Combination therapy involving GREM1 antagonists for cancer treatment

Abstract

The present invention provides a combination therapy comprising a GREM1 antagonist and one or more therapies selected from a) anti-angiogenic therapy, b) chemotherapy, and c) immunotherapy. The invention also provides a method for selecting a subpopulation from subjects resistant to immunotherapy that may respond to the immunotherapy using a GREM1 antagonist.

Description

Technical Field

[0001] The present disclosure generally relates to cancer therapies involving anti-angiogenic therapy and / or chemotherapy and / or immunotherapy in combination with a gremlin 1 (GREM1) antagonist for treating cancer, particularly GREM1-expressing cancer.

Background Art

[0002] GREM1 is closely associated with several tumor types including fibrosis lesions of the kidney, lung, liver, retina, and pancreatic cancer, colorectal cancer, lung cancer, glioma, gastric cancer, prostate cancer (Sneddon et al., PNAS 2006 Oct; 103(40): 14842-14847). For example, abnormal upregulation of gremlin 1 confers tumorigenic ability to colorectal cells outside the stem cell niche. It has also been found that tumor stem cells highly express and secrete gremlin 1 in glioma to maintain their stemness (Yan, K. et al., Genes Dev 28, 1085-1100 (2014)). Therefore, gremlin 1 has been used as a therapeutic target in the treatment of gremlin-related diseases.

[0003] Although GREM1 antagonists have shown certain therapeutic effects against several GREM1-expressing diseases, effective combination therapies involving GREM1 antagonists for treating GREM1-expressing cancer are still rare. Therefore, in order to meet clinical needs, improved combination therapies for GREM1-expressing cancer are strongly demanded.

Summary of the Invention

[0004] The present disclosure particularly provides a method for treating GREM1-expressing cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

[0005] In one embodiment, the anti-angiogenic therapy includes a VEGFA or VEGFR antagonist.

[0006] In one embodiment, the VEGFA antagonist is an anti-VEFRA antibody such as bevacizumab (Avastin).

[0007] In one embodiment, the VEGFR antagonist is either a small molecule VEGFR inhibitor or a large molecule VEGFR inhibitor.

[0008] In one embodiment, VEGFR is VEGFR-1, VEGFR-2, or VEGFR-3.

[0009] In one embodiment, the VEGFR antagonist is a large molecule VEGFR inhibitor, such as an anti-VEGFR-2 antibody. In one embodiment, the anti-VEGFR-2 antibody is selected from the group consisting of ramucirumab, olinvacimab, gentuximab, alacizumab pegol, vulinacimab, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, and HLX12.

[0010] In one embodiment, the VEGFR antagonist is a small molecule VEGFR inhibitor such as citravatinib, anlotinib, apatinib, telatinib, altiratinib, kanitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), and / or regorafenib (Stivarga®).

[0011] In one embodiment, the method includes administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with an anti-VEGFA antibody, an anti-VEGFR-2 antibody, or a small molecule VEGFR inhibitor.

[0012] In one embodiment, the cancer is colorectal cancer, stomach cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0013] In one embodiment, the cancer is determined to be resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or to have low or absent PD-L1 expression in the cancerous lesion tissue.

[0014] In one embodiment, chemotherapy includes a combination of chemotherapeutic agents.

[0015] In one embodiment, the combination of chemotherapeutic agents includes leucovorin calcium (folic acid), fluorouracil, and irinotecan hydrochloride (FOLFIRI).

[0016] In one embodiment, the method comprises administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with a combination of chemotherapeutic agents and an anti-VEGFR-2 antibody, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0017] In one embodiment, the cancer is determined to be resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or to have low or absent PD-L1 expression in the cancerous lesion tissue.

[0018] In one embodiment, the immunotherapy includes a PD-1 / PD-L1 axis inhibitor.

[0019] In one embodiment, the PD-1 / PD-L1 axis inhibitor comprises a PD-1 inhibitor selected from the group consisting of antibodies, small molecules, and combinations thereof.

[0020] In one embodiment, the PD-1 inhibitor is nivolumab (OPDIVO, BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA, MK-3475), MEDI0680 (AMP-514), MEDI4736, BI754091, pidilizumab (CT-011), cemiplimab (LIBTAYO, REGN28) 10) Spartalizumab (PDR001), Cetrerimab (JNJ63723283), Tripalimab (JS001), PF-06801591, Tislelizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, Lambrolizumab, Camrelizuma (SHR-1210), Sintilimab (Tyvyt, IBI308), Penplimab (AK105), Zimberelimab, Retifanlimab, Serplulimab, Balstilimab, Geptanolimab, Prolgol This includes an anti-PD-1 antibody selected from the group consisting of imab), Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofazinlimab, Sindelizumab, MGA404, Sym021, BAT1306, and HX008.

[0021] In one embodiment, the PD-1 inhibitor is nivolumab (OPDIVO, BMS-936558).

[0022] In one embodiment, the PD-1 / PD-L1 axis inhibitor comprises a PD-L1 inhibitor selected from the group consisting of antibodies, small molecules, and combinations thereof.

[0023] In one embodiment, the PD-L1 inhibitor is atezolizumab (TECENTRIQ, R05541267, MPDL3280A, RG7446), BMS-936559, avelumab (bavencio), rodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, envafolimab (KN035), MDX-1105, STI-1040, CS1001, and adebre. This product contains an anti-PD-L1 antibody selected from the group consisting of Adebrelimab (SHR-1316), SHR-1701, TOB2450, Bintrafusp, LP002, STI-3031, Cosibelimab, Pacmilimab, NM01, LDP, AMP-224, Garivulimab (BGB-A333), A167, SCD-135, Opucolimab, and GR1405.

[0024] In one embodiment, the cancer is determined to be a) resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or b) have low or no PD-L1 expression in the lesional tissue of GREM1-expressing cancer.

[0025] In one embodiment, the method includes administering a GREM1 antagonist to a subject for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer cells, followed by administering a PD-1 / PD-L1 axis inhibitor to the subject.

[0026] In one embodiment, the method involves administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with a chemotherapeutic agent (e.g., FOLFIRI) and a PD-1 / PD-L1 axis inhibitor (e.g., a PD-1 inhibitor or nivolumab), wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0027] In one embodiment, the cancer is determined to be resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or to have low or absent PD-L1 expression in the cancerous lesion tissue.

[0028] In one embodiment, the method comprises administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with an anti-VEGFR-2 antibody, a combination of chemotherapeutic agents, and a PD-1 / PD-L1 axis inhibitor, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0029] In one embodiment, the cancer is determined to be resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or to have low or absent PD-L1 expression in the cancerous lesion tissue.

[0030] In one embodiment, the subject is further determined to have low, moderate, or high GREM1 expression in the lesional tissue of a GREM1-expressing cancer. In another embodiment, the subject is further determined to have 10–20% of tumor cells GREM1-positive, as measured by IHC.

[0031] In another aspect, the present disclosure provides a method for improving a subject's response to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject is determined to have GREM1 present in a biological sample of the subject's lesional tissue, or the subject is determined to have a threshold level of GREM1 expression in a biological sample of the subject's lesional tissue, and the method is a) This includes administering a therapeutically effective dose of a GREM1 antagonist to the subject to increase PD-L1 expression in the lesional tissue, thereby improving the subject's responsiveness to the PD-1 / PD-L1 axis inhibitor.

[0032] In one embodiment, the subject is determined to be a) resistant to or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) to have low or absent PD-L1 expression in the diseased tissue.

[0033] In one embodiment, the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

[0034] In one embodiment, the diseased tissue is cancerous tissue.

[0035] In one embodiment, the cancer is colorectal cancer, stomach cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0036] In one embodiment, the method further comprises administering a therapeutically effective dose of a PD-1 / PD-L1 axis inhibitor to the subject after PD-L1 expression has increased in the subject's lesional tissue.

[0037] In another aspect, the Disclosure provides a method for determining whether a subject is likely to be eligible for or responsive to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor, the method being: a) including determining the presence or expression level of GREM1 in a biological sample of diseased tissue from the subject, The presence or absence of GREM1, or its expression level, indicates whether the subject is eligible for or likely to respond to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0038] In one embodiment, the presence of GREM1 or an expression level of GREM1 exceeding a threshold level, as determined in step a), indicates that the subject is likely to be eligible for or respond to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0039] In one embodiment, the absence of GREM1 or a GREM1 expression level below the threshold determined in step a) indicates that the subject is not eligible for or unlikely to respond to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0040] In one embodiment, the method provided herein, before step a), The procedure further includes step i) bringing a biological sample of diseased tissue from a subject into contact with a GREM1 diagnostic agent under conditions that enable the detection of GREM1 expression levels in the sample.

[0041] In one embodiment, the subject is determined to be a) resistant to or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) to have low or absent PD-L1 expression in the diseased tissue.

[0042] In one embodiment, the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

[0043] In one embodiment, the method provided herein further comprises administering a therapeutically effective dose of a GREM1 antagonist to the subject for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer cells, followed by administering a PD-1 / PD-L1 axis inhibitor to the subject.

[0044] In one embodiment, GREM1 expression is detected by a GREM1 diagnostic reagent containing an anti-GREM1 antibody or its antigen-binding fragment.

[0045] In one embodiment, the GREM1 antagonist comprises an anti-GREM1 antibody or its antigen-binding fragment.

[0046] In one embodiment, the anti-GREM1 antibody or its antigen-binding fragment comprises heavy chains HCDR1, HCDR2, HCDR3 and / or light chains LCDR1, LCDR2, LCDR3. HCDR1 contains an amino acid sequence including TYGMA (SEQ ID NO: 1), or a homologous sequence having at least 80% sequence identity thereto. HCDR2 contains an amino acid sequence including WINTLSGEPTYADDFKG (SEQ ID NO: 2), or a homologous sequence having at least 80% sequence identity thereto. HCDR3 contains an amino acid sequence including EPMDY (SEQ ID NO: 3), or a homologous sequence having at least 80% sequence identity thereto. LCDR1 contains an amino acid sequence including KSSQSLLDSDGKTYLS (SEQ ID NO: 4), or a homologous sequence having at least 80% sequence identity thereto. LCDR2 includes an amino acid sequence containing LVSKLDS (SEQ ID NO: 5), or a homologous sequence having at least 80% sequence identity thereto. LCDR3 contains an amino acid sequence including WQGAHFPLT (SEQ ID NO: 6), or a homologous sequence having at least 80% sequence identity thereto.

[0047] In one embodiment, the anti-GREM1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7, The light chain variable region contains the amino acid sequence of SEQ ID NO: 8.

[0048] In one embodiment, the method further involves the substitution or modification of one or more amino acid residues, but still retains specific binding specificity or affinity for hGREM1.

[0049] In one embodiment, at least one of the substitutions or modifications is present in one or more CDR sequences and / or one or more non-CDR regions of VH or VL sequences.

[0050] In one embodiment, the method further comprises an immunoglobulin constant region, and optionally a human IgG constant region.

[0051] In one embodiment, the steady region includes a steady region of human IgG1, IgG2, IgG3, or IgG4, and optionally, the steady region includes a heavy chain steady region containing the sequence of SEQ ID NO: 9 and / or a light chain steady region containing the sequence of SEQ ID NO: 10.

[0052] In one embodiment, a GREM1 antagonist or anti-GREM1 diagnostic reagent is linked to one or more conjugate portions.

[0053] In some embodiments, the conjugate moiety includes clearance modifiers, therapeutic agents (e.g., chemotherapeutic agents), toxins, radioisotopes, detectable labels (e.g., lantanides, luminescence labels, fluorescent labels, biotin / avidin, or enzyme substrate labels), pharmacokinetic modifiers, DNA alkylating agents, topoisomerase inhibitors, tubulin conjugates, and other anticancer agents such as androgen receptor inhibitors.

[0054] In one embodiment, the subject is a human.

[0055] In some embodiments, administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular dose.

[0056] In one embodiment, the GREM1 antagonist is administered before, simultaneously with, or after the administration of an anti-angiogenic agent and / or chemotherapeutic agent and / or immunotherapeutic agent.

[0057] In another aspect, the Disclosure provides the use of a GREM1 antagonist in the manufacture of a drug for treating GREM1-expressing cancer in a subject as required, the treatment comprising administering the drug to a subject in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

[0058] In another aspect, the disclosure provides the use of GREM1 antagonists in the manufacture of pharmaceuticals to improve the subject's response to treatment with PD-1 / PD-L1 axis inhibitors. The subject has been determined to have GREM1 present in a biological sample of the subject's diseased tissue, or the subject has been determined to have a GREM1 expression level that has reached a threshold level in a biological sample of the subject's diseased tissue. The improvement includes a) administering a therapeutically effective dose of a GREM1 antagonist to the subject so as to increase PD-L1 expression in the lesional tissue, thereby improving the subject's responsiveness to the PD-1 / PD-L1 axis inhibitor.

[0059] In another aspect, the disclosure provides the use of an anti-GREM1 diagnostic reagent in the manufacture of a kit for determining whether a subject is eligible for or responsive to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor. The anti-GREM1 diagnostic reagent can determine the presence or expression level of GREM1 in a biological sample of lesional tissue from the subject. The presence or absence of GREM1, or its expression level, indicates whether the subject is eligible for or likely to respond to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0060] In one embodiment, the subject is determined to be a) resistant to or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) to have low or absent PD-L1 expression in the diseased tissue.

[0061] In one embodiment, the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

[0062] In another aspect, the Disclosure provides a kit useful for treating GREM1-expressing cancer in a subject as needed, comprising a GREM1 antagonist and a package insert, the package insert including instructions for using the GREM1 antagonist in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

[0063] In one embodiment, GREM1-expressing cancer is characterized by a) resistance or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or b) low or absent expression of PD-L1 in the lesional tissue, and / or c) expression of GREM1 in the lesional tissue.

[0064] In another embodiment, the present disclosure provides a method for improving tumor-infiltrating lymphocytes in a subject having a solid tumor, the method comprising administering a therapeutically effective dose of a GREM1 antagonist as described in any one of the prior claims to the subject.

[0065] In one embodiment, a solid tumor is a cold tumor.

[0066] In one embodiment, the subject is resistant to or refractory to anticancer therapies such as immunotherapy, for example, immune checkpoint inhibitors.

[0067] In another embodiment, the present disclosure provides a method for promoting the transformation of a cold tumor to a hot tumor in a subject having a solid tumor, the method comprising administering a therapeutically effective dose of a GREM1 antagonist as described in any one of the prior claims to the subject.

[0068] In another embodiment, the present disclosure provides a method for treating cancer in a subject having a cold tumor, the method comprising administering to the subject a therapeutically effective dose of a GREM1 antagonist as described in any one of the prior claims.

[0069] In one embodiment, the subject is resistant to or refractory to anticancer therapies such as immunotherapy, for example, immune checkpoint inhibitors.

[0070] In one embodiment, the method provided herein further includes administering one or more therapies to a subject.

[0071] In one embodiment, one or more therapies can promote T cell proliferation, activation, and / or tumor invasion.

[0072] In one embodiment, the T cells are CD3+ T cells or CD8+ T cells.

[0073] In some embodiments, one or more therapies are anti-angiogenic therapies, immunotherapies, and / or chemotherapy according to any one of the preceding claims.

[0074] In one embodiment, the subject is determined to have moderate or high PD-L1 expression in the lesional tissue. It should be understood that both the general description above and the detailed description below are merely illustrative and explanatory and do not limit the invention. Furthermore, the accompanying drawings incorporated herein and constituting part thereof illustrate embodiments of the invention and, together with this specification, help to illustrate the principles of the invention.

[0075] [Brief description of the drawing] The drawings referenced herein constitute part of this specification. The features shown in the drawings illustrate only some embodiments of this application, and not all embodiments, unless explicitly indicated otherwise in the detailed description. Readers of this specification should not take this to the contrary.

[0076] Throughout the drawing, the same reference number is used for identical or similar parts. [Brief explanation of the drawing]

[0077] [Figure 1A] This figure shows that anti-GREM1 antibody used in combination with DC101 inhibited CRC PDX tumor growth (mean ± SEM, n=8). [Figure 1B] This figure shows pathological information of BZ-CRC-01 PDX tumors in which gremlin 1 was detected in 10-20% of tumor cells (A) by IHC. [Figure 2]This figure shows that anti-GREM1 antibody, used in combination with FOLFIRI and DC101, inhibited CRC PDX tumor growth (mean ± SEM, n=8). [Figure 3] This figure shows that anti-GREM1 antibodies used in combination with FOLFIRI, nivolumab, and DC101 inhibited CRC PDX tumor growth (mean ± SEM, n=8). [Figure 4] This figure shows a representative IHC image of GREM1 expression in BZ-CRC-01 PDX tumor sections. [Figure 5] This figure shows representative IHC images of PD-L1 expression before and after Hu14E3 HaLa treatment of BZ-CRC-01 PDX tumor sections. [Figure 6] This figure shows a representative IHC image of tumor-infiltrating lymphocytes (TILs) after Hu14E3 HaLa treatment of BZ-CRC-01 PDX tumor sections. [Figure 7] This figure shows the safety profile of Hu14E3 HaLa in NHP. [Modes for carrying out the invention]

[0078] The following descriptions of this disclosure are merely illustrative of various embodiments of this disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. Those skilled in the art will see that various equivalents, changes, and modifications may be made without departing from the scope of this disclosure, and that such equivalent embodiments are also included herein. All references cited herein (including publications, patents, and patent applications) are incorporated herein by reference in their entirety.

[0079] definition

[0080] When used herein, the terms “one,” “one,” “the,” and similar terms used in the context of the present invention (particularly in the context of the claims) should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this interpretation.

[0081] As used herein, the term “antagonist” in relation to GREM1 means any molecule that partially or completely inhibits, blocks, or neutralizes the biological activity of GREM1. Suitable GREM1 antagonists include, but are not limited to, antibodies, antisense oligonucleotides, peptides, and small organic molecules. In some embodiments, the GREM1 antagonist is an anti-GREM1 antibody.

[0082] As used herein, the term “antibody” includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific or bispecific antibody that binds to a particular antigen, or any polypeptide peptide that mimics an antibody in terms of its ability to bind to a particular antigen. Naturally intact antibodies consist of two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each consisting of a variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3, respectively). Mammalian light chains are classified as λ or κ, each consisting of a variable region (VL) and a constant region. Antibodies are “Y”-shaped, with the stem of the Y consisting of a second and third constant region of two heavy chains linked to each other by disulfide bonds. Each arm of the Y chain contains a variable region of a single light chain, a variable region of a single heavy chain bound to a constant region, and a first constant region. The variable regions of the light and heavy chains play a role in antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (the light chain CDRs contain LCDR1, LCDR2, and LCDR3, while the heavy chain CDRs contain HCDR1, HCDR2, and HCDR3).The CDR boundaries of the antibody and antigen-binding domains disclosed herein are those of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J.Mol.Biol., 273(4), 927(1997); Chothia, C. et al., JMolBiol. Dec5; 186(3): 651-63(1985); Chothia, C. and Lesk, AM, J.Mol.Biol., 196, 901(1987); NRWhitelegg et al., Prote in Engineering, v13(12), 819-824(2000); Chothia, C. et al., Nature. Dec21-28; 342(6252): 877-83(1989); Kabat E.A. et al., National Institutes of The antibody may be defined or identified by the conventions of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015). The three CDRs are more conserved than the CDRs and are sandwiched between adjacent stretches known as framework regions (FRs), which form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exert various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of the heavy chain. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of alpha, delta, epsilon, gamma, and muon heavy chains, respectively. Some of the main antibody classes are further divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), and IgA2 (α2 heavy chain).In one embodiment, the antibody provided herein includes any antigen-binding fragment thereof.

[0083] As used herein, the terms “antigen-binding fragment” or “antigen-binding moiety” mean a fragment formed from an antibody fragment containing one or more CDRs (e.g., an antibody fragment), or any other moiety that binds to an antigen but does not contain an intact native antibody structure (e.g., an antibody moiety). Examples of antigen-binding fragments / moies 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-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. Antigen-binding fragments / moies are capable of binding to the same antigen to which the parent antibody binds. In some embodiments, an antigen-binding fragment / moiety may contain one or more CDRs from a particular parent antibody.

[0084] 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) linked by disulfide bonds to a variable region and a first constant region of a single heavy chain. Fab is obtained by papain digestion of the antibody at a residue adjacent to the N-terminus of the disulfide bond between the heavy chains in the hinge region.

[0085] "Fab'" refers to a Fab fragment that includes part of the hinge region, and it differs from Fab in a few residues (including one or more cysteines) in the hinge region because it is obtained by pepsin digestion of the antibody at residues close to the C-terminus of the disulfide bond between the heavy chains of the hinge region.

[0086] "F(ab')2" refers to a Fab' dimer that contains two light chains and parts of two heavy chains.

[0087] In relation to antibodies, "Fv" refers to the smallest fragment of an antibody that carries a complete antigen-binding site. An Fv fragment consists of a single variable region of a light chain bound to a single variable region of a heavy chain. "dsFv" refers to a disulfide-stabilized Fv fragment in which the linkage between the single variable region of the light chain and the single variable region of the heavy chain is a disulfide bond.

[0088] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). "scFv dimer" refers to a single chain containing two heavy chain variable regions and two light chain variable regions via a linker. In some embodiments, the "scFv dimer" is a bivalent diabody or bivalent ScFv (BsFv) containing a dimerized VH-VL (linked by a peptide linker) with another VH-VL moiety, such that one VH moiety cooperates with the other VL moiety to form two binding sites that can target the same antigen (or epitope) or a different antigen (or epitope). In another embodiment, the "scFv dimer" is a bispecific diabody containing VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (linked by a peptide linker), such that VH1 and VL1 cooperate, VH2 and VL2 cooperate, and each cooperated pair has different antigen specificity.

[0089] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of scFv units attached to the Fc region of the antibody.

[0090] "Camelized single-domain antibody," "heavy-chain antibody," "nanobody," or "HCAb" refers to an antibody that contains two VH domains and does not contain a light chain (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38(1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302(2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy-chain antibodies were originally obtained from camelids (camels, dromedaries, and llamas). Camelized antibodies, while lacking a light chain, possess a genuine antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun3;363(6428):446-8(1993); Nguyen VK. et al., “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation,” Immunogenetics. Apr;54(1):39-47(2002); Nguyen VK. et al., Immunology. May;109(1):93-101(2003)). The variable domain (VHH domain) of heavy-chain antibodies is the smallest known antigen-binding unit produced by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15(2007)). A "diabody dAb" contains a small antibody fragment with two antigen-binding sites, and these fragments contain a VH domain (VH-VL or VL-VH) attached to a VL domain in a single polypeptide chain (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15;90(14):6444-8 (1993), EP404097, WO93 / 11161). Because the linker is too short, the two domains on the same chain cannot pair up, and therefore these domains are forced to pair with a complementary domain on another chain, thereby forming two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes).

[0091] 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 some embodiments, two or more VH domains are covalently linked by a peptide linker to form a bivalent or polyvalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

[0092] In one embodiment, "(dsFv)2" comprises three peptide chains, i.e., two VH moieties linked by a peptide linker and attached to two VL moieties by disulfide bonds.

[0093] In one embodiment, the "bispecific ds diabody" includes VH1-VL2 (linked by a peptide linker) linked to VL1-VH2 (linked by a peptide linker) via a disulfide crosslink between VH1 and VL1.

[0094] In one embodiment, "bispecific dsFv" or "dsFv-dsFv'" contains three peptide chains, i.e., a VH1-VH2 moiety in which the heavy chain is linked by a peptide linker (e.g., a long, mobile linker) and pairs with the VL1 and VL2 moieties, respectively, via disulfide crosslinks. Each disulfide-paired heavy and light chain has a different antigen specificity.

[0095] As used herein, the term “humanized” means that an antibody or antigen-binding fragment comprises a CDR derived from a non-human animal, an FR region derived from humans, and, where applicable, a constant region derived from humans. In some embodiments, amino acid residues of the variable region framework of a humanized gremlin antibody are substituted for sequence optimization. In some 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.

[0096] As used herein, the term "anti-GREM1 antibody" means an antibody that can specifically bind to GREM1 (e.g., human GREM1 or non-human GREM1) with sufficient specificity and / or affinity for diagnostic and / or therapeutic use.

[0097] As used herein, the term "affinity" means the strength of non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0098] As used herein, the term "specific binding" or "specifically binds" means a non-random binding reaction between two molecules, such as between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human and / or non-human gremlin 1 with a binding affinity (KD) of 10 -6 M or less (e.g., 5 × 10 -7 M or less, 2 × 10 -7 M or less, 10 -7 M or less, 5 × 10 -8 M or less, 2 × 10 -8 M or less, 10 -8 M or less, 5 × 10 -9 M or less, 4 × 10 -9 M or less, 3 × 10 -9 M or less, 2 × 10 -9 M or less, or 10 -9 M or less). As used herein, KD is the ratio of the dissociation rate to the association rate (k off / k onThe KD value refers to any conventional method known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). In some embodiments, the KD value can be appropriately determined using flow cytometry. Various immunoassay formats may be used to select antibodies that specifically react with a particular protein. For example, solid-phase ELISA immunoassay is routinely used to select antibodies that specifically react with a protein (e.g., Harlow and 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 produce a signal at least twice the background signal, more typically at least 10 to 100 times the background signal.

[0099] The "percentage of sequence identity (%)" for an amino acid sequence (or nucleic acid sequence) is defined as the proportion of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues of the reference sequence, after the sequences have been aligned and gaps introduced as necessary to achieve maximum correspondence. Alignment aimed at 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 National Center for Biotechnology Information (NCBI) website; see also Altschul S.F. 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 Institute for Bioinformatics website; see also Higgins DGet al, Methods in Enzymology, 266:383-402 (1996), Larkin MA et al, Bioinformatics (Oxford, UK), 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 to suit alignment, for example, by selecting an appropriate algorithm. In some embodiments, the positions of non-identical residues may differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be up-adjusted to compensate for the conservative nature of the substitutions. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is incorporated herein by reference.

[0100] 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.

[0101] The term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals (non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles). Unless otherwise specified, the terms "patient" and "subject" are used synonymously herein.

[0102] As used herein, "treating" a medical condition or "treatment" includes preventing or alleviating a medical condition, slowing the onset or progression of a medical condition, reducing the risk of progression of a medical condition, preventing or slowing the progression of symptoms associated with a medical condition, reducing or terminating symptoms associated with a medical condition, causing complete or partial regression of a medical condition, curing a medical condition, or a combination thereof.

[0103] As used herein, “cancer” means any medical condition characterized by malignant cell proliferation or neoplasm, abnormal growth, invasion, or metastasis, and includes both solid tumors and non-solid cancers such as leukemia (e.g., hematopoietic malignancies). As used herein, “solid tumor” means a solid mass of neoplastic and / or malignant cells.

[0104] The terms “therapeutic effective dose” or “effective dose” refer to the amount of a pharmaceutical formulation that produces several desired local or systemic therapeutic effects in a reasonable benefit-to-risk ratio applicable to any treatment, whether alone or in combination with further administrations. In the case of treatment of a particular disease, the desired local or systemic therapeutic effect is preferably related to inhibiting the course of the disease. This includes slowing the progression of the disease, in particular interrupting or reversing its progression. When administered to prevent a disease, the amount is sufficient to avoid or delay the onset of the disease. A therapeutic effective dose or effective dose does not need to cure or prevent the onset of a disease or condition. The effective dose of a pharmaceutical formulation described herein depends on the condition being treated, the severity of the disease, the individual parameters of the patient (including age, physiological status, body size, and weight), the duration of treatment, the type of supplemental therapy (if any), the specific route of administration, and similar factors. Therefore, the administered dose of a pharmaceutical formulation described herein may depend on such various parameters. If the patient does not respond adequately to the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used. In some embodiments, the therapeutically effective dose of the pharmaceutical formulation depends on its therapeutic index, solubility, etc.

[0105] In this specification, any reference to a value or parameter with the term "approximately" includes (describes) embodiments relating to that value or parameter itself. For example, the statement "approximately X" includes the statement "X". A numerical range includes the numerical value that defines the range. Generally, the term "approximately" means the given value of a variable and all values ​​of the variable that are within the experimental error of the given value (e.g., within the 95% confidence interval of the mean) or within 10% of the given value, whichever is greater. When the term "approximately" is used in the context of a period (year, month, week, day, etc.), the term "approximately" means that period ± one quantity of the next dependent period (e.g., approximately one year means 11 to 13 months, approximately six months means 6 months ± one week, approximately one week means 6 to 8 days, etc.), or within 10% of the given value, whichever is greater.

[0106] I. Methods to improve tumor-infiltrating lymphocytes

[0107] This disclosure provides a method for improving tumor-infiltrating lymphocytes in a subject having a solid tumor, the method comprising administering a therapeutically effective dose of a GREM1 antagonist to the subject. In one embodiment, the solid tumor is a cold tumor. In one embodiment, the subject is resistant or refractory to anticancer therapies such as immunotherapy, e.g., immune checkpoint inhibitors (e.g., PD-1 / PD-L1 axis inhibitors).

[0108] This disclosure also provides a method for promoting the transformation from a cold tumor to a hot tumor in a subject having a solid tumor, the method comprising administering a therapeutically effective dose of a GREM1 antagonist to the subject. As used herein, the term “cold tumor,” which is used synonymously with the terms “immune desert tumor” or “immune elimination tumor,” refers to a tumor lacking innate immunity or ineffective innate antitumor immune features, characterized by 1) lack of T cell infiltration, 2) low mutagenesis, 3) low major histocompatibility complex (MHC) class I expression, 4) low PD-L1 expression, and / or 5) the presence of immunosuppressive cell populations (e.g., tumor-associated macrophages, regulatory T cells, myeloid suppressor cells). As used herein, the term “hot tumor” means a tumor characterized by 1) high T cell infiltration, 2) increased interferon-γ signaling, 3) PD-L1 expression, and / or 4) high tumor mutagenesis.

[0109] This disclosure further provides a method for treating cancer in a subject having a cold tumor, the method comprising administering a therapeutically effective dose of a GREM1 antagonist to the subject.

[0110] In one embodiment, the subject is resistant to or refractory to anticancer therapies such as immunotherapy, for example, immune checkpoint inhibitors.

[0111] In some embodiments, the method further comprises administering one or more therapies to a subject. In some embodiments, one or more therapies are capable of promoting T cell proliferation, activation, and / or tumor infiltration. In some embodiments, the T cells are CD3+ T cells or CD8+ T cells. In some embodiments, one or more therapies are anti-angiogenic therapies, immunotherapies, and / or chemotherapy.

[0112] II. Combination Therapy

[0113] This disclosure provides a method for treating GREM1-expressing cancer in a subject as required. In one embodiment, the method includes administering a therapeutically effective dose of a GREM1 antagonist in combination with a second treatment method.

[0114] In another embodiment, the Disclosure provides a method for treating GREM1-expressing cancer in a subject as required. In one embodiment, the method comprises administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

[0115] In another aspect, the Disclosure provides the use of a GREM1 antagonist in the manufacture of a pharmaceutical / pharmaceutical composition for treating a GREM1-expressing cancer in a subject as required, the treatment comprising administering the pharmaceutical / pharmaceutical composition to a subject in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

[0116] 1. Immunotherapy

[0117] In some embodiments, the immunotherapy used in the methods provided herein comprises a PD-1 / PD-L1 axis inhibitor. The term "PD-1 / PD-L1 axis inhibitor" refers to a molecule (e.g., small molecule, antibody, etc.) that inhibits the interaction between PD-1 and PD-L1 axis binding partners, such as PD-1 and PD-L1, thereby reducing or mitigating the inhibitory effects on T cell function (e.g., proliferation, cytokine production, and target cell elimination) resulting from signaling on the PD-1 / PD-L1 signaling axis. PD-1 / PD-L1 axis inhibitors may include PD-1 inhibitors or PD-L1 inhibitors.

[0118] In some embodiments, PD-1 / PD-L1 axis inhibitors include PD-1 inhibitors. As used herein, the term “PD-1 inhibitor” means a molecule that reduces, inactivates, inhibits, blocks, or interferes with signaling resulting from the interaction of PD-1 with one or more of its binding partners (e.g., PD-L1). In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its binding partner (e.g., PD-L1). For example, a PD-1 inhibitor may be an anti-PD-1 antibody or its antigen-binding fragment, a fusion protein, an oligopeptide, an immunoadhesin, and other molecules that reduce, inactivates, inhibits, blocks, or interferes with signaling resulting from the interaction of PD-1 and PD-L1.

[0119] In some embodiments, the PD-1 inhibitor is nivolumab (OPDIVO, BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA, MK-3475), MEDI0680 (AMP-514), MEDI4736, BI754091, pizilizumab (CT-011), semiprimab (LIBTAYO, REGN2810), spartalizumab (PDR001), cetrelimab (JNJ63723283), tripalimab (JS001), PF-06801591, tislerizumab (BGB-A31) 7) An anti-PD-1 antibody selected from the group consisting of AMP-224 (GSK-2661380), ABBV-181, lambrolizumab, camrelizuma (SHR-1210), cintilimab (Tyvyt, IBI308), penprimab (AK105), zimberelimab, letifanlimab, serpullimab, valstilimab, geptanolimab, prorugolimab, ezabenlimab, sasanlimab, pimivalimab, budigalimab, nofadinlimab, syndelizumab, MGA404, Sym021, BAT1306, and HX008.

[0120] In one embodiment, the PD-1 inhibitor is nivolumab. Nivolumab (Bristol-Myers Squibb / Ono Pharmaceutical Co., Ltd.), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006 / 121168.

[0121] In one embodiment, the anti-PD-1 antibody used in the method provided herein comprises a heavy chain and a light chain sequence, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 19 and the light chain comprises the amino acid sequence of SEQ ID NO: 20.

[0122] In some embodiments, the anti-PD-1 antibody used in the method provided herein comprises six CDRs from SEQ ID NO: 17 and SEQ ID NO: 18 (e.g., three heavy chain CDRs from SEQ ID NO: 17 and three light chain CDRs from SEQ ID NO: 18). In some embodiments, the anti-PD-1 antibody used in the method provided herein comprises a heavy chain variable domain from SEQ ID NO: 19 and a light chain variable domain from SEQ ID NO: 20. In some embodiments, the anti-PD-1 antibody used in the method provided herein comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 18.

[0123] In one embodiment, the anti-PD-1 antibody used in the method provided herein comprises a heavy chain HCDR1, HCDR2, HCDR3 sequence and / or a light chain LCDR1, LCDR2, LCDR3 sequence, wherein the HCDR1 sequence comprises SEQ ID NO: 11 or a homologous sequence having at least 80% sequence identity; the HCDR2 sequence comprises SEQ ID NO: 12 or a homologous sequence having at least 80% sequence identity; the HCDR3 sequence comprises SEQ ID NO: 13 or a homologous sequence having at least 80% sequence identity; the LCDR1 sequence comprises SEQ ID NO: 14 or a homologous sequence having at least 80% sequence identity; the LCDR2 sequence comprises SEQ ID NO: 15 or a homologous sequence having at least 80% sequence identity; and the LCDR3 sequence comprises SEQ ID NO: 16 or a homologous sequence having at least 80% sequence identity.

[0124] In one embodiment, the anti-PD-1 antibody (e.g., nivolumab) used in the method provided herein is administered intravenously in doses of 120 mg, 160 mg, 200 mg, 240 mg, 280 mg, 320 mg, 360 mg, 400 mg, 480 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, or 1400 mg, or intravenously in doses of 2 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, or 24 mg / kg. Nivolumab may be administered intravenously in accordance with facility guidelines, published guidelines, and the respective product prescribing information, and may be administered according to this protocol.

[0125] In one embodiment, the PD-1 / PD-L1 axis inhibitor comprises a PD-L1 inhibitor selected from the group consisting of antibodies, small molecules, and combinations thereof. In one embodiment, the PD-L1 inhibitor is atezolizumab (TECENTRIQ, R05541267, MPDL3280A, RG7446), BMS-936559, avelumab (bavencio), rodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, emvafolimab (KN035), MDX- This product contains an anti-PD-L1 antibody selected from the group consisting of 1105, STI-1040, CS1001, adebrelimab (SHR-1316), SHR-1701, TOB2450, vintrafusp, LP002, STI-3031, cosivelimab, pacumimab, NM01, LDP, AMP-224, galibrimab (BGB-A333), A167, SCD-135, opcolimab, and GR1405.

[0126] In one embodiment, the anti-PD-L1 antibody used in the method provided herein is administered intravenously in doses of 120 mg, 160 mg, 200 mg, 240 mg, 280 mg, 320 mg, 360 mg, 400 mg, 480 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, or 1400 mg, or intravenously in doses of 2 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 12 mg / kg, 14 mg / kg, 16 mg / kg, 18 mg / kg, 20 mg / kg, 22 mg / kg, or 24 mg / kg.

[0127] 2.Chemotherapy

[0128] In some embodiments, the chemotherapy used in the methods provided herein is a chemotherapy regimen (or combination of chemotherapy agents). In some embodiments, the chemotherapy used in the methods provided herein includes a combination of chemotherapy agents. The term "chemotherapeutic agent" refers to a biological (high molecular weight) compound or a chemical (small molecular weight) compound that can be used to treat cancer. Examples of chemotherapy agents 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 chemotherapy drugs include erlotinib, afatinib, docetaxel, adriamycin, 5-FU (5-fluorouracil), panobinostat, gemcitabine, cisplatin, pemetrexed, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, metformin, temozolomide, tamoxifen, oteracil, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, trametinib, tegafur, gimeracil, leucovorin calcium (folic acid) (LV), irinotecan hydrochloride (CPT-11), platinum (such as cisplatin), epirubicin, oxaliplatin, and capecitabine. In one embodiment, the chemotherapy comprises a combination of chemotherapeutic agents selected from the group consisting of LV, 5-FU, CPT-11, epirubicin, oxaliplatin, capecitabine, platinum (e.g., cisplatin), tegafur, gimeracil, oteracil, docetaxel, and pemetrexed. In one embodiment, the combination of chemotherapeutic agents comprises LV, 5-FU, and CPT-11. In another embodiment, the combination of chemotherapeutic agents consists of LV, 5-FU, and CPT-11.

[0129] In one embodiment, the chemotherapy regimen (or combination of chemotherapy agents) may be selected from the group consisting of FOLFIRI chemotherapy, EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, CAPOX chemotherapy, FOLFOX chemotherapy, DCF chemotherapy, SOX chemotherapy, and FLOT chemotherapy. The combination of drugs used in FOLFIRI chemotherapy includes or consists of LV, 5-FU, and CPT-11. The combination of drugs used in EOX chemotherapy includes or consists of epirubicin, oxaliplatin, and capecitabine. The combination of drugs used in ECF chemotherapy includes or consists of epirubicin, cisplatin, and 5-FU. The combination of drugs used in ECX chemotherapy includes or consists of epirubicin, cisplatin, and capecitabine. The combination of drugs used in EOF chemotherapy includes or consists of epirubicin, oxaliplatin, and 5-FU. The drug combination used in FLO chemotherapy includes or consists of 5-FU, LV, and oxaliplatin. The drug combination used in SOX chemotherapy includes or consists of tegafur, gimeracil, oteracil, and oxaliplatin.

[0130] In one embodiment, the chemotherapy dose was 20 mg / m². 2 ~400mg / m 2 (For example, 40 mg / m²) 2 , 60 mg / m² 2 , 80 mg / m² 2 , 100 mg / m² 2 , 120 mg / m² 2 , 140 mg / m² 2 , 160 mg / m² 2 , 180 mg / m² 2 , 200 mg / m² 2 , 220 mg / m² 2 , 240 mg / m² 2 , 300 mg / m² 2 340 mg / m² 2 , 380 mg / m² 2, or 400 mg / m² 2 ) contains CPT-11. In one embodiment, the chemotherapy is 20 mg / m² 2 ~400mg / m 2 (For example, 40 mg / m²) 2 , 60 mg / m² 2 , 80 mg / m² 2 , 100 mg / m² 2 , 120 mg / m² 2 , 140 mg / m² 2 , 160 mg / m² 2 , 180 mg / m² 2 , 200 mg / m² 2 , 220 mg / m² 2 , 240 mg / m² 2 , 300 mg / m² 2 340 mg / m² 2 , 380 mg / m² 2 , or 400 mg / m² 2 ) contains LV. In one embodiment, chemotherapy is 100 mg / m2 to 800 mg / m2. 2 (For example, 100 mg / m²) 2 , 120 mg / m² 2 , 140 mg / m² 2 , 160 mg / m² 2 , 180 mg / m² 2 , 200 mg / m² 2 , 220 mg / m² 2 , 240 mg / m² 2 , 300 mg / m² 2 340 mg / m² 2 , 380 mg / m² 2 , 400 mg / m² 2 500 mg / m² 2 , 600 mg / m² 2 700 mg / m² 2 , or 800 mg / m² 2 ) contains a bolus dose of 5-FU.

[0131] In one embodiment, the chemotherapy used in the method provided herein is the FOLFIRI regimen. In one embodiment, the FOLFIRI regimen is CPT-11 180 mg / m² on day 1. 2 90-minute infusion, LV 200 mg / m² during CPT-11 infusion.2 A 2-hour infusion, followed immediately by 5-FU 400 mg / m² every two weeks. 2 Bolus administration of 2,400 mg / m² 2 This consists of a 46-hour continuous infusion. In one embodiment, the recommended dosing schedule for FOLFIRI, given every two weeks, is as follows: Day 1: CTP-11 180 mg / m² 2 IV infusion and LV 400mg / m² 2 IV infusion of [unclear] followed by 5-FU 400 mg / m² 2 Rapid intravenous administration of 5-FU 2400 mg / m² followed by continuous infusion for 46 hours. 2 IV infusion. There are several different FOLFIRI regimens, each with different dosages and methods of administering the three drugs.

[0132] Other chemotherapy regimens described above are described in PCT patent application PCT / JP2022 / 017017, which is incorporated herein by reference.

[0133] 3. Anti-angiogenic therapy

[0134] In some embodiments, the anti-angiogenic therapy used in the methods provided herein includes an anti-angiogenic agent that can block the growth of blood vessels that support tumor growth. Some anti-angiogenic agents target vascular endothelial growth factor (VEGF) or its receptor VEGFR. In some embodiments, the anti-angiogenic therapy used in the methods provided herein includes a vascular endothelial growth factor A (VEGFA) antagonist. In some embodiments, the VEGFA antagonist is an anti-VEGFRA antibody such as bevacizumab (Avastin®).

[0135] In some embodiments, the anti-angiogenic therapy used in the methods provided herein includes VEGFR antagonists such as VEGFR-1, VEGFR-2, and VEGFR-3. The term "VEGFR antagonist," used synonymously with the term "VEGFR inhibitor," refers to a molecule (e.g., small or large molecule (e.g., antibody)) that inhibits the interaction between VEGFR ligands and VEGF ligands (e.g., those secreted by tumors, e.g., solid tumors), thereby inhibiting angiogenesis and interfering with the blood supply to the tumor.

[0136] In one embodiment, the VEGFR antagonist is an anti-VEGFR-2 antibody. In one embodiment, the anti-VEGFR-2 antibody is selected from the group consisting of ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacirumab, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, and HLX12.

[0137] In one embodiment, the VEGFR antagonist (VEGFR inhibitor) is a small molecule VEGFR inhibitor selected from the group consisting of citravatinib, anlotinib, apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta) (trademark), cabozantinib (Cabometyx) (trademark), fluquintinib (ELUNATE) (trademark), and regorafenib (Stivarga) (trademark).

[0138] In one embodiment, anti-angiogenic therapies used in the methods provided herein (e.g., bevacizumab (Avastin®), ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, citravatinib, anlotinib, apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sora Fenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), or regorafenib (Stivarga®) may be administered intravenously in doses of 120 mg, 160 mg, 200 mg, 240 mg, 280 mg, 320 mg, 360 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, or 1400 mg. It is administered intravenously in doses of 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, or 14 mg / kg. Anti-angiogenic therapies used in the methods provided herein (e.g., bevacizumab (Avastin®), ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, cyto Lavatinib, anlotinib, apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), or regorafenib (Stivarga®) may be administered intravenously in accordance with facility guidelines, public guidelines, and their respective product prescribing information, and may be administered according to this protocol.

[0139] 4. Specific combination therapies

[0140] In one embodiment, the method involves delivering a therapeutically effective dose of a GREM1 antagonist to a VEGFR inhibitor (e.g., bevacizumab (Avastin®), ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, citravatinib). This includes administering to a subject in combination with anlotinib, apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), or regorafenib (Stivarga®). In some embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or its antigen-binding fragment provided herein. In some embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody 14E3 or its humanized variant (e.g., hu14E3, hu14E3 HaLa).

[0141] In one embodiment, the method involves delivering a therapeutically effective dose of a GREM1 antagonist to a VEGFR inhibitor (e.g., bevacizumab (Avastin®), ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, citravatinib, anlotinib, apatinib, teratinib, alti). The drug is administered to subjects in combination with latinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), or regorafenib (Stivarga®), and the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0142] In one embodiment, the method involves administering a therapeutically effective dose of a GREM1 antagonist in combination with a chemotherapy agent (or regimen) (e.g., FOLFIRI) and a VEGFR inhibitor (e.g., bevacizumab (Avastin®), ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, m Ab-04, HLX12, citravatinib, anlotinib, apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), or regorafenib (Stivarga®) are administered to the subject in combination. In some embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or its antigen-binding fragment provided herein. In some embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody 14E3 or its humanized variant (e.g., hu14E3, hu14E3 HaLa).

[0143] In one embodiment, the method involves administering a therapeutically effective dose of a GREM1 antagonist in combination with a chemotherapy agent (or regimen) (e.g., FOLFIRI) and a VEGFR inhibitor (e.g., bevacizumab (Avastin®), ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, citravatinib, and anlotinib). The drug is administered to the subject in combination with apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), and / or regorafenib (Stivarga®), and the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0144] In one embodiment, the method involves administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with a combination of chemotherapeutic agents (e.g., FOLFIRI) and a PD-1 / PD-L1 axis inhibitor (e.g., nivolumab). In one embodiment, the GREM1 antagonist comprises an anti-GREM1 antibody or its antigen-binding fragment provided herein. In one embodiment, the GREM1 antagonist comprises an anti-GREM1 antibody 14E3 or its humanized variant (e.g., hu14E3, hu14E3 HaLa).

[0145] In one embodiment, the method involves administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with a chemotherapeutic agent (e.g., FOLFIRI) and a PD-1 / PD-L1 axis inhibitor (e.g., nivolumab), wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0146] In one embodiment, the method involves delivering a therapeutically effective dose of a GREM1 antagonist, such as a VEGFR inhibitor (e.g., citravatinib, anlotinib, apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®)), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), or regorafenib (Stivarga®). The treatment involves administering to a subject in combination with bevacizumab (Avastin®), ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12), a combination of chemotherapeutic agents (e.g., FOLFIRI), and a PD-1 / PD-L1 axis inhibitor (e.g., nivolumab). In some embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or its antigen-binding fragment provided herein. In some embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody 14E3 or its humanized variant (e.g., hu14E3, hu14E3 HaLa).

[0147] In one embodiment, the method involves delivering a therapeutically effective dose of a GREM1 antagonist from a VEGFR inhibitor (e.g., citravatinib, anlotinib, apatinib, teratinib, artiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®)), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), or regorafenib (Stivarga®) to bevacizumab (Avastin®) The treatment involves administering to the subject in combination with ramucirumab, olimbacimab, gentuximab, aracizumab pegol, brinacirumab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12), a combination of chemotherapy agents (e.g., FOLFIRI), and a PD-1 / PD-L1 axis inhibitor (e.g., nivolumab), wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0148] 5. GREM1-expressing cancers

[0149] As used herein, the term “GREM1-expressing cancer” means any cancer or tumor involving cancer cells that express or secrete GREM1 (e.g., low, moderate, or high expression).

[0150] GREM1 expression can be determined using methods known in the art, including but not limited to protein-based assays such as immunohistochemistry and ELISA, or nucleic acid-based assays such as amplification assays, hybridization assays, or sequencing assays. In some embodiments, GREM1 expression can be determined using methods or antibodies provided herein. In some embodiments, the subject is further determined to have low, moderate, or high GREM1 expression in lesional tissue of a GREM1-expressing cancer.

[0151] In one embodiment, a subject is determined to have GREM1 expression in the lesional tissue (e.g., the presence of GREM1 or a GREM1 expression level exceeding a threshold level). As used herein, “threshold level” means a GREM1 expression level at an intensity of 1+, 2+, or 3+ as measured by IHC.

[0152] In one embodiment, GREM1 expression is determined from diseased tissue (e.g., a biological sample).

[0153] The presence and / or expression level of GREM1 in diseased tissue (e.g., cancerous or tumorous tissue) can be determined by various methods known in the art. In some embodiments, the biological sample may be further processed to isolate analytes such as nucleic acids or proteins. The presence and / or expression level of GREM1 can be determined, for example, by quantitative fluorescence cytometry, immunohistochemistry (IHC), or nucleic acid-based methods. For example, a biological sample from a subject may be exposed to an anti-GREM1 diagnostic reagent that binds to and detects expressed GREM1 proteins.

[0154] In one embodiment, GREM1 expression in diseased tissue (e.g., cancerous or tumorous tissue) is determined or measured by IHC. In one embodiment, the expression level of human GREM1 protein in cancerous or tumorous tissue from a subject may be determined according to the method described in Example 4 provided herein.

[0155] In one embodiment, a subject is determined to have high GREM1 expression in diseased tissue (e.g., cancerous or tumorous tissue) derived from the subject. In another embodiment, diseased tissue is determined to have GREM1 expression that is higher or comparable to that in healthy or non-cancerous cells. High GREM1 expression in a biological sample such as diseased tissue (e.g., cancerous or tumorous tissue) means GREM1 expression at an intensity of at least 2+ (e.g., 2+ or 3+) as measured by IHC.

[0156] In one embodiment, a subject is determined to have moderate GREM1 expression in diseased tissue (e.g., cancerous or tumorous tissue) derived from the subject. Moderate GREM1 expression in a biological sample such as diseased tissue (e.g., cancerous or tumorous tissue) means GREM1 expression at an intensity of at least 1+ and less than 2+ as measured by IHC.

[0157] In one embodiment, a subject is determined to have low GREM1 expression in lesional tissue (e.g., cancerous or tumorous tissue) derived from the subject. In another embodiment, lesional tissue is determined to have GREM1 expression comparable to that in healthy tissue and detectable by an anti-GREM1 diagnostic reagent. Low GREM1 expression in a biological sample such as lesional tissue (e.g., cancerous or tumorous tissue) means GREM1 expression at an intensity greater than 0+ but less than 1+, as measured by IHC.

[0158] 6. GREM1-expressing cancers that are resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or have low or absent PD-L1 expression.

[0159] In one embodiment, GREM1-expressing cancer is determined to be resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or to have low or absent PD-L1 expression in the cancerous lesion tissue (i.e., low or no PD-L1 expression).

[0160] This disclosure reveals, surprisingly, that treating tumors with GREM1 antagonists increases the expression of programmed death ligand 1 (PD-L1) in the tumors. PD-L1 is a protein that interacts with programmed death protein 1 (PD-1) and is expressed in tumor tissue, for example, on immune cells and tumor cells. Studies have investigated the correlation between tumor PD-L1 expression and the therapeutic effect of anti-PD-1 antibodies, showing that PD-L1 overexpression is associated with a significantly higher objective response rate (ORR) (Gettinger et al., Over all survival and long-term safety of nivolumab (anti-programmed death 1 antibody, BMS-936558, ONO-4538) in patients with previously treated advanced non-small-cell lung cancer. J Clin Oncol. 2015;33:2004-12.; Garon et al., Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018-28. doi:10.1056 / NEJMoa1501824). Publications and reports have also shown that patients with tumors that have low PD-L1 expression may receive less benefit from treatment with PD-1 / PD-L1 axis inhibitors.

[0161] Based at least in part on the above findings, the methods provided herein have unexpected effects in the treatment of GREM1-expressing cancers that are resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or are determined to have low or no PD-L1 expression in cancerous lesion tissue (i.e., low or no PD-L1 expression).

[0162] As used herein, “resistance” means that a disease or condition is unresponsive to treatment (which may be either spontaneous or treatment-induced).

[0163] As used herein, “refractory” means that a disease or condition is resistant to or unresponsive to treatment (e.g., the number of new cells increases even when treatment is administered). Unless otherwise indicated, “refractory” means resistance to or unresponsiveness to any prior treatment with PD-1 / PD-L1 axis inhibitors.

[0164] PD-L1 expression can be determined using methods known in the art, including but not limited to immunohistochemistry and protein-based assays such as ELISA, or nucleic acid-based assays such as amplification assays, hybridization assays, or sequencing assays. The methods described above for detecting GREM1 expression can also be used for detecting PD-L1 expression, except that the detection reagents must be replaced with reagents for PD-L1 detection.

[0165] In some embodiments, PD-L1 expression may be determined using the method described in PCT / CN2022 / 131820, which is incorporated herein by reference. In some embodiments, PD-L1 expression may be determined according to the method described in Example 4 provided herein. The reagents used throughout this specification to detect PD-L1 expression may be anti-PD-L1 diagnostic antibodies, e.g., 22C3 as described in US20170285037A1 (the contents of which are incorporated herein by reference in their entirety) and clone 28-8, a commercially available monoclonal rabbit anti-PD-L1 antibody. In some embodiments, the reagents used throughout this specification to detect PD-L1 expression are a polypeptide containing the antigen-binding moiety of 22C3 as described in US20170285037A1 (the contents of which are incorporated herein by reference in their entirety) and clone 28-8, a commercially available monoclonal rabbit anti-PD-L1 antibody.

[0166] As used herein, the term "low PD-L1 expression" means a PD-L1 expression level that is lower than or equal to a reference level.

[0167] In relation to PD-L1 expression, the term "reference level" refers to the threshold (e.g., minimum) expression level of PD-L1 in a biological sample, such as diseased tissue (e.g., cancerous or tumorous tissue), derived from a subject responding to treatment with a PD-1 / PD-L1 axis inhibitor.

[0168] In one embodiment, cancers with low PD-L1 expression have an intensity of 1+ or less or 2+ or less, as measured by IHC using antibody 22C3 or antibody clone 28-8, or have a PD-L1 positivity rate of 5% or less in tumor cells in CPS.

[0169] As used herein, the term “absence of PD-L1 expression” means a PD-L1 expression level below the baseline threshold level. In some embodiments, a biological sample without PD-L1 expression (e.g., cancer cells) does not have a detectable PD-L1 signal that can be detected by an anti-PD-L1 diagnostic antibody using proven techniques such as IHC.

[0170] In relation to PD-L1 expression, the term "baseline threshold level" refers to the detectable threshold expression level of PD-L1 in a biological sample.

[0171] PD-L1 expression may be measured by the method described in PCT / CN2022 / 131820, which is incorporated herein by reference. Depending on the tumor type and / or the use of different PD-L1 detection assays, the threshold expression level for PD-L1 may differ and may be determined using methods known in the art.

[0172] In some embodiments, PD-L1 expression in lesional tissue is low or undetectable by anti-PD-L1 diagnostic antibodies.

[0173] In one embodiment, the method provided herein includes administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with a PD-1 / PD-L1 axis inhibitor. In one embodiment, the method includes administering a GREM1 antagonist to a subject for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer tissue or cancer cells, and then administering a PD-1 / PD-L1 axis inhibitor to the subject. In one embodiment, the subject is administered the GREM1 antagonist for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer tissue / cancer cells.

[0174] In one embodiment, GREM1-expressing cancers are colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0175] In one embodiment, the subject is a human.

[0176] 7. Pharmaceutical composition and route of administration

[0177] The GREM1 antagonists, chemotherapeutic agents, immunotherapeutic agents, or anti-angiogenic agents described above may each be administered in the form of any suitable pharmaceutical composition. The term “pharmaceutical composition” means a formulation containing an effective therapeutic agent (e.g., the GREM1 antagonists, chemotherapeutic agents, immunotherapeutic agents, or anti-angiogenic agents described above) preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration to a subject.

[0178] Pharmaceutical compositions are typically provided in a uniform dosage form and may be prepared by methods known in the art. Pharmaceutical compositions may be in liquid dosage forms, such as solutions or suspensions, or solid dosage forms, such as tablets and capsules. Pharmaceutical compositions described herein are generally referred to as “therapeutic doses” and “pharmaceutically acceptable formulations.” As used herein, the term “pharmaceutically acceptable” means non-toxic materials that do not interact with the action of the active ingredient of the pharmaceutical composition.

[0179] The pharmaceutical compositions described herein may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical composition of this disclosure comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0180] In some embodiments, administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular dose.

[0181] In one embodiment, the GREM1 antagonist is administered before, simultaneously with, or after the administration of an anti-angiogenic agent and / or chemotherapeutic agent and / or combination of chemotherapeutic agents and / or immunotherapy.

[0182] In one embodiment, the method includes administering a GREM1 antagonist to a subject for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer tissue / cells, and then administering a PD-1 / PD-L1 axis inhibitor to the subject. In one embodiment, the subject is administered a GREM1 antagonist for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer tissue / cancer cells.

[0183] III. GREM1 as a biomarker for immunotherapy eligibility

[0184] In another aspect, the Disclosure provides a method for determining whether a subject is likely to be eligible for or responsive to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor, the method being: b) including determining the presence or expression level of GREM1 in a biological sample of diseased tissue from the subject, The presence or absence of GREM1, or its expression level, indicates whether the subject is eligible for or likely to respond to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0185] In one embodiment, the presence of GREM1 or an expression level of GREM1 exceeding a threshold level, as determined in step a), indicates that the subject is likely to be eligible for or respond to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0186] In one embodiment, the absence of GREM1 or a GREM1 expression level below the threshold determined in step a) indicates that the subject is not eligible for or unlikely to respond to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0187] In one embodiment, the method proceeds before step a), The procedure further includes step i) bringing a biological sample of diseased tissue from a subject into contact with a GREM1 diagnostic agent under conditions that enable the detection of GREM1 expression levels in the sample.

[0188] In one embodiment, the subject is determined to be a) resistant to or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) to have low or no PD-L1 expression in the lesional tissue. In another embodiment, the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

[0189] In one embodiment, the method further comprises administering a therapeutically effective dose of a GREM1 antagonist to the subject for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer tissue / cells, followed by administering a PD-1 / PD-L1 axis inhibitor to the subject.

[0190] In some embodiments, GREM1 expression is detected by a GREM1 diagnostic reagent. In some embodiments, the GREM1 diagnostic reagent / agent comprises a polypeptide containing an antigen-binding portion of an anti-GREM1 antibody or its antigen-binding fragment. In some embodiments, the GREM1 diagnostic reagent / agent comprises an anti-GREM1 antibody or its antigen-binding fragment, e.g., an anti-GREM1 antibody or its antigen-binding fragment provided herein (e.g., its humanized variant such as 14E3 or hu14E3 HaLa). Detailed embodiments of the detection method are described in the following sections.

[0191] In another aspect, the Disclosure provides a method for improving a subject's responsiveness to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject is determined to have GREM1 present in a biological sample of the subject's lesional tissue, or the subject is determined to have a threshold level of GREM1 expression in a biological sample of the subject's lesional tissue, and the method comprises a) administering a therapeutically effective dose of a GREM1 antagonist to the subject so as to increase PD-L1 expression in the lesional tissue, thereby improving the subject's responsiveness to the PD-1 / PD-L1 axis inhibitor.

[0192] In some embodiments, the subject is determined to be a) resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) to have low or no PD-L1 expression in the lesional tissue. In some embodiments, the subject is determined to have moderate or high PD-L1 expression in the lesional tissue. PD-L1 expression may be determined using the method described in PCT / CN2022 / 131820, which is incorporated herein by reference. In some embodiments, PD-L1 expression may be determined according to the method described in Example 4 provided herein. Throughout this specification, reagents used to detect PD-L1 expression may be anti-PD-L1 diagnostic antibodies, e.g., 22C3 described in US20170285037A1 (this disclosure is incorporated herein by reference in its entirety) and clone 28-8, a commercially available monoclonal rabbit anti-PD-L1 antibody. In one embodiment, the reagent used throughout this specification to detect PD-L1 expression is a polypeptide containing the antigen-binding moiety of 22C3 as described in US20170285037A1 (the disclosure thereof is incorporated herein in whole by reference) and clone 28-8, a commercially available monoclonal rabbit anti-PD-L1 antibody.

[0193] In one embodiment, the diseased tissue is cancerous tissue. In one embodiment, the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0194] In one embodiment, the method further comprises administering a therapeutically effective dose of a PD-1 / PD-L1 axis inhibitor to the subject after PD-L1 expression has increased in the subject's lesional tissue.

[0195] In another aspect, the Disclosure provides the use of a GREM1 antagonist in the manufacture of a pharmaceutical product to improve a subject's responsiveness to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject is determined to have GREM1 present in a biological sample of the subject's lesional tissue, or the subject is determined to have a threshold level of GREM1 expression in a biological sample of the subject's lesional tissue, and the improvement comprises a) administering a therapeutically effective dose of the GREM1 antagonist to the subject so as to increase PD-L1 expression in the lesional tissue, thereby improving the subject's responsiveness to the PD-1 / PD-L1 axis inhibitor.

[0196] In another aspect, the Disclosure provides the use of an anti-GREM1 diagnostic reagent in the manufacture of a kit for determining whether a subject is likely to be eligible for or responsive to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor, the anti-GREM1 diagnostic reagent being capable of determining the presence or expression level of GREM1 in a biological sample of the subject's lesional tissue, the presence or absence or expression level of GREM1 indicating whether the subject is likely to be eligible for or responsive to treatment with a GREM1 antagonist in combination with a PD-1 / PD-L1 axis inhibitor.

[0197] 1. Determination of GREM1 expression

[0198] The methods or uses provided herein involve the determination of GREM1 expression. In one embodiment, a subject is determined to have GREM1 expression in lesional tissue (e.g., the presence of GREM1 or a GREM1 expression level above a threshold level). As used herein, “threshold level” means a GREM1 expression level at an intensity of 1+, 2+, or 3+ as measured by IHC.

[0199] Appropriate methods known in this field may be used, and are described in detail below.

[0200] i. Sample preparation

[0201] In one embodiment, the subject is a human. In one embodiment, the method provided herein further comprises providing a biological sample from the subject, the biological sample comprising diseased tissue (e.g., cancerous tissue or tumor tissue).

[0202] Any biological specimen suitable for performing the methods provided herein can be obtained from a subject. As used herein, “biological specimen” means a biological sample (optionally subjected to additional processing) taken from a subject by sampling. The collection of a specimen from a subject is performed according to standard protocols commonly followed in hospitals or clinics, such as during a biopsy.

[0203] In some embodiments, the sample may be a biological sample containing cancer cells or non-cancerous cells (e.g., stromal fibroblasts). For example, the non-cancerous cells may originate from the same tissue or organ in which the cancer cells are found. In some embodiments, the biological sample containing or suspected of containing cancer cells is obtained from a subject. In some embodiments, the biological sample may originate from cancer cells, cancerous tissue, or tumor-infiltrating immune cells. In some embodiments, the biological sample is tumor tissue.

[0204] In some embodiments, the biological sample is a fresh or stored sample obtained from tumor tissue, for example, by tumor biopsy or fine-needle aspiration. In some embodiments, the sample may be any biological fluid containing cancer cells or non-cancerous cells (e.g., peripheral blood mononuclear cells (PBMCs)).

[0205] Examples of biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal lavage fluid, intrapleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, and tissues such as biopsy tissue (e.g., biopsy bone tissue, bone marrow, breast tissue, gastrointestinal tissue, lung tissue, colon tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, colon tissue, kidney tissue, endometrial tissue, cervical tissue, lymph node tissue, muscle tissue, or skin tissue), and paraffin-embedded tissue. In further embodiments, biological samples include cells, tissues, blood, plasma, serum, urine, mouthwash, feces, saliva, and any combination thereof.

[0206] In one embodiment, the sample may be further processed by a desired method for determining the expression level of at least one biomarker, such as GREM1.

[0207] ii. Determination of GREM1 expression

[0208] In one embodiment, GREM1 expression is determined from diseased tissue (e.g., a biological sample).

[0209] The presence and / or expression level of GREM1 in diseased tissue (e.g., cancerous or tumorous tissue) can be determined by various methods known in the art. In some embodiments, the biological sample may be further processed to isolate analytes such as nucleic acids or proteins. The presence and / or expression level of GREM1 can be determined, for example, by quantitative fluorescence cytometry, immunohistochemistry (IHC), or nucleic acid-based methods. For example, a biological sample from a subject may be exposed to an anti-GREM1 diagnostic reagent that binds to and detects expressed GREM1 proteins.

[0210] In one embodiment, GREM1 expression in diseased tissue (e.g., cancerous or tumorous tissue) is determined or measured by IHC. In one embodiment, the expression level of human GREM1 protein in cancerous or tumorous tissue from a subject may be determined according to the method described in Example 4 provided herein.

[0211] In one embodiment, a subject is determined to have high GREM1 expression in diseased tissue (e.g., cancerous or tumorous tissue) derived from the subject. In another embodiment, diseased tissue is determined to have GREM1 expression that is higher or comparable to that in healthy or non-cancerous cells. High GREM1 expression in a biological sample such as diseased tissue (e.g., cancerous or tumorous tissue) means GREM1 expression at an intensity of at least 2+ (e.g., 2+ or 3+) as measured by IHC.

[0212] In one embodiment, a subject is determined to have moderate GREM1 expression in diseased tissue (e.g., cancerous or tumorous tissue) derived from the subject. Moderate GREM1 expression in a biological sample such as diseased tissue (e.g., cancerous or tumorous tissue) means GREM1 expression at an intensity of at least 1+ and less than 2+ as measured by IHC.

[0213] In one embodiment, a subject is determined to have low GREM1 expression in lesional tissue (e.g., cancerous or tumorous tissue) derived from the subject. In another embodiment, lesional tissue is determined to have GREM1 expression comparable to that in healthy tissue and detectable by an anti-GREM1 diagnostic reagent. Low GREM1 expression in a biological sample such as lesional tissue (e.g., cancerous or tumorous tissue) means GREM1 expression at an intensity greater than 0+ but less than 1+, as measured by IHC.

[0214] IV. GREM1 antagonists

[0215] The GREM1 antagonists used in the methods provided herein are capable of inducing PD-L1 expression in the diseased tissue of a subject. In some embodiments, the GREM1 antagonist used in the methods provided herein comprises an anti-GREM1 antibody or its antigen-binding fragment, for example, one of those described in PCT Patent Application PCT / CN2022 / 072297 (which is incorporated herein by reference). In some embodiments, the GREM1 antagonist used in the methods provided herein is an anti-GREM1 antibody having equivalent properties to Hu14E3 HaLa described in PCT Patent Application PCT / CN2022 / 072297 (which is incorporated herein by reference). As used herein, the term "Hu14E3 HaLa" refers to a humanized anti-GREM1 antibody comprising a heavy chain variable region (Hu14E3-Ha VH) and a light chain variable region (Hu14E3-Ha VL), wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.

[0216] In some embodiments, the GREM1 antagonist used in the method provided herein comprises an antigen-binding moiety of Hu14E3 HaLa. In some embodiments, the GREM1 antagonist used in the method provided herein comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region being the same as those of Hu14E3 HaLa, respectively. In some embodiments, the GREM1 antagonist used in the method provided herein comprises a heavy chain HCDR1, HCDR2, HCDR3, and / or a light chain LCDR1, LCDR2, LCDR3, the heavy chain HCDR1, HCDR2, HCDR3, and / or a light chain LCDR1, LCDR2, LCDR3 being the same as those of Hu14E3 HaLa, respectively.

[0217] In one embodiment, the anti-GREM1 antibody or its antigen-binding fragment comprises a heavy chain HCDR1, HCDR2, HCDR3, and / or a light chain LCDR1, LCDR2, LCDR3, wherein HCDR1 comprises an amino acid sequence containing TYGMA (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity thereto, HCDR2 comprises an amino acid sequence containing WINTLSGEPTYADDFKG (SEQ ID NO: 2) or a homologous sequence having at least 80% sequence identity thereto, and HCDR3 comprises an amino acid sequence containing EPMDY (SEQ ID NO: 3) Each contains an amino acid sequence or a homologous sequence having at least 80% sequence identity thereto. LCDR1 contains an amino acid sequence containing KSSQSLLDSDGKTYLS (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity thereto. LCDR2 contains an amino acid sequence containing LVSKLDS (SEQ ID NO: 5) or a homologous sequence having at least 80% sequence identity thereto. LCDR3 contains an amino acid sequence containing WQGAHFPLT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity thereto.

[0218] While CDRs are known to play a role in antigen binding, it has been found that not all six CDRs are necessarily essential or immutable. In other words, in anti-GREM1 antibodies, substituting, altering, or modifying one, two, or three CDRs may substantially maintain specific binding affinity to GREM1.

[0219] In one embodiment, the anti-GREM1 antibody also contains the EPMDY (SEQ ID NO: 3) heavy chain CDR3 sequence in its antigen-binding fragment. Because the heavy chain CDR3 region is centrally located in the antigen-binding site, it is thought to have the most contact with the antigen and supply the most free energy to the antibody-antigen affinity. Furthermore, the heavy chain CDR3 is considered to be the most diverse of the antigen-binding CDRs in terms of length, amino acid composition, and three-dimensional structure due to multiple diversification mechanisms (Tonegawa S. Nature. 302:575-81). The diversity of the heavy chain CDR3 is sufficient to produce the highest antibody specificity (Xu JL, Davis MM. Immunity. 13:37-45) and the desired antigen-binding affinity (Schier R, etc. J Mol Biol. 263:551-67).

[0220] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises 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-GREM1 antibody is a single-domain antibody comprising all or part of the heavy chain variable domains provided herein. Further information on such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0221] In one embodiment, an anti-GREM1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0222] In one embodiment, the anti-GREM1 antibody or its antigen-binding fragment further comprises substitutions or modifications of one or more amino acid residues, but still retains specific binding specificity or affinity for GREM1 (e.g., human GREM1, i.e., hGREM1).

[0223] In one embodiment, at least one of the substitutions or modifications is present in one or more CDR sequences and / or one or more non-CDR regions of VH or VL sequences.

[0224] In one embodiment, the anti-GREM1 antibody or its antigen-binding fragment further comprises an immunoglobulin constant region, optionally a human IgG constant region. In one embodiment, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region, and optionally, the constant region comprises a heavy chain constant region containing the sequence of SEQ ID NO: 9 and / or a light chain constant region containing the sequence of SEQ ID NO: 10.

[0225] In some embodiments, a GREM1 antagonist is conjugated to one or more conjugate moieties. In some embodiments, the conjugate moieties include clearance modifiers, therapeutic agents (e.g., chemotherapeutic agents), toxins, radioisotopes, detectable labels (e.g., lantanides, luminescence labels, fluorescent labels, or enzyme substrate labels), pharmacokinetic modification moieties, DNA alkylating agents, topoisomerase inhibitors, tubulin conjugates, and other anticancer agents, such as those described in PCT Patent Application PCT / CN2022 / 072297 (which is incorporated herein by reference).

[0226] V.GREM1 Diagnostic Reagent / Drug

[0227] The GREM1 diagnostic reagents / agents used in the methods provided herein are capable of specifically detecting low, moderate, or high expression of GREM1 in vivo (e.g., in the body of a subject) or in vitro (e.g., in a biological sample of diseased tissue from a subject). In some embodiments, the subject is further determined to have 5–50% (e.g., 10–40%, 10–30%, 10–20%, or 15%) of tumor cells positive for GREM1, as measured by IHC. In some embodiments, the GREM1 diagnostic reagents / agents used in the methods provided herein are polypeptides comprising an antigen-binding moiety of either an anti-GREM1 antibody or its antigen-binding fragment as described in PCT Patent Application PCT / CN2022 / 072297 (which is incorporated herein by reference). In one embodiment, the GREM1 diagnostic reagent / agent used in the method provided herein comprises one of the anti-GREM1 antibodies or antigen-binding fragments described in PCT patent application PCT / CN2022 / 072297 (which is incorporated herein by reference).

[0228] In one embodiment, the GREM1 diagnostic reagent / agent is a polypeptide comprising an antigen-binding fragment of Hu14E3 HaLa as described in PCT / CN2022 / 072297 (which is incorporated herein by reference). In one embodiment, the antigen-binding fragment comprises a heavy chain HCDR1, HCDR2, HCDR3 and / or a light chain LCDR1, LCDR2, LCDR3, wherein HCDR1 comprises an amino acid sequence containing TYGMA (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity thereto, HCDR2 comprises an amino acid sequence containing WINTLSGEPTYADDFKG (SEQ ID NO: 2) or a homologous sequence having at least 80% sequence identity thereto, and HCDR3 comprises an amino acid sequence containing EPMDY (SEQ ID NO: 3), The antigen-binding fragment includes a homologous sequence having at least 80% sequence identity, where LCDR1 includes an amino acid sequence containing KSSQSLLDSDGKTYLS (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity thereto, LCDR2 includes an amino acid sequence containing LVSKLDS (SEQ ID NO: 5) or a homologous sequence having at least 80% sequence identity thereto, and LCDR3 includes an amino acid sequence containing WQGAHFPLT (SEQ ID NO: 6) or a homologous sequence having at least 80% sequence identity thereto. In one embodiment, the antigen-binding fragment includes the heavy chain CDR3 sequence of EPMDY (SEQ ID NO: 3).

[0229] In one embodiment, the antigen-binding fragment includes a heavy chain variable region and a light chain variable region, the heavy chain variable region including the amino acid sequence of SEQ ID NO: 7, and the light chain variable region including the amino acid sequence of SEQ ID NO: 8.

[0230] In one embodiment, the GREM1 diagnostic reagent / agent further comprises an immunoglobulin constant region, optionally a human IgG constant region. In one embodiment, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region, and optionally, the constant region comprises a heavy chain constant region containing the sequence of SEQ ID NO: 9 and / or a light chain constant region containing the sequence of SEQ ID NO: 10.

[0231] In some embodiments, the GREM1 diagnostic reagent / agent comprises all or some of the heavy chain variable domains and / or all or some of the light chain variable domains. In one embodiment, the GREM1 diagnostic reagent / agent is a single-domain antibody comprising all or some of the heavy chain variable domains provided herein. Further information on such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0232] In one embodiment, the GREM1 diagnostic reagent / agent comprises Hu14E3 HaLa or its antigen-binding fragment.

[0233] In one embodiment, the GREM1 diagnostic reagent / agent further comprises one or more conjugate moieties conjugated to the polypeptide, Hu14E3 HaLa, or its antigen-binding fragment. In one embodiment, the conjugate moieties include a detectable label (e.g., lantanide, luminescence label, fluorescent label, biotin / avidin, or enzyme substrate label).

[0234] VI. Kit

[0235] In another aspect, the Disclosure provides a kit useful for treating GREM1-expressing cancer in a subject as needed, comprising a GREM1 antagonist and a package insert, the package insert including instructions for using the GREM1 antagonist in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

[0236] In one embodiment, GREM1-expressing cancer is characterized by a) resistance or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or b) low or absent PD-L1 expression in the lesional tissue, and / or c) GREM1 expression in the lesional tissue. In one embodiment, the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

[0237] As used herein, the term “packaging instructions” means instructions contained in a commercially available drug package, which include, for example, information on indications, dosage, usage, method of administration, contraindications, other drugs to be used in combination with the packaged product, and / or warnings regarding the use of such drugs. In some embodiments, the instructions include selecting a subpopulation that is a) resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or b) has low or no expression of PD-L1 in the diseased tissue, and / or c) has GREM1 expression in the diseased tissue, and / or d) has been administered a GREM1 antagonist for a period of time sufficient to increase the level of PD-L1 expression in the subject’s cancer tissue / cancer cells. In some embodiments, the instructions include administering a therapeutically effective dose of a GREM1 antagonist to the subject for a period of time sufficient to increase the level of PD-L1 expression in the subject’s cancer tissue / cells, followed by administration of a PD-1 / PD-L1 axis inhibitor to the subject.

[0238] The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filter media, needles, and syringes.

[0239] VII. Cancer

[0240] In one embodiment, the cancer in the method provided herein is a GREM1-expressing cancer. In one embodiment, the cancer is selected from the group consisting of solid tumors or hematological malignancies. In one embodiment, the solid tumors are 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 neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, Burkitt lymphoma, cervical cancer, colon cancer, colorectal cancer, pulmonary cancer, endometrial cancer, esophageal cancer, and Ewing's sarcoma. These include retinoblastoma, gastric cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas), 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.

[0241] In one embodiment, the cancer is selected from the group consisting of colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0242] In one embodiment, the cancer is colorectal cancer. In another embodiment, the cancer is a subtype of colorectal cancer such as CMS1, CMS2, CMS3, CMS4, etc. A detailed description of the subtypes of colorectal cancer can be found, for example, in PCT patent application PCT / US2022 / 076717, which is incorporated herein by reference. [Examples]

[0243] While this disclosure is specifically illustrated and described with reference to certain embodiments (some of which are preferred embodiments), those skilled in the art should understand that various modifications may be made herein without departing from the spirit and scope of this disclosure, as disclosed herein.

[0244] Example 1: Efficacy of anti-gremlin 1 antibody in combination with DC101 against a BZ-CRC-0001 PDX tumor model in NOG mice.

[0245] This example demonstrates that a synergistic therapeutic effect was achieved in the treatment of colorectal cancer (CRC) by combining an anti-gremlin 1 antibody (i.e., anti-GREM1 antibody) with anti-angiogenic therapy. The anti-GREM1 antibody used in this example is Hu14E3 HaLa, as described herein. The safety profile of Hu14E3 HaLa is shown in Figure 7. The anti-angiogenic therapy includes anti-VEGFR-2 antibody DC101, a monoclonal antibody that reacts with mouse VEGFR-2, and is commercially available (e.g., BioXell, catalog number BE0060).

[0246] The BZ-CRC-0001 colorectal cancer PDX was obtained from the passage of NODSCID mice at Beijing Tumor Hospital, and a PDX bank was established. Pathological information for BZ-CRC-0001 colorectal cancer is shown in Figure 1B. Each NOG mouse was subcutaneously inoculated with a small tumor tissue block, approximately 3 mm in diameter, excised from tumor dissections of tumor-carrying mice. Nineteen days after inoculation, the tumor size was approximately 50 mm. 3 The animals were selected and randomly divided into four groups of eight mice each. The animals were given 5 × 10⁶ of 0.1 mL of chlorine. 6 Human PBMCs were administered intravenously. Animals were selected and administered one week after human PBMC infusion. Animals in groups 1 to 4 were administered 10 mg / kg of hIgG1 control, 10 mg / kg of anti-gremlin 1 antibody, 5 mg / kg of DC101, and a combination of 10 mg / kg of anti-gremlin 1 antibody and 5 mg / kg of DC101.

[0247] hIgG1 control, Hu14E3 HaLa, and DC101 were administered intraperitoneally twice weekly for 4 weeks. Animals were sacrificed by CO2 inhalation at the end of the study. Tumor size was measured two-dimensionally two or three times weekly using calipers (INSIZE), and volume was measured in mm using the formula V = 0.5 a*b^2. 3The formula is expressed as follows, where a and b are the longest and shortest diameters of the tumor, respectively. The results were analyzed using Prism GraphPad, and the mean value is expressed as SEM. A t-test was performed to compare the two groups, and the difference was considered significant if p < 0.05 and ** < 0.01. As shown in Figure 1A, the tumor growth inhibition rates for anti-gremlin 1 antibody alone, DC101, and the combination of anti-gremlin 1 antibody and DC101 were 46.80%, 54.12%, and 66.53% on the sacrifice day, respectively. The combination of anti-gremlin 1 antibody and DC101 showed superior antitumor activity compared to anti-gremlin 1 antibody alone, and improved antitumor activity compared to DC101 alone.

[0248] Example 2: Efficacy of anti-gremlin 1 antibody in combination with FOLFIRI and DC101 in a BZ-CRC-0001 PDX tumor model in NOG mice

[0249] This example demonstrates that a synergistic therapeutic effect was achieved in the treatment of CRC by combining an anti-gremlin 1 antibody (i.e., anti-GREM1 antibody), chemotherapy, and anti-angiogenic therapy. The anti-GREM1 antibody used in this example is Hu14E3 HaLa as described herein. The chemotherapy is FOLFIRI. The anti-angiogenic therapy includes anti-VEGFR-2 antibody DC101, a monoclonal antibody that reacts with mouse VEGFR-2, and is commercially available (e.g., BioXell, catalog number BE0060).

[0250] The BZ-CRC-0001 colorectal cancer PDX was obtained from the passage of NODSCID mice at Beijing Tumor Hospital, and a PDX bank was established. Each NOG mouse was subcutaneously inoculated with a small tumor tissue block, approximately 3 mm in diameter, excised from tumor fragments of tumor-carrying mice. Eighteen days after inoculation, the tumor size was approximately 50 mm. 3 We selected animals and randomly divided them into 5 groups of 8 mice each. The tumor-bearing mice were given 5 × 10⁴ 6Human PBMCs were administered intravenously. Animals were selected and administered one week after human PBMC infusion. Animals in groups 1 to 5 were administered 35 mg / kg isotype control and vehicle, 30 mg / kg anti-gremlin 1 antibody, 30 mg / kg anti-gremlin 1 antibody combined with 5 mg / kg DC101, FOLFIRI (5-fluorouracil (5-FU): 7.5 mg / kg, leucovorin (LV): 22.5 mg / kg, irinotecan (CPT-11): 4 mg / kg) combined with 5 mg / kg DC101, 30 mg / kg anti-gremlin 1 antibody, and FOLFIRI (5-FU: 7.5 mg / kg, LV: 22.5 mg / kg, CPT-11: 4 mg / kg) combined with 5 mg / kg DC101.

[0251] Isotype control, anti-gremlin 1 antibody, and DC101 were administered intraperitoneally twice weekly for 4 weeks. LV was administered intraperitoneally once weekly for 4 weeks, and 5-Fu and CPT-11 were administered intravenously once weekly for 4 weeks. Animals were sacrificed by CO2 inhalation at the end of the study. Tumor size was measured two-dimensionally two or three times weekly using calipers (INSIZE), and volume was measured in mm using the formula V = 0.5 a*b^2. 3 The formula is expressed as follows, where a and b are the longest and shortest diameters of the tumor, respectively. The results were analyzed using Prism GraphPad, and the mean value is expressed as SEM. A t-test was performed to compare the two groups, and the difference was considered significant if p < 0.05 and ** < 0.01. As shown in Figure 2, the tumor growth inhibition rates for anti-gremlin 1 antibody 30 mg / kg, anti-gremlin 1 antibody and DC101 combination, FOLFIRI and DC101 combination, and anti-gremlin 1 antibody, FOLFIRI, and DC101 combination on the sacrifice day were 30.10%, 47.47%, 58.75%, and 68.91%, respectively. The combination of anti-gremlin 1 antibody and DC101 showed superior antitumor activity compared to anti-gremlin 1 antibody alone. The combination of anti-gremlin 1 antibody, FOLFIRI, and DC101 showed superior antitumor activity compared to anti-gremlin 1 antibody alone, and improved antitumor activity compared to the combination of FOLFIRI and DC101.

[0252] Example 3: Efficacy of Anti-Gremmlin 1 Antibody in Combination with FOLFIRI, DC101, and Nivolumab against BZ-CRC-0001 PDX Tumor Model in NOG Mice

[0253] This example shows that a synergistic therapeutic effect was achieved in the treatment of CRC by the combined use of an anti-gremmlin 1 antibody (i.e., anti-GREM1 antibody), chemotherapy, anti-angiogenesis therapy, and immunotherapy. The anti-GREM1 antibody used in this example is Hu14E3 HaLa described herein. The chemotherapy is FOLFIRI. The anti-angiogenesis therapy includes the anti-VEGFR-2 antibody DC101, a monoclonal antibody reactive with mouse VEGFR-2, which is commercially available (e.g., manufactured by BioXell, catalog number BE0060). The immunotherapy includes nivolumab, an anti-PD-1 antibody.

[0254] BZ-CRC-0001 colorectal cancer PDX was obtained from the passage of NODSCID mice in Beijing Cancer Hospital, and a PDX bank was established. Each NOG mouse was subcutaneously inoculated with a small tumor tissue block with a diameter of about 3 mm cut from the tumor exfoliated pieces of tumor-bearing mice. 18 days after inoculation, animals with a tumor size of about 50 mm 3 were selected and randomly divided into 4 groups of 8 mice each. The animals were intravenously inoculated with 5×10 6 human PBMCs in 0.1 mL. One week after the human PBMC injection, the animals were selected and administered. The animals in groups 1 to 4 were administered a combination of 43 mg / kg hIgG1 control and vehicle, 30 mg / kg anti-gremmlin 1 antibody, 10 mg / kg nivolumab, and FOLFIRI (5-FU: 5 mg / kg, LV: 20 mg / kg, CPT-11: 3 mg / kg), 3 mg / kg DC101, 10 mg / kg nivolumab (commercially available anti-PD-1 antibody), and 30 mg / kg anti-gremmlin 1 antibody.

[0255] hIgG1 control, anti-gremlin 1 antibody, nivolumab, and DC101 were administered intraperitoneally twice weekly for 4 weeks. LV was administered intraperitoneally twice at weeks 1 and 3, and 5-Fu and CPT11 were administered intravenously twice at weeks 1 and 3. Animals were sacrificed by CO2 inhalation at the end of the study. Tumor size was measured two-dimensionally two or three times weekly using calipers (INSIZE), and volume was measured in mm using the formula V = 0.5 a*b^2. 3 The formula is expressed as follows, where a and b are the longest and shortest diameters of the tumor, respectively. The results were analyzed using Prism GraphPad, and the mean value is expressed as SEM. A t-test was performed to compare the two groups, and the difference was considered significant if p < 0.05 and ** < 0.01. As shown in Figure 3, the tumor growth inhibition rates for anti-gremlin 1 antibody alone, nivolumab, and combination therapy of FOLFIRI, DC101, nivolumab, and anti-gremlin 1 antibody on the sacrificial day were 56.03%, 26.86%, and 80.72%, respectively. The combination therapy group showed superior antitumor effects compared to anti-gremlin 1 antibody alone and nivolumab alone.

[0256] Example 4: Evaluation of tumor-infiltrating lymphocyte (TIL), gremlin, and PD-L1 expression in BZ-CRC-0001 PDX tumor model sections using IHC assay.

[0257] This example demonstrates that the anti-GREM1 antibody Hu14E3 HaLa can be used as a diagnostic reagent to detect GREM1 expression in diseased tissue. This example also shows that CRC tumor tissue exhibits moderate to high GREM1 expression, while PD-L1 expression is low or absent. This example further demonstrates that treatment with the anti-GREM1 antibody Hu14E3 HaLa may increase PD-L1 expression levels in a dose-dependent manner.

[0258] 1. Gremlin 1 expression status in the BZ-CRC-0001 PDX tumor model

[0259] To investigate the expression levels and staining patterns of gremlin 1 in BZ-CRC-01 PDX tumor samples, biotinylated 14E3 (Hu14E3 HaLa biotin) was prepared and used for detection. In short, EZ-Link TM Sulfo-NHS-LC-Biotin (Thermo Fisher, A39257) was dissolved in ultrapure water to prepare a 10 mM biotin reagent solution. 27 μL of biotin solution was added for every 2 mg of 14E3 antibody to be labeled, and the mixture was gradually mixed at room temperature for 0.5 hours. Low molecular weight reaction products were processed according to the manufacturer's instructions using Zeba. TM The substance was removed by desalting using a spin desalting column (Thermo Fisher, 89890).

[0260] Immunohistochemical staining (IHC) was performed on slides of paraffin-embedded PDX specimens fixed in 4% neutral buffered formalin. After deparaffinization and rehydration, all slides were scanned using EnVision. TM Antigen retrieval was performed by boiling the FLEX target retrieval solution (Dako, K8002) at 97-99°C for 25 minutes. Afterward, the cells were quenched and blocked using an IHC biotin block kit (MaiXin, BLK-0001) according to the instructions. The cells were then incubated with 20 μg / mL of our proprietary biotinylated monoclonal mouse anti-gremlin 1 (Hu14E3 HaLa biotin) at 37°C for 30 minutes. Antibody binding was performed using horseradish peroxidase-labeled streptavidin (MaiXin, SP KIT-D1) and EnVision. TM Visualization was performed using FLEX substrate working solution (Dako, K8002). The sections were counterstained with hematoxylin and mounted using permanent mounting medium.

[0261] All viable stromal fibroblasts / tumor cells on the entire slide were evaluated under bright-field microscopy and incorporated into the scoring method. Generally, at least 100 viable stromal fibroblasts and / or tumor cells were shown as a percentage score. Gremlin 1 positivity was defined as viable stromal fibroblasts / tumor cells showing partial or complete cytoplasmic staining in stromal fibroblasts / tumor cells. The percentage of stromal fibroblasts / tumor cells at four different staining intensities was estimated as 0 (no staining), 1+ (weak), 2+ (moderate), and 3+ (strong). The sum of the four percentages should have been 100%. For each specimen, the total positivity rate (defined as the percentage of viable tumor cells showing staining intensity 1+ or higher) was determined. In the BZ-CRC-01 PDX model, gremlin 1 positivity showing punctate cytoplasmic staining was observed at moderate to strong staining intensities (see Figure 4).

[0262] 2. Regulation of PD-L1 expression after treatment with anti-gremlin 1 antibody (Hu14E3 HaLa) in the BZ-CRC-0001 PDX tumor model.

[0263] To gain a deeper understanding of immunomodulation before and after anti-gremlin 1 antibody treatment in BZ-CRC-01 PDX tumor samples, PD-L1 expression status was also evaluated (see Examples 1 and 2). Immunohistochemical staining (IHC) was performed on paraffin-embedded (FFPE) tumor sections fixed in 4% neutral buffered formalin using a commercially available rabbit anti-human PD-L1 (SP263) monoclonal antibody. After deparaffinization and rehydration, all slides were antigen-retrieved in BOND epitope retrieval solution 2 (Leica, AR9640) at 97-99°C for 30 minutes. Subsequently, the slides were quenched, blocked with a peroxidase inhibitor, and incubated with appropriately diluted SP263 antibody (0.2 μg / mL) at room temperature (RT) for 30 minutes. Antibody binding was visualized for 8 minutes using BOND polymer detection (Leica, DS9800) on a Leica BOND III automated staining system. The sections were finally counterstained with hematoxylin and mounted using a permanent mounting medium.

[0264] As shown in Table 1, all samples were scored by the combined positive score (CPS) of stained immunocells and tumor cells for all visible tumor cells, using membrane staining of different intensities (negative (0), weak (1+), moderate (2+), strong (3+)). Interestingly, the staining results showed that treatment with anti-gremlin 1 antibody increased PD-L1 expression levels compared to isotype controls, and in the BZ-CRC-01 PDX model, the staining ratio increased as the dose increased from 10 mg / kg to 30 mg / kg, as shown in Figure 5 and Table 2.

[0265] Table 1 Interpretation of PD-L1 IHC results [Table 1]

[0266] Table 2 Scoring of PD-L1 IHC results on BZ-CRC-01 PDX tumor sections across Hu14E3 HaLa dose levels [Table 2]

[0267] 3. Invasion of CD3+ and CD8+ TILs after treatment with anti-gremlin 1 antibody (Hu14E3 HaLa) in the BZ-CRC-0001 PDX tumor model.

[0268] Immunocytes on BZ-CRC-01 PDX tumor sections were stained with CD3 and CD8 biomarkers to observe T lymphocyte infiltration in the tumor microenvironment before and after anti-gremlin 1 antibody treatment (see 1 and 2). These paraffin-embedded (FFPE) tumor sections fixed in 4% neutral buffered formalin were stained with commercially available CD8α (D8A8Y) rabbit mAb (CST, 85336S) and CD3ε (D7A6E TMImmunohistochemistry (IHC) was performed using rabbit mAb (CST, 85061S). After deparaffinization and rehydration, all slides were antigen-activated in BOND epitope retrieval solution 2 (Leica, AR9640) at 97 - 99 °C for 30 minutes. Then, they were quenched, blocked with a peroxidase inhibitor, and incubated with appropriately diluted CD8α (1:200) and CD3ε (1:200) respectively at room temperature (RT) for 30 minutes. Antibody binding was visualized for 8 minutes using BOND polymer detection (Leica, DS9800) on an automated stainer, Leica BOND III. Sections were finally counterstained with hematoxylin and mounted using a permanent mounting medium.

[0269] All samples were analyzed with immune cells that were completely stained at all intensities. Notably, as shown in Figure 6, the results indicated that treatment with the anti-gremlin 1 antibody may have facilitated the infiltration of CD3 and CD8 positive T cells compared to the isotype control.

[0270] Various embodiments have been described herein with reference to the accompanying drawings. However, it will be apparent that various modifications and changes may be made, and additional embodiments may be implemented, without departing from the broader scope of the invention as set forth in the following claims. Further, by considering the specification and implementation of one or more embodiments of the invention disclosed herein, other embodiments will be apparent to those skilled in the art. Accordingly, the applications and examples of this specification are regarded as illustrative, and the true scope and spirit of the invention are to be shown by the following listing of exemplary claims.

[0271] Table 3 Sequences referred to or used in this application

Table 3-1

Table 3-2

Table 3-3

Claims

1. A method for treating GREM1-expressing cancer in a subject that requires it, A method comprising administering a therapeutically effective dose of a GREM1 antagonist to a subject in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

2. The method according to claim 1, wherein the anti-angiogenic therapy comprises an antagonist of VEGFA or VEGFR (e.g., VEGFR-1, VEGFR-2, and VEGFR-3).

3. The method according to claim 2, wherein the antagonist of VEGFA is an anti-VEFRA antibody such as bevacizumab (Avastin (trademark)).

4. The method according to claim 2, wherein the antagonist of VEGFR is an anti-VEGFR-2 antibody such as ramucirumab, olimbacimab, gentuximab, alacizumab pegol, blinacimab, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, and / or HLX12.

5. The aforementioned antagonists of VEGFR include citravatinib, anlotinib, apatinib, telatinib, altiratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, and vandetanib. The method according to claim 2, wherein the small molecule VEGFR inhibitor is one of the following: ib), axitinib (Inlyta®), cabozantinib (Cabometyx®), fluquintinib (ELUNATE®), and / or regorafenib (Stivarga®).

6. The method according to claim 2, comprising administering a therapeutically effective amount of the GREM1 antagonist to the subject in combination with the anti-VEFRA antibody, the anti-VEGFR-2 antibody, or the small molecule VEGFR inhibitor.

7. The method according to any one of claims 2 to 6, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

8. The method according to claim 7, wherein the cancer is determined to be resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or to have low or no PD-L1 expression in the lesional tissue of the cancer.

9. The method according to any one of claims 1 to 8, wherein the chemotherapy includes a combination of chemotherapeutic agents.

10. The method according to claim 9, wherein the combination of chemotherapeutic agents comprises leucovorin calcium (folic acid), fluorouracil, and irinotecan hydrochloride (FOLFIRI).

11. The method according to claim 9 or 10, comprising administering a therapeutically effective amount of the GREM1 antagonist to the subject in combination with the combination of chemotherapeutic agents and the anti-VEFRA antibody or anti-VEGFR-2 antibody or small molecule VEGFR inhibitor, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

12. The method according to claim 11, wherein the cancer is determined to be resistant or refractory to the treatment with a PD-1 / PD-L1 axis inhibitor, and / or to have low or no PD-L1 expression in the lesional tissue of the cancer.

13. The method according to any one of claims 1 to 12, wherein the immunotherapy comprises a PD-1 / PD-L1 axis inhibitor.

14. The method according to claim 13, wherein the PD-1 / PD-L1 axis inhibitor comprises a PD-1 inhibitor selected from the group consisting of antibodies, small molecules, and combinations thereof.

15. The aforementioned PD-1 inhibitors include nivolumab (OPDIVO; BMS-936558), dostallimab (TSR-042), pembrolizumab (KEYTRUDA; MK-3475), MEDI0680 (AMP-514), MEDI4736, BI754091, pidilizumab (CT-011), cemiprimab (LIBTAYO, REGN2810), and Spartali Zumab (Spartalizumab) (PDR001), Cetrerimab (JNJ63723283), Tripalimab (JS001), PF-06801591, Tislerizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, Lambrolizumab, Camrelizuma (SHR-1210) ), Sintilimab (Tyvyt, IBI308), Penplimab (AK105), Zimberelimab, Retifanlimab, Serplulimab, Balstilimab, Geptanolimab, Prolgolimab, Ezabenli The method according to claim 14, comprising an anti-PD-1 antibody selected from the group consisting of mab (Ezabenlimab), sasanlimab, pimivalimab, budigalimab, nofazinlimab, sindelizumab, MGA404, Sym021, BAT1306, and HX008.

16. The method according to claim 15, wherein the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558).

17. The method according to claim 13, wherein the PD-1 / PD-L1 axis inhibitor comprises a PD-L1 inhibitor selected from the group consisting of antibodies, small molecules, and combinations thereof.

18. The aforementioned PD-L1 inhibitors include atezolizumab (TECENTRIQ, R05541267, MPDL3280A, RG7446), BMS-936559, avelumab (bavencio), rodapolimab (LODAPOLIMAB) (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, emvafolimab (KN035), MDX-1105, STI-1040, CS1001, and adebrerimab (Adebr The method according to claim 17, comprising an anti-PD-L1 antibody selected from the group consisting of elimai (SHR-1316), SHR-1701, TOB2450, Bintrafusp, LP002, STI-3031, Cosibelimai, Pacmilimai, NM01, LDP, AMP-224, Garivulimab (BGB-A333), A167, SCD-135, Opcolimab, and GR1405.

19. The method according to any one of claims 1 to 18, wherein the cancer is a) resistant to or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or b) PD-L1 expression is determined to be low or absent in the lesional tissue of the GREM1-expressing cancer.

20. The method according to any one of claims 1 to 19, comprising administering the GREM1 antagonist to the subject and, after a period of time sufficient to increase the expression level of PD-L1 in the cancer cells of the subject, administering the PD-1 / PD-L1 axis inhibitor to the subject.

21. The method according to any one of claims 1 to 20, comprising administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the chemotherapeutic agent (e.g., FOLFIRI) and the PD-1 / PD-L1 axis inhibitor (e.g., a PD-1 inhibitor or nivolumab), wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

22. The method according to claim 21, wherein the cancer is determined to be resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or to have low or no PD-L1 expression in the lesional tissue of the cancer.

23. The method according to any one of claims 1 to 22, comprising administering a therapeutically effective amount of the GREM1 antagonist to the subject in combination with the anti-VEFRA antibody or anti-VEGFR-2 antibody or small molecule VEGFR inhibitor, the chemotherapeutic agent, and the PD-1 / PD-L1 axis inhibitor, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

24. The method according to claim 23, wherein the cancer is determined to be resistant or refractory to the treatment with a PD-1 / PD-L1 axis inhibitor, and / or to have low or no PD-L1 expression in the lesional tissue of the cancer.

25. The method according to any one of claims 1 to 24, wherein the subject is further determined to have low, moderate, or high GREM1 expression in the lesional tissue of the GREM1-expressing cancer.

26. The method according to claim 25, wherein the subject is further determined to have 10-20% of its tumor cells GREM1-positive, as measured by IHC.

27. A method for improving the response of a subject to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject is determined to have GREM1 present in a biological sample of the subject's diseased tissue, or the subject is determined to have a GREM1 expression level that has reached a threshold level in the biological sample of the diseased tissue, and the method is a) A method comprising administering a therapeutically effective dose of a GREM1 antagonist to a subject so as to increase the expression of PD-L1 in lesional tissue, thereby improving the subject's responsiveness to a PD-1 / PD-L1 axis inhibitor.

28. The method according to claim 27, wherein the subject is determined to be a) resistant to or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) has low or no expression of PD-L1 in the diseased tissue.

29. The method according to claim 27, wherein the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

30. The method according to any one of claims 27 to 29, wherein the lesional tissue is cancerous tissue.

31. The method according to claim 27, wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

32. The method according to any one of claims 27 to 31, further comprising administering a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor to the subject after the expression of PD-L1 has increased in the lesional tissue of the subject.

33. A method for determining the suitability of a subject for treatment with a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor, or the likelihood of responding to said treatment, wherein the method is: c) Determining the presence or expression level of GREM1 in a biological sample of the lesional tissue of the subject, A method for indicating whether the presence or absence or expression level of GREM1 indicates whether the subject is eligible for or likely to respond to treatment with a combination of the GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor.

34. The method according to claim 33, wherein the presence of GREM1 or the expression level of GREM1 exceeding a threshold level, as determined in step a), indicates that the subject is eligible for or likely to respond to treatment with a combination of the GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor.

35. The method according to claim 33, wherein the absence of GREM1 or the GREM1 expression level not exceeding a threshold, as determined in step a), indicates that the subject is not eligible for or unlikely to respond to treatment with the GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor in combination.

36. Before step a), The method according to any one of claims 33 to 35, further comprising step i) contacting the biological sample of the lesional tissue of the subject with a GREM1 diagnostic agent under conditions that enable detection of the expression level of GREM1 in the sample.

37. The method according to any one of claims 33 to 36, wherein the subject is determined to be a) resistant to or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) has low or no expression of PD-L1 in the diseased tissue.

38. The method according to any one of claims 33 to 36, wherein the subject is determined to have moderate or high PD-L1 expression in the diseased tissue.

39. The method according to any one of claims 33 to 38, further comprising administering a therapeutically effective amount of the GREM1 antagonist to the subject for a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer cells, followed by administering the PD-1 / PD-L1 axis inhibitor to the subject.

40. The method according to any one of claims 33 to 39, wherein the GREM1 expression is detected by a GREM1 diagnostic reagent comprising an anti-GREM1 antibody or an antigen-binding fragment thereof.

41. The method according to any one of claims 1 to 40, wherein the GREM1 antagonist comprises an anti-GREM1 antibody or an antigen-binding fragment thereof.

42. The anti-GREM1 antibody or its antigen-binding fragment comprises heavy chains HCDR1, HCDR2, HCDR3, and / or light chains LCDR1, LCDR2, LCDR3. The HCDR1 comprises an amino acid sequence containing TYGMA (SEQ ID NO: 1), or a homologous sequence having at least 80% sequence identity therewith. The HCDR2 comprises an amino acid sequence containing WINTLSGEPTYADDFKG (SEQ ID NO: 2), or a homologous sequence having at least 80% sequence identity therewith. The HCDR3 comprises an amino acid sequence containing EPMDY (SEQ ID NO: 3), or a homologous sequence having at least 80% sequence identity therewith. The LCDR1 includes an amino acid sequence containing KSSQSLLLDSDGKTYLS (SEQ ID NO: 4), or a homologous sequence having at least 80% sequence identity therewith. The aforementioned LCDR2 includes an amino acid sequence containing LVSKLDS (SEQ ID NO: 5), or a homologous sequence having at least 80% sequence identity therewith. The method according to claim 40 or 41, wherein the LCDR3 comprises an amino acid sequence containing WQGAHFPLT (SEQ ID NO: 6), or a homologous sequence having at least 80% sequence identity thereto.

43. The anti-GREM1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, The method according to claim 42, wherein the light chain variable region includes the amino acid sequence of SEQ ID NO:

8.

44. The method according to claim 42 or 43, further comprising substitution or modification of one or more amino acid residues, but still retaining specific binding specificity or affinity for hGREM1.

45. The method according to claim 44, wherein at least one of the substitutions or modifications is present in one or more of the CDR sequences and / or in one or more of the non-CDR regions of the VH or VL sequences.

46. The method according to any one of claims 42 to 45, further comprising an immunoglobulin constant region, optionally further comprising a human IgG constant region.

47. The method according to claim 46, wherein the constant region includes a constant region of human IgG1, IgG2, IgG3, or IgG4, and optionally the constant region includes a heavy chain constant region containing the sequence of SEQ ID NO: 9 and / or a light chain constant region containing the sequence of SEQ ID NO:

10.

48. The method according to any one of claims 1 to 47, wherein the GREM1 antagonist or the anti-GREM1 diagnostic reagent is linked to one or more conjugate portions.

49. The method according to claim 48, wherein the conjugate portion comprises a clearance modifier, a therapeutic agent (e.g., a chemotherapeutic agent), a toxin, a radioisotope, a detectable label (e.g., lantanide, luminescence label, fluorescent label, biotin / avidin, or enzyme substrate label), a pharmacokinetic modifier, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, and other anticancer drugs such as an androgen receptor inhibitor.

50. The method according to any one of claims 1 to 49, wherein the subject is a human.

51. The method according to any one of claims 1 to 50, wherein the administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

52. The method according to any one of claims 1 to 51, wherein the administration of the GREM1 antagonist is performed before, simultaneously with, or after the administration of the anti-angiogenic agent and / or the chemotherapeutic agent and / or the immunotherapy agent.

53. Use of a GREM1 antagonist in the manufacture of a drug for treating GREM1-expressing cancer in a subject, wherein the treatment comprises administering the drug to the subject in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

54. The use of GREM1 antagonists in the manufacture of pharmaceuticals to improve the response of subjects to treatment with PD-1 / PD-L1 axis inhibitors, The subject has been determined to have GREM1 present in a biological sample of the subject's lesional tissue, or the subject has been determined to have a GREM1 expression level that has reached a threshold level in a biological sample of the subject's lesional tissue. The improvement includes a) administering a therapeutically effective dose of a GREM1 antagonist to the subject so as to increase PD-L1 expression in the lesional tissue, thereby improving the subject's responsiveness to a PD-1 / PD-L1 axis inhibitor.

55. The use of an anti-GREM1 diagnostic reagent in the manufacture of a kit for determining the suitability of a subject for treatment with a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor, or the likelihood of responding to said treatment, The anti-GREM1 diagnostic reagent is capable of determining the presence or expression level of GREM1 in a biological sample of the lesional tissue of the subject, Uses of the GREM1 antagonist, the absence of GREM1, or its expression level, indicating whether the subject is eligible for or likely to respond to treatment with a combination of the GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor.

56. The use according to claim 55, wherein the subject is determined to be a) resistant to or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) has low or no expression of PD-L1 in the lesional tissue.

57. The use according to claim 55, wherein the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

58. A kit useful for treating GREM1-expressing cancer in a subject requiring treatment, comprising a GREM1 antagonist and a package insert, wherein the package insert includes instructions for using the GREM1 antagonist in combination with a) anti-angiogenic therapy, b) chemotherapy, c) immunotherapy, d) anti-angiogenic therapy and chemotherapy, e) chemotherapy and immunotherapy, f) anti-angiogenic therapy and immunotherapy, or g) anti-angiogenic therapy, chemotherapy and immunotherapy.

59. The kit according to claim 58, characterized in that the GREM1-expressing cancer is a) resistant to or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or b) has low or no expression of PD-L1 in the lesional tissue, and / or c) has expression of GREM1 in the lesional tissue.

60. The kit according to claim 58, wherein the subject is determined to have moderate or high PD-L1 expression in the lesional tissue.

61. A method for improving tumor-infiltrating lymphocytes in a subject having a solid tumor, comprising administering a therapeutically effective amount of a GREM1 antagonist according to any one of claims 1 to 60 to the subject.

62. The method according to claim 61, wherein the solid tumor is a cold tumor.

63. The method according to claim 62, wherein the subject is resistant to or refractory to anticancer therapies such as immunotherapy, for example, immune checkpoint inhibitors.

64. A method for promoting the transformation of a subject having a solid tumor from a cold tumor to a hot tumor, comprising administering a therapeutically effective dose of a GREM1 antagonist according to any one of claims 1 to 63 to the subject.

65. A method for treating cancer in a subject having a cold tumor, comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist according to any one of claims 1 to 64.

66. The method according to claim 65, wherein the subject is resistant to or refractory to anticancer therapies such as immunotherapy, for example, immune checkpoint inhibitors.

67. The method according to any one of claims 61 to 65, further comprising administering one or more therapies to the subject.

68. The method according to claim 67, wherein one or more of the therapies can promote the proliferation, activation, and / or tumor infiltration of T cells.

69. The method according to claim 68, wherein the T cell is a CD3+ T cell or a CD8+ T cell.

70. The method according to claim 67, wherein the one or more therapies are anti-angiogenic therapies, immunotherapies, and / or chemotherapy according to any one of claims 1 to 69.

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