG2b isotype.

[0167]

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

[0168]

[0182] Antibody variant

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

[0169]

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

[0170]

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

[0186] Affinity variants either retain the specific binding affinity of the parent antibody to hGREM1, or even possess an improved hGREM1-specific binding affinity superior to that of the parent antibody. Various methods known in the art can be used to achieve this objective. 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 their binding affinity to human GREM1. In another example, computer software can be used to virtually simulate the binding of an antibody to human GREM1 and identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided in substitutions to prevent a decrease in binding affinity, or they may be 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 are conservative substitutions.

[0172]

[0188] In certain embodiments, the anti-hGREM1 antibody or antigen-binding fragment provided herein contains 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 variant contains a total of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer substitutions in one or more CDR sequences and / or FR sequences.

[0173]

[0189] In a particular embodiment, the anti-hGREM1 antibody and its antigen-binding fragment comprises 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 with the CDR sequences (one or more) listed in Table 1, while simultaneously maintaining a binding affinity to hGREM1 at a level similar to or even higher than that of its parent antibody. ii. Glycosylated variants

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

[0174]

[0191] Antibody glycosylation is typically N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue, such as in tripeptide sequences like 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 removing 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 either the antibody or the Fc sequence. Alternatively, in certain embodiments, antibody glycosylation variants may be obtained by producing the antibody in a host cell line in which the selected sugar group cannot be added to the mature core carbohydrate structure of the antibody. iii. Cysteine-manipulated variant

[0193] The anti-hGREM1 antibodies and antigen-binding fragments provided herein also include cysteine-manipulated 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 crosslink. Cysteine-manipulated variants are useful for conjugation with, for example, cytotoxic compounds and / or imaging compounds, labels, or radioisotopes, via maleimide or haloacetyl at the manipulated cysteine ​​site. Methods for introducing free cysteine ​​residues by manipulating antibody or antigen-binding fragments 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 fragments 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, or bivalent domain antibodies.

[0179]

[0198] Various techniques can be used for the production of such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as Escherichia coli (e.g., in the case of Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., in the case of 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 the production of antibody fragments will be apparent to those skilled in the art.

[0180]

[0199] In certain embodiments, the antigen-binding fragment is an scFv. The generation of scFvs is described, for example, in WO93 / 16185; U.S. Patent Nos. 5,571,894; and 5,587,458. The scFv can be fused to an effector protein at either its amino or carboxyl terminus to yield a fusion protein (see, for example, Antibody Engineering, edited by Borrebaeck).

[0181]

[0200] In certain embodiments, the anti-hGREM1 antibody and its antigen-binding fragment provided herein are divalent, tetravalent, hexavalent, or polyvalent. As used herein, “valency” refers to the presence of a specified number of antigen-binding sites in a given molecule. Thus, the terms “divalent,” “tetravalent,” and “hexavalent” represent the presence of two, four, and six binding sites, respectively, in the antigen-binding molecule. Any molecule with more than two binding sites is considered polyvalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0182]

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

[0183]

[0202] A bivalent molecule can also be bispecific if its two binding sites are specific to different antigens or epitopes. This also applies to polyvalent molecules. For example, a trivalent molecule can be bispecific if two binding sites are monospecific to a first antigen (or epitope) and a third binding site is specific to a second antigen (or epitope).

[0184]

[0203] Epitope

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

[0185]

[0205] As used herein, the term “epitope” refers to a specific group of atoms or amino acids of an antigen to which an antibody binds. An epitope may include a specific amino acid, sugar side chain, phosphoryl group, or sulfonyl group that is in direct contact with the antibody. Those skilled in the art will understand that, without excessive experimentation, it is possible to determine whether a given antibody and one of the antibodies of this disclosure compete for binding to the GREM1 antigen polypeptide, thereby determining whether that antibody binds to the same epitope, a duplicate epitope, or an adjacent epitope as the antibodies of this disclosure (e.g., each of the hybridoma / chimeric or humanized antibodies 14E3, 69H5, 22F1, 56C11, 36F5, 42B9, and 67G11 provided herein, as well as any of their chimeric and humanized variants).

[0186]

[0206] As used herein with respect to two antigen-binding proteins (e.g., antibodies), the term “competing for binding” means that, as determined by a competitive binding assay, one antigen-binding protein blocks or reduces the binding of the other antigen-binding protein to an antigen (e.g., human / mouse GREM1). Competitive binding assays are well known in the art and include, for example, direct or indirect radioimmunoassays (RIAs), direct or indirect enzyme immunoassays (EIAs), Fortebio, competitive ELISA assays, and sandwich competitive assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9: pp. 242-253). Typically, such assays involve the use of purified antigen or antigen-carrying 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, they bind to the same epitope, a duplicate epitope, or an adjacent epitope that is close enough to the epitope bound by the other antibody to cause steric hindrance. Typically, if competing antibodies are present in excess, they will inhibit (e.g., reduce) the specific binding of the test antibody to the 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 their biological characteristics

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

[0188]

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

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

[0189]

[0211] This disclosure 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 does not exhibit such inhibition, or exhibits only very limited inhibition, in non-cancer cells.

[0190]

[0212] Eliminating GREM1 and / or reducing its activity in cancer cells is desirable because it has inhibitory effects on cancer cell proliferation and sphere formation, as well as inducing apoptosis. However, conventional GREM1 knockout in mice can lead to abnormal intestinal development and hematopoietic disorders, making GREM1 inhibition generally undesirable (Rowan, SC et al. Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure. J Pathol 251, pp. 117-122). This suggests that conventional elimination and / or reduction of GREM1 activity inevitably causes adverse effects on other normal tissues. In this regard, the unexpected cancer-specific modulation of BMP signaling by GREM1-mediated inhibition by the anti-GREM1 antibody provided herein is beneficial because it avoids undesirable side effects on normal tissues.

[0191]

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

[0192]

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

[0193]

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

[0194]

[0216] GREM1-mediated inhibition of BMP signaling can be determined by measuring BMP signaling of BMP ligands in the presence and absence of GREM1, respectively, in which case the difference indicates GREM1-mediated inhibition. However, anti-GREM1 antibodies can reduce GREM1-mediated inhibition of BMP signaling, or in other words, restore BMP signaling. The reduction or restoration of GREM1-mediated inhibition of BMP signaling can be calculated as the increase in BMP signaling in the presence of the anti-GREM1 antibody compared to BMP signaling in the absence of the anti-GREM1 antibody. The percentage of such reduction or restoration can be calculated as the ratio of the reduction in GREM1-mediated inhibition to total GREM1-mediated inhibition. A 100% reduction in 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 antibody provided herein can bind to a chimeric GREM1 (also referred to herein as "XM5") containing the amino acid sequence of SEQ ID NO: 68. Chimeric GREM1 XM5 is a mutant version of hGREM1, comprising 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 DAN (FSYSVPNTFPQSTESLVHCDS, SEQ ID NO: 64), which do not bind to BMP. Therefore, this chimeric hGREM1 does not bind to BMP. Certain existing anti-GREM1 antibodies cannot bind to this chimeric hGREM1, which suggests that their binding to hGREM1 requires a BMP binding loop in hGREM1. In contrast, the anti-GREM1 antibodies provided herein can bind to this chimeric hGREM1, indicating that the anti-GREM1 antibodies bind to hGREM1 at an epitope outside this BMP-binding loop.

[0197]

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

[0198]

[0221] The benchmark antibody 6245P cannot bind to X5.

[0199]

[0222] iii) Antibodies that cross-react with or do not cross-react with mouse GREM1

[0223] In certain embodiments, some of the anti-GREM1 antibodies provided herein can bind to hGREM1 but cannot specifically bind to mouse GREM1.

[0200]

[0224] hGREM1 and mouse GREM1 share 98% sequence identity, with different amino acid residues found only in the N-terminal region of hGREM1, including Gln27 and Asn33 of hGREM1, in which case the residue numbering follows SEQ ID NO: 69. Therefore, in the case of an anti-GREM1 antibody that does not cross-react with mouse GREM1, the antibody may bind to hGREM1 with an epitope containing Gln27 and / or Asn33 of hGREM1, in which case the residue numbering follows SEQ ID NO: 69, or the antibody may bind to an hGREM1 fragment containing residues Gln27 and / or Asn33, and optionally the hGREM1 fragment is expected to have a length of at least 3 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acid residues. The epitope may be a structural epitope or a linear epitope.

[0201]

[0225] In certain embodiments, the antibodies provided herein include heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, as well as light chain CDR1 (LCDR1), LCDR2, and LCDR3, which are antibodies that can bind to hGREM1 but cannot specifically bind to mouse gremlin 1, and are selected from the group consisting of 69H5, 22F1, and 14E3.

[0202]

[0226] In certain embodiments, the anti-hGREM1 antibodies provided herein are cross-reactive to mouse GREM1 and include heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, as well as light chain CDR1 (LCDR1), LCDR2, and LCDR3, 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 can block the binding of hGREM1 to BMP7 by at least 50% of the maximum blocking percentage when measured by ELISA.

[0205]

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

[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 an inhibitor (e.g., an anti-gremlin 1 antibody) to block the interaction between two proteins (e.g., human gremlin 1 and BMP7). Generally, the blocking percentage increases with increasing concentration of the inhibitor (e.g., anti-gremlin 1 antibody), but may reach a plateau where no further blocking can be achieved despite further increases in the concentration of the inhibitor (e.g., anti-gremlin 1 antibody). A higher maximum blocking percentage indicates more effective blocking. The maximum blocking percentage may vary to some extent depending on the different assays, such as competitive ELISA assays and competitive FACS assays.

[0207]

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

[0209]

[0234] BMP-7 is a homodimeric protein with a cysteine ​​knot, and it is selectively expressed only in certain 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 kidneys is highest in adult organs and is downregulated in the 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, 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, 2808-2820, 1995; Simon et al., Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney. Am. J. Physiol. 276, F382-F389, 1999; Wang et al., Loss of tubular bone morphogenetic protein-7 in diabetic nephropathy. J. Am. Soc. Nephrol. 12, 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, 202-214., 1998; Simon et al., Expression of bone morphogenetic protein-7 mRNA in normal and ischemic adult rat kidney. Am. J. Physiol. 276, F382-F389., 1999).

[0210]

[0235] BMP7, as one of the key cytokines in the TGFβ superfamily, plays an anti-fibrotic role in chronic kidney disease by balancing the TGF-beta signaling pathway, which mediates renal fibrosis by increasing the production of the extracellular matrix (ECM) and decreasing its degradation (Rui et al., Role of bone morphogenetic protein-7 in renal fibrosis, Front. Physiol., April 23, 2015). In several animal models of renal injury, BMP7 treatment has been reported to reverse renal fibrosis and restore renal function (Hruska et al., Osteogenic protein-1 prevents renal fibrogenesis associated with ureteral obstruction. Am.J.Physiol.Renal Physiol.279, pp. 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, BMP7 activity in the kidney is determined not only by the availability of BMP7 itself, but also by the balance between 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) must be considered in relation to the efficacy of the treatment (Michael et al., Reversal of experimental renal fibrosis by BMP7 provides insights into novel therapeutic strategies for chronic kidney disease, Pediatr Nephrol. September 2008; 23(9): pp. 1395-1398).

[0212]

[0237] This disclosure shows that gremlin 1 binds to BMP7, and that the anti-gremlin 1 antibodies provided herein (e.g., 42B9, 36F5, 67G11, and 14E3HaLa) have more potent blocking activity for the binding of gremlin 1 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 constructed according to the sequence of H4H6245P disclosed in WO2014159010, which incorporates the entire disclosure 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). Therefore, it can be reasonably expected that the anti-gremlin 1 antibodies provided herein (e.g., 42B9, 36F5, 67G11, and 14E3HaLa) may improve the therapeutic efficacy of treatments for fibrotic diseases and kidney diseases (e.g., renal fibrosis).

[0213]

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

[0214]

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

[0240] In certain embodiments, the antibodies provided herein can block the 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 include heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, as well as light chain CDR1 (LCDR1), LCDR2, and LCDR3, selected from the group consisting of 14E3, 42B9, 67G11, and 36F5. These antibodies are particularly useful in methods for treating conditions or diseases related to GREM1 binding to FGFR or FGFR activity.

[0215]

[0241] In certain embodiments, the antibodies provided herein do not block the 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 include heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, as well as light chain CDR1 (LCDR1), LCDR2, and LCDR3 antibodies selected from the group consisting of 56C11.

[0216]

[0242] In certain embodiments, the antibodies provided herein partially block the 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 include heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, as well as 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 critical signaling 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 via the epidermal growth factor (EGF) receptor mediated by its ligand called EGF, or via the fibroblast growth factor (FGF) receptor mediated by its ligand called FGF. Surprisingly, this disclosure has found that GREM1 appears to play a role in the activation of MAPK signaling, possibly functioning as a novel ligand for FGFR. This disclosure further finds that the anti-GREM1 antibody provided herein can reduce GREM1-mediated activation of MAPK signaling, and, in particular, can block the interaction between GREM1 and FGFR.

[0218]

[0245] vii) Antibodies that bind to both hGREM1 and DAN

[0246] The antibodies provided in this disclosure can specifically bind to one or more (e.g., one, two, three, or more) DAN family members, including GREM1. In certain embodiments, the antibodies provided herein can bind to both hGREM1 and DAN. As used herein, the term “DAN” refers to the founding members of the DAN family (also known as NbI1 and DAND1), which are moderate antagonists for modulating BMP signaling. DAN originally acted as tumor suppressor genes 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. Gremlins act as potent antagonists, while DANs function as moderate antagonists. Both can antagonize BMP2, BMP4, and BMP7, but share only about 20% identity.

[0219]

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

[0220]

[0248] In certain embodiments, the anti-hGREM1 antibody provided herein is capable of binding to hGREM1 but not to DAN, and comprises heavy chain CDR1 (HCDR1), HCDR2, and HCDR3, as well as light chain CDR1 (LCDR1), LCDR2, and LCDR3 of antibody 14E3, 22F1, 56C11, or 69H5.

[0221]

[0249] bispecific antibody

[0250] In certain embodiments, the antibodies and antigen-binding fragments provided herein are bispecific. As used herein, “bispecific” includes molecules having three or more specificities, and molecules having three or more specificities, i.e., multispecific. In certain embodiments, the bispecific antibodies and antigen-binding fragments provided herein can specifically bind to first and second epitopes of hGREM1, or can specifically bind to hGREM1 and a second antigen. In certain embodiments, the first and second epitopes of hGREM1 are entirely different from or do not overlap with each other. In certain embodiments, the bispecific antibodies and antigen-binding fragments can bind to both the first and second epitopes simultaneously. 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 targets encompass biomolecules involved in the generation, inhibition, or modulation of immune responses, optionally, cellular immune responses. Examples of immune-related targets include immune checkpoint molecules.

[0223]

[0252] Immune checkpoint molecules can amplify immune responses through co-stimulatory signals or suppress immune responses through co-inhibitory signals. 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 includes PD-1, PD-L1, CTLA-4, or LAG-3.

[0224]

[0253] In certain embodiments, the second antigen includes a tumor antigen. As used herein, “tumor antigen” means tumor-specific antigen (e.g., an antigen unique to tumor cells and not commonly found in non-tumor cells) and tumor-associated antigen (e.g., an antigen found in both tumor and non-tumor cells but expressed differently in tumor cells or found in the tumor microenvironment). Tumor-specific antigens may also include tumor neoantigens (e.g., expressed in cancer cells because somatic mutations alter the protein sequence or create a fusion protein 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), and estrogen receptors. Examples include the body, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2, GPC-3, nectin-4, ROR1, metoserin, 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.

[0226]

[0255] In certain embodiments, tumor antigens include 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-related mucin, VEGF, VEGFR (e.g., VEGFR3), and estrogen receptor Contains phosphate, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2, GPC-3, nectin-4, ROR1, metoserin, 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 their antigen-binding fragments provided herein may be in appropriate formats known in the art. For example, exemplary bispecific formats include bispecific diabodies, scFv-based bispecific formats, IgG-scFv fusions, bivariable domain (DVD)-Ig, quadromas, knob-into-holes, common light chains (e.g., common light chains with knob-into-holes), BiTE, CrossMab, CrossFab, Duobody, SEEDbody, leucine zipper, dual-acting Fab (DAF)-IgG, and Mab 2 It can be a bispecific format (see, for example, Brinkmann et al. 2017, Mabs, 9(2): pp. 182-212). The bispecific molecule may have a symmetric or asymmetric structure.

[0228]

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

[0229]

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

[0230]

[0259] In another embodiment, sequences encoding the antibody heavy chain variable domains for two specificities are fused to immunoglobulin constant domain sequences, respectively, and subsequently inserted into one or more expression vectors, which, along with the expression vector for the light chain sequence, are co-transfected into host cells suitable for recombinant expression of the bispecific antibody (see, e.g., WO94 / 04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be recombinantly constructed and expressed from host cells (see, e.g., Gruber et al., J.Immunol., 152:5368 (1994)).

[0231]

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

[0232]

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

[0233]

[0262] Bispecific antigen-binding fragments can be generated from bispecific antibodies, for example, by proteolytic cleavage or by chemical crosslinking. For example, an antigen-binding fragment of an antibody (e.g., Fab') can be prepared, 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 provided herein can be manipulated at the interface so that a knob-into-hole association can be formed to promote heterodimerization of two different antigen-binding sites. Such actions can maximize the percentage of heterodimers recovered from recombinant cell cultures. As used herein, “knob-into-hole” refers to an interaction between two polypeptides (e.g., Fc), in which one polypeptide has a protrusion (i.e., a “knob”) due to the presence of a bulky side-chain amino acid residue (e.g., tyrosine or tryptophan), and the other polypeptide has a recess (i.e., a “hole”) where a small side-chain amino acid residue (e.g., alanine or threonine) is present, but the protrusion can be positioned in the recess to promote the interaction between the two polypeptides, resulting in the formation of a heterodimer or complex. Methods for generating polypeptides having a knob-into-hole are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0235]

[0264] Conjugate

[0265] In some embodiments, an anti-hGREM1 antibody and its antigen-binding fragment are linked to one or more conjugate portions. A conjugate is a portion that can be attached to an antibody or its antigen-binding fragment. Various conjugates are intended to be linked to the antibodies or antigen-binding fragments provided herein (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, JMCruse and RE Lewis, Jr. (eds.), Carger Press, New York, 1989). Such conjugates may be linked to the antibody or antigen-binding fragment by means of covalent bond, affinity bond, intercalation, coordination bond, complex formation, association, mixing, 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, or a thioether linker.

[0236]

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

[0237]

[0267] Conjugates may be clearance modifiers, therapeutic agents (e.g., chemotherapeutic agents), toxins, radioisotopes, detectable labels (e.g., lantanides, luminescence labels, fluorescent labels, or enzyme substrate labels), pharmacokinetic modifiers, DNA alkylating agents, topoisomerase inhibitors, tubulin conjugates, or other anticancer drugs such as androgen receptor inhibitors.

[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 lantanides, luminescence labels, chromophore moieties, digoxigenin, biotin / avidin, DNA molecules for detection, or gold.

[0239]

[0269] Examples of radioactive isotopes 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 P can be cited. Radioisotope-labeled antibodies are useful in receptor-targeted imaging experiments.

[0240]

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

[0241]

[0271] In certain embodiments, the conjugate may be a purified portion such as magnetic beads or nanoparticles.

[0242]

[0272] Pharmaceutical composition

[0273] This disclosure provides a pharmaceutical composition comprising an anti-hGREM1 antibody or its antigen-binding fragment and one or more pharmaceutically acceptable carriers.

[0243]

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

[0244]

[0275] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may 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. Inclusion of one or more antioxidants, such as methionine, in a composition comprising an antibody or antigen-binding fragment and conjugate provided herein, as disclosed herein, reduces the oxidation of the antibody or antigen-binding fragment. Such reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Accordingly, in certain embodiments, a composition is provided comprising one or more antibodies or antigen-binding fragments disclosed herein and one or more antioxidants, such as methionine. Furthermore, a method is provided for preventing oxidation of an antibody or antigen-binding fragment, extending its shelf life, and / or improving its effectiveness, by mixing the antibody or antigen-binding fragment provided herein with one or more antioxidants such as methionine.

[0245]

[0276] To further illustrate, pharmaceutically 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 solution; non-aqueous vehicles such as non-volatile oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungiostatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphoric acid or citrate buffers; antioxidants such as sodium bisulfate; and local anesthetics. The pharmaceutical composition may include a drug, such as procaine hydrochloride; a suspending and dispersing agent, such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; an emulsifier, such as polysorbate 80 (TWEEN®-80); a chelating agent, 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 used as carriers may be added to the pharmaceutical composition in a multi-dose container, including phenol or cresol; mercury agents; benzyl alcohol; chlorobutanol; methyl and propyl p-hydroxybenzoic acid esters; thimerosal; benzalkonium chloride; and benzethonium chloride. Suitable excipients may include, for example, water, brine, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or pharmaceuticals such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0246]

[0277] Pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may contain standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0247]

[0278] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for producing a liquid solution, suspension, or emulsion. Preparations for injection may include sterile and / or nonpyrogenic solutions ready for injection, sterile dry soluble materials such as lyophilized powders ready for combination with a solvent immediately before use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble materials ready for combination with a vehicle immediately before use, and sterile and / or nonpyrogenic emulsions. The solutions may be aqueous or non-aqueous.

[0248]

[0279] In certain embodiments, a unit dose of parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. As is known and practiced in the art, all preparations intended 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 the 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, e.g., citrate, sodium phosphate, or potassium, or other such buffers known to those skilled in the art, in one embodiment, at a neutral pH. Subsequent sterile filtration of the solution and subsequent freeze-drying under standard conditions known to those skilled in the art yield the desired formulation. In one embodiment, the resulting solution will be distributed into vials for freeze-drying. Each vial may contain a single dose or multiple doses of the anti-hGREM1 antibody or its antigen-binding fragment or composition thereof. Overfilling vials by a small amount (e.g., about 10%) beyond the amount required for a dose or series of doses is acceptable to facilitate accurate sampling and accurate dosing. Lyophilized powders can be stored under suitable conditions, such as at about 4°C to room temperature.

[0250]

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

[0251]

[0282] In a particular embodiment, the pharmaceutical composition further comprises a second therapeutic agent.

[0252]

[0283] In certain embodiments, the second therapeutic agent may be a drug for treating cancer, such as a chemotherapeutic agent, an anticancer drug, radiotherapy, immunotherapy, an anti-angiogenic agent (e.g., VEGFR antagonists such as VEGFR-1, VEGFR-2, and VEGFR-3), targeted therapy, cell therapy, gene therapy agent, hormone therapy agent, cytokine, palliative care, surgery for treating cancer (e.g., tumor resection), or one or more antiemetic agents or other treatments for complications arising 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 may be a drug for treating fibrotic diseases.

[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 Recombination Methods

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

[0257]

[0289] In certain embodiments, the isolated polynucleotide comprises one or more nucleotide sequences represented by 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 sequences having at least 80% (e.g., at least 85%, 88%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereof, and / or variants thereof having only degenerate substitutions, wherein the isolated polynucleotide encodes a variable region of an exemplary antibody provided herein.

[0258]

[0290] The DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0259]

[0291] This disclosure provides vectors (e.g., expression vectors) comprising isolated polynucleotides provided herein. In certain embodiments, the expression vectors provided herein comprise a polynucleotide encoding an antibody or its antigen-binding fragment, at least one promoter (e.g., SV40, CMV, EF-1α) operably ligated to the polynucleotide sequence, and at least one selection marker. Examples of vectors, but not limited to, include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages, and M13 phages, plasmids (e.g., 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 comprising a polynucleotide sequence encoding an antibody or its antigen-binding fragment 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 prokaryotic cells, yeast cells, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, e.g., Gram-negative or Gram-positive bacteria, e.g., Enterobacteriaceae, e.g., Escherichia, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Sigella, as well as Bacillus, e.g., B. subtilis and B. richeniformis, Pseudomonas, e.g., Pseudomonas, and Streptomyces.

[0261]

[0293] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also 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 also commonly available and useful herein, e.g., Schizosaccharomyces pombe; Kluiveromyces hosts, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC24,178), K. warthii (ATCC56,500), K. drosophila These include *A. mu* (ATCC36,906), *K. thermotolerance*, and *K. marxianas*; *Yarrowia* (EP402,226); *Pichia pastris* (EP183,070); *Candida*; *Trichoderma resia* (EP244,234); *Neurospora crassae*; *Schwaniomyces*, e.g., *Schwaniomyces occidentalis*; as well as filamentous fungi, e.g., *Neurospora*, *Penicillium*, *Tripocladium*, and Aspergillus hosts, e.g., *A. nidurans* and *A. niger*.

[0262]

[0294] Host cells suitable for the expression of glycosylated antibodies or antigen fragments provided herein are derived from multicellular organisms such as invertebrate cells, plant cells, and insect cells. Numerous baculovirus strains and variants and corresponding insect-acceptable host cells have been identified from hosts such as the fall armyworm (caterpillar), Aedes aegypti (mosquito), Aedes japonica (mosquito), Drosophila melanogaster (fruit fly), and silkworm moth. Various virus strains for transfection, such as the L-1 variant of the Coenagrionidae NPV and the Bm-5 strain of the silkworm NPV, are publicly available and such viruses may be used herein in accordance with the present invention, particularly for transfection of fall armyworm cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

[0263]

[0295] However, interest in vertebrate cells is greatest, and the proliferation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: p. 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: p. 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: p. 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer 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 cells (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad.Sci.383: pp. 44-68 (1982)); MRC5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2). In some preferred embodiments, the host cells are mammalian cultured cell lines, e.g., CHO, BHK, NS0, 293, and their derivatives.

[0264]

[0296] Host cells are transformed with the above-mentioned expression vector or cloning vector for anti-hGREM1 antibody production and cultured in a conventional nutrient medium modified to be suitable for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. In another embodiment, the antibody may be produced by homologous recombination 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. Commercial media, such as Ham F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma), are suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: p. 44 (1979), Barnes et al., Anal. Biochem. 102: p. 255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655, or 5,122,469; WO90 / 03430; WO87 / 00195; or U.S. Patent No. Re. 30,985 may be used as culture media for host cells. Any of these media may be supplemented as appropriate 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), e.g., (HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN® drugs), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations that would be known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with host cells selected for expression and would be obvious to those skilled in the art.

[0266]

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

[0267]

[0299] Anti-hGREM1 antibodies and their antigen-binding fragments 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 their antigen-binding fragments. 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:pp. 1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J.5:pp. 1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Mechanically stable matrices such as controlled porous 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 used. ABX® resin (JTBaker, 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 anion or cation-exchange resins with heparin SEPHAROSE® chromatography (e.g., polyaspartate 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 impurities may be subjected to low-pH hydrophobic interaction chromatography using an elution buffer at a pH between approximately 2.5 and 4.5, preferably carried out at a low salt concentration (e.g., approximately 0–0.25 M salt).

[0270]

[0302] How to use

[0303] In one embodiment, this disclosure provides therapeutic applications for antibodies provided herein.

[0271]

[0304] In certain embodiments, the Disclosure provides a method for treating or preventing a GREM1-related disease or condition in a subject requiring treatment or prevention of a GREM1-related disease or condition, comprising the steps of 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 for treating a GREM1-related disease or condition in a subject requiring treatment, comprising the step of administering a therapeutically effective amount of an anti-human GREM1 antibody or its antigen-binding fragment to the subject, wherein the anti-human GREM1 antibody or its antigen-binding fragment is: a) An epitope containing residue Gln27 and / or residue Asn33 that can bind to hGREM1, where the residue numbers follow SEQ ID NO: 69, and / or b) It can bind to an hGREM1 fragment containing 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; and / or c) It can selectively reduce hGREM1-mediated inhibition of BMP signaling in cancer cells compared to non-cancer cells; and / or d) Non-cancer cells exhibit a reduction of 50% or less in hGREM1-mediated inhibition of BMP signaling; and / or e) Can bind to a chimeric hGREM1 containing the amino acid sequence of SEQ ID NO: 68; and / or g) When measured with Fortebio, K is less than 1 nM. D It can be bound to hGREM1; and / or h) When measured by ELISA, the binding of hGREM1 to BMP7 can be blocked at a maximum blockage percentage of more than 50%; and / or i) The interaction between GREM1 (e.g., hFGFR1 or mGREM1) and FGFR (e.g., FGFR1, preferably human FGFR1 (hFGFR1) or mouse FGFR1 (mFGFR1)) can be blocked.

[0273]

[0306] In another aspect, the Disclosure provides a method for inhibiting FGFR1 activity in a subject requiring inhibition of FGFR1 activity, or a method for treating a disease or condition associated with GREM1-mediated FGFR1 activation, comprising the step of administering a therapeutically effective amount of an anti-human GREM1 antibody or its antigen-binding fragment to the subject, wherein the anti-human GREM1 antibody or its antigen-binding fragment includes: a) HCDR1 containing the sequence of sequence number 1, HCDR2 containing the sequence of sequence number 2, and HCDR3 containing the sequence of sequence number 3; LCDR1 containing the sequence of sequence number 4, LCDR2 containing the sequence of sequence number 5, and LCDR3 containing the sequence of sequence number 6; b) HCDR1 containing the sequence of sequence number 11, HCDR2 containing the sequence of sequence number 12, and HCDR3 containing the sequence of sequence number 13; LCDR1 containing the sequence of sequence number 14, LCDR2 containing the sequence of sequence number 15, and LCDR3 containing the sequence of sequence number 16; c) HCDR1 containing the sequence of sequence number 21, HCDR2 containing the sequence of sequence number 22, and HCDR3 containing the sequence of sequence number 23; LCDR1 containing the sequence of sequence number 24, LCDR2 containing the sequence of sequence number 25, and LCDR3 containing the sequence of sequence number 26; d) HCDR1 containing the sequence of sequence number 31, HCDR2 containing the sequence of sequence number 32, HCDR3 containing the sequence of sequence number 33; LCDR1 containing the sequence of sequence number 34, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 36; e) HCDR1 containing the sequence of sequence number 114, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 116; LCDR1 containing the sequence of sequence number 117, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; f) HCDR1 containing the sequence of sequence number 119, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 120; LCDR1 containing the sequence of sequence number 121, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; or g) HCDR1 containing the sequence of sequence number 119, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 120; LCDR1 containing the sequence of sequence number 122, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118.

[0274]

[0307] In another aspect, the Disclosure provides a method for inhibiting FGFR1 activity in a subject requiring inhibition of FGFR1 activity, or a method for treating a disease or condition associated with GREM1-mediated FGFR1 activation, comprising the step of administering a therapeutically effective amount of an anti-human GREM1 antibody or its antigen-binding fragment to the subject, wherein the anti-human GREM1 antibody or its antigen-binding fragment includes: a) HCDR1 containing the sequence of sequence number 123, HCDR2 containing the sequence of sequence number 115, HCDR3 containing the sequence of sequence number 124; LCDR1 containing the sequence of sequence number 125, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118, b) HCDR1 containing the sequence of sequence number 114, HCDR2 containing the sequence of sequence number 115, HCDR3 containing the sequence of sequence number 116; LCDR1 containing the sequence of sequence number 117, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; c) HCDR1 containing the sequence of sequence number 119, HCDR2 containing the sequence of sequence number 115, HCDR3 containing the sequence of sequence number 120; LCDR1 containing the sequence of sequence number 121, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; or d) HCDR1 containing the sequence of sequence number 119, HCDR2 containing the sequence of sequence number 115, HCDR3 containing the sequence of sequence number 120; LCDR1 containing the sequence of sequence number 122, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118.

[0275]

[0308] GREM1-related diseases or conditions may be conditions that benefit from modulation of GREM1 activity (e.g., reduction of GREM1 activity). In some embodiments, GREM1-related diseases or conditions are characterized by GREM1 expression or overexpression.

[0276]

[0309] As used herein, the term “overexpression” of GREM1 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, at choice, 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 healthy subjects. In some embodiments, GREM1-expressing cancers have a GREM1 expression level at least 10% higher than the reference level (e.g., at least 15%, 20%, 30%, 35%, 40%, 50%, or 1x, 2x, 3x, or even higher).

[0277]

[0310] GREM1 expression can be determined based on either the nucleic acid level or the protein level. The expression level of GREM1 may be measured at the nucleic acid level by any method known in the art, for example, but not limited to amplification assays (e.g., polymerase chain reaction, quantitative real-time PCR, rolling circle replication, isothermal amplification, etc.), hybridization assays (e.g., Northern blotting, microarrays, fluorescence insight hybridization (FISH), etc.), or sequencing assays (e.g., RNA sequencing). Alternatively, the expression level of GREM1 may be measured at the protein level by any method known in the art, for example, but not limited to immunoassays (e.g., Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme-mediated immunoassay (EIA), radioimmunoassay (RIA), sandwich assays, competitive assays, immunofluorescence staining and imaging, immunohistochemistry (IHC), and fluorescence-activated cell sorting (FACS)).

[0278]

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

[0279]

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

[0280]

[0313] i. Cancer treatment

[0314] In certain embodiments, this disclosure provides a method for treating or preventing cancer using antibodies provided herein.

[0281]

[0315] In some embodiments, the cancer is a GREM1-expressing cancer. As used herein, “GREM1-expressing cancer” means a cancer characterized by having GREM1-expressing cancer cells and / or having GREM1 expression in the tumor microenvironment. In some embodiments, a GREM1-expressing cancer has GREM1 overexpression in cancer cells and / or in the tumor microenvironment.

[0282]

[0316] GREM1 acts through an autocrine mechanism to promote the proliferation of tumor cells that express GREM1. GREM1 may also be secreted by non-cancerous cells present within or surrounding the tumor microenvironment, even if cancer cells themselves do not necessarily need to express GREM1, creating a niche suitable for the proliferation or survival of cancer cells.

[0283]

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

[0284]

[0318] In certain embodiments, the subject is identified as having GREM1-expressing cancer cells or exhibiting GREM1 expression in the tumor microenvironment. The presence and / or expression level of GREM1 on cancer cells or in the tumor microenvironment may be determined using a biological sample obtained from the subject by various methods known in the art or provided herein. A biological sample containing or suspected of containing cancer cells, or derived from the tumor microenvironment, may be obtained from or derived from the subject, e.g., formalin-fixed paraffin-embedded (FFPE) tissue, fresh biopsy, blood (suspected of containing circulating tumor cells), or other bodily fluids. In some embodiments, cancer cells, stromal cells, and / or extracellular matrix may be separated from the biological sample. In certain embodiments, the biological sample may be further processed to isolate analytes, e.g., nucleic acids or proteins.

[0285]

[0319] GREM1-expressing cancers can be any type of cancer. In certain embodiments, the cancer is selected from solid tumors or hematological malignancies. In certain embodiments, solid tumors include adrenocortical carcinoma, anal cancer, astrocytoma, pediatric cerebellar or cerebral cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma / gliomas, ependymoma, medulloblastoma, supratentorial primitive neuroectoderm tumor, visual tract and hypothalamic glioma, breast cancer, Burkitt lymphoma, cervical cancer, colon cancer, emphysema, endometrial cancer, esophageal cancer, Ewing's sarcoma, retinoblastoma, These include stomach cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (pancreatic endocrine cancer), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, liver cancer, lung cancer, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, Ewing family tumors, skin cancer, gastric cancer, testicular cancer, throat cancer, thyroid cancer, or vaginal cancer.

[0286]

[0320] In certain embodiments, GREM1-expressing cancer is also PD-L1-expressing. In certain embodiments, GREM1-expressing cancer is not PD-L1-expressing cancer. As used herein, the term "PD-L1-expressing" refers to cancer that is positive for PD-L1 expression using any detection method known in the art, e.g., immunohistochemistry (IHC), flow cytometry (e.g., FACS). For example, PD-L1-expressing cancer can refer to 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 exhibiting PD-L1 cell surface membrane staining being at least 1%, 2%, 3%, 4%, or 5% of all 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 cancer may refer to cancers that are positive for PD-L1 expression using the scoring process described in WO2014165422A1. In certain embodiments, GREM1-expressing cancers are resistant to or unresponsive to treatment with PD-1 / PD-L1 axis inhibitors.

[0287]

[0321] In certain embodiments, hematological malignancies are leukemias (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphomas (e.g., Hodgkin lymphoma, or non-Hodgkin lymphoma (e.g., Waldenström macroglobulinemia (WM))), or myelomas (e.g., multiple myeloma (MM)).

[0288]

[0322] In certain embodiments, cancers include 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, and glial cancer. These include blastomas, astrocytomas, melanomas, myelodysplastic syndromes, sarcomas, teratomas, gliomas, adenocarcinomas, leukemias (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphomas (e.g., Hodgkin lymphoma, or non-Hodgkin lymphoma (e.g., Waldenström macroglobulinemia (WM)), or myelomas (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, cancer is metastatic. In certain embodiments, this disclosure further provides methods for treating or preventing cancer metastasis using antibodies provided herein. Cancer metastasis is the process by which cancer cells spread within the body from their original site to another site.

[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. Each term, “triple-negative breast cancer” or “TNBC,” refers to breast cancer that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein. TNBC may be unresponsive to HER2-targeted hormone therapy or drugs.

[0293]

[0327] In certain embodiments, the cancer is multiple myeloma (MM). GREM1 is known to be secreted in large quantities by a subset of bone marrow (BM) mesenchymal stromal cells and is considered to play a crucial role in the development of MM disease. Quantitative PCR analysis of human and mouse BM stromal samples 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, p. 2149).

[0294]

[0328] ii. Treatment of fibrotic diseases

[0329] In some embodiments, GREM1-related diseases or conditions are fibrotic diseases. Fibrotic diseases are diseases or conditions characterized by fibrosis. Fibrosis is a scarring process that is a common feature of chronic organ injury, such as in the lungs, liver, kidneys, skin, heart, intestines, or muscles. Fibrosis is characterized by increased activity of transforming growth factor beta (TGF-beta), which results in increased and altered deposition of the 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 for 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 of the lungs, liver, kidneys, eyes, skin, heart, intestines, or muscles. Examples of pulmonary fibrotic diseases include pulmonary fibrosis, cystic fibrosis, pulmonary hypertension, progressive massive fibrosis, bronchiolitis obstructive, and airway remodeling associated with chronic asthma or idiopathic lung. Examples of hepatic fibrotic diseases include cirrhosis or non-alcoholic steatohepatitis. Examples of renal fibrotic diseases include renal fibrosis, ischemic nephropathy, tubulointerstitial fibrosis, diabetic nephropathy, nephrosclerosis, or nephrotoxicity. Examples of ocular fibrotic diseases include corneal fibrosis and subretinal fibrosis. Examples of cutaneous fibrotic diseases include nephrogenic systemic fibrosis, keloids, or scleroderma. Examples of cardiac fibrotic diseases include myocardial fibrosis or old myocardial infarction.

[0296]

[0331] In another aspect, the Disclosure also provides a method for improving the effectiveness of BMP7 treatment in treating a fibrotic disease (e.g., renal fibrosis) in a subject requiring treatment for the fibrotic disease, comprising the step of administering a therapeutically effective dose of an anti-GREM1 antibody provided herein to the subject. In a particular embodiment, the subject is the subject of the BMP7 treatment. In another aspect, the Disclosure also provides a method for treating a fibrotic disease (e.g., renal fibrosis) in a subject requiring treatment for the fibrotic disease, comprising the step of administering a therapeutically effective dose of an anti-gremlin 1 antibody provided herein to the subject in combination with a BMP7 treatment. As used herein, “BMP7 treatment” can mean any treatment that involves increasing the level of BMP7 in a subject requiring an increase in the level of BMP7. For example, a BMP7 treatment may be the administration of recombinant BMP7 or the administration of a BMP7 peptide mimetic. BMP7 treatment may also include restoring endogenous BMP7, for example, by reducing the levels and / or activity of a BMP7 antagonist (e.g., noggin, or uterine sensitization-associated gene-1 (USAG-1)) or by increasing the levels and / or activity of a BMP7 agonist (e.g., keyrin / cordin-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. September 2008; 23(9): pp. 1395-1398). In certain embodiments,

[0332] In another embodiment, the Disclosure also provides a method for treating a disease in which it is possible to benefit from increasing BMP7 activity or decreasing gremlin-mediated inhibition of BMP7 activity, the method comprising the step of administering a therapeutically effective dose of an anti-gremlin 1 antibody provided herein to the target. In certain embodiments, the disease is a fibrotic disease and / or kidney disease. In certain embodiments, the disease is selected from the group consisting of ischemia-reperfusion injury, ischemic acute renal 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, GREM1-related disease or condition is pulmonary arterial hypertension (PAH). The term “pulmonary arterial hypertension” (“PAH”) refers to progressive lung injury characterized by persistently elevated pulmonary artery pressure. GREM1 has been found to be elevated in the walls of intrapulmonary microvessels in hypoxic mice. Anti-GREM1 antibodies have been found to alleviate or improve 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 measurements in subjects with PAH, such as 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. 20180057580A1 for further details).

[0298]

[0335] In some embodiments, GREM1-related disease or condition is osteoarthritis (OA). GREM1 has been reported as a mechanical load inducer in chondrocytes and is detected at high levels in the mid- and deep layers of cartilage after periodic tension or hydrostatic loads. GREM1 has been reported to be upregulated in osteoarthritis, and serum and synovial fluid GREM1 concentrations 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 has been reported to slow the development of osteoarthritis (see SHChang et al., Nature Communications, (2019)10:1442).

[0299]

[0336] In some embodiments, GREM1-related diseases or conditions are angiogenesis. GREM1 is an agonist of vascular endothelial growth factor receptor-2 (VEGFR-2), a major pro-angiogenic receptor. Heparan sulfate (HS) and heparin, glycosaminoglycans (GAGs) known for their anticoagulant effects, have been shown to bind to GREM1. GREM1 binds to heparin and activates VEGFR-2 in a BMP-independent manner (Chiodelli et al. 2011; Arterioscler. Thromb. Vasc. Biol. 31: e116-e127). Anti-GREM1 antibodies have been found to alleviate or improve one or more symptoms associated with angiogenesis or heparin-mediated angiogenesis (see U.S. Patent Application No. 20200157194 for details).

[0300]

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

[0301]

[0338] In some embodiments, the GREM1-related disease or condition is a retinal disease. In some embodiments, the GREM1-related disease or condition is a kidney disease.

[0302]

[0339] Route of administration and dosage

[0340] The antibodies or antigen-binding fragments provided herein may be administered in therapeutically effective doses. The therapeutically effective dose of the antibodies or antigen-binding fragments provided herein will depend on various factors known in the art, such as body weight, age, medical history, current prescriptions, the subject's health status and potential for cross-reactivity, allergies, susceptibility and adverse side effects, as well as the route of administration and the extent of disease development. The dose may be increased or decreased proportionally by those skilled in the art (e.g., physicians or veterinarians) as indicated by these and other circumstances or requirements.

[0303]

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

[0304]

[0342] The dosage may be adjusted to produce the optimal desired response (e.g., a therapeutic response). For example, a single dose may be administered, or divided doses may be administered over time.

[0305]

[0343] The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, for example, by parenteral (e.g., intravenous, intramuscular, or intradermal injection, including subcutaneous, intraperitoneal, and intravenous infusion) 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 means 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 in combination with a second anticancer agent to treat cancer, for example, chemotherapeutic agents, anticancer drugs, radiotherapy, immunotherapy, anti-angiogenic agents, targeted therapies, cell therapies, gene therapy agents, hormone therapy agents, cytokines, palliative care, surgery for cancer treatment (e.g., tumor resection), one or more antiemetics, treatment of complications arising from chemotherapy, or nutritional supplements for cancer patients, or agents that modulate the tumor microenvironment.

[0308]

[0347] The term "chemotherapeutic drug" refers to a biological (high molecular weight) or chemical (small molecular weight) compound that can be used to treat cancer. Types of chemotherapeutic drugs include, but are not limited to, histone deacetylase inhibitors (HDACIs), alkylating agents, antimetabolites, alkaloids, cytotoxic / anticancer antibiotics, topoisomerase inhibitors, tubulin inhibitors, proteins, antibodies, and kinase inhibitors. Examples of chemotherapeutic drugs 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 drug is cisplatin.

[0309]

[0348] As used herein, the term “immunotherapy” refers to one type of treatment that stimulates the immune system to fight diseases such as cancer, or more generally, one type of treatment that boosts the immune system. Immunotherapy includes passive immunotherapy (e.g., antibody therapy or CAR-T cell therapy) by delivering drugs (e.g., effector cells) that have established tumor immunoreactivity and can directly or indirectly mediate antitumor effects without necessarily relying on the intact host immune system. Immunotherapy may further include active immunotherapy, in which the treatment utilizes in vivo stimulation of the endogenous host immune system to respond to disease cells by administering an immune response modifier.

[0310]

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

[0311]

[0350] Checkpoint modulators can interfere with cancer cells' ability to evade immune system attacks and help the immune system respond more strongly to tumors. Immune checkpoint molecules may either enhance or suppress the immune response by mediating co-stimulatory signals. 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 includes a PD-1 / PD-L1 axis inhibitor.

[0312]

[0351] Adoptive cell transplantation is a procedure that attempts to boost the natural ability of T cells to fight cancer. In this procedure, T cells are harvested from the patient, grown, and activated in vitro. In certain embodiments, the T cells are modified into CAR-T cells in vitro. The T cells or CAR-T cells that are most active against cancer are cultured in large batches in vitro for 2 to 8 weeks. During this period, the patient undergoes treatments such as chemotherapy and radiation therapy to reduce the body's immunity. After these treatments, the T cells or CAR-T cells cultured in vitro are 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 interferons and interleukins. Examples of cytokine therapy include, but are not limited to, interferons, e.g., interferon-α, -β, and -γ; colony-stimulating factors, e.g., macrophage CSF, granulocyte-macrophage CSF, and granulocyte CSF; insulin growth factor (IGF-1); vascular endothelial growth factor (VEGF); transforming growth factor (TGF); fibroblast growth factor (FGF); interleukins, e.g., 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, e.g., 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, thereby causing tumor cell lysis and the subsequent release of large amounts of tumor antigens. This release activates the immune system, which targets and eliminates cancer cells containing such tumor antigens. Examples of oncolytic viruses, though not limited to them, include talimogene raharpalebeck.

[0315]

[0354] Therapeutic vaccines act against cancer by boosting the immune system's response to cancer cells. Therapeutic vaccines can contain non-pathogenic microorganisms (e.g., Mycobacterium boviscalmet, BCG) and genetically modified viruses that target tumor cells or one or more immunogenic components. For example, BCG can be inserted directly into the bladder using a catheter to induce 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 certain proteins present in cancer cells but not in normal cells, or proteins that are more abundant in cancer cells, or target molecules in the tumor microenvironment that contribute to cancer growth and survival. Targeted therapy directs the therapeutic agent towards the target tumor, thereby protecting normal tissue from the effects of the therapeutic agent.

[0318]

[0357] Targeted therapies can target, for example, tyrosine kinase receptors and nuclear receptors. 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, such as Erk and PI3K / Akt, AP-2α, AP-2β, AP-2γ, mitogen-activated protein kinase (MAPK), PTEN, p53, p19ARF, Rb, Apaf-1, CD-95 / Fas, TRAIL-R1 / R2, caspase-8, forkhead, Box03A, MDM2, IAPs, NF-κB, 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 therapies can also target tumor-associated ligands, such as estrogen, estradiol (E2), progesterone, androgens, glucocorticoids, prolactin, thyroid hormones, insulin, P70 S6 kinase protein (PS6), Survivin, fibroblast growth factor (FGF), EGF, Neu differentiation factor (NDF), transforming growth factor alpha (TGF-α), IL-1A, TGF-beta, IGF-1, IGF-II, IGFBP, IGFBP protease, and IL-10.

[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 moiety and the extracellular domain of a TGF-beta receptor.

[0322]

[0361] In some embodiments, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with a second anticancer agent to treat prostate cancer. In certain embodiments, the anticancer agent includes an antiprostate cancer agent. In some embodiments, the antiprostate cancer agent includes androgen axis inhibitors; androgen synthesis inhibitors; ADP-ribose polymerase (PARP) inhibitors; or a combination thereof.

[0323]

[0362] In certain embodiments, the androgen axis inhibitor is selected from the group consisting of luteinizing hormone-releasing hormone (LHRH) agonists, LHRH antagonists, and androgen receptor antagonists.

[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, Elleada (apalutamide), filmagon (degarelix), flutamide, goserelin acetate, Jevtana (cabazitaxel), leuprolide acetate, Leupron (leuprolide acetate), Leupron Depot (leuprolide acetate), Lympa The following are selected from the group consisting of -za (olaparib), mitoxantrone hydrochloride, nilandrone (nilutamide), nilutamide, Nubequa (darolutamide), olaparib, Provenzi (cypuroicell-T), radium-223 chloride, Rubraca (lucaparib cansylate), lucaparib cansylate, cypuroicell-T, Taxotere (docetaxel), Zofigo (radium-223 chloride), Extanzi (enzalutamide), Zoladex (goserelin acetate), and Zytiga (abiraterone acetate).

[0328]

[0367] In certain embodiments, a dietary supplement for cancer patients may be a suitable supplement that has a protective effect against cancer. In certain embodiments, the dietary supplement contains indole-3-carbinol or a derivative thereof that produces indole-3-carbinol after ingestion. Indole-3-carbinol is thought to have a protective effect against cancer and may also be preventive against precancerous conditions.

[0329]

[0368] In certain embodiments, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with indole-3-carbinol, or derivatives thereof that produce indole-3-carbinol after ingestion. In certain embodiments, such combinations are useful for treating gremlin-related diseases. In certain embodiments, such combinations are useful for treating cancers, such as breast cancer, hepatocellular carcinoma, and colorectal cancer. In certain embodiments, such combinations are useful for treating breast cancer, such as triple-negative breast cancer.

[0330]

[0369] In some embodiments, the antibodies or antigen-binding fragments 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 disorders. In certain embodiments, the second antifibrotic agent may be an ACE inhibitor (or ARB), an anti-MASP2 antibody, an endothelin receptor antagonist, an NRF2 inhibitory steroid, a CTLA4-IgG, or a TNF inhibitor.

[0331]

[0370] In another embodiment, the second therapeutic agent is selected from the group consisting of antifibrotic agents such as pirfenidone, anti-inflammatory drugs, NSAIDs, corticosteroids such as prednisone, nutritional supplements, vascular endothelial growth factor (VEGF) antagonists [e.g., "VEGF traps," e.g., aflibercept or other VEGF inhibitory fusion proteins described in U.S. Patent No. 7,087,411, or anti-VEGF antibodies or their antigen-binding fragments (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 mimetic of BMP7), and any other palliative therapies useful in improving at least one symptom associated with fibrosis-related conditions or cancer. In a particular embodiment, 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 embodiments of these embodiments, an antibody or antigen-binding fragment disclosed herein, administered in combination with one or more additional therapeutic agents, may be administered simultaneously with one or more additional therapeutic agents, and in certain embodiments of these embodiments, the antibody or antigen-binding fragment and the additional therapeutic agents may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered “in combination” with another therapeutic agent does not have to be administered simultaneously with that agent or in the same composition as that agent. An antibody or antigen-binding fragment administered before or after another agent is considered to be administered “in combination” with that agent, even if the antibody or antigen-binding fragment and the second agent are administered via different routes, where the phrase “in combination” is used herein. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein should be administered according to the schedule set out on the product information sheet for the additional therapeutic agent, or according to Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57; Medical Economics Company; ISBN: 1563634457; 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 its antigen-binding fragment provided herein and a second therapeutic agent—which may be formulated in one composition or in different compositions. Instructions for use or labeling may be further included to provide information on how the combination therapy is carried out.

[0335]

[0374] Methods of detection and / or diagnosis

[0375] In some embodiments, the present disclosure provides a method for detecting the presence or amount of GREM1 in a sample derived from a subject, comprising the steps of contacting the sample with an antibody or its antigen-binding fragment, 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, though not limited to, by measuring the mRNA level of GREM1 using techniques such as 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. 22-29). In short, RNA-seq involves reverse transcription of target mRNA into cDNA, fragmentation and sequencing of the cDNA, and analysis of sequence data for mRNA quantification; RNAscope involves insight hybridizing target mRNA with one or more oligonucleotides conjugated with a fluorescent probe and detecting mRNA levels by measuring fluorescence intensity.

[0337]

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

[0338]

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

[0339]

[0379] In some embodiments, the Disclosure provides a kit comprising an antibody or its antigen-binding fragment provided herein, conjugated with an optionally detectable portion. The kit may be useful in detecting the presence or amount of GREM1 in a biological sample, or in a diagnostic method provided herein.

[0340]

[0380] In some embodiments, this disclosure provides a kit comprising an antibody or its antigen-binding fragment provided herein and a second therapeutic agent. The kit may be useful in the treatment, prevention, and / or improvement of GREM1-related diseases.

[0341]

[0381] In some embodiments, the Disclosure also provides the use of antibodies or antigen-binding fragments provided herein in the manufacture of pharmaceuticals for treating or diagnosing GREM1-related diseases or conditions in subjects. [Examples]

[0342]

[0382] While this disclosure has been specifically shown and described with reference to specific embodiments (some of which are preferred embodiments), those skilled in the art will understand that various modifications can be made in form and detail without departing from the spirit and scope of this disclosure as disclosed herein.

[0343] Example 1: Preparation of antigen

[0383] HGREM1-His (R&D): Recombinant HGREM1 protein (accession number O60565) was expressed in NS-0 cells. In short, the coding region of the hGREM1 gene from Lys25~Asp184, which has a 10×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 an 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 number O70326) Lys25~Asp184 was fused with a 10×His tag at the C-terminus and produced in NS-0 cells. The transfection supernatant was purified using a His-tag affinity column. The purified protein was characterized using an SDS-PAGE gel. The protein was purchased from R&D Systems (catalog number 956-GR).

[0345]

[0385] HGREM1-His(ACRO): Recombinant hGREM1 proteins Lys25~Asp184 (accession number NP_037504) were fused with a polyhistidine tag at the C-terminus and produced in human 293 cells (HEK293). The transfection supernatant from HEK293 cells was purified using a His-tag affinity column. The purified protein was characterized using an SDS-PAGE gel. This protein was purchased from ACRO Biosystems (catalog number GR1-H52H3).

[0346]

[0386] HGREM1-Fc (ACRO): Recombinant hGREM1 protein Lys25~Asp184 (accession number NP_037504) was fused with an hIgG1 Fc tag at its C-terminus and produced in human 293 cells (HEK293). The purified protein was characterized using SDS-PAGE gel. This protein was purchased from ACRO Biosystems (catalog number GR1-H5254).

[0347]

[0387] The above-mentioned gremlin protein was used in the following experiment.

[0348] Example 2: Antibody generation

[0388] 1. Antigen conjugation and immunity

[0389] In the case of immunotherapy, recombinant h-gremlin-His protein was conjugated with various MabSpace immunoenhancing peptides. In short, a 2-8 molar excess of the peptide was mixed with Sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate, Pierce, catalog number 22322) activated h-gremlin protein and incubated at room temperature for 1 hour. The reaction was stopped, and the conjugated protein was analyzed using an SDS-PAGE gel for quality control.

[0349]

[0390] The above conjugated h-gremlin-His proteins were emulsified in a 1:1 ratio using complete Freund's adjuvant (Pierce, catalog no. 77140) and then immunized C57B / L6 mice subcutaneously and intraperitoneally. Additional immunizations were performed using CpG and alum to preserve the native three-dimensional structure of the proteins. Immunizations were performed at least every two weeks, and antiserum from the mice was collected after the first immunization for analysis of anti-h-gremlin titer by ELISA assay.

[0350]

[0391] To determine serum titer, 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 of hGREM1-His in high-pH coating buffer (0.16% Na2CO3, 0.3% NaHCO3, pH 9.8). The 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 at room temperature for 2 hours. Next, the blocking buffer was aspirated, and 100 μl of serum, serially diluted with dilution buffer (PBS + 1% BSA + 1% goat serum + 0.01% Tween® 20), was transferred to each well of the ELISA plate and incubated at room temperature for 60 minutes. The plate was then washed three times using the method described above. Next, 100 μl / well of a solution of HRP-conjugated goat anti-mouse Fc antibody (Abcam, catalog no. Ab98808) diluted with dilution buffer was added to each well of the plate. The ELISA plate was then incubated at RT for 60 minutes, and the plate was washed three times with 250 μl / well of washing 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 using a Thermo Multiscan FC.

[0351]

[0392] 2.Fusion

[0393] Four days prior to fusion, each mouse was intraperitoneally boosted with non-conjugate h-gremlin-His protein in PBS. On the day of fusion, the spleen was aseptically removed and the organ was processed into a single-cell suspension. Red blood cells were lysed and the splenocytes were washed with DMEM (Gibco). Live myeloma cells in the logarithmic growth phase (SP2 / 0) were mixed with mouse splenocytes in a 1:4 ratio. The cells were then washed twice before fusion with PEG. After fusion, the cells were washed with DMEM and suspended in cell growth medium supplemented with 10% FBS + HFCS + OPI + 1×HAT. 200 μl of this cell suspension per well 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, after which the growth medium was aspirated from the wells and replaced with fresh growth medium. Screening of the hybridoma supernatant was initiated 2-3 days after the medium change.

[0352]

[0394] 3. Antibody screening by ELISA assay

[0395] The same protocol as for serum titer measurement described above was used. Briefly, the plates were coated with 1 μg / ml of h-gremlin-His at 4°C overnight. After washing, 100 μl of hybridoma supernatant was added and fully bound. Next, HRP-conjugated goat anti-mouse Fc antibody was added to detect the bound gremlin antibody. Finally, after the TMB reaction and H2SO4 termination, the plates were read at 450 nM using a Thermo Multiscan FC. Subsequently, cells from the ELISA-positive hybridoma wells were grown 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 with the desired binding profile and blocking activity were selected from ELISA-positive hybridoma wells and plated into 96-well plates using limiting dilutions. These cells were grown for 7 days. Once a suitable cell volume was reached, the supernatant was collected from each well and re-screened 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 grown in a further limiting dilution in 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 at least three times. If more than 90 wells showed a positive binding signal, the two clones with the highest antigen-binding activity were identified, transferred to a 24-well plate containing medium, and grown for a further 2 days. Once the 24-well plate reached confluence, the cells were transferred to a 6-well plate. After 5 days of incubation, a portion of the cells were frozen. The remaining cells were transferred to a flask and grown. Once the flask reached confluence, half of the cells were frozen for further backup (3 vials per clone). The remaining half were further grown in a flask containing medium for antibody production. Isotypes were 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 the hybridoma cell cultures as follows. All purification processes were performed at room temperature. Various mAbs were purified using a single purification scheme and affinity chromatography.

[0356]

[0401] The host cell culture medium (CCF) was centrifuged to remove cell debris. The CCF supernatant was then filtered, diluted, and loaded onto a Protein G High Performance (Bio-Rad) column for equilibration.

[0357]

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

[0358]

[0403] Following purification, the gremlin mAbs were formulated in PBS by dialysis using a 10,000 MWCO membrane (Pierce Slide-A-Lyzer or dialysis tube). Following formulation, the gremlin mAbs were filtered.

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

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

[0360]

[0405] The humanized antibodies provided herein (e.g., Hu14E3, Hu22F1, and Hu56C11) showed similarly high binding affinity to hGREM1 and / or mGREM1.

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

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

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

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

[0363]

[0408] Mutant versions of gremlin XM5 were also tested for their ability to directly bind to BMP2 / 4 / 7 with gremlin. XM5 was constructed and expressed by Mabspace, in which amino acids 123–143 of gremlin (NSFYIPRHIRKEEGSFQSCSF, SEQ ID NO: 63), known as the BMP binding loop, were replaced with amino acids 63–83 of DAN (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, an 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 BMP (BMP2 / 4 / 7) containing his-tag was added, and then the secondary antibody anti-his-HRP was used for binding detection.

[0364]

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

[0365]

[0410] In addition, the ability of serial dilutions of the antibodies provided herein to block the binding of gremlin to human BMP2 / 4 / 7 was also examined via ELISA. Plates were coated overnight with recombinant human BMP2 / 4 / 7 (0.5 μg / ml), then serial dilutions of the antibodies were incubated with modified human gremlin-his 1 μg / ml for 1 hour at RT, and this complex was then added to the coated plates and incubated for a further 1 hour at RT. The plates were then washed, and anti-hisHRP (GenScript) was added to the plates. The plates were then developed with TMB solution and stopped by adding stop solution. The plates were read at 450 nm with a plate reader. The results are shown in Figures 3E to 3H, demonstrating 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 the 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 the binding of gremlin to BMP2 and BMP4 (see Figures 3E and 3F). The chimeric anti-gremlin 1 antibody (56C11-chi) can inhibit the binding of gremlin to BMP2 (see Figure 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, showing a maximum blockage percentage of at least 50%, and even at least 70%. In contrast, the benchmark antibody 6245P showed a maximum blockage of less than 30% against the binding of human gremlin to BMP7, suggesting that 6245P was far less effective in 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 following day, as shown on the x-axis of Figure 4, cells were stimulated with either control medium or medium containing BMP4 (BMP4 30) 30 ng / ml or BMP4 30 ng / ml + gremlin 200 ng / ml (B 30 + gremlin 200); or BMP4 30 ng / ml + gremlin 200 ng / ml (B + G + 0.096 / 0.048 / 0.24 / 1.2 / 6 / 30 μg / ml) with various concentrations of antibodies provided herein. Luciferase activity of cells in each well was measured using a plate reader (Thermo Scientific Varioskan Flash) after incubation at 37°C and 5% CO2 for 3 hours.

[0368]

[0413] As shown in Figure 4, in contrast to the benchmark antibody, the antibody showed no activity whatsoever in reducing gremlin-mediated inhibition of BMP signaling in reporter cells that were not of cancer cell origin, whereas the benchmark antibody could 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] Purified hybridoma antibodies were also tested for their ability to reduce gremlin-mediated inhibition of BMP signaling using BMP4-inducible ATDC-5 cell differentiation. ATDC-5 is a chondroblastic cell line that can differentiate in response to BMP4 signaling. Differentiation of chondroblastic cell lines can be blocked by gremlin, a known BMP inhibitor. In this assay, blockade of gremlin leads to a reversal of BMP4 inhibition. Differentiation can be measured colorimetrically and quantitatively by using a substrate that detects the endogenous expression of alkaline phosphatase (ALP), an early marker of osteoblast differentiation. The level of differentiation can positively reflect the activity of BMP4 signaling.

[0370]

[0415] ATDC-5 cells were plated in 96-well plates at a density of 3000 cells / well and grown in DMEM / F12, 10% FBS + 1% PS at a volume of 100 μl / well, incubated overnight at 37°C and 5% CO2. The following day, hGREM1 was mixed with serially diluted antibodies in serum-free medium and incubated at 37°C for 30 minutes. Human BMP4 (Peprotech), diluted in serum-free medium, was added to the gremlin / antibody mixture and incubated for a further 30 minutes at 37°C. After incubation, 100 μl of the mixture was added to ATDC-5 cells plated in 100 μl of complete medium. The final concentrations of hBMP4 and h-gremlin on the cells in each well were 100 ng / ml and 400 ng / 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) + protein inhibitor (Roche). ALP was measured using p-nitrophenyl phosphate (PNPP) (Sigma). OD405 was measured using a Multiscan FC microplate reader (Thermo Scientific).

[0371]

[0416] As shown in Figure 5, the benchmark antibody 6245P was able to reduce gremlin-mediated inhibition of BMP signaling in cell differentiation, while antibodies 14E3 (Figure 5A), 22F1 (Figure 5B), 56C11 (Figure 5C), and 69H5 (Figure 5D) showed no such effect. In other words, the antibodies provided herein were unable to restore gremlin inhibition of BMP signaling involved in the differentiation of non-cancer cells.

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

[0417] PC3 prostate cancer cells were plated at 100,000 cells / well in DMEM / 10% FBS, 1% PS (complete medium) into 12-well plates and grown to 90% confluence at 37°C and 5% CO2. After starvation overnight in serum-free medium, PC3 cells were stimulated with BMP4 with or without gremlin for 30 minutes. For Western blotting analysis, cells were lysed in RIPA buffer (CST). Cell lysates were separated using 4%-12% SDS-PAGE (Genscript) and transferred to PVDF membranes (Millipore). The membranes were incubated overnight at 4°C with antibodies specific to smad phosphorylation (p-smad1 / 5 / 9) (1:1000, CST), which positively reflects BMP signaling activity, and with antibodies specific to 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 visualization was performed using Cheniluminescent Imager (MiniChemi, Sagecreation).

[0373]

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

[0374]

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

[0375]

[0420] As shown in Figure 6B, the strength of p-smad1 / 5 / 9 or pSMAD1 / 5 / 9 recovered from GREM1-mediated inhibition increased with increasing concentrations of the 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 anti-gremlin 1 antibodies provided herein in various cell types.

[0377]

[0421] Since gremlins are expressed in multiple physiological tissues and affect multiple cell types, the inventors evaluated whether the anti-gremlin 1 antibodies provided herein had similar effects on various cell types responsive to gremlin-mediated inhibition of BMP signaling. Briefly, multiple cell types of different origins, including osteoblasts ATDC-5, renal fibroblasts NRK49F, renal epithelial cells HK2, and tumor cells PC3, were stimulated with BMP4 or with BPM4 in conjunction with gremlin. Cells stimulated with both BMP4 and gremlin were then supplemented with either a gremlin antibody or control IgG 1 or 10 μg / ml. The antibodies tested here include 14E3 and benchmark antibody 6245P. As shown in Figure 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, while 14E3 reverses gremlin-mediated inhibition of BMP4 signaling only in tumor cells such as PC3 cells, and does not reverse it 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) differ significantly from benchmark antibodies in their biological activity, which is consistent with their differential activity using the ALP assay in ATDC-5 cells, as described in Example 8. Such selectivity directed towards BMP signaling reversion 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 being largely toxic to non-cancer cells.

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

[0422] Four lead antibodies exhibiting the desired profile were selected for gene cloning. Sequences of the variable regions of the mouse anti-hGREM1 light and heavy chains were obtained using polymerase chain reaction (PCR) amplification techniques 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 primers. The variable regions of the mouse IgG gene were cloned by PCR using MuIgG VH3'-2 and MuIg-5' reader primers for the heavy chain variable region, and MuIgK VL3'-1 and MuIg-5” reader primers (NOVAGEN) for the light chain variable region. The resulting bands were cloned into the TOPO TA cloning vector, and DNA from more than 10 clones was sequenced using an ABI DNA sequencer (Perkin Elmer). The consensus sequence was determined using vector NTI Advanced 10 software (Invitrogen). After sequence analysis and confirmation, the variable regions of the gremlin gene were cloned into recombinant expression vectors (VL to pCP-mCK; VH to 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 antibody secretion, a signal peptide (MGWSCIILFLVATGVHS (SEQ ID NO: 65)) was fused to the N-terminus of the antibody.

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

[0381]

[0424] Recombinant antibody protein expression and purification were performed by the following method: 1 × 10⁶ in Freestyle 293 Expression Medium containing 10% Pluronic F-68. 6 HEK293E cells cultured at 100 cells / ml were transfected with equal volumes of heavy-chain and light-chain vector DNA at a final concentration of 0.5 μg / ml and 1.0 μg / ml of PEI (polyethyleneimine-linear, Polyscience). The ratio of DNA to PEI was 1:2. The formation time of the DNA-PEI complex by Optimal MEM should be 15 minutes at room temperature. The transfected cells were cultured in a flask at 5% CO2, 37°C, and a shaking rate of 125 rpm. 1% peptone medium was added 22-26 hours after transfection. The conditioned medium was collected on day 6, and the supernatant was centrifuged at 3000 rpm for 30 minutes. The clarified conditioned medium was then loaded onto an nProtein A column (GEHealthcare), washed with PBS + 0.1% Triton-X100, and finally, bound IgG was eluted with a 0.1 M glycine-containing solution at pH 3.5. The eluted antibody protein was dialyzed to PBS and stored at -80°C. To remove endotoxins, the purified protein was further processed by passing it through a Hitrap DEAE Sepharose FF column, and the resulting antibody was analyzed to determine its purity level using size exclusion chromatography (Superdex 200 5 / 150 GL, GEHealthcare).

[0382]

[0425] Figure 8 shows the binding analysis of recombinant chimeric anti-gremlin 1 antibodies prepared according to the method described above. In this case, 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 selective antibodies for in vivo research

[0426] Each of these cloned antibodies was scaled up to produce large quantities of antibodies for in vivo testing in a renal failure model.

[0384]

[0427] Hybridoma cells are cultured in roller bottles containing in vitro production medium, and the monoclonal antibodies produced in the conditioned medium are then processed and purified by a protein A affinity column using a low-endotoxin procedure.

[0385]

[0428] In short, hybridoma cells were harvested and grown, adapted to grow in hybridoma-producing medium (DMEM + 2% Low IgG FBS), and inoculated into roller bottles containing 300 ml of culture medium. The cells were then cultured in the roller bottles for 2-3 weeks, and the culture medium was harvested and clarified before purification. The mAbs produced from the culture medium were then purified using a protein A affinity column, dialyzed against PBS (pH 7.4), and concentrated to 1.0 mg / ml or higher as needed. The following parameters were measured for quality control: purity of the antibody product, endotoxin level, agglutination level, and 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 higher as determined by SDS-PAGE and HPLC. d. Aggregation rate: Less than 10% by HPLC e. Endotoxin: 3 EU / mg or less Example 14: Characterization of the binding affinity measurement of selected gremlin antibodies to captured hGREM1

[0430] Antibody affinity was measured at 25°C using Biacore T200. HGREM1 (Peprotech) was immobilized on the Biacore sensor chip. Reaction kinetics experiments were performed using HBS-EP+ as both the running buffer and sample buffer. The antibody-antigen association rate was measured by injecting antibodies of various concentrations (ranging from 12.5 to 400 nM, 2-fold dilution) onto the captured hGREM1 surface. Antibody-antigen association was monitored for 180 seconds, while dissociation in the buffer was monitored for 360 seconds. Reaction kinetics analysis was performed using Biacore T200 evaluation software to determine the Ka and Kd values. KD was calculated from the experimentally determined Ka and Kd values ​​as KD = Kd / Ka. As shown in Figure 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 Research

[0431] Epitope binning analysis using competitive ELISA assays

[0432] Epitope binning of anti-gremlin hybridoma antibodies was performed using a competitive ELISA assay. Clear polystyrene plates (BEAVER) were coated overnight at 4°C with 100 μl / well of high-pH coating buffer containing 0.5 μg / ml hGREM1 (ACRO). The plates were then washed once in an automated plate washer using PBS + 0.1% Tween® 20 (Sigma). 100 μl of block solution consisting of PBS + 1% BSA + 1% normal goat serum + 0.05% Tween® 20 (Sigma) was added to each well and incubated at room temperature for 2 hours. Subsequently, 50 μl / well of antibody dilution buffer (PBS + 1% BSA + 1% normal goat serum + 0.01% Tween® 20) containing 20 μg / ml saturated antibody (hybridoma antibody) was added to each well of the plate and incubated for 1 hour. Next, 50 μl / well of a 40 ng / ml competing antibody (chimeric antibody, e.g., 14E3-C, 22F1-C, 56C11-C, and 69H5-C) was added to each well of the plate and incubated at room temperature for another 1 hour. Then, they were 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 incubated at room temperature for 1 hour. Finally, 100 μl / well of TMB (Pierce) was added to each well, and after 5 minutes, 50 μl of stop solution was added to each well. The plates were read at 450 nm with a plate reader. Failure of the second antibody to bind in the presence of a saturated amount of the first antibody indicates that the two antibodies were present in the same epitope bin; successful binding of the second antibody in the presence of a saturated amount of the first antibody indicates that the two antibodies were present in different epitope bins. Based on the complete dataset, multiple antibody bins exist. 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 present in a single epitope bin. On the other hand, the binding of 56C11 to gremlin was not competed for by 14E3, 22F1, or 69H5, indicating that 56C11 was present in a different epitope bin than 14E3, 22F1, and 69H5. This result reiterates the data shown in Figure 1, which indicated that 56C11 was able to bind to mouse gremlin, while 14E3, 22F1, and 69H5 were not.

[0390]

[0434] Cross-competition experiments were also conducted to analyze whether 14E3 / 22F1 binds to an epitope different from that of the benchmark antibody 6245P. In short, a fixed amount of either the chimeric antibody 14E3-C / 22F1-C or 6245P was added, followed by gradually increasing amounts of 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 even in a 100-fold excess, they could not compete with 6245P binding. This indicates that 14E3-C and 22F1-C bind to an epitope 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), in this case numbered according to SEQ ID NOs. 69 and 70, respectively. 14E3 binds to human gremlin but not to mouse gremlin, and therefore we hypothesize that these two different amino acids may be key amino acids influencing antibody binding. To verify the two key residues for 14E3 binding, we cloned a signal peptide (SEQ ID NOs. 71) and four variants with a C-terminal His-tag, including human gremlin_WT, human gremlin_Q27P, human gremlin_N33T, and human gremlin_Q27P / N33T, into a pcDNA3.1(+) vector, and all constructs were confirmed by DNA sequencing. Plasmids of four constructs, purified using the QIAGEN Plasmid Midi Kit, were transfected into ExpiCHO cells in 3 ml scale using the ExpiCHO transfection kit. Transfected cells were cultured in a shaking flask at 125 rpm in an incubator at 8% CO2 and 37°C. Cell cultures were harvested on day 4, centrifuged at 8000 rpm for 30 minutes, and the supernatant was used for antibody binding assays.

[0392]

[0437] Human (Huam) gremlin 1 sequence without signal peptide: KKKGSQGAIPPPDKAQHNDSEQTQSP Q QPGSR N RGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD(Sequence ID 69)

[0438] Mouse gremlin 1 sequence without signal peptide: KKKGSQGAIPPPDKAQHNDSEQTQSP PQPGSR T RGRGQGRGTAMPGEEVLESSQEALHVTERKYLKRDWCKTQPLKQTIHEEGCNSRTIINRFCYGQCNSFYIPRHIRKEEGSFQSCSFCKPKKFTTMMVTLNCPELQPPTKKKRVTRVKQCRCISIDLD(Sequence ID 70)

[0439] Note: Two different amino acids between human and mouse gremlin are shown in bold and underlined. The signal peptide sequence for human gremlin 1 is described in SEQ ID NO: 71, and the signal peptide sequence for mouse gremlin 1 is described in SEQ ID NO: 72.

[0393]

[0440] Biolayer Interference (BLI) Assay

[0441] Human gremlin Ab, 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 μl per well in the Loading Column of a 96-well half-area microplate (Greiner Bio-one). The supernatants of the gremlin WT and mutants to be tested were added to the Association Column of the plate at 100 μl per well. Medium or KD buffer was used as a reference control. The AHC sensor was placed in the first Baseline Column for 60 seconds to obtain the first baseline, and 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 the second baseline. The sensor was then placed in the Association Column for 300 seconds to fully associate the gremlin / gremlin Ab, and finally in the dissociation Column for 300 seconds. We will analyze group data from ForteBio (Octet96).

[0394]

[0442] Figure 10D shows that 14E3 can still bind to human gremlin 1 variants with Thr-mediated Asn33 substitution (i.e., N33T), although binding was partially reduced. However, Pro-mediated Gln27 substitution (i.e., Q27P) in human gremlin 1 significantly reduced 14E3 binding, a reduction also observed in human gremlin 1 variants with both N33T and Q27P substitutions. In contrast, neither a single N33T mutation nor a single Q27P mutation in human gremlin 1 significantly reduced 6245P binding, suggesting that 6245P binds to epitopes in human gremlin 1 that do not contain N33 or Q27. This is consistent with the binding results observed in human gremlin 1 variants with both N33T and Q27P mutations. These data suggest that 14E3 binds to epitopes containing Q27, as shown in SEQ ID NO: 69. The epitopes to which 14E3 binds may, to a lesser extent, include N33 of sequence number 69.

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

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

[0396]

[0444] The binding of the chimeric antibody 14E3 to XM5 or gremlin was tested using a similar ELISA protocol as described above. Briefly, 14E3-C or 6245P was coated (1 μg / ml) and serially diluted XM5-his supernatant or gremlin-his (2 μg / ml to 0.49 μg / ml) was added for binding. The 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 was able to bind to XM5, while 6245P was 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) was used for coating. 100 μl of block solution consisting of PBS + 1% BSA + 1% normal goat serum + 0.5% Tween® 20 (Sigma) was added to each well and incubated at room temperature for 2 hours. Four-fold serial antibody dilutions starting from 2 μg / ml were added. Subsequently, the plates were washed three times with PBS + 0.1% Tween® 20 200 μl, followed by the addition of 1:10000 goat anti-mouse IgG-HRP (abcam) 100 μl / well and incubated at room temperature for 1 hour. Then, they were washed 3× with PBS + 0.1% Tween® 20. Finally, 100 μl of TMB (InnoReagents) 100 μl / well was added to each well, and 50 μl of stop solution was added to each well after 2 minutes. The plates were read at 450 nM using a Multiscan FC microplate reader (Thermo Scientific). The EC50 values ​​are shown in the table 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 to XM5 with EC50 values ​​ranging from 0.006 ng / ml to 0.008 ng / ml.

[0399] Example 17: Epitope analysis using Fortebio

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

[0400]

[0448] If the second anti-gremlin 1 antibody is unable to bind to gremlin, this indicates that the second anti-gremlin 1 antibody binds to a similar epitope as the first anti-gremlin 1 antibody; if the second anti-gremlin 1 antibody can bind without any effect from the first anti-gremlin 1 antibody, this indicates that the epitopes of those antibodies are distinct. 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 was unable to bind to gremlin in the presence of 14E3, indicating that 22F1 / 69H5 and 14E3 may bind to similar epitope sites. Conversely, 6245P, as the first anti-gremlin 1 antibody, failed to block the binding of 14E3 / 22F1 to the antigen, but completely blocked the binding of 56C11 / 69H5 (Figure 11B). Therefore, depending on the type of epitope, there are probably three groups: one group containing 14E3 and 22F1, and another group containing 56C11 and 69H5, and these epitopes may overlap with the epitopes of 6245P (Table 6).

[0401]

[0449]

[0402] [Table 7]

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

[0450] Humanized 14E3

[0451] Humanized antibodies for 14E3 were designed using the following protocol, employing three-dimensional structural simulation and humanization via CDR transplantation.

[0404]

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

[0405]

[0453] The inventors also simulated the structure of CDR-transplanted 14E3 to guide the design of reverse mutations and the evaluation of the feasibility and stability of developing humanized antibodies. Structural simulations were performed in a similar manner.

[0406]

[0454] Humanization was performed by CDR transplantation. After a BLAST search of the mouse 14E3 sequence in the IMGT human immunoglobulin gene database, the human germline framework sequence IGHV / 7-4 was used for the heavy chain and IGKV / 2-30 for the light chain, respectively, for CDR transplantation to obtain humanized 14E3 without reverse mutations. To further maintain the activity of humanized 14E3, the inventors aligned the framework sequence of the humanized antibody with the framework sequence of the corresponding mouse antibody. Various residues were double-checked using a mouse antibody structural model: if any of these residues are present in a position that interacts with and may affect the CDR residues, it should be reverse-mutated to a mouse residue. This disclosure describes the acquisition of three humanized heavy chain variable regions with different reverse mutations, labeled Hu14E3_Ha VH, Hu14E3_Hb VH, and Hu14E3_Hc VH, as well as two humanized light chain variable regions with different reverse mutations, labeled Hu14E3-La V and Hu14E3-Lb VL (see Table 5). The cDNA of these humanized heavy and light chain variable regions was 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 the same as for the chimeric antibodies.

[0407]

[0455] Humanized 22F1

[0456] Humanized antibodies for 22F1 were designed using a similar protocol. Briefly, human germline framework sequences IGHV / 1-46 were used for the heavy chain and IGKV / 2-30 for the light chain, respectively, for CDR transplantation. Then, humanized variants with CDR transplantation and reverse mutations were designed using computer modeling. This disclosure yields four humanized heavy chain variable regions with reverse mutations: Hu22F1_Ha VH, Hu22F1_Hb VH, Hu22F1_Hc VH, and Hu22F1_Hd VH, and two light chain variable regions with different reverse mutations: Hu22F1-La VL and Hu22F1-Lb VL. (See Table 5). Each humanized heavy chain was co-expressed with a humanized light chain to obtain eight versions of humanized antibodies for 22F1: Hu22F1_HaLa, Hu22F1_HaLb, Hu22F1_HbLa, Hu22F1_HbLb, Hu22F1_HcLa, Hu22F1_HcLb, Hu22F1_HdLa, and Hu22F1_HdLb. The expression and purification procedures were the same as for the chimeric antibodies.

[0408]

[0457] Recombinant antibody protein expression and purification were carried out in the following steps: ExpiCHO cells were expressed in 5-6 × 10⁶ cells. 6ExpiCHO Expression Medium was inoculated at a concentration of 10 μg / ml. Subsequently, ExpiCHO cells were transfected with equal volumes of heavy-chain and light-chain vector DNA at a final concentration of 1.0 μg / ml using the ExpiCHO transfection kit. Transfected cells were cultured in a 37°C incubator supplemented with 8% CO2 in a shaking flask at 125 rpm. ExpiCHO feed was added 18-22 hours after transfection. Cell cultures were collected on day 10. The collected cell culture medium (HCCF) was obtained by centrifugation. The HCCF was then loaded onto an rProtein A column (GEHealthcare) and washed with PBS. The final IgG antibody was eluted using a solution containing 20 mM citrate at pH 3.2. Finally, the eluted antibody protein was neutralized and stored at -80°C for long-term use. The generated antibodies were analyzed to determine their purity levels using SDS-PAGE and size exclusion chromatography (TSKgel G3000SWXL, TOSOH).

[0409]

[0458] Humanized 56C11

[0459] Humanized antibodies for 56C11 were designed using a similar protocol. Briefly, the human germline framework sequence IGHV1-2*02 was used for the heavy chain and IGKV2-30*02 for the light chain, respectively, for CDR transplantation.

[0410]

[0460] Heavy chain (HC) variants 1, 2, 3, and 4 were obtained by directly transplanting three CDRs into germline sequences. The above combinations of heavy chain and light chain variable regions produce 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, and 56C11-HcLb.

[0411]

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

[0462] Human IgG monohelic acid 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 described 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 collected when the cell viability was approximately 60%. 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 levels were controlled to less than 1 EU / mg. Next, the purified antibodies were characterized by SDS-PAGE and SEC-HPLC.

[0412] Example 19: Conjugation of humanized antibodies to h-gremlin in ELISA and Fortebio

[0467] The same protocol was used in Example 4. As shown in Figure 12A, the humanized variants of 14E3 retained similar binding activity to chimeric 14E3 (e.g., 14E3 hIgG1 or 14E3-C), indicating that the biological activity was likely unaffected by humanization. However, most humanized variants of 22F1 significantly lost binding, with only 22F1-HdLa and 22F1-HdLb still exhibiting good affinity (Figures 12B and C).

[0413]

[0468] The affinity of the humanized antibody was also measured using Fortebio. Human gremlin protein was diluted in kinetic buffer to a concentration of 2 μg / ml. 0 nM was used as a reference control. The antibody to be tested was 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 on an NTA biosensor. Baseline was detected for 60 seconds, followed by detection of anti-gremlin antibody association for 120 seconds. on Factor data was obtained. Subsequently, the data was dissociated in dynamic buffer for 90 seconds and 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, which is much lower than the KD value of the benchmark antibody.

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

[0469] In short, human prostate cancer PC3 cells were maintained in vitro as monolayer cultures at 37°C in a 5% CO2 atmosphere in RPMI1640 medium (Thermo Fisher) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, 100 ug / ml streptomycin (Hyclone), and 1 ug / ml puromycin (Gibco). Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment (Hyclone). Cells growing in the logarithmic growth phase were collected and counted for tumor inoculation. SPF-grade male nude mice were subcutaneously inoculated with a mixture of 1 x 10^6 PC3 cells containing 50% Matrigel. Ten days later, the inoculated mice were castrated by orchiectomy. When tumor size reached approximately 200 mm³, tumor-bearing mice were selected and randomized into two groups (n=8). For three weeks, every four days, animals were intraperitoneally injected with 10 mg / kg of hIgG1 control and 10 mg / kg of Hu14E3_HaLa. Tumor size was measured two-dimensionally every four days using a caliper (INSIZE), and the volume was expressed in mm³ using the formula: V = 0.5a*b² (where a and b are the major and minor diameters of the tumor, respectively). Results were analyzed using Prism GraphPad and expressed as mean ± SEM. Comparison between the two groups was performed by t-test, but p was... * <0.05 and ** A difference is considered statistically significant if it is <0.01. As a result, Hu14E3_HaLa was able to effectively inhibit tumor growth by either volume or weight (Figures 13A and 13B).

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

[0470] Since gremlin 1 can be expressed in both tumor cells and stromal fibroblasts, the inventors evaluated the contribution of gremlin 1 to modulating the tumor microenvironment and its potential to modulate tumor growth, either alone or in combination with checkpoint inhibitors. Accordingly, the inventors evaluated the antitumor activity of 56C11 against the syngeneic tumor model, the CT-26 model. In short, 24 female Balb / c mice aged 5-6 weeks were given 2 × 10⁶ mice. 6 Individual CT26 tumor cells were inoculated. The tumor volume was approximately 100 mm². 3 When this was reached, the animals were randomized and divided into groups as follows: 1) isotype control at 20 mg / kg, and 2) 56C11 at 20 mg / kg. The antibody was injected intraperitoneally (IP) twice weekly for two weeks. The results shown in Figure 14 demonstrate that the anti-GREM1 antibody 56C11 (which is cross-reactive to mouse GREM1) alone can have significant antitumor activity without significant impact on total body weight. The humanized anti-GREM1 antibodies provided herein (e.g., Hu14E3, Hu14E3, Hu22F1, and Hu56C11) are expected to exhibit similar technical effects in humans as their chimeric counterparts in demonstrating significant antitumor activity without significant impact on total body weight.

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

[0471] To further investigate the antitumor efficacy of the combination of an immune checkpoint inhibitor and an anti-GREM1 antibody, the inventors evaluated the antitumor activity of combining MPDL-3280A with a surrogate anti-mouse GREM1 antibody (anti-mGREM1 antibody) in a CT26 tumor model. In short, 5 × 10⁶ CT26 cells were introduced into 5-6 week old female Balb / c mice. 5 Each mouse was inoculated. Animals were randomized. Tumor volume was approximately 100 mm². 3When this was reached, mice were divided into four groups and treated twice weekly for two weeks with IP (intracellular therapy) with: 1) control IgG1 alone at 10 mpk, 2) MPDL3280A alone at 3 mpk, 3) anti-mGREM1 antibody alone at 10 mpk, or 4) a combination of MPDL3280A at 3 mpk and anti-mGREM1 antibody at 10 mpk. As shown in Figures 15A and 15B, the combination of anti-mGREM1 antibody with an immune checkpoint inhibitor (e.g., an antibody against PD-L1) resulted in significantly higher antitumor activity (in terms of either tumor volume or tumor weight) compared to anti-mGREM1 antibody alone or immune checkpoint inhibitor alone. This suggests that anti-mGREM1 antibody can enhance the antitumor activity of immune checkpoint inhibitors.

[0417] Example 23: Efficacy of humanized 14E3 in combination with cisplatin in an esophageal cancer PDX model.

[0472] Esophageal tumor tissue (E7) specifically positive for human gremlin IHC was obtained from the passaged and established PDX bank of NOD / SCID mice at Beijing Cancer Hospital. The inventors tested GREM1 expression and PD-L1 expression in the E7 esophageal PDX model by immunohistochemistry using either an anti-GREM1 antibody (14E3) or an anti-PD-L1 antibody (22C3). Figure 16 shows that the esophageal cancer PDX model E7 was positive for GREM1 expression but lacked PD-L1 expression.

[0418]

[0473] Each mouse was subcutaneously inoculated with a small tumor tissue mass, approximately 3 mm in diameter, sheared from a one-piece tumor deskin wound derived from a tumor-bearing mouse. Eighteen days after inoculation, the tumor was approximately 70 mm in diameter. 3Animals with tumor sizes were selected and randomly divided into four groups, each consisting of eight mice. The mice were then treated with an 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. The isotype control and hzd14E3 were administered twice weekly for four weeks by intravenous injection and PBS, while cisplatin was administered once weekly for four weeks by intravenous injection. Animals were euthanized by CO2 inhalation at the end of the study. Tumor size was measured two-dimensionally two or three times weekly using a caliper (INSIZE), and the volume was expressed in mm³ using the formula: V = 0.5a*b² (where a and b are the major and minor diameters of the tumor, respectively). Results were analyzed using Prism GraphPad and expressed as mean ± SEM. A t-test was performed to compare the two groups, but p was... * <0.05 and ** If the difference is <0.01, it is considered statistically significant.

[0419]

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

[0420]

[0475] To date, first-line therapies for esophageal cancer generally include esophagectomy, chemotherapy, targeted therapy, immunotherapy (e.g., targeting PD-1 or PD-L1), and / or combinations thereof. Second-line and subsequent therapies for esophageal cancer may involve targeted therapies such as ramucirumab targeting the vascular endothelial growth factor (VEGF) receptor or trastuzumab for metastatic adenocarcinoma overexpressing HER2 (NCCN Clinical Practice Guidelines in Oncology. Esophageal and Esophagogastric Junction Cancers. National Comprehensive Cancer Network. V1.2020). The inventors' data above demonstrate that the anti-GREM1 antibody provided herein can effectively treat tumors that do not express PD-L1, such as esophageal cancer that does not overexpress PD-L1, and can achieve further 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 therapies for esophageal cancer.

[0421] Example 24: Efficacy of hzd14E3 (in combination with DC101) and 6245P for an esophageal cancer PDX model.

[0476] Esophageal tumor tissue (E7) specifically positive for human gremlin IHC was obtained from NOD / SCID mice at Beijing Cancer Hospital and from an established PDX bank. Each mouse was subcutaneously inoculated with small tumor tissue fragments approximately 3 mm in diameter, sheared from a one-piece tumor descathed wound derived from a tumor-bearing mouse. Eighteen days after inoculation, approximately 70 mm 3Animals with tumor sizes were selected and randomly divided into four groups, each consisting of eight mice. The mice were then treated with an 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 commercially available monoclonal antibody that reacts with mouse VEGFR-2 (e.g., under BioXell catalog number BE0060). The control and test substances were administered by intravenous injection twice weekly for four weeks. Animals were euthanized by CO2 inhalation at the end of the study. Tumor size was measured two-dimensionally two or three times weekly using a caliper (INSIZE), and the formula was: V = 0.5a*b 2 The volume was expressed in mm³ using the formula (where a and b are the major and minor diameters of the tumor, respectively). The results were analyzed using Prism GraphPad and expressed as mean ± SEM. A t-test was performed to compare the two groups, but p was... * <0.05 and ** A difference is considered significant if it is <0.01. Other humanized anti-GREM1 antibodies provided herein (e.g., Hu22F1 and Hu56C11) are expected to exhibit similar technical effects.

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

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

[0423]

[0478] Next, 0.25 μg / ml of gremlin was selected to test its blocking activity. The ability of the antibody to block gremlin binding to human FGFR1-Fc was investigated via ELISA. Plates were coated overnight with recombinant FGFR1-Fc (2 μg / ml), then serial dilutions of the antibody were incubated with 0.25 μg / ml of modified human gremlin-his for 1 hour at RT. This complex was then added to the coated plates and incubated for a further 1 hour at RT. The plates were then washed, and anti-hisHRP (GenScript) was added to the plates. The plates were then developed with TMB solution and stopped by adding stop solution. The plates were read at 450 nm with 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 exhibited partial blocking activity; in this case, 69H5-chi was able to block the binding of hGREM1 to FGFR1 with an IC50 of 7.138 nM, and 22F1-chi was able to block the binding of hGREM1 to FGFR1 with an IC50 of 5.117 nM. The humanized anti-GREM1 antibodies provided herein (e.g., Hu14E3, Hu22F1) are expected to exhibit similar technical effects to their chimeric counterparts in blocking the binding of hGREM1 to FGFR1.

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

[0479] Transparent polystyrene plates (BEAVER) were coated overnight at 4°C with 100 μl / well of high-pH coating buffer containing human gremlin (ACRO) and DAN (Sinobiological) 0.5 μg / ml. The plates were then washed once in an automated plate washer using PBS + 0.1% Tween® 20 (Sigma). 100 μl of block solution consisting of PBS + 1% BSA + 1% normal goat serum + 0.5% Tween® 20 (Sigma) was added to each well and incubated at room temperature for 2 hours. Next, 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 2 μg / ml (13.33 nM), were added to each well of the plate, followed by 100 μl of a 4-fold dilution, and incubated at room temperature for 1 hour. Subsequently, the plates were washed three times with PBS + 0.1% Tween® 20 20, followed by the addition of 1:10000 goat anti-mouse IgG-HRP or mouse anti-human IgG-HRP (abcam) 100 μl / well, and incubated at room temperature for 1 hour. They were then washed 3× with PBS + 0.1% Tween® 20. Finally, 100 μl / well of TMB (InnoReagents) was added to each well, and after 2 minutes, 50 μl of stop solution was added to each well. The plates were read at 450 nM using 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 exhibited similar gremlin binding activity to the DAN protein.

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

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

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

[0481] A mouse mammary cancer cell line, EMT6, transfected with the human PD-L1 gene and screened for stable expression of human PD-L1, was named EMT6 / hPD-L1. EMT6 / hPD-L1 cells were maintained in vitro as monolayer cultures at 37°C in a 5% CO2 atmosphere in DMEM medium (Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, and 100 ug / ml streptomycin (Hyclone). Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment (Hyclone). Cells growing in the logarithmic growth phase were collected and counted for tumor inoculation. SPF-grade male BABL / c mice were inoculated with 2*10^6 EMT6 / hPD-L1 cells mixed with 50% Matrigel. In the first study, tumor-bearing mice with a tumor size of approximately 80 mm³ were selected and randomized into two groups (n=10). The animals were treated twice weekly for 4 weeks by intravenous injection with either an hIgG1 control at 24.9 mg / kg or AM4B6 at 24.9 mg / kg. In the second study, tumor-bearing mice with a tumor size of approximately 70 mm³ were selected and randomized into two groups (n=8). The animals were treated twice weekly for 3 weeks by intravenous injection with either an hIgG1 control at 10 mg / kg or 36F5 at 10 mg / kg. Tumor size was measured twice weekly in two dimensions using a caliper (INSIZE), and the volume was expressed in mm³ using the formula: V = 0.5a*b² (where a and b are the major and minor diameters of the tumor, respectively). Results were analyzed using Prism GraphPad and expressed as mean ± SEM. A t-test was performed to compare the two groups, but p was... * <0.05 and **A difference is considered statistically significant if <0.01. Table 7 and Figure 21A show the results of the first study. The results indicate that the anti-PD-L1 antibody (AM4B6) did not have antitumor activity against the EMT6 / hPD-L1 tumor model. The EMT6 / hPD-L1 tumor model exhibits poor response to PD-L1 antibodies. Table 8 and Figure 21B show the results of the second study. The results indicate that the anti-Gemlin1 antibody (36F5) has promising antitumor activity against the EMT6 / hPD-L1 tumor model that exhibits poor response to PD-L1 antibodies.

[0427]

[0482]

[0428] [Table 8]

[0429]

[0483]

[0430] [Table 9]

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

[0484] E7 is a human gremlin-highly expressing esophageal cancer PDX obtained from the Beijing Cancer Hospital's passaged and established PDX bank of NOD / SCID mice. NOG mice were severely immunodeficient and purchased from Vital River. Each mouse was subcutaneously inoculated with small tumor tissue fragments approximately 3 mm in diameter, sheared from a one-piece tumor descathed wound derived from a tumor-bearing mouse (moue). 27 days after inoculation, animals with a tumor size of approximately 50 mm³ were selected and intravenously injected with 5 × 10⁶ human PBMCs per mouse. One week later, the animals were screened for reconstruction profiling and randomly divided into 6 groups, each consisting of 8 mice. The animals were treated twice weekly for 5 weeks by intravenous injection with isotype control 30 mg / kg, 14E3 30 mg / kg, 36F5 30 mg / kg, and nivolumab 10 mg / kg. Tumor size was measured in two dimensions twice a week using a caliper (INSIZE), and the volume was expressed in mm³ using the formula: V = 0.5a*b^2 (where a and b are the major and minor diameters of the tumor, respectively). The results were analyzed using Prism GraphPad and expressed as mean ± SEM. A t-test was performed to compare the two groups, but p was... * <0.05 and ** A difference is considered statistically significant if <0.01. Table 9, Figure 22A, and Figure 22B show the results of the study. The results indicate that the anti-PD-1 antibody (nivolumab) did not have antitumor activity against the E7 tumor model. The anti-Gemlin1 antibodies 36F5 and 14E3 showed promising antitumor activity against the E7 tumor model which was poorly responsive to PD-1 antibodies.

[0432]

[0485]

[0433] [Table 10]

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

[0486] A mouse colon cancer cell line, MC38, transfected with the human PD-L1 gene and screened for stable expression of human PD-L1, was named MC38 / hPD-L1. MC38 / hPD-L1 cells were maintained in vitro as monolayer cultures at 37°C in a 5% CO2 atmosphere in 1640 medium (Hyclone) supplemented with 10% heat-inactivated fetal bovine serum (ExCell Biology), 100 U / ml penicillin, and 100 ug / ml streptomycin (Hyclone). Tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment (Hyclone). Cells growing in the logarithmic growth phase were collected and counted for tumor inoculation. SPF-grade female C57BL / 6 mice were inoculated with 2 x 10^6 MC38 / hPD-L1 cells containing 50% Matrigel. When the tumor size was approximately 120 mm³, tumor-bearing mice were selected and randomized into four groups (n=8). Animals were treated twice weekly for 3 weeks by ip injection with either hIgG1 control (3 mg / kg), 56C11 (20 mg / kg), 23F11 (anti-PDL1 antibody) (3 mg / kg), or a combination of 56C11 (20 mg / kg) and 23F11 (3 mg / kg). Tumor size was measured twice weekly in two dimensions using a caliper (INSIZE), and the volume was expressed in mm³ using the formula: V = 0.5a*b² (where a and b are the major and minor 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-tests, but p was... * <0.05 and ** A difference is considered significant if <0.01. The combination of 56C11 with anti-PDL1 antibody enhances antitumor 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] Embodiments of the Invention [Aspect 1] An isolated antibody against human gremlin 1 (hGREM1) or its antigen-binding fragment, characterized by the following: a) It can selectively reduce hGREM1-mediated inhibition of BMP signaling in cancer cells compared to non-cancer cells; b) Non-cancer cells exhibit a reduction of less than 50% in hGREM1-mediated inhibition of BMP signaling; c) Can bind to chimeric hGREM1 containing the amino acid sequence of SEQ ID NO: 68; d) It can bind to hGREM1 but cannot specifically bind to mouse gremlin 1, or alternatively, it is cross-reactive to mouse gremlin 1; e) Binding to hGREM1 with an epitope containing residue Gln27 and / or residue Asn33, where the residue numbers follow SEQ ID NO: 69, or binding to an hGREM1 fragment containing residue Gln27 and / or residue Asn33, wherein the hGREM1 fragment optionally has a length of at least 3 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acid residues; f) When measured with Fortebio, K is less than 1 nM. D It can then bind to hGREM1; h) When measured by ELISA, the binding of hGREM1 to BMP7 can be blocked at a maximum blockage percentage exceeding 50%; i) The interaction between GREM1 and FGFR can be blocked; and / or j) It can be coupled to both GREM1 and DAN. An isolated antibody or its antigen-binding fragment having at least one of the following. [Aspect 2] An isolated antibody against hGREM1 or its antigen-binding fragment according to Aspect 1, wherein the epitope is a linear epitope or a three-dimensional epitope. [Aspect 3] An isolated antibody against human gremlin 1 (hGREM1) or an antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, wherein the heavy chain variable region is: a) HCDR1 containing a sequence selected from the group consisting of sequence numbers 1, 11, 21, 31, 114, 119 and 123, b) HCDR2 containing sequences selected from the group consisting of sequence numbers 2, 12, 22, 32 and 115, and c) HCDR3 containing a sequence selected from the group consisting of sequence numbers 3, 13, 23, 33, 116, 120, and 124. including and / or The aforementioned light chain variable region is: d) LCDR1 containing a sequence selected from the group consisting of sequence numbers 4, 14, 24, 34, 117, 121, 122 and 125, e) LCDR2 containing a sequence selected from the group consisting of sequence numbers 5, 15, 25, and 35, and f) LCDR3 containing a sequence selected from the group consisting of sequence numbers 6, 16, 26, 36 and 118, An isolated antibody or its antigen-binding fragment, containing [the specified substance]. [Aspect 4] The heavy chain variable region is: a) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 1, HCDR2 containing the sequence of sequence number 2, and HCDR3 containing the sequence of sequence number 3; b) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 11, HCDR2 containing the sequence of sequence number 12, and HCDR3 containing the sequence of sequence number 13; c) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 21, HCDR2 containing the sequence of sequence number 22, and HCDR3 containing the sequence of sequence number 23; d) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 31, HCDR2 containing the sequence of sequence number 32, and HCDR3 containing the sequence of sequence number 33; e) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 114, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 116; and f) Heavy chain variable regions including HCDR1 containing the sequence of sequence number 119, HCDR2 containing the sequence of sequence number 115, and HCDR3 containing the sequence of sequence number 120; and g) Heavy chain variable region including HCDR1 containing the sequence of SEQ ID NO: 123, HCDR2 containing the sequence of SEQ ID NO: 115, and HCDR3 containing the sequence of SEQ ID NO: 124. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, selected from the group consisting of the following. [Aspect 5] The variable region of the light chain is: a) Light chain variable region including LCDR1 containing the sequence of sequence number 4, LCDR2 containing the sequence of sequence number 5, and LCDR3 containing the sequence of sequence number 6; b) Light chain variable regions including LCDR1 containing the sequence of sequence number 14, LCDR2 containing the sequence of sequence number 15, and LCDR3 containing the sequence of sequence number 16; c) Light chain variable region containing LCDR1 containing the sequence of sequence number 24, LCDR2 containing the sequence of sequence number 25, and LCDR3 containing the sequence of sequence number 26; d) Light chain variable regions including LCDR1 containing the sequence of sequence number 34, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 36; e) Light chain variable region containing LCDR1 containing the sequence of sequence number 117, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; f) Light chain variable region containing LCDR1 containing the sequence of sequence number 121, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; i) Light chain variable regions including LCDR1 containing the sequence of sequence number 122, LCDR2 containing the sequence of sequence number 35, and LCDR3 containing the sequence of sequence number 118; and j) Light chain variable region including LCDR1 containing the sequence of sequence number 1...

Claims

1. An isolated antibody against human gremlin 1 (hGREM1) or its antigen-binding fragment, comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein, An antibody or its antigen-binding fragment, wherein the heavy chain variable region comprises HCDR1 containing the sequence of SEQ ID NO: 1, HCDR2 containing the sequence of SEQ ID NO: 2, and HCDR3 containing the sequence of SEQ ID NO: 3; and the light chain variable region comprises LCDR1 containing the sequence of SEQ ID NO: 4, LCDR2 containing the sequence of SEQ ID NO: 5, and LCDR3 containing the sequence of SEQ ID NO:

6.

2. a) A heavy chain variable region containing the sequence of Sequence ID 7 and a light chain variable region containing the sequence of Sequence ID 8; or b) A heavy chain variable region containing the sequence of sequence number 41 and a light chain variable region containing the sequence of sequence number 47; or c) A heavy chain variable region containing the sequence of sequence number 41 and a light chain variable region containing the sequence of sequence number 49; or d) A heavy chain variable region containing the sequence of sequence number 43 and a light chain variable region containing the sequence of sequence number 47; or e) A heavy chain variable region containing the sequence of sequence number 43 and a light chain variable region containing the sequence of sequence number 49; or f) A heavy chain variable region containing the sequence of sequence number 45 and a light chain variable region containing the sequence of sequence number 47; or g) Heavy chain variable region containing the sequence of Sequence ID No. 45 and light chain variable region containing the sequence of Sequence ID No. 49 The antibody or antigen-binding fragment according to claim 1, comprising:

3. The antibody or antigen-binding fragment according to claim 1, further comprising an immunoglobulin constant region, or further comprising a human IgG constant region, 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 its antigen-binding fragment according to claim 1, which is humanized or chimeric.

5. Diabody, Fab, Fab', F(ab') ２ Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv) ２ The antibody or antigen-binding fragment according to claim 1, which is a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), or a multispecific antibody.

6. The antibody or antigen-binding fragment according to claim 1, which is bispecific, or can specifically bind to a first and second epitope of a gremlin, or can specifically bind 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, 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-related mucin, VEGF, VEGFR (e.g., VEGFR-1, VEGFR-2, VEGFR-3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, claudin 18.2 An antibody or antigen-binding fragment thereof according to claim 6, selected from the group consisting of GPC-3, Nectin-4, ROR1, Metserine, 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 according to claim 1, which is linked to one or more conjugate portions.

9. A pharmaceutical composition or kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

10. An isolated polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

11. A vector comprising an isolated polynucleotide as described in claim 10.

12. A host cell comprising the vector according to claim 11.

13. A method for expressing an antibody or an antigen-binding fragment according to any one of claims 1 to 8, comprising the step of culturing a host cell containing a vector under conditions in which a vector containing an isolated polynucleotide encoding an antibody or an antigen-binding fragment according to any one of claims 1 to 8 is expressed.

14. A pharmaceutical composition for treating a GREM1-related disease or condition in a subject, inhibiting FGFR1 activation in a subject requiring inhibition of FGFR1 activation, or treating a disease or condition associated with GREM1-mediated FGFR1 activation, comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8.

15. The pharmaceutical composition according to 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 an elevated level 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 according to claim 15, wherein the cancer is a GREM1-expressing cancer.

17. The pharmaceutical composition according to claim 16, wherein the cancer is a PD-L1 expressing cancer, or is not a PD-L1 expressing cancer, or is resistant to or unresponsive to treatment with a PD-1 / PD-L1 axis inhibitor.

18. The pharmaceutical composition according to claim 14, wherein the subject is identified as having GREM1-expressing cancer cells or having GREM1 expression in the tumor microenvironment.

19. The aforementioned cancers include 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, and bone marrow cancer. The pharmaceutical composition according to claim 15, which is a dysplastic syndrome, sarcoma, teratoma, glioma, adenocarcinoma, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML)), lymphoma (e.g., Hodgkin lymphoma, or non-Hodgkin lymphoma (e.g., Waldenström macroglobulinemia (WM))), or myeloma (e.g., multiple myeloma (MM)).

20. The pharmaceutical composition according to claim 15, wherein the breast cancer is triple-negative breast cancer.

21. The pharmaceutical composition according to claim 15, wherein the cancer is esophageal cancer.

22. The pharmaceutical composition according to claim 21, wherein the cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, or resistant or refractory to treatment with an anti-PD-1 antibody (e.g., nivolumab).

23. The pharmaceutical composition according to claim 15, wherein the fibrotic disease is a fibrotic disease of the lungs, liver, kidneys, eyes, skin, heart, intestines, or muscles.

24. The pharmaceutical composition according to claim 14, wherein the subject is a human.

25. The pharmaceutical composition according to claim 14, which is administered in combination with a second therapeutic agent, wherein the second therapeutic agent comprises anticancer therapy.

26. The anticancer therapy comprises a chemotherapeutic agent (e.g., cisplatin), radiotherapy, immunotherapy agents (e.g., immune checkpoint modulators, e.g., PD-1 / PD-L1 axis inhibitors, TGF-beta inhibitors), anti-angiogenic agents (e.g., VEGFR antagonists 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 resection), one or more antiemetics, and chemotherapy. The pharmaceutical composition according to claim 25, selected from treatment of complications arising from the procedure, nutritional supplements for cancer patients (e.g., indole-3-carbinol), agents that modulate the tumor microenvironment (e.g., a bifunctional molecule comprising a PD-L1 binding moiety and the extracellular domain of a TGF-beta receptor), or antifibrotic therapies (e.g., BMP7 treatment, ACE inhibitors (or ARBs), anti-MASP2 antibodies, endothelin receptor antagonists, NRF2 inhibitory steroids, CTLA4-IgG or TNF inhibitors).

27. The pharmaceutical composition according to claim 25, wherein the anticancer therapy comprises an androgen axis inhibitor; an androgen synthesis inhibitor; a PARP inhibitor; or a combination thereof for the treatment of prostate cancer, or comprises abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, darolutamide, degarelix, docetaxel, enzalutamide, flutamide, goserelin acetate, leuprolide acetate, mitoxantrone hydrochloride, nilutamide, olaparib, radium-223 dichloride, lucaparib cansylate, or cypureucel-T for the treatment of prostate cancer.

28. The pharmaceutical composition according to claim 27, wherein the androgen axis inhibitor is degarelix, bicalutamide, flutamide, nilutamide, apalutamide, darolutamide, enzalutamide, or abiraterone.

29. A method for detecting the presence or amount of gremlin in a sample, comprising the steps of contacting the sample with an antibody or antigen-binding fragment described in any one of claims 1 to 8, and determining the presence or amount of gremlin in the sample.

30. A pharmaceutical composition for treating diseases in which it is beneficial to increase BMP7 activity or decrease gremlin-mediated inhibition of BMP7 activity, or for improving the efficacy of BMP7 treatment in subjects requiring improved efficacy of BMP7 treatment, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

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