Combination therapy involving GREM1 antagonists for treatment of cancer

Abstract

The present disclosure provides herein a combination therapy of a GREM1 antagonist and one or more therapies selected from the group consisting of a) an anti-angiogenesis therapy, b) a chemotherapy, and c) an immunotherapy The disclosure also provides a method of selecting a sub-population from subjects resistant to immunotherapy that can be susceptible to said immunotherapy by a GREM1 antagonist.

Description

Technical Field

[0001] This disclosure generally relates to cancer therapies associated with gremlin1 (GREM1) antagonists in combination with anti-angiogenic therapies and / or chemotherapy and / or immunotherapy for the treatment of cancer, particularly cancers expressing GRM1. Background Technology

[0002] GREM1 is closely associated with fibrotic lesions of the kidneys, lungs, liver, and retina, as well as several tumor types, including pancreatic cancer, colon cancer, lung cancer, glioma, gastric cancer, and prostate cancer. Sneddon et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS) PNAS ) October 2006; 103(40): 14842-14847) For example, aberrant upregulation of gremlin1 confers tumorigenicity on colon cells outside the stem cell niche. It has also been found that tumor stem cells highly express and secrete gremlin1 in gliomas to maintain their stemness. Yan, K. et al., Genes and Development, 28, 1085-1100 (2014) Therefore, gremlin1 has been used as a therapeutic target for treating gremlin-related diseases.

[0003] Although GREM1 antagonists have shown some therapeutic efficacy against several GREM1-expressing diseases, effective combination therapies involving GREM1 antagonists for treating GREM1-expressing cancers remain rare. Therefore, there is an urgent need for improved combination therapies for GREM1-expressing cancers to meet clinical needs. Summary of the Invention

[0004] Among other things, this disclosure provides a method for treating cancer expressing GREM1 in a subject in need, 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 some embodiments, the anti-angiogenic therapy includes an antagonist of VEGFA or VEGFR.

[0006] In some embodiments, the antagonist of the VEGFA is an anti-VEFRA antibody, such as bevacizumab (Avastin®).

[0007] In some embodiments, the VEGFR antagonist is a small molecule VEGFR inhibitor or a large molecule VEGFR inhibitor.

[0008] In some implementations, the VEGFR is VEGFR-1, VEGFR-2, or VEGFR-3.

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

[0010] In some embodiments, the VEGFR antagonist is a small molecule VEGFR inhibitor, such as sitravatinib, anlotinib, apatinib, telatinib, atiratinib, kanitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fruquitinib (ELUNATE®), and / or regorafenib (Stivarga®).

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

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

[0013] In some embodiments, the cancer is resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

[0014] In some embodiments, the chemotherapy includes a combination of chemotherapeutic agents.

[0015] In some embodiments, the combination of chemotherapeutic agents includes calcium leucovorin (leucovorin), fluorouracil, and irinotecan hydrochloride (FOLFIRI).

[0016] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the chemotherapeutic agent and the 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 some embodiments, the cancer is resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

[0018] In some embodiments, the immunotherapy includes PD-1 / PD-L1 axis inhibitors.

[0019] In some embodiments, the PD-1 / PD-L1 axis inhibitor includes PD-1 inhibitors selected from the group consisting of antibodies, small molecules, and combinations thereof.

[0020] In some embodiments, the PD-1 inhibitor comprises an anti-PD-1 antibody selected from the group consisting of: nivolumab (OPDIVO; BMS-936558), dostarlimab (TSR-042), pembrolizumab (KEYTRUDA; MK-3475), MEDI0680 (AMP-514), MEDI4736, BI 754091, pitilizumab (CT-011), cimiplimab (LIBTAYO; REGN2810), spartalizumab (PDR001), and cetrelimab (JNJ). 63723283), Toripalimab (JS001), PF-06801591, Tislelizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, Lamborizumab, Camrelizumab (SHR-1210), Sintilimab (Tyvyt, IBI308), Penpulimab (AK105), Zimberelimab. Retifanlimab, Serplulimab, Balstilimab, Geptanolimab, Prolgolimab, Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofazinlimab, Sindelizumab, MGA404, Sym021, BAT1306, and HX008.

[0021] In some embodiments, the PD-1 inhibitor is nivolumab (OPDIVO; BMS-936558).

[0022] In some embodiments, the PD-1 / PD-L1 axis inhibitor includes PD-L1 inhibitors selected from the group consisting of antibodies, small molecules, and combinations thereof.

[0023] In some embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody selected from the group consisting of: atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446), BMS-936559, avelumab (bavencio), lodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, and envalizumab (Envafo). The following are listed: limab (KN035), MDX-1105, STI-1040, CS1001, 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 some implementations, a) the cancer is resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or b) the cancer is identified as having low or no PD-L1 expression in disease tissue of the cancer expressing GREM1.

[0025] In some embodiments, the method includes administering the GREM1 antagonist to the subject, and then administering the PD-1 / PD-L1 axis inhibitor to the subject after a period of time sufficient to increase the expression level of PD-L1 in the subject's cancer cells.

[0026] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the chemotherapy agent (e.g., FOLFIRI) and the PD-1 / PD-L1 axis inhibitor (e.g., a PD-1 inhibitor or nivolumab), and 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 some embodiments, the cancer is resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

[0028] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the anti-VEGFR-2 antibody, the chemotherapy combination, 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.

[0029] In some embodiments, the cancer is resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

[0030] In some embodiments, the object is further identified as having low, intermediate, or high GREM1 expression in the diseased tissue of the cancer expressing GREM1. In some embodiments, the object is further identified as having 10-20% GREM1-positive tumor cells, as measured by IHC.

[0031] On the other hand, this disclosure provides a method for improving the responsiveness of a subject to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject has been identified as having GREM1 in a biological sample of the subject's disease tissue, or the subject has been identified as having GREM1 expression levels in the biological sample of the disease tissue reaching a threshold level, the method comprising: a) Administering a therapeutically effective amount of a GREM1 antagonist to the subject to increase PD-L1 expression in diseased tissues, thereby improving the subject's responsiveness to PD-1 / PD-L1 axis inhibitors.

[0032] In some implementations, a) the object is identified as resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) the object is identified as having low or no PD-L1 expression in diseased tissue.

[0033] In some implementations, the object is identified as having moderate or high PD-L1 expression in diseased tissue.

[0034] In some implementations, the diseased tissue is cancerous tissue.

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

[0036] In some embodiments, the method further includes administering a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor to the subject after an increase in PD-L1 expression in the diseased tissue of the subject.

[0037] On the other hand, this disclosure provides a method for determining eligibility or responsiveness to treatment in subjects using a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor, the method comprising: a) Determine the presence or expression level of GREM1 in biological samples of the diseased tissue of the subject. The presence or absence of GREM1 or its expression level indicates whether the subject is likely to be eligible for treatment with the combination of the GREM1 antagonist and the PD-1 / PD-L1 axis inhibitor or whether the subject is likely to respond to the treatment.

[0038] In some implementations, the presence of GREM1 determined in step a) or the expression level of GREM1 being above a threshold level indicates that the subject may be eligible for treatment with the combination of the GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor or may be responsive to the treatment.

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

[0040] In some embodiments, the method provided herein further includes step i) prior to step a). i) Contact the sample with a GREM1 diagnostic agent under conditions that allow detection of the expression level of GREM1 in the biological sample of the diseased tissue of the subject.

[0041] In some implementations, a) the object is identified as resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) the object is identified as having low or no PD-L1 expression in diseased tissue.

[0042] In some implementations, the object is identified as having moderate or high PD-L1 expression in diseased tissue.

[0043] In some embodiments, the method provided herein further includes administering a therapeutically effective amount of the GREM1 antagonist to the subject for a duration sufficient to increase the expression level of PD-L1 in the subject's cancer cells, and then administering the PD-1 / PD-L1 axis inhibitor to the subject.

[0044] In some embodiments, the GREM1 expression is detected by a GREM1 diagnostic reagent comprising an anti-GREM1 antibody or an antigen-binding fragment thereof.

[0045] In some embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or an antigen-binding fragment thereof.

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

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

[0048] In some embodiments, the method further includes substitution or modification of one or more amino acid residues, while still retaining the specific binding specificity and / or affinity for hGREM1.

[0049] In some embodiments, at least one of the substitutions or modifications substitutes or modifies one or more CDR sequences in the CDR sequence and / or one or more non-CDR regions in the non-CDR regions of the VH or VL sequence.

[0050] In some embodiments, the method further includes an immunoglobulin constant region, optionally a constant region of human IgG.

[0051] In some embodiments, 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.

[0052] In some embodiments, the GREM1 antagonist or the anti-GREM1 diagnostic reagent is linked to one or more conjugate moieties.

[0053] In some embodiments, the conjugate portion includes a scavenging modifier, a therapeutic agent (e.g., a chemotherapeutic agent), a toxin, a radioisotope, a detectable label (e.g., a lanthanide element, a luminescent label, a fluorescent label, a biotin / avidin, or an enzyme-substrate label), a pharmacokinetic modification portion, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anticancer drugs (such as androgen receptor inhibitors).

[0054] In some implementations, the object is a person.

[0055] In some embodiments, the administration is performed orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

[0056] In some embodiments, 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 immunotherapeutic agent.

[0057] On the other hand, this disclosure provides the use of GREM1 antagonists in the preparation of medicaments for treating cancers expressing GREM1 in subjects of need, wherein the treatment comprises administering the medicament 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.

[0058] On the other hand, this disclosure provides the use of GREM1 antagonists in the preparation of medicaments for improving the response of subjects to treatment with PD-1 / PD-L1 axis inhibitors. The object has been identified as having GREM1 present in a biological sample of the object's diseased tissue, or the object has been identified as having GREM1 expression levels in the biological sample of the diseased tissue reaching a threshold level. The improvement described therein includes administering a therapeutically effective amount of a GREM1 antagonist to the subject, thereby increasing the expression of PD-L1 in diseased tissues and thus improving the subject's responsiveness to PD-1 / PD-L1 axis inhibitors.

[0059] On the other hand, this disclosure provides the use of anti-GREM1 diagnostic reagents in the preparation of kits for determining the eligibility or responsiveness to treatment in subjects using a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor. The anti-GREM1 diagnostic reagent is capable of determining the presence or expression level of GREM1 in biological samples of the diseased tissue of the subject. The presence or absence of GREM1 or its expression level indicates whether the subject is likely to be eligible for treatment with the combination of the GREM1 antagonist and the PD-1 / PD-L1 axis inhibitor or whether the subject is likely to respond to the treatment.

[0060] In some implementations, a) the object is identified as resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) the object is identified as having low or no PD-L1 expression in diseased tissue.

[0061] In some implementations, the object is identified as having moderate or high PD-L1 expression in diseased tissue.

[0062] On the other hand, this disclosure provides a kit for treating cancers expressing GREM1 in subjects of need, the kit comprising a GREM1 antagonist and a packaging insert comprising 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 some embodiments, the cancer expressing GREM1 is characterized by: a) resistance or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or b) low or no PD-L1 expression in the disease tissue, and / or c) GREM1 expression in the disease tissue.

[0064] On the other hand, this disclosure provides a method for improving tumor-infiltrating lymphocytes in a subject with a solid tumor, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist as defined in this disclosure.

[0065] In some implementations, the solid tumor is a cold tumor.

[0066] In some implementations, the object is resistant to or refractory to anticancer therapies, such as immunotherapy, for example, immune checkpoint inhibitors.

[0067] On the other hand, this disclosure provides a method for promoting the transformation of a cold tumor into a hot tumor in a subject with a solid tumor, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist as defined in this disclosure.

[0068] On the other hand, this disclosure provides a method for treating cancer in a subject suffering from a cold tumor, the method comprising: administering a therapeutically effective amount of a GREM1 antagonist as defined in this disclosure to the subject.

[0069] In some implementations, the object is resistant to or refractory to anticancer therapies, such as immunotherapy, for example, immune checkpoint inhibitors.

[0070] In some embodiments, the methods provided herein further include administering one or more therapies to the subject.

[0071] In some embodiments, the one or more therapies can promote T cell proliferation, activation, and / or tumor infiltration.

[0072] In some embodiments, the T cells are CD3+ T cells or CD8+ T cells.

[0073] In some embodiments, the one or more therapies are anti-angiogenic therapies, immunotherapies, and / or chemotherapy as defined in this disclosure.

[0074] In some embodiments, the object is identified as having moderate or high PD-L1 expression in diseased tissue. It should be understood that both the above general description and the following detailed description are exemplary and explanatory only and do not limit the invention. Furthermore, the accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. Attached Figure Description

[0075] The accompanying drawings cited herein form part of the specification. Unless otherwise expressly stated in the detailed description, the features shown in the drawings illustrate only some embodiments of this application, and not all embodiments of this application, and readers of the specification should not draw the contrary inferences.

[0076] Figure 1A shows the inhibition of CRC PDX tumor growth by combination of anti-GREM1 antibody and DC101 (mean ± SEM, n=8).

[0077] Figure 1B shows the pathological information of BZ-CRC-01 PDX tumors, in which Gremlin-1 was detected in 10-20% of tumor cells (A) by IHC.

[0078] Figure 2 shows the inhibition of CRC PDX tumor growth by combination of anti-GREM1 antibody with FOLFIRI and DC101 (mean ± SEM, n = 8).

[0079] Figure 3 shows the inhibition of CRC PDX tumor growth by combination of anti-GREM1 antibody with FOLFIRI, nivolumab and DC101 (mean ± SEM, n = 8).

[0080] Figure 4 shows a representative IHC image of GREM1 expression on a BZ-CRC-01 PDX tumor slice.

[0081] Figure 5 shows representative IHC images of PD-L1 expression before and after Hu14E3 HaLa treatment on BZ-CRC-01 PDX tumor slices.

[0082] Figure 6 shows a representative IHC image of tumor-infiltrating lymphocytes (TILs) treated with Hu14E3 HaLa on a BZ-CRC-01 PDX tumor slice.

[0083] Figure 7 illustrates the safety characteristics of Hu14E3 HaLa in NHP.

[0084] The same reference numerals will be used throughout the accompanying drawings to denote the same or similar parts. Detailed Implementation

[0085] The following description of this disclosure is intended only to illustrate various embodiments of this disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalent forms, variations, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including disclosures, patents, and patent applications, are incorporated herein by reference in their entirety.

[0086] definition As used herein, unless otherwise stated herein or explicitly contradicted by the context, the terms “a / an,” “the,” and similar terms used in the context of this invention (especially in the context of the claims) shall be interpreted to encompass both the singular and the plural.

[0087] As used herein, the term "antagonist" in relation to GREM1 refers to any molecule that partially or completely inhibits, blocks, or neutralizes the biological activity of GREM1. Suitable GREM1 antagonists may 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.

[0088] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen, or any polypeptide that mimics an antibody in its ability to bind to a specific antigen. A natural, intact antibody consists of two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region (VH) and a first, second, and third constant region (CH1, CH2, 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 the second and third constant regions of two heavy chains linked together by disulfide bonds. Each arm of the Y includes the variable and constant regions of a single light chain and the variable and first constant regions of a single heavy chain that bind to the variable and constant regions of the single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of two chains typically include three highly variable rings, called complementarity-determining regions (CDRs) (the CDRs for light chains include LCDR1, LCDR2, and LCDR3, and the CDRs for heavy chains include HCDR1, HCDR2, and HCDR3).The CDR boundaries of the antibody and antigen binding domains disclosed in this paper can be defined or identified by the Kabat, IMGT, AbM, Chothia, or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol., 186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); NR Whitelegg et al., Protein Engineering, Vol. 13(12), 819-824 (2000); Chothia, C. et al., Nature, Dec 21–28; 342(6252):877–83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27: 55–77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (2nd ed.), Chapter 26, 481–514, (2015). Three CDRs are interposed between side extensions called framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are classified into several classes based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are further subdivided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In some embodiments, the antibodies provided herein encompass any of their antigen-binding fragments.

[0089] As used herein, the terms "antigen-binding fragment" or "antigen-binding moiety" refer to a fragment formed from an antibody fragment comprising one or more CDRs (e.g., an antibody fragment), or any other portion (e.g., an antibody fragment) that binds to an antigen but does not include the complete structure of the native antibody. Examples of antigen-binding fragments / migrations include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent bifunctional antibodies), multispecific antibodies, camelified single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding fragment / migration is capable of binding to the same antigen that the parent antibody binds to. In some embodiments, the antigen-binding fragment / migration may include one or more CDRs from a particular parent antibody.

[0090] The term "Fab" in antibody refers to a monovalent antigen-binding fragment consisting of a single light chain (both the variable and constant regions) linked to the variable and first constant regions of a single heavy chain via disulfide bonds. Fab can be obtained by digesting the antibody with papain at the N-terminal residues of the disulfide bonds between the heavy chains near the hinge region.

[0091] "Fab" refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of an antibody at a C-terminal residue of the disulfide bond between the heavy chains near the hinge region, and thus differs from Fab in a few residues (including one or more cysteine ​​residues) in the hinge region.

[0092] “F(ab')2” refers to a dimer of Fab’ that includes a portion of two light chains and two heavy chains.

[0093] The term "Fv" in antibody refers to the smallest fragment of an antibody carrying an intact antigen-binding site. An Fv fragment consists of the variable region of a single light chain linked to the variable region of a single heavy chain. "dsFv" refers to a disulfide-bonded Fv fragment, where the variable regions of the light and heavy chains are connected by disulfide bonds.

[0094] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of light chain variable regions directly linked to heavy chain variable regions or linked to each other via peptide linker sequences (Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)). "scFv dimer" refers to a single chain comprising two heavy chain variable regions, two light chain variable regions, and a linker. In some embodiments, "scFv dimer" is a bivalent bifunctional antibody or bivalent scFv (BsFv) comprising two dimerized VH-VLs (linked by peptide linkers) and another partially dimerized VH-VL, such that one VH portion coordinates with the other VL portion to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). In other embodiments, the “scFv dimer” is a bispecific bifunctional antibody comprising conjugated VH1-VL2 (linked by a peptide linker) and VL1-VH2 (also linked by a peptide linker), such that VH1 coordinates to VL1 and VH2 coordinates to VL2, and each coordination pair has a different antigen specificity.

[0095] "Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody composed of scFv linked to the Fc region of the antibody.

[0096] "Camelized single-domain antibody," "heavy chain antibody," "nanobody," or "HCAb" refers to an antibody that includes two VH domains but not 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 derived from the Camelidae family (camel, dromedary camel, and llama). Although lacking light chains, camel-like antibodies possess a true antigen-binding library (Hamers-Casterman C. et al., Nature, June 3;363(6428):446-8 (1993); Nguyen VK. et al., “Heavy-chain antibodies in Camelidae: a case of evolutionary innovation”, Immunogenetics, April;54(1):39-47 (2002); Nguyen VK. et al., Immunology, May;109(1):93-101 (2003)). The variable domain (VHH domain) of a heavy chain antibody represents the smallest known antigen-binding unit generated by an adaptive immune response (Koch-Nolte F. et al., FASEB J, Nov; 21(13):3490-8. e.g., June 15, 2007 (2007)). A “bifunctional antibody” comprises a small antibody fragment having two antigen-binding sites, wherein the fragment comprises a VH domain and a VL domain linked in a single polypeptide chain (VH-VL or VL-VH) (see, for example, Holliger P. et al., PNAS, July 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161). Because the linker is too short, the two domains on the same chain cannot pair, thus forcing the domain to pair with a complementary domain of another chain, thereby creating two antigen-binding sites. Antigen binding sites can target the same or different antigens (or epitopes).

[0097] "Domain-specific antibodies" refer to antibody fragments containing only heavy chain variable regions or light chain variable regions. In some embodiments, two or more VH domains are covalently linked by peptide linkers to generate bivalent or multivalent domain antibodies. The two VH domains of a bivalent domain antibody can target the same or different antigens.

[0098] In some implementations, “(dsFv)2” comprises three peptide chains: two VH moieties linked by peptide linkers and bound to two VL moieties by disulfide bridges.

[0099] In some implementations, the “bispecific ds bifunctional antibody” includes VH1-VL2 (also linked via a peptide linker) which binds to VL1-VH2 (linked via a peptide linker) through a disulfide bridge between VH1 and VL1.

[0100] In some embodiments, a “bispecific dsFv” or “dsFv-dsFv” comprises three peptide chains: VH1-VH2 moieties, wherein the heavy chain is bound by a peptide linker (e.g., a long, flexible linker) and paired with the VL1 and VL2 moieties respectively via disulfide bridges. Each pair of disulfide-paired heavy and light chains exhibits different antigen specificities.

[0101] As used herein, the term "humanized" means that the antibody or antigen-binding fragment includes a CDR derived from a non-human animal, a FR region derived from a human, and a constant region derived from a human (where applicable). In some embodiments, amino acid residues of the variable region framework of the humanized gremlin antibody are substituted to achieve sequence optimization. In some embodiments, the variable region framework sequence of the 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.

[0102] As used herein, “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 (e.g., to provide diagnostic and / or therapeutic use).

[0103] As used herein, the term “affinity” refers to the strength of the non-covalent interaction between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0104] As used herein, the terms "specific binding" or "specifically binding" refer to a non-random binding reaction between two molecules, such as an antibody and an antigen. In some embodiments, the antibody or antigen-binding fragments provided herein specifically bind to human and / or non-human grimlin1, wherein the binding affinity (KD) is ≤10. -6M (for example, ≤ 5 × 10) -7 M, ≤ 2 × 10 -7 M, ≤ 10 -7 M, ≤5 × 10 -8 M, ≤2 × 10 -8 M, ≤ 10 -8 M, ≤5 × 10 -9 M, ≤4 × 10 -9 M, ≤3 × 10 -9 M, ≤2 × 10 -9 M or ≤ 10 -9 M). In this paper, KD refers to the ratio of dissociation rate to association rate (k...). off / k on The ratio can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance, microthermophoresis, HPLC-MS, and flow cytometry (such as FACS). In some embodiments, the KD value can be suitably determined using flow cytometry. A variety of immunoassay formats can be used to select antibodies that specifically react with a particular protein. For example, solid-phase ELISA is routinely used to select antibodies that specifically react with a protein (see, for example, Harlow and Lane, Using Antibodies, A Laboratory Manual (1998), to describe immunoassay methods and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective binding reaction will produce a signal at least twice the background signal, and more typically at least 10 to 100 times the background signal.

[0105] The “sequence identity percentage (%)” for an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment and, where necessary, the introduction of vacancies to achieve maximum correspondence. For example, publicly available tools such as BLASTN and BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Molecular Biology, 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389–3402 (1997)) and ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8) can be used. Alignment can be performed using software such as ALIGN or Megalign (DNASTAR) (2007) to determine the percentage of sequence identity between amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters provided by these tools, or can customize parameters suitable for alignment, for example, by selecting an appropriate algorithm. In some implementations, different residue positions may differ due to conserved amino acid substitutions. A “conserved amino acid substitution” means that one amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitutions do not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to conserved substitutions, the percentage or degree of similarity can be adjusted upwards to correct for the conservatism of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, for example, Pearson, (1994) Methods in Molecular Biology, 24: 307-331, which is incorporated herein by reference.

[0106] As used herein, a “homologous sequence” means that a polynucleotide sequence (or its complementary strand) or amino acid sequence has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optionally aligned.

[0107] The term "object" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "object" are used interchangeably in this document.

[0108] As used in this article, “treating” a symptom includes preventing or alleviating the symptom, slowing the onset or progression of the symptom, reducing the risk of developing the symptom, preventing or delaying the development of symptoms associated with the symptom, reducing or ending symptoms associated with the symptom, producing complete or partial remission of the symptom, curing the symptom, or some combination thereof.

[0109] As used herein, “cancer” means any medical condition characterized by malignant cell growth or proliferation, abnormal proliferation, invasion, or metastasis, and includes both solid tumors and non-solid carcinomas (e.g., hematologic malignancies), such as leukemia. As used herein, “solid tumor” means a solid mass of proliferating and / or malignant cells.

[0110] The term "therapeutic effective amount" or "effective amount" refers to the amount of a pharmaceutical agent that produces a certain desired local or systemic therapeutic effect with a reasonable benefit / risk ratio suitable for any treatment alone or in combination with other doses. In the case of treating a specific disease, the desired local or systemic therapeutic effect is preferably related to inhibiting the progression of the disease. This includes slowing the progression of the disease, and in particular, interrupting or reversing the progression of the disease. When administered for disease prevention, the dose is sufficient to avoid or delay the onset of the disease. A therapeutic effective amount or effective amount does not require treatment or prevention of the occurrence of the disease or symptom. The effective amount of the pharmaceutical agent described herein will depend on the symptom to be treated, the severity of the disease, the individual parameters of the patient, including age, physical condition, size and weight, duration of treatment, type of concomitant therapy (if present), specific route of administration, and similar factors. Therefore, the dosage of the pharmaceutical agent described herein can depend on a variety of such parameters. If the initial dose is insufficient to elicit a response in the patient, a higher dose may be used (or a higher dose may be effectively obtained through a different, more local route of administration). In some embodiments, the therapeutic effective amount of the pharmaceutical agent will depend on its therapeutic index, solubility, etc.

[0111] In this document, the reference to "about" a value or parameter includes (and describes) the implementation of said value or parameter itself. For example, a reference to "about X" includes "X". A range of numbers includes the number that defines the range. Generally, the term "about" refers to the indicated value of a variable and all values ​​of the variable that are within the experimental error range of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10% of the indicated value (whichever is greater). When the term "about" is used in the context of a time period (year, month, week, day, etc.), the term "about" means the time period plus or minus the next subordinate time period (e.g., about 1 year means 11–13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6–8 days; etc.), or within 10% of the indicated value, whichever is greater.

[0112] I. Methods to improve tumor-infiltrating lymphocytes This disclosure provides a method for improving tumor-infiltrating lymphocytes in a subject with a solid tumor, the method comprising administering a therapeutically effective amount of a GREM1 antagonist to the subject. In some embodiments, the solid tumor is a cold tumor. In some embodiments, the subject is resistant or refractory to anticancer therapies, such as immunotherapy, for example, immune checkpoint inhibitors (e.g., PD-1 / PD-L1 axis inhibitors).

[0113] This disclosure also provides a method for promoting the transformation of a cold tumor into a hot tumor in a subject with a solid tumor, the method comprising administering a therapeutically effective amount of a GREM1 antagonist to the subject. As used herein, the term “cold tumor” is used interchangeably with the terms “immune desert tumor” or “immune rejection tumor”, referring to a tumor lacking innate immunity or having ineffective innate antitumor immune features, characterized by 1) lack of T cell infiltration; 2) low mutational load; 3) low expression of major histocompatibility complex (MHC) class I; 4) low PD-L1 expression; and / or 5) the presence of immunosuppressive cell populations (e.g., tumor-associated macrophages, T regulatory cells, myeloid-derived suppressor cells). As used herein, the term “hot tumor” refers to a tumor characterized by 1) high T cell infiltration; 2) increased interferon-γ signaling; 3) expression of PD-L1 and / or 4) high tumor mutational load.

[0114] This disclosure further provides a method for treating cancer in a subject with a cold tumor, the method comprising: administering a therapeutically effective amount of a GREM1 antagonist to the subject.

[0115] In some implementations, the object is resistant to or refractory to anticancer therapies, such as immunotherapy, for example, immune checkpoint inhibitors.

[0116] In some embodiments, the method further includes administering one or more therapies to the subject. In some embodiments, the one or more therapies are capable of promoting T cell proliferation, activation, and / or tumor invasion. In some embodiments, the T cells are CD3+ cells. + T cells or CD8 + T cells. In some embodiments, the one or more therapies are anti-angiogenic therapy, immunotherapy, and / or chemotherapy.

[0117] II. Combination Therapy This disclosure provides a method for treating cancers expressing GREM1 in subjects of need. In some embodiments, the method includes administering a therapeutically effective amount of a GREM1 antagonist in combination with a second therapy.

[0118] On the other hand, this disclosure provides a method for treating cancers expressing GREM1 in a subject in need. In some embodiments, the method includes 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.

[0119] On the other hand, this disclosure provides the use of a GREM1 antagonist in the preparation of a medicament / medical composition for treating cancers expressing GREM1 in a subject of need, wherein the treatment comprises administering the medicament / medical composition 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.

[0120] 1. Immunotherapy In some embodiments, the immunotherapy used in the methods provided herein includes a PD-1 / PD-L1 axis inhibitor. The term "PD-1 / PD-L1 axis inhibitor" refers to a molecule (e.g., a small molecule, antibody, etc.) that inhibits the interaction between PD-1 / PD-L1 axis-binding ligands (such as PD-1 and PD-L1) to reduce or eliminate the inhibitory effects of T cell functions (e.g., proliferation, cytokine production, and target cell killing) resulting from signaling along the PD-1 / PD-L1 signaling axis. PD-1 / PD-L1 axis inhibitors may include either PD-1 inhibitors or PD-L1 inhibitors.

[0121] In some embodiments, PD-1 / PD-L1 axis inhibitors include PD-1 inhibitors. As used herein, the term "PD-1 inhibitor" refers to a molecule that reduces, eliminates, inhibits, blocks, or interferes with signal transduction resulting from the interaction of PD-1 with one or more binding ligands (e.g., PD-L1). In some embodiments, a PD-1 inhibitor is a molecule that blocks the binding of PD-1 to its binding ligand (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, eliminate, inhibit, block, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1.

[0122] In some implementations, the PD-1 inhibitor is an anti-PD-1 antibody selected from the group consisting of: nivolumab (OPDIVO; BMS-936558), dotalimab (TSR-042), pembrolizumab (KEYTRUDA; MK-3475), MEDI0680 (AMP-514), MEDI4736, BI 754091, pildizumab (CT-011), cimiprimab (LIBTAYO; REGN2810), spartazolizumab (PDR001), and cilizumab (JNJ). 63723283), toripalimab (JS001), PF-06801591, tislelizumab (BGB-A317), AMP-224 (GSK-2661380), ABBV-181, ramborizumab, camrelizumab (SHR-1210), sintilimab (Tyvyt, IBI308), penaprilimab (AK105), cepalimab, refulimab, slulilimab, bartilimab, genolimab, pugulimab, itazolizumab, sasanlimab, pivimab, buglimab, novalimab, sintolizumab, MGA404, Sym021, BAT1306 and HX008.

[0123] In some implementations, the PD-1 inhibitor is nivolumab. Nivolumab (Bristol-Myers Squibb / Ono) is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO. ® This is the anti-PD-1 antibody described in WO2006 / 121168.

[0124] In some embodiments, the anti-PD-1 antibody used in the methods provided herein comprises a heavy chain sequence 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.

[0125] In some embodiments, the anti-PD-1 antibody used in the methods 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 methods 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 methods 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.

[0126] In some embodiments, the anti-PD-1 antibody used in the methods provided herein comprises heavy chain HCDR1, HCDR2, and HCDR3 and / or light chain LCDR1, LCDR2, and LCDR3 sequences, wherein: the HCDR1 sequence comprises SEQ ID NO: 11 or a homologous sequence having at least 80% sequence identity with it; the HCDR2 sequence comprises SEQ ID NO: 12 or a homologous sequence having at least 80% sequence identity with it; the HCDR3 sequence comprises SEQ ID NO: 13 or a homologous sequence having at least 80% sequence identity with it; the LCDR1 sequence comprises SEQ ID NO: 14 or a homologous sequence having at least 80% sequence identity with it; the LCDR2 sequence comprises SEQ ID NO: 15 or a homologous sequence having at least 80% sequence identity with it; and the LCDR3 sequence comprises SEQ ID NO: 16 or a homologous sequence having at least 80% sequence identity with it.

[0127] In some embodiments, the anti-PD-1 antibody (e.g., nivolumab) used in the methods provided herein is administered intravenously at 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 at 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 according to institutional guidelines, published guidelines, and relevant product prescribing information, and according to this regimen.

[0128] In some embodiments, the PD-1 / PD-L1 axis inhibitor comprises a PD-L1 inhibitor selected from antibodies, small molecules, and combinations thereof. In some embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody selected from the group consisting of: atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446), BMS-936559, avermab (bavencio), lodalimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ05. 3. Envorimab (KN035), MDX-1105, STI-1040, CS1001, Adiberismab (SHR-1316), SHR-1701, TOB2450, Bintrafusp, LP002, STI-3031, Cochilimab, Parkimilimab, NM01, LDP, AMP-224, Grelimab (BGB-A333), A167, SCD-135, Oplacimab, and GR1405.

[0129] In some embodiments, the anti-PD-1 antibody used in the methods provided herein is administered intravenously at 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 at 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.

[0130] 2. Chemotherapy In some embodiments, the chemotherapy used in the methods provided herein is a chemotherapy regimen (or a combination of chemotherapy agents). In some embodiments, the chemotherapy used in the methods provided herein includes a combination of chemotherapy agents. The term "chemotherapy agent" is a biological (macromolecule) or chemical (small molecule) compound that can be used to treat cancer. Types 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, kinase inhibitors, etc. Examples of chemotherapy drugs include erlotinib, afatinib, docetaxel, doxorubicin, 5-FU (5-fluorouracil), panobinostat, gemcitabine, cisplatin, pemetrexed, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, and metformin. Formin, temozolomide, tamoxifen, oteracil, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, trametinib, tegafur, gimeracil, leucovorin (LV), irinotecan hydrochloride (CPT-11), platinum (e.g., cisplatin), epirubicin, oxaliplatin, and capecitabine. In some embodiments, 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 some embodiments, the combination of chemotherapy agents includes LV, 5-FU, and CPT-11.

[0131] In some embodiments, 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 drug combination for FOLFIRI chemotherapy includes or consists of: LV, 5-FU, and CPT-11. The drug combination for EOX chemotherapy includes or consists of: epirubicin, oxaliplatin, and capecitabine. The drug combination for ECF chemotherapy includes or consists of: epirubicin, cisplatin, and 5-FU. The drug combination for ECX chemotherapy includes or consists of: epirubicin, oxaliplatin, and capecitabine. The drug combination for EOF chemotherapy includes or consists of: epirubicin, oxaliplatin, and 5-FU. The drug combination for FLO chemotherapy includes or consists of the following: 5-FU, LV, and oxaliplatin. The drug combination for SOX chemotherapy includes or consists of the following: tegafur, gemmelaxyl, otetracil, and oxaliplatin.

[0132] In some implementations, chemotherapy includes 20 mg / m². 2 Up to 400 mg / m 2 (For example, 40 mg / m 2 60 mg / m 2 80 mg / m 2 100 mg / m 2 120 mg / m 2 140 mg / m 2 160 mg / m 2 180 mg / m 2 200 mg / m 2 220 mg / m 2 240 mg / m 2 300 mg / m 2 340 mg / m 2 380 mg / m 2 Or 400 mg / m 2 CPT-11. In some embodiments, chemotherapy includes 20 mg / m². 2 Up to 400 mg / m 2 (For example, 40 mg / m 2 60 mg / m 2 80 mg / m 2 100 mg / m 2 120 mg / m2 140 mg / m 2 160 mg / m 2 180 mg / m 2 200 mg / m 2 220 mg / m 2 240 mg / m 2 300 mg / m 2 340 mg / m 2 380 mg / m 2 Or 400 mg / m 2 LV. In some implementations, chemotherapy comprises a single bolus dose of 100 mg / m². 2 Up to 800 mg / m 2 (For example, 100 mg / m 2 120 mg / m 2 140 mg / m 2 160 mg / m 2 180 mg / m 2 200 mg / m 2 220 mg / m 2 240 mg / m 2 300 mg / m 2 340 mg / m 2 380 mg / m 2 400 mg / m 2 500 mg / m 2 600 mg / m 2 700 mg / m 2 Or 800 mg / m 2 ) of 5-FU.

[0133] In some embodiments, the chemotherapy used in the methods provided herein is the FOLFIRI regimen. In some embodiments, the FOLFIRI regimen consists of the following: on day one, an infusion of 180 mg / m². 2 CPT-11 (infusion time 90 minutes), and during CPT-11 infusion, 200 mg / m² should be administered. 2 LV (infusion time 90 minutes), followed by a single infusion of 400 mg / m². 2 5-FU and continuous infusion of 2,400 mg / m² 2 5-FU (infusion over 46 hours), administered as described above every two weeks. In some embodiments, the recommended dosing schedule for FOLFIRI every two weeks is as follows: Day 1: 180 mg / m² 2CTP-11 IV infusion and 400 mg / m² 2 LV IV infusion, followed by 400 mg / m² 2 5-FU intravenous bolus, followed by 2400 mg / m². 2 5-FU IV infusion (continuous infusion over 46 hours). Several different FOLFIRI regimens vary in the dosage and route of administration of the three drugs.

[0134] The other chemotherapy regimens described above are described in PCT patent application PCT / JP2022 / 017017, which is incorporated herein by reference.

[0135] 3. Anti-angiogenic therapy In some embodiments, the anti-angiogenic therapy used in the methods provided herein includes an anti-angiogenic agent that can inhibit the growth of blood vessels supporting tumor growth. Some of these 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 an antagonist of vascular endothelial growth factor A (VEGFA). In some embodiments, the VEGFA antagonist is an anti-VEGFA antibody, such as bevacizumab (Avastin). ® ).

[0136] In some embodiments, the anti-angiogenic therapies used in the methods provided herein include antagonists of VEGFR, such as VEGFR-1, VEGFR-2, and VEGFR-3. The term “VEGFR antagonist” is used interchangeably with the term “VEGFR inhibitor,” which is a molecule (e.g., a small molecule or a large molecule (e.g., an antibody) that inhibits the interaction between VEGFR and VEGF ligands (e.g., tumor-secreted ligands, such as those in solid tumors), thereby inhibiting angiogenesis and blocking tumor blood supply.

[0137] In some embodiments, the VEGFR antagonist is an anti-VEGFR-2 antibody. In some embodiments, the anti-VEGFR-2 antibody is selected from the group consisting of: ramucirumab, ovasiximab, montuximab, pego-araciizumab, furincimab, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, and HLX12.

[0138] In some embodiments, the VEGFR antagonist (or VEGFR inhibitor) is a small molecule VEGFR inhibitor selected from the group consisting of: cetravatinib, anlotinib, apatinib, tellatinib, ateratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, and axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) and Regorafenib (Stivarga ® ).

[0139] In some embodiments, the anti-angiogenic therapy used in the methods provided herein (e.g., bevacizumab (Avastin)) ® ), Ramucirumab, Ovaximab, Cimetuzumab, Pego-Alasicizumab, Flinximab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Cetravatinib, Anlotinib, Apatinib, Tellatinib, Ateratinib, Canitinib, Lenvatinib Mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) or Regorafenib (Stivarga ® )) Administer intravenously at 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; or intravenously at doses of 2 mg / kg, 4 mg / kg, 6 mg / kg, 8 mg / kg, 10 mg / kg, 12 mg / kg, or 14 mg / kg. The anti-angiogenic therapy used in the methods described herein (e.g., bevacizumab (Avastin)) ®), Ramucirumab, Ovaximab, Cimetuzumab, Pego-Alasicizumab, Flinximab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Cetravatinib, Anlotinib, Apatinib, Tellatinib, Ateratinib, Canitinib, Lenvatinib Mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) or Regorafenib (Stivarga ® It can be administered intravenously according to institutional guidelines, published guidelines and relevant product prescription information, and the dosage should be followed accordingly.

[0140] 4. Specific combination therapies In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist and a VEGFR inhibitor (e.g., bevacizumab (Avastin)). ® ), Ramucirumab, Ovaximab, Cimetuzumab, Pego-Alasicizumab, Flinximab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Cetravatinib, Anlotinib, Apatinib, Tellatinib, Ateratinib, Canitinib, Lenvatinib Mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) or Regorafenib (Stivarga ® A combination of [various methods]. In some embodiments, the GREM1 antagonist comprises the anti-GREM1 antibody or its antigen-binding fragment provided herein. In some embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or its humanized variants (e.g., hu14E3, hu14E3 HaLa).

[0141] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist and a VEGFR inhibitor (e.g., bevacizumab (Avastin)). ®), Ramucirumab, Ovaximab, Cimetuzumab, Pego-Alasicizumab, Flinximab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Cetravatinib, Anlotinib, Apatinib, Tellatinib, Ateratinib, Canitinib, Lenvatinib Mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) or Regorafenib (Stivarga ® The combination of the cancers, and the cancers described therein are colorectal cancer, stomach cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0142] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the chemotherapeutic agent (or chemotherapy regimen) (e.g., FOLFIRI) and a VEGFR inhibitor (e.g., bevacizumab (Avastin®), ramucirumab, ovavasirumab, montuximab, pego-araciizumab, furincimab, regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, cetravatinib, anlotinib, apatinib, tellatinib, ateratinib, canitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) or Regorafenib (Stivarga ® A combination of [various methods]. In some embodiments, the GREM1 antagonist comprises the anti-GREM1 antibody or its antigen-binding fragment provided herein. In some embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or its humanized variants (e.g., hu14E3, hu14E3 HaLa).

[0143] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the chemotherapeutic agent (or chemotherapy regimen) (e.g., FOLFIRI) and a VEGFR inhibitor (e.g., bevacizumab (Avastin)). ®), Ramucirumab, Ovaximab, Cimetuzumab, Pego-Alasicizumab, Flinximab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Cetravatinib, Anlotinib, Apatinib, Tellatinib, Ateratinib, Canitinib, Lenvatinib Mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) and / or Regorafenib (Stivarga ® The combination of the cancers, and the cancers described therein are colorectal cancer, stomach cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0144] In some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the chemotherapy agent (e.g., FOLFIRI) and a PD-1 / PD-L1 axis inhibitor (e.g., nivolumab). In some embodiments, the GREM1 antagonist comprises the anti-GREM1 antibody or its antigen-binding fragment provided herein. In some embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or its humanized variant (e.g., hu14E3, hu14E3 HaLa).

[0145] In some embodiments, the method includes administering to the subject a therapeutically effective amount of a combination of the GREM1 antagonist and the chemotherapeutic agent (e.g., FOLFIRI) and the PD-1 / PD-L1 axis inhibitor (e.g., nivolumab), and 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 some embodiments, the method includes administering to the subject a therapeutically effective amount of the GREM1 antagonist and a VEGFR inhibitor (e.g., bevacizumab (Avastin)). ® ), Ramucirumab, Ovaximab, Cimetuzumab, Pego-Alasicizumab, Flinximab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Cetravatinib, Anlotinib, Apatinib, Tellatinib, Ateratinib, Canitinib, Lenvatinib Mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib (Inlyta ®Cabometyx ® fruquintinib (ELUNATE) ® ) or Regorafenib (Stivarga ® This can be a combination of a chemotherapeutic agent (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 an antigen-binding fragment thereof as provided herein. In some embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or a humanized variant thereof (e.g., hu14E3, hu14E3 HaLa).

[0147] In some embodiments, the method includes administering to the subject a therapeutically effective amount of a combination of the GREM1 antagonist and a VEGFR inhibitor (e.g., bevacizumab (Avastin)). ® ), Ramucirumab, Ovaximab, Cimetuzumab, Pego-Alasicizumab, Flinximab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Cetravatinib, Anlotinib, Apatinib, Tellatinib, Ateratinib, Canitinib, Lenvatinib Mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib (Inlyta ® Cabometyx ® fruquintinib (ELUNATE) ® ) or Regorafenib (Stivarga ® The combination of chemotherapy agents (e.g., FOLFIRI) and the 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. Cancers expressing GREM1 As used in this article, “GREM1-expressing cancer” refers to any cancer or tumor involving cancer cells that express or secrete GREM1 (e.g., low, moderate, or high expression).

[0149] 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 the methods or antibodies provided herein. In some embodiments, the object is further identified as having low, intermediate, or high GREM1 expression in diseased tissues of cancer expressing GREM1.

[0150] In some implementations, the object is identified as having GREM1 expression in diseased tissue (e.g., the presence of GREM1 or an expression level of GREM1 above a threshold level). As used herein, "threshold level" refers to an expression level of GREM1 with an intensity of 1+, 2+, or 3+ as measured by IHC.

[0151] In some implementations, GREM1 expression is determined from diseased tissue (e.g., from biological samples).

[0152] The presence and / or expression level of GREM1 in diseased tissues (e.g., cancerous or tumor tissues) can be determined by various methods known in the art. In some embodiments, the biological sample may be further processed to, for example, isolate analytes such as nucleic acids or proteins. The presence and / or expression level of GREM1 can be determined by, for example, quantitative fluorescent blood cell counting, 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 the expressed GREM1 protein.

[0153] In some embodiments, the expression of GREM1 in diseased tissues (e.g., cancerous or tumor tissues) is determined or measured by IHC. In some embodiments, the expression level of human GREM1 protein in cancerous or tumor tissues from a subject can be determined according to the method described in Example 4 provided herein.

[0154] In some embodiments, the object is identified or has been identified as having high GREM1 expression in diseased tissue (e.g., cancerous or tumor tissue) derived from the object. In some embodiments, GREM1 expression in the diseased tissue is identified or has been identified as higher than or comparable to expression in healthy or non-cancerous cells. High GREM1 expression in a biological sample, such as diseased tissue (e.g., cancerous or tumor tissue), is defined as a GREM1 expression intensity of at least 2+ (e.g., 2+ or 3+ as measured by IHC).

[0155] In some implementations, the object is identified or has been identified as having moderate GREM1 expression in diseased tissue (e.g., cancerous or tumor tissue) derived from the object. Moderate GREM1 expression in a biological sample, such as diseased tissue (e.g., cancerous or tumor tissue), means that the expression intensity of GREM1 is at least 1+ and less than 2+ as measured by IHC.

[0156] In some embodiments, the object is identified or has been identified as having low GREM1 expression in diseased tissue (e.g., cancerous or tumor tissue) derived from the object. In some embodiments, the diseased tissue is identified or has been identified as having GREM1 expression comparable to that in healthy tissue and can be detected by an anti-GREM1 diagnostic reagent. Low GREM1 expression in a biological sample, such as diseased tissue (e.g., cancerous or tumor tissue), is defined as a GREM1 expression intensity greater than 0 but less than 1+ degrees, as measured by IHC.

[0157] 6. Cancers expressing GREM1 that are resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors and / or have low or no PD-L1 expression. In some embodiments, the cancer expressing GREM1 is resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer (i.e., low or no PD-L1 expression).

[0158] This publication unexpectedly found that treatment of tumors with GREM1 antagonists led to upregulation of programmed death ligand 1 (PD-L1) expression in tumors. PD-L1 is a protein that interacts with programmed death protein 1 (PD-1) and is expressed on immune cells and tumor cells, such as in tumor tissues. Studies have investigated the correlation between tumor PD-L1 expression and the efficacy of anti-PD-1 antibody therapy, and have shown that PD-L1 overexpression is associated with significantly higher objective response rates (ORR). Gettinger et al., nivolumab (anti-programmed death 1 antibody, BMS-936558, ONO-4538) in patients with previously treated Overall survival and long-term safety in patients with advanced non-small cell lung cancer ( Overall 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 ) Journal of Clinical Oncology (J Clin Oncol.) 2015;33:2004-12.; Garon et al., for the treatment of non-small cell lung cancer. Pembrolizumab for the treatment of non-small-cell lung cancer (Cancer) *The New England Journal of Medicine* 2015;372:2018–28. doi: 10.1056 / NEJMoa1501824. Public records and reports also indicate that cancer patients with low PD-L1 expression may benefit less from treatment with PD-1 / PD-L1 axis inhibitors.

[0159] Based at least in part on the findings described above, the methods presented herein have unexpected effects in treating cancers that express GREM1, which are resistant or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or are identified as having low or no PD-L1 expression in disease tissues of the cancer (i.e., low or no PD-L1 expression).

[0160] As used in this article, “resistance” refers to the lack of response of a disease or symptom to treatment (which can be natural non-response or treatment-induced non-response).

[0161] As used herein, “refractory” refers to the resistance or lack of response of a disease or symptom to treatment (e.g., an increase in the number of vegetation cells even with treatment). Unless otherwise stated, the term “refractory” refers to resistance or lack of response to any prior treatment with a PD-1 / PD-L1 axis inhibitor.

[0162] PD-L1 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). The methods described above for detecting GREM1 expression can also be used to detect PD-L1 expression, the difference being that the assay reagents should be replaced with reagents used for detecting PD-L1.

[0163] In some embodiments, PD-L1 expression can be determined using the method described in PCT / CN2022 / 131820, which is incorporated herein by reference. In some embodiments, PD-L1 expression can be determined according to the method described in Example 4 provided herein. Throughout this specification, the reagent used to detect PD-L1 expression can be an anti-PD-L1 diagnostic antibody, such as 22C3, as described in US20170285037A1, the disclosure of which is incorporated herein by reference in its entirety, and a commercially available monoclonal rabbit anti-PD-L1, namely clone 28-8. In some embodiments, throughout this specification, the reagent used to detect PD-L1 expression is a polypeptide comprising the antigen-binding portion of 22C3, as described in US20170285037A1, the disclosure of which is incorporated herein by reference in its entirety, and a commercially available monoclonal rabbit anti-PD-L1, namely clone 28-8.

[0164] As used in this article, the term “low PD-L1 expression” refers to a PD-L1 expression level that is below or does not exceed the reference level.

[0165] The term "reference level" in relation to PD-L1 expression refers to a threshold (e.g., minimum) level of PD-L1 expression in a biological sample (such as diseased tissue (e.g., cancerous or tumor tissue)) derived from a subject that has responded to treatment with a PD-1 / PD-L1 axis inhibitor.

[0166] In some implementations, cancers with low PD-L1 expression, such as those measured by IHC using antibody 22C3 or antibody clone 28-8, have an intensity of no more than 1+ or no more than 2+, or a PD-L1 positivity percentage in tumor cells of no more than 5% in CPS.

[0167] As used herein, the term "no PD-L1 expression" means a PD-L1 expression level below a baseline threshold level. In some implementations, biological samples without PD-L1 expression (e.g., cancer cells) lack any detectable PD-L1 signal, as can be detected by anti-PD-L1 diagnostic antibodies using proven techniques such as IHC.

[0168] The term “baseline threshold level” for PD-L1 expression refers to the threshold detectable expression level of PD-L1 in a biological sample.

[0169] PD-L1 expression can be measured using the method described in PCT / CN2022 / 131820, which is incorporated herein by reference. The threshold expression level for PD-L1 expression may vary for different tumor types and / or when using different PD-L1 assays, and can be determined using methods known in the art.

[0170] In some embodiments, the PD-L1 expression in the diseased tissue is low or cannot be detected by anti-PD-L1 diagnostic antibodies.

[0171] In some embodiments, the method provided herein includes administering a therapeutically effective amount of a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor to the subject. In some embodiments, the method includes administering the GREM1 antagonist to the subject, and then administering the PD-1 / PD-L1 axis inhibitor to the subject after a period of time sufficient to increase PD-L1 expression levels in the subject's cancer tissue / cancer cells. In some embodiments, the GREM1 antagonist has been administered to the subject for a period of time sufficient to increase PD-L1 expression levels in the subject's cancer tissue / cancer cells.

[0172] In some embodiments, the cancer expressing GREM1 is colorectal cancer, gastric cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0173] In some implementations, the object is a person.

[0174] 7. Pharmaceutical composition and route of administration The aforementioned GREM1 antagonists, chemotherapeutic agents, immunotherapeutic agents, or antiangiogenic agents can each be administered in the form of any suitable pharmaceutical composition. The term "pharmaceutical composition" refers to a formulation comprising a therapeutically effective agent (e.g., the aforementioned GREM1 antagonists, chemotherapeutic agents, immunotherapeutic agents, or antiangiogenic agents), preferably together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition can be used to treat, prevent, or reduce the severity of a disease or condition by administering the pharmaceutical composition to a subject.

[0175] Pharmaceutical compositions are typically provided in homogeneous dosage forms and can be prepared in ways known in the art. Pharmaceutical compositions may be, for example, liquid dosage forms, such as solutions or suspensions, or solid dosage forms, such as tablets and capsules. The pharmaceutical compositions described herein are generally applied in terms of “therapeuticly effective amounts” and “pharmaceutically acceptable preparations.” As used herein, the term “pharmaceuticalally acceptable” means the non-toxicity of materials that do not interact with the action of the active component of the pharmaceutical composition.

[0176] The pharmaceutical compositions described herein may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions disclosed herein include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0177] In some embodiments, the administration is performed orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

[0178] In some embodiments, the administration of the GREM1 antagonist is performed before, simultaneously with, or after the administration of the anti-angiogenic agent, and / or the combination of chemotherapeutic agents and / or the immunotherapy.

[0179] In some embodiments, the method includes administering the GREM1 antagonist to the subject, and then administering the PD-1 / PD-L1 axis inhibitor to the subject after a period of time sufficient to increase PD-L1 expression levels in the subject's cancer tissue / cells. In some embodiments, the GREM1 antagonist has already been administered to the subject for a period of time sufficient to increase PD-L1 expression levels in the subject's cancer tissue / cancer cells.

[0180] III. GREM1 as a biomarker for immunotherapy eligibility On the other hand, this disclosure provides a method for determining eligibility or responsiveness to treatment in subjects using a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor, the method comprising: b) Determine the presence or expression level of GREM1 in biological samples of the diseased tissue of the subject. The presence or absence of GREM1 or its expression level indicates whether the subject is likely to be eligible for treatment with the combination of the GREM1 antagonist and the PD-1 / PD-L1 axis inhibitor or whether the subject is likely to respond to the treatment.

[0181] In some implementations, the presence of GREM1 determined in step a) or the expression level of GREM1 being above a threshold level indicates that the subject may be eligible for treatment with the combination of the GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor or may be responsive to the treatment.

[0182] In some implementations, the absence of GREM1 or the expression level of GREM1 not exceeding a threshold level as determined in step a) indicates that the subject is not eligible for treatment with the combination of the GREM1 antagonist and the PD-1 / PD-L1 axis inhibitor or is unlikely to respond to the treatment.

[0183] In some implementations, the method further includes step i) prior to step a). i) Contact the sample with a GREM1 diagnostic agent under conditions that allow detection of the expression level of GREM1 in the biological sample of the diseased tissue of the subject.

[0184] In some embodiments, a) the subject has been identified as resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) the subject has been identified as having low or no PD-L1 expression in diseased tissues. In some embodiments, the subject has been identified as having intermediate or high PD-L1 expression in diseased tissues.

[0185] In some embodiments, the method further includes administering a therapeutically effective amount of the GREM1 antagonist to the subject for a duration sufficient to increase the expression level of PD-L1 in the subject's cancerous tissue / cells for a certain period of time, and then administering the PD-1 / PD-L1 axis inhibitor to the subject.

[0186] In some embodiments, GREM1 expression is detected using a GREM1 diagnostic reagent. In some embodiments, the GREM1 diagnostic reagent / pharmaceutical comprises a polypeptide comprising an antigen-binding portion of an anti-GREM1 antibody or an antigen-binding fragment thereof. In some embodiments, the GREM1 diagnostic reagent / pharmaceutical comprises an anti-GREM1 antibody or an antigen-binding fragment thereof, such as the anti-GREM1 antibody or antibody-binding fragment thereof provided herein (e.g., 14E3 or its humanized variant, such as hu14E3HaLa). Detailed embodiments of the detection methods are described in the following sections.

[0187] On the other hand, this disclosure provides a method for improving the responsiveness of a subject to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject has been identified as having GREM1 in a biological sample of the subject's disease tissue, or the subject has been identified as having GREM1 expression at a threshold level in the biological sample of the disease tissue, the method comprising: a) administering a therapeutically effective amount of a GREM1 antagonist to the subject to increase PD-L1 expression in the disease tissue, thereby improving the subject's responsiveness to the PD-1 / PD-L1 axis inhibitor.

[0188] In some embodiments, a) the subject has been identified as resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) the subject has been identified as having low or no PD-L1 expression in diseased tissue. In some embodiments, the subject has been identified as having intermediate or high PD-L1 expression in diseased tissue. PD-L1 expression can be determined using the method described in PCT / CN2022 / 131820, which is incorporated herein by reference. In some embodiments, PD-L1 expression can be determined according to the method described in Example 4 provided herein. Throughout this specification, reagents used for detecting PD-L1 expression can be anti-PD-L1 diagnostic antibodies, such as 22C3, as described in US20170285037A1, the disclosure of which is incorporated herein by reference in its entirety, and commercially available monoclonal rabbit anti-PD-L1, namely clone 28-8. In some embodiments, throughout the specification, the reagent used to detect PD-L1 expression is a polypeptide comprising the antigen-binding moiety of 22C3, as described in US20170285037A1, the disclosure of which is incorporated herein by reference in its entirety, and a commercially available monoclonal rabbit anti-PD-L1, namely clone 28-8.

[0189] In some embodiments, the diseased tissue is cancerous tissue. In some embodiments, the cancer is colorectal cancer, stomach cancer, lung cancer, endometrial cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.

[0190] In some embodiments, the method further includes administering a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor to the subject after PD-L1 expression has increased in the subject's diseased tissue.

[0191] On the other hand, this disclosure provides the use of a GREM1 antagonist in the preparation of a medicament for improving the responsiveness of a subject to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject has been identified as having GREM1 present in a biological sample of the subject's disease tissue, or the subject has been identified as having GREM1 expression at a threshold level in the biological sample of the disease tissue, wherein the improvement comprises administering a therapeutically effective amount of the GREM1 antagonist to the subject, thereby increasing PD-L1 expression in the disease tissue and thereby improving the subject's responsiveness to the PD-1 / PD-L1 axis inhibitor.

[0192] On the other hand, this disclosure provides the use of an anti-GREM1 diagnostic reagent in the preparation of a kit for determining in a subject the eligibility or responsiveness to treatment with a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor, wherein the anti-GREM1 diagnostic reagent is capable of determining the presence or expression level of GREM1 in a biological sample of the subject’s diseased tissue, wherein the presence or absence or expression level of GREM1 indicates whether the subject may be eligible for treatment with the combination of the GREM1 antagonist and the PD-1 / PD-L1 axis inhibitor or whether the subject may respond to the treatment.

[0193] 1. Determine GREM1 expression The methods or uses provided herein relate to determining GREM1 expression. In some embodiments, the object is determined to have GREM1 expression in diseased tissue (e.g., the presence of GREM1 or an expression level of GREM1 above a threshold level). As used herein, a “threshold level” refers to an expression level of GREM1 with an intensity of 1+, 2+, or 3+ as measured by IHC.

[0194] Suitable methods known in the art can be used, and these methods are described in detail below.

[0195] i. Sample preparation In some embodiments, the object is a human being. In some embodiments, the method provided herein further includes providing a biological sample from the object, wherein the biological sample includes diseased tissue (e.g., cancerous tissue or tumor tissue).

[0196] Any biological sample suitable for performing the methods described herein can be obtained from the object. As used herein, "biological sample" means a biological sample obtained by sampling from the object, optionally with additional processing. Sample collection from the object is performed according to standard protocols generally followed in hospitals or clinics, such as during a biopsy.

[0197] In some embodiments, the sample may be a biological sample comprising cancer cells or non-cancer cells (e.g., mesenchymal fibroblasts). For example, the non-cancer cells may originate from the same tissue or organ as the cancer cells found. In some embodiments, a biological sample containing or suspected of containing cancer cells may be obtained from the object. In some embodiments, the biological sample may be derived from cancer cells or cancerous tissue or tumor-infiltrating immune cells. In some embodiments, the biological sample is tumor tissue.

[0198] In some embodiments, the biological sample is a fresh or archived sample obtained from tumor tissue, for example, by tumor biopsy or fine-needle aspiration. In some embodiments, the sample can be any biological fluid containing cancer cells or non-cancer cells (e.g., peripheral blood mononuclear cells (PBMCs)).

[0199] 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, pleural effusion, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, etc., and tissues such as biopsy tissues (e.g., biopsied 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 tissues. In another embodiment, biological samples include cells, tissues, blood, plasma, serum, urine, mouthwash, feces, saliva, and any combination thereof.

[0200] In some implementations, the sample may be further processed using desired methods for determining the expression level of at least one biomarker, such as GREM1.

[0201] ii. Determination of GREM1 expression In some implementations, GREM1 expression is determined from diseased tissue (e.g., from biological samples).

[0202] The presence and / or expression level of GREM1 in diseased tissues (e.g., cancerous or tumor tissues) can be determined by various methods known in the art. In some embodiments, the biological sample may be further processed to, for example, isolate analytes such as nucleic acids or proteins. The presence and / or expression level of GREM1 can be determined by, for example, quantitative fluorescent blood cell counting, 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 the expressed GREM1 protein.

[0203] In some embodiments, the expression of GREM1 in diseased tissues (e.g., cancerous or tumor tissues) is determined or measured by IHC. In some embodiments, the expression level of human GREM1 protein on cancerous or tumor tissues from a subject can be determined according to the method described in Example 4 provided herein.

[0204] In some embodiments, the object is identified or has been identified as having high GREM1 expression in diseased tissue (e.g., cancerous or tumor tissue) derived from the object. In some embodiments, GREM1 expression in the diseased tissue is identified or has been identified as higher than or comparable to expression in healthy or non-cancerous cells. High GREM1 expression in a biological sample, such as diseased tissue (e.g., cancerous or tumor tissue), is defined as a GREM1 expression intensity of at least 2+ (e.g., 2+ or 3+ as measured by IHC).

[0205] In some implementations, the object is identified or has been identified as having moderate GREM1 expression in diseased tissue (e.g., cancerous or tumor tissue) derived from the object. Moderate GREM1 expression in a biological sample, such as diseased tissue (e.g., cancerous or tumor tissue), means that the expression intensity of GREM1 is at least 1+ and less than 2+ as measured by IHC.

[0206] In some embodiments, the object is identified or has been identified as having low GREM1 expression in diseased tissue (e.g., cancerous or tumor tissue) derived from the object. In some embodiments, the diseased tissue is identified or has been identified as having GREM1 expression comparable to that in healthy tissue and can be detected by an anti-GREM1 diagnostic reagent. Low GREM1 expression in a biological sample, such as diseased tissue (e.g., cancerous or tumor tissue), is defined as a GREM1 expression intensity greater than 0 but less than 1+, as measured by IHC.

[0207] IV. GREM1 antagonists The GREM1 antagonist used in the methods provided herein is capable of inducing PD-L1 expression in the diseased tissues of the subject. In some embodiments, the GREM1 antagonist used in the methods provided herein comprises an anti-GREM1 antibody or an antigen-binding fragment thereof, such as any one 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 properties equivalent to those of 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.

[0208] In some embodiments, the GREM1 antagonist used in the methods provided herein comprises the antigen-binding portion of Hu14E3 HaLa. In some embodiments, the GREM1 antagonist used in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are identical to those of Hu14E3 HaLa. In some embodiments, the GREM1 antagonist used in the methods provided herein comprises heavy chains HCDR1, HCDR2, and HCDR3 and / or light chains LCDR1, LCDR2, and LCDR3, wherein the heavy chains HCDR1, HCDR2, and HCDR3 and / or the light chains LCDR1, LCDR2, and LCDR3 are identical to those of Hu14E3 HaLa.

[0209] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises heavy chains HCDR1, HCDR2, and HCDR3 and / or light chains LCDR1, LCDR2, and LCDR3, wherein: HCDR1 comprises an amino acid sequence containing TYGMA (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity with it; HCDR2 comprises an amino acid sequence containing WINTLSGEPTYADDFKG (SEQ ID NO: 2) or a homologous sequence having at least 80% sequence identity with it; HCDR3 comprises an amino acid sequence containing EPMDY (SEQ ID NO: 3) or a homologous sequence having at least 80% sequence identity with it; LCDR1 comprises an amino acid sequence containing KSSQSLLDSDGKTYLS (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity with it; LCDR2 comprises an amino acid sequence containing LVSKLDS (SEQ ID NO: 5) or a homologous sequence having at least 80% sequence identity with it; and LCDR3 comprises an amino acid sequence containing WQGAHFPLT (SEQ ID NO: 5). 6) The amino acid sequence of or a homologous sequence that has at least 80% sequence identity with it.

[0210] It is known that CDRs are responsible for antigen binding; however, it has been found that not all six CDRs are necessarily essential or immutable. In other words, one, two, or three CDRs in an anti-GREM1 antibody can be substituted, altered, or modified, while still essentially retaining the specific binding affinity for GREM1.

[0211] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises the heavy chain CDR3 sequence of EPMDY (SEQ ID NO: 3). The heavy chain CDR3 region is located at the center of the antigen-binding site and is therefore considered to be in the most contact with the antigen, providing the most free energy for the antibody's affinity for the antigen. It is also believed that the heavy chain CDR3 is the most diverse CDR to date in terms of length, amino acid composition, and conformation of the antigen-binding site through a variety of diverse mechanisms (Tonegawa S. Nature 302:575-81). This diversity of the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity 13:37-45) and desirable antigen-binding affinity (Schier R et al., Journal of Molecular Biology 263:551-67).

[0212] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment comprises all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-GREM1 antibody is a single-domain antibody composed of all or a portion of the heavy chain variable domain provided herein. More information about such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0213] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 8.

[0214] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment further includes one or more amino acid residue substitutions or modifications, but still retains specific binding specificity or affinity for GREM1 (e.g., human GREM1, i.e., hGREM1).

[0215] In some embodiments, at least one of the substitutions or modifications substitutes or modifies one or more CDR sequences in the CDR sequence and / or one or more non-CDR regions in the non-CDR regions of the VH or VL sequence.

[0216] In some embodiments, the anti-GREM1 antibody or its antigen-binding fragment further includes an immunoglobulin constant region, optionally a constant region of human IgG. In some embodiments, 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 comprising the sequence of SEQ ID NO: 9 and / or a light chain constant region comprising the sequence of SEQ ID NO: 10.

[0217] In some embodiments, the anti-GREM1 antagonist is linked to one or more conjugate moieties. In some embodiments, the conjugate moieties include scavenging modifiers, therapeutic agents (e.g., chemotherapeutic agents), toxins, radioisotopes, detectable labels (e.g., lanthanides, luminescent labels, fluorescent labels, or enzyme-substrate labels), pharmacokinetic modifying moieties, DNA alkylating agents, topoisomerase inhibitors, tubulin binding agents, and other anticancer drugs (such as androgen receptor inhibitors), as described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference.

[0218] V. GREM1 Diagnostic Reagent / Pharmaceutical The GREM1 diagnostic reagents / pharmaceuticals used in the methods provided herein are capable of specifically detecting low, intermediate, or high GREM1 expression in vivo (e.g., in the subject) or in vitro (e.g., in a biological sample of the subject's diseased tissue). In some embodiments, the subject is further identified as having 5-50% (e.g., 10-40%, 10-30%, 10-20%, or 15%) GREM1-positive tumor cells as measured by IHC. In some embodiments, the GREM1 diagnostic reagents / pharmaceuticals used in the methods provided herein are peptides comprising any one of the antigen-binding portions or antigen-binding fragments of the anti-GREM1 antibody described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference. In some embodiments, the GREM1 diagnostic reagents / pharmaceuticals used in the methods provided herein comprise any one of the anti-GREM1 antibodies or antigen-binding fragments of the anti-GREM1 antibody described in PCT patent application PCT / CN2022 / 072297, which is incorporated herein by reference.

[0219] In some embodiments, the GREM1 diagnostic reagent / pharmaceutical is a polypeptide comprising an antigen-binding fragment of Hu14E3 HaLa, as described in PCT / CN2022 / 072297, which is incorporated herein by reference. In some embodiments, the antigen-binding fragment includes heavy chains HCDR1, HCDR2, and HCDR3 and / or light chains LCDR1, LCDR2, and LCDR3, wherein: HCDR1 includes an amino acid sequence containing TYGMA (SEQ ID NO: 1) or a homologous sequence having at least 80% sequence identity with it; HCDR2 includes an amino acid sequence containing WINTLSGEPTYADDFKG (SEQ ID NO: 2) or a homologous sequence having at least 80% sequence identity with it; HCDR3 includes an amino acid sequence containing EPMDY (SEQ ID NO: 3) or a homologous sequence having at least 80% sequence identity with it; LCDR1 includes an amino acid sequence containing KSSQSLLDSDGKTYLS (SEQ ID NO: 4) or a homologous sequence having at least 80% sequence identity with it; LCDR2 includes an amino acid sequence containing LVSKLDS (SEQ ID NO: 5) or a homologous sequence having at least 80% sequence identity with it; and LCDR3 includes an amino acid sequence containing WQGAHFPLT (SEQ ID NO: 1). 6) The amino acid sequence of or a homologous sequence having at least 80% sequence identity with the antigen-binding fragment. In some embodiments, the antigen-binding fragment includes the heavy chain CDR3 sequence of EPMDY (SEQ ID NO: 3).

[0220] In some embodiments, the antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 8.

[0221] In some embodiments, the GREM1 diagnostic reagent / pharmaceutical further includes an immunoglobulin constant region, optionally a constant region of human IgG. In some embodiments, 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 comprising the sequence of SEQ ID NO: 9 and / or a light chain constant region comprising the sequence of SEQ ID NO: 10.

[0222] In some embodiments, the GREM1 diagnostic reagent / pharmaceutical includes all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the GREM1 diagnostic reagent / pharmaceutical is a single-domain antibody composed of all or a portion of the heavy chain variable domain provided herein. More information about such single-domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0223] In some implementations, the GREM1 diagnostic reagent / pharmaceutical includes Hu14E3 HaLa or its antigen-binding fragment.

[0224] In some embodiments, the GREM1 diagnostic reagent / pharmaceutical further comprises one or more conjugate moieties linked to the aforementioned polypeptide or Hu14E3 HaLa or its antigen-binding fragment. In some embodiments, the conjugate moieties comprise detectable markers (e.g., lanthanides, luminescent markers, fluorescent markers, biotin / avidin, or enzyme-substrate markers).

[0225] VI. Reagent Kit On the other hand, this disclosure provides a kit for treating cancers expressing GREM1 in subjects of need, the kit comprising a GREM1 antagonist and a packaging insert comprising 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.

[0226] In some embodiments, the cancer expressing GREM1 is characterized by: a) resistance or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or b) low or no PD-L1 expression in the disease tissue, and / or c) GREM1 expression in the disease tissue. In some embodiments, the subject is identified as having intermediate or high PD-L1 expression in the disease tissue.

[0227] As used herein, the term "packaging instructions" refers to instructions included in the commercial packaging of a pharmaceutical product, including information such as indications, dosage, usage, administration, contraindications, other pharmaceutical products used with the packaged product, and / or warnings regarding the use of such pharmaceutical products. In some embodiments, the instructions include selecting subgroups that are resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or b) have low or no PD-L1 expression in the diseased tissue, and / or c) have GREM1 expression in the diseased tissue, and / or d) have been administered the GREM1 antagonist for a sustained period of time sufficient to increase PD-L1 expression levels in the cancerous tissue / cancer cells of the subject. In some embodiments, the instructions include administering a therapeutically effective amount of the GREM1 antagonist for a sustained period of time sufficient to increase PD-L1 expression levels in the cancerous tissue / cells of the subject, and then administering the PD-1 / PD-L1 axis inhibitor to the subject.

[0228] The kit may further include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0229] VII. Cancer In some embodiments, the cancer in the methods provided herein is a cancer expressing GREM1. In some embodiments, the cancer is selected from the group consisting of solid tumors or hematologic malignancies. In some implementations, solid tumors include adrenocortical carcinoma, anal cancer, astrocytoma, cerebellar or brain cancer in children, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumors, brain cancer, cerebellar astrocytoma, cerebellar astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, Burkitt's lymphoma, cervical cancer, colon cancer, colorectal cancer, emphysema, endometrial cancer, esophageal cancer, Ewing's sarcoma, retinoblastoma, gastric / stomach cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas), and Kaposi's sarcoma. Sarcoma), kidney cancer (renal cell carcinoma), laryngeal cancer, liver cancer, lung cancer, neuroblastoma and non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, Ewing family tumors, skin cancer, stomach cancer, testicular cancer, laryngeal cancer, thyroid cancer, or vaginal cancer.

[0230] In some embodiments, 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.

[0231] In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is a subtype of colorectal cancer, such as CMS1, CMS2, CMS3, or CMS4. Detailed descriptions of colorectal cancer subtypes can be found, for example, in PCT patent application PCT / US2022 / 076717, which is incorporated herein by reference.

[0232] Example While this disclosure has been specifically shown and described with reference to specific embodiments, some of which are preferred embodiments, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the spirit and scope of this disclosure as disclosed herein.

[0233] Example 1: Efficacy of the combination of anti-Glemlin1 antibody and DC101 in the BZ-CRC-0001 PDX tumor model in NOG mice. This example demonstrates that the combined use of an anti-Gremlin 1 antibody (i.e., an anti-GREM1 antibody) and anti-angiogenic therapy achieves a synergistic therapeutic effect in the treatment of colorectal cancer (CRC). 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 the anti-VEGFR-2 antibody DC101, which is a monoclonal antibody that reacts with mouse VEGFR-2 and is commercially available (e.g., catalog number #BE0060 from BioXell).

[0234] BZ-CRC-0001 colorectal cancer PDX was obtained from the NODSCID mouse channel at Beijing Cancer Hospital, and a PDX library was established. Pathological information of BZ-CRC-0001 colorectal cancer is shown in Figure 1B. Small tumor tissue blocks, approximately 3 mm in diameter, were subcutaneously inoculated into each NOG mouse. These small tumor tissue blocks were cut from tumor exfoliates from tumor-bearing mice. Nineteen days post-inoculation, animals with tumors approximately 50 mm^3 in size were selected and randomly divided into four groups of eight mice each. Animals were intravenously inoculated with 0.1 mL of 5*10^6 human PBMCs. Animals were selected and administered the drug one week after the human PBMC infusion. Groups 1 through 4 were administered 10 mg / kg hIgG1 control, 10 mg / kg anti-Gremlin1 antibody, 5 mg / kg DC101, and a combination of 10 mg / kg anti-Gremlin1 antibody and 5 mg / kg DC101.

[0235] hIgG1 control, Hu14E3 HaLa, and DC101 were administered via intraperitoneal injection twice weekly for 4 weeks. Animals were euthanized by CO2 inhalation at the end of the study. Tumor size was measured two or three times weekly in two dimensions using calipers and expressed in mm using the following formula. 3 Volume is expressed in units: V = 0.5 a*b^2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean = SEM. Comparisons between groups were performed using a t-test, and a difference was considered significant if p* < 0.05 and ** < 0.01. As shown in Figure 1A, on the day of sacrifice, the tumor growth inhibition rates of single anti-Gremlin1 antibody, DC101, and the combination of anti-Gremlin1 antibody and DC101 were 46.80%, 54.12%, and 66.53%, respectively. The combination of anti-Gremlin1 antibody and DC101 showed better anti-tumor activity compared to anti-Gremlin1 antibody alone, and also improved anti-tumor activity compared to DC101 alone.

[0236] Example 2: Efficacy of the combination of anti-gremlin1 antibody with FOLFIRI and DC101 in the BZ-CRC-0001PDX tumor model in NOG mice This example demonstrates that the combined use of anti-Gremlin 1 antibody (i.e., anti-GREM1 antibody), chemotherapy, and anti-angiogenic therapy achieves a synergistic therapeutic effect in the treatment of CRC. The anti-GREM1 antibody used in this example is Hu14E3 HaLa, as described herein. The chemotherapy is FOLFIRI. The anti-angiogenic therapy comprises the anti-VEGFR-2 antibody DC101, a monoclonal antibody that reacts with mouse VEGFR-2 and is commercially available (e.g., catalog number #BE0060 from BioXell).

[0237] BZ-CRC-0001 colorectal cancer PDX was obtained from the NODSCID mouse channel at Beijing Cancer Hospital, and a PDX library was established. Small tumor tissue blocks, approximately 3 mm in diameter, were subcutaneously inoculated into each NOG mouse. These small tumor tissue blocks were cut from tumor exfoliates from tumor-bearing mice. Eighteen days post-inoculation, animals with tumors approximately 50 mm^3 in size were selected and randomly divided into 5 groups of 8 mice each. Tumor-bearing mice were intravenously inoculated with 0.1 mL of 5*10^6 human PBMCs. Animals were selected and administered the drug one week after the human PBMC infusion. Animals from groups 1 through 5 were administered 35 mg / kg of isotype control and mediator, 30 mg / kg of anti-gremlin1 antibody, a combination of 30 mg / kg of anti-gremlin1 antibody and 5 mg / kg of DC101, a combination of FOLFIRI (5-fluorouracil (5-FU): 7.5 mg / kg, leucovorin (LV): 22.5 mg / kg, irinotecan (CPT-11): 4 mg / kg) and 5 mg / kg of DC101, and a combination of 30 mg / kg of anti-gremlin1 antibody, FOLFIRI (5-FU: 7.5 mg / kg, LV: 22.5 mg / kg, CPT-11: 4 mg / kg) and 5 mg / kg of DC101.

[0238] Allotype control, anti-gremlin1 antibody, and DC101 were administered via intraperitoneal injection twice weekly for 4 weeks. LV was administered via intraperitoneal injection once weekly for 4 weeks; 5-FU and CPT-11 were administered via intravenous injection once weekly for 4 weeks. Animals were euthanized by CO2 inhalation at the end of the study. Tumor size was measured two or three times weekly in two dimensions using calipers and expressed in mm using the following formula. 3Volume is expressed in units: V = 0.5 a*b^2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean = SEM. Comparisons between groups were performed using a t-test, and a difference was considered significant if p* < 0.05 and ** < 0.01. As shown in Figure 2, on the day of sacrifice, the tumor growth inhibition rates of 30 mg / kg anti-gremlin1 antibody, the combination of anti-gremlin1 antibody and DC101, the combination of FOLFIRI and DC101, and the combination of anti-gremlin1 antibody and FOLFIRI and DC101 were 30.10%, 47.47%, 58.75%, and 68.91%, respectively. The combination of anti-gremlin1 antibody and DC101 showed better anti-tumor activity than anti-gremlin1 antibody alone, and the combination of anti-gremlin1 antibody with FOLFIRI and DC101 showed better anti-tumor activity than anti-gremlin1 antibody alone, and the combination of FOLFIRI and DC101 improved anti-tumor activity.

[0239] Example 3: Efficacy of anti-gremlin1 antibody combined with FOLFIRI, DC101, and Nivolumab in the BZ-CRC-0001 PDX tumor model in NOG mice. This example demonstrates that the combined use of anti-Gremlin 1 antibody (i.e., anti-GREM1 antibody), chemotherapy, anti-angiogenic therapy, and immunotherapy achieves a synergistic therapeutic effect in the treatment of CRC. The anti-GREM1 antibody used in this example is Hu14E3 HaLa, as described herein. The chemotherapy is FOLFIRI. The anti-angiogenic therapy includes the anti-VEGFR-2 antibody DC101, a monoclonal antibody that reacts with mouse VEGFR-2 and is commercially available (e.g., catalog number #BE0060 from BioXell). The immunotherapy includes the anti-PD-1 antibody nivolumab.

[0240] BZ-CRC-0001 colorectal cancer PDX was obtained from the NODSCID mouse channel at Beijing Cancer Hospital, and a PDX library was established. Small tumor tissue blocks, approximately 3 mm in diameter, were subcutaneously inoculated into each NOG mouse. These small tumor tissue blocks were cut from tumor exfoliates from tumor-bearing mice. Eighteen days post-inoculation, animals with tumors approximately 50 mm^3 in size were selected and randomly divided into four groups of eight mice each. Animals were intravenously inoculated with 0.1 mL of 5*10^6 human PBMCs. Animals were selected and administered the drug one week after the human PBMC infusion. Animals from groups 1 through 4 were administered a combination of 43 mg / kg hIgG1 control and mediator, 30 mg / kg anti-gremlin1 antibody, 10 mg / kg nivolumab, 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-gremlin1 antibody.

[0241] Animals were administered hIgG1 control, anti-gremlin1 antibody, nivolumab, and DC101 via intraperitoneal injection twice weekly for 4 weeks; LV was administered twice intraperitoneally at weeks 1 and 3; and 5-FU and CPT-11 were administered twice intravenously at weeks 1 and 3. Animals were euthanized by CO2 inhalation at the end of the study. Tumor size was measured two or three times weekly in two dimensions using calipers and expressed in mm using the following formula. 3 Volume is expressed in units: V = 0.5 a*b^2, where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean = SEM. Comparisons between groups were performed using a t-test, and a p-value of * < 0.05 and ** < 0.01 was considered statistically significant. As shown in Figure 3, on the day of sacrifice, the tumor growth inhibition rates of single anti-gremlin1 antibody, nivolumab, FOLFIRI, DC101, and the combination of nivolumab and anti-gremlin1 antibody were 56.03%, 26.86%, and 80.72%, respectively. The combination group showed superior anti-tumor efficacy compared to single anti-gremlin1 antibody and single nivolumab.

[0242] Example 4: Evaluation of tumor-infiltrating lymphocyte (TIL), Gremlin, and PD-L1 expression in the BZ-CRC-0001 PDX tumor model using IHC assay This example demonstrates that the anti-GREM1 antibody Hu14E3 HaLa can be used as a diagnostic reagent for detecting GREM1 expression in diseased tissues. This example also shows that CRC tumor tissues exhibit intermediate to high GREM1 expression and low or no PD-L1 expression. This example further demonstrates that treatment with the anti-GREM1 antibody Hu14E3 HaLa can upregulate PD-L1 expression levels in a dose-dependent manner.

[0243] 1. Gremlin1 expression status in the BZ-CRC-0001 PDX tumor model To investigate the expression level and staining pattern of Gremlin 1 in BZ-CRC-01 PDX tumor samples, biotinylated 14E3 (Hu14E3 HaLa biotin) was prepared and used for detection. Briefly, EZ-Link™ Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, A39257) was dissolved in ultrapure water to prepare a 10 mM biotin reagent solution. 27 µL of biotin solution was added to every 2 mg of 14E3 antibody for labeling, and the mixture was gently mixed at room temperature for 0.5 h. Low molecular weight reaction products were removed by desalting on a Zeba™ rotary desalting column (Thermo Fisher Scientific, 89890) according to the manufacturer's instructions.

[0244] Immunohistochemistry (IHC) was performed on paraffin-embedded PDX slides fixed in 4% neutral buffered formalin. After dewaxing and rehydration, all slides underwent antigen retrieval by boiling in EnVision™ FLEX target retrieval solution (Dako, K8002) at 97–99 °C for 25 min, followed by quenching and blocking with an IHC biotin-blocking kit (MaiXin, BLK-0001) as directed, and incubation at 37 °C for 30 min with 20 μg / mL internally biotinylated monoclonal mouse anti-Gremlin1 (Hu14E3 HaLa biotin). Antibody binding was visualized using horseradish peroxidase-labeled streptavidin (MaiXin, SP KIT-D1) and EnVision™ FLEX substrate working solution (Dako, K8002). Finally, sections were counterstained with hematoxylin and mounted with a permanent mounting agent.

[0245] All viable stromal fibroblasts / tumor cells on the entire slide were assessed under a light field using a microscope and incorporated into the scoring method. Typically, a percentage scoring of at least 100 viable stromal fibroblasts and / or tumor cells is recommended. Positive gremlin1 expression was defined as viable stromal fibroblasts / tumor cells showing partial or complete cytoplasmic staining within the stromal fibroblast / tumor cell population. 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 all four percentages should equal 100%. The total positive percentage for each sample was determined, defined as the percentage of viable tumor cells showing ≥ 1+ staining intensity. Gremlin1 positivity with moderate to strong intensities was observed in the BZ-CRC-01 PDX model, indicating punctate cytoplasmic staining (see Figure 4).

[0246] 2. Regulation of PD-L1 expression after treatment with anti-gremlin1 antibody (Hu14E3 HaLa) in the BZ-CRC-0001 PDX tumor model PD-L1 expression status was also assessed to better understand immunomodulation in BZ-CRC-01 PDX tumor samples prior to and after anti-gremlin1 antibody treatment (see Examples 1 and 2). Immunohistochemistry (IHC) was performed on these 4% neutral buffered formalin-fixed paraffin-embedded (FFPE) tumor sections using a commercially available rabbit anti-human PD-L1 (SP263) monoclonal antibody. After dewaxing and rehydration, all slides were subjected to antigen retrieval in BOND epitope retrieval solution 2 (Leica, AR9640) at 97–99°C for 30 min. They were then quenched, blocked with a peroxidase inhibitor, and incubated at room temperature (RT) for 30 min with an appropriately diluted SP263 (0.2 μg / mL) antibody. Antibody binding was visualized for 8 min on an automated Leica BOND III stained slide using BOND polymer detection (Leica, DS9800). Finally, the sections were counterstained with hematoxylin and mounted with a permanent mounting agent.

[0247] All samples were scored using a combined positive score (CPS) of total PD-L1 staining by immune cells and tumor cells relative to all visible tumor cells, stained with membranes of varying intensities (negative (0), weak (1+), moderate (2+), and strong (3+)), as depicted in Table 1. Interestingly, the staining results showed that treatment with anti-gremlin1 antibody upregulated PD-L1 expression levels in the BZ-CRC-01 PDX model compared to the isotype control, and the staining ratio increased with increasing dose from 10 mg / kg to 30 mg / kg, as depicted in Figure 5 and Table 2.

[0248] Table 1. Interpretation of PD-L1 IHC results Table 2. PD-L1 IHC scores across Hu14E3 HaLa dose levels on BZ-CRC-01 PDX tumor sections. 3. CD3 precipitation after treatment of BZ-CRC-0001 PDX tumor model with anti-gremlin1 antibody (Hu14E3 HaLa) + and CD8 + TIL penetration On BZ-CRC-01 PDX tumor sections, immune cells were stained with CD3 and CD8 biomarkers to observe T lymphocyte infiltration in the tumor microenvironment before and after anti-gremlin1 antibody treatment (references 1 and 2). Immunohistochemistry (IHC) was performed on these 4% neutral buffered formalin-fixed paraffin-embedded (FFPE) tumor sections using commercially available CD8α (D8A8Y) rabbit mAb (CST, 85336S) and CD3ε (D7A6E™) rabbit mAb (CST, 85061S). After dewaxing and rehydration, all slides were subjected to antigen retrieval in BOND epitope retrieval solution 2 (Leica, AR9640) at 97–99°C for 30 min. Subsequently, they were quenched, blocked with a peroxidase inhibitor, and incubated at room temperature (RT) for 30 min with appropriately diluted CD8α (1:200) and CD3ε (1:200) antibodies, respectively. Antibody binding was visualized for 8 minutes on an automatically stained Leica BOND III using BOND polymer detection (Leica, DS9800). Finally, the sections were counterstained with hematoxylin and mounted with a permanent mounting agent.

[0249] All samples were analyzed using fully stained immune cells of any intensity. Notably, the results showed that treatment with anti-gremlin1 antibody promoted the infiltration of CD3 and CD8 positive T cells relative to the isotype control, as depicted in Figure 6.

[0250] Various embodiments have been described herein with reference to the accompanying drawings. However, it will be apparent that various modifications and changes can be made thereto, and other embodiments can be implemented without departing from the broader scope of the invention as set forth in the appended claims. Furthermore, other embodiments will be apparent to those skilled in the art upon consideration of the description and practice of one or more embodiments of the invention disclosed herein. Therefore, the embodiments in this application and herein are intended to be considered exemplary only, and the true scope and spirit of the invention are indicated by the following list of exemplary claims.

[0251] Table 3. Sequences mentioned or used in this application

Claims

1. A method for treating cancer expressing GREM1 in a subject of need, the method comprising: Administering a therapeutically effective amount of the GREM1 antagonist to the subject in combination with the following: 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 of 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 of claim 2, wherein the VEGFA antagonist is an anti-VEFRA antibody, such as bevacizumab (Avastin®).

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

5. The method according to claim 2, wherein the VEGFR antagonist is a small molecule VEGFR inhibitor, such as sitravatinib, anlotinib, apatinib, telatinib, atiratinib, kanitinib, lenvatinib mesylate, pazopanib, sorafenib, sunitinib, vandetanib, axitinib (Inlyta®), cabozantinib (Cabometyx®), fruquitinib (ELUNATE®), and / or regorafenib (Stivarga®).

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

7. The method according to any one of claims 2-6, 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 of claim 7, wherein the cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

9. The method according to any one of the preceding claims, wherein the chemotherapy comprises a combination of chemotherapeutic agents.

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

11. The method of claim 9 or 10, wherein the method comprises administering to the subject a therapeutically effective amount of the combination of the GREM1 antagonist and the chemotherapeutic agent and the combination of the anti-VEFRA antibody or the anti-VEGFR-2 antibody or the small molecule VEGFR inhibitor, and 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 of claim 11, wherein the cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

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

14. The method of 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 method of claim 14, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody selected from the group consisting of: nivolumab (OPDIVO; BMS-936558), dostarlimab (TSR-042), pembrolizumab (KEYTRUDA; MK-3475), MEDI0680 (AMP-514), MEDI4736, BI754091, and pildizumab (Pi dilizumab (CT-011), cimiplimab (LIBTAYO, REGN2810), spartalizumab (PDR001), cetrelimab (JNJ63723283), toripalimab (JS001), PF-06801591, tislelizumab (BGB-A317), AMP-224 (GSK- 2661380), ABBV-181, Lamborizumab, Camrelizumab (SHR-1210), Sintilimab (Tyvyt, IBI308), Penpulimab (AK105), Zimberelimab, Retifanlimab, Serplulimab, Batilimab ( Balstilimab, Geptanolimab, Prolgolimab, Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofazinlimab, Sindelizumab, MGA404, Sym021, BAT1306, and HX008.

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

17. The method of 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 method of claim 17, wherein the PD-L1 inhibitor comprises an anti-PD-L1 antibody selected from the group consisting of: atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446), BMS-936559, avelumab (bavencio), lodapolimab (LY3300054), durvalumab (MEDI4736), CX-072 (Proclaim-CX-072), FAZ053, envorimab (E (nvafolimab, KN035), MDX-1105, STI-1040, CS1001, 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.

19. The method according to any one of the preceding claims, wherein a) the cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or b) the cancer is identified as having low or no PD-L1 expression in the disease tissue of the cancer expressing GREM1.

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

21. The method according to any one of the preceding claims, wherein the method comprises 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), and 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 of claim 21, wherein the cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

23. The method according to any one of the preceding claims, wherein the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the anti-VEFRA antibody or anti-VEGFR-2 antibody or small molecule VEGFR inhibitor, the combination of the chemotherapy agent and the PD-1 / PD-L1 axis inhibitor, and 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 of claim 23, wherein the cancer is resistant or refractory to treatment with a PD-1 / PD-L1 axis inhibitor, and / or is identified as having low or no PD-L1 expression in the diseased tissue of the cancer.

25. The method according to any one of the preceding claims, wherein the object is further identified as having low, intermediate or high GREM1 expression in the diseased tissue of the cancer expressing GREM1.

26. The method of claim 25, wherein the object is further identified as having 10-20% GREM1 positive tumor cells as measured by IHC.

27. A method for improving the responsiveness of a subject to treatment with a PD-1 / PD-L1 axis inhibitor, wherein the subject has been identified as having GREM1 in a biological sample of diseased tissue, or the subject has been identified as having GREM1 expression at a threshold level in the biological sample of diseased tissue, the method comprising: a) Administering a therapeutically effective amount of a GREM1 antagonist to the subject to increase PD-L1 expression in diseased tissues, thereby improving the subject's responsiveness to PD-1 / PD-L1 axis inhibitors.

28. The method of claim 27, wherein a) the object is identified as resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) the object is identified as having low or no PD-L1 expression in diseased tissue.

29. The method of claim 27, wherein the object is identified as having moderate or high PD-L1 expression in diseased tissue.

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

31. The method of 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, wherein the method further comprises administering a therapeutically effective amount of a PD-1 / PD-L1 axis inhibitor to the subject after an increase in PD-L1 expression in the diseased tissue of the subject.

33. A method for determining eligibility or responsiveness to treatment in subjects using a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor, the method comprising: c) Determine the presence or expression level of GREM1 in biological samples of the diseased tissue of the subject. The presence or absence of GREM1 or its expression level indicates whether the subject is likely to be eligible for treatment with the combination of the GREM1 antagonist and the PD-1 / PD-L1 axis inhibitor or whether the subject is likely to respond to the treatment.

34. The method of claim 33, wherein the presence of GREM1 or the expression level of GREM1 above a threshold level determined in step a) indicates that the subject may be eligible for treatment with the combination of the GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor or may be responsive to the treatment.

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

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

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

38. The method according to any one of claims 33 to 36, wherein the object is identified as having moderate or high PD-L1 expression in 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 duration sufficient to increase the expression level of PD-L1 in the cancer cells of the subject, and then 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 the preceding claims, wherein the GREM1 antagonist comprises an anti-GREM1 antibody or an antigen-binding fragment thereof.

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

43. The method of claim 42, wherein the anti-GREM1 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein The heavy chain variable region includes the amino acid sequence of SEQ ID NO: 7, and 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, while still retaining the specific binding specificity or affinity for hGREM1.

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

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

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

10.

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

49. The method of claim 48, wherein the conjugate portion comprises a scavenging modifier, a therapeutic agent (e.g., a chemotherapeutic agent), a toxin, a radioisotope, a detectable label (e.g., a lanthanide element, a luminescent label, a fluorescent label, a biotin / avidin, or an enzyme-substrate label), a pharmacokinetic modification portion, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anticancer drugs (e.g., androgen receptor inhibitors).

50. The method according to any one of the preceding claims, wherein the object is a person.

51. The method according to any one of the preceding claims, wherein the administration is performed orally, nasally, intravenously, subcutaneously, sublingually, or intramuscularly.

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

53. Use of a GREM1 antagonist in the preparation of a medicament for treating cancers expressing GREM1 in a subject in need, wherein said treatment comprises administering the medicament 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. Use of GREM1 antagonists in the preparation of medicaments for improving the response of subjects to treatment with PD-1 / PD-L1 axis inhibitors. The object has been identified as having GREM1 present in a biological sample of the object's diseased tissue, or the object has been identified as having GREM1 expression levels in the biological sample of the diseased tissue reaching a threshold level. The improvement described therein includes administering a therapeutically effective amount of a GREM1 antagonist to the subject, thereby increasing the expression of PD-L1 in diseased tissues and thus improving the subject's responsiveness to PD-1 / PD-L1 axis inhibitors.

55. Use of anti-GREM1 diagnostic reagents in the preparation of kits for determining in subjects the eligibility or responsiveness to treatment with a combination of a GREM1 antagonist and a PD-1 / PD-L1 axis inhibitor. The anti-GREM1 diagnostic reagent is capable of determining the presence or expression level of GREM1 in biological samples of the diseased tissue of the subject. The presence or absence of GREM1 or its expression level indicates whether the subject is likely to be eligible for treatment with the combination of the GREM1 antagonist and the PD-1 / PD-L1 axis inhibitor or whether the subject is likely to respond to the treatment.

56. The use according to claim 55, wherein a) the object is identified as resistant or refractory to PD-1 / PD-L1 axis inhibitors, and / or b) the object is identified as having low or no PD-L1 expression in diseased tissue.

57. The use according to claim 55, wherein the object is identified as having moderate or high PD-L1 expression in diseased tissue.

58. A kit for treating cancers expressing GREM1 in subjects of need, the kit comprising a GREM1 antagonist and a packaging insert comprising 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, wherein the cancer expressing GREM1 is characterized by: a) resistance or refractory to treatment with PD-1 / PD-L1 axis inhibitors, and / or b) low or no PD-L1 expression in the disease tissue, and / or c) GREM1 expression in the disease tissue.

60. The kit of claim 58, wherein the object is identified as having moderate or high PD-L1 expression in diseased tissue.

61. A method for improving tumor-infiltrating lymphocytes in a subject with a solid tumor, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist as defined in any one of the preceding claims.

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

63. The method of claim 62, wherein the object 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 cold tumor into a hot tumor in a subject with a solid tumor, the method comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist as defined in any one of the preceding claims.

65. A method for treating cancer in a subject suffering from a cold tumor, the method comprising: Administer a therapeutically effective amount of the GREM1 antagonist as defined in any of the preceding claims to the subject.

66. The method of claim 65, wherein the object 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 of claim 67, wherein one or more of the therapies can promote T cell proliferation, activation, and / or tumor invasion.

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

70. The method of claim 67, wherein the one or more therapies are anti-angiogenic therapies, immunotherapies, and / or chemotherapy as defined in any of the preceding claims.

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