Anti-GDF15 Antibodies for Use in Combination Treatment of Specific Patient Groups and Dosage Regimens for the Treatment of Cancer

Abstract

The present invention relates to the treatment of specific patient populations using anti-GDF15 antibodies in combination with checkpoint inhibitors. Additionally, the present invention also provides dosing regimens for the treatment of cancer in human patients using anti-GDF15 antibodies.

Description

[Technical Field]

[0001] The present invention relates to the treatment of specific patient populations using anti-GDF15 antibodies in combination with checkpoint inhibitors. Additionally, the present invention also provides dosing regimens for the treatment of cancer in human subjects using anti-GDF15 antibodies. [Background technology]

[0002] GDF-15 is a divergent member of the TGF-beta superfamily, and its functions in appetite regulation, metabolism, inflammation, cell and tissue survival, and immune tolerance have been described. GDF-15 is a homodimer produced as a proprotein, which is cleaved into a 25 kDa (2 × 112 amino acids) dimeric mature GDF-15 and a 2 × 18 kDa (2 × 167 amino acids) propeptide, which are present in tissues (Tsai, 2018) and released into the bloodstream.

[0003] To date, two major categories of GDF-15 activity have been described. The first category relates to metabolic effects: GDF-15 mediates cachexia by altering food intake and inducing anorexia (Johnen, 2007). This effect is mediated by a brainstem-specific receptor named GFRAL, which was described in late 2017 (Emmerson, 2017). In contrast, the second category relates to immunomodulatory effects: GDF-15 has been shown to be a mediator of immune tolerance in pregnancy (Tong et al., 2004), tissue injury (Chung et al., 2017), inflammation (Abulizi, 2016), autoimmune disease, and tumor evasion. GDF-15 inhibits leukocyte integrin activation, thereby preventing their infiltration (Kempf, 2011).

[0004] In recent years, growing evidence has emerged that GDF-15 may play a crucial immunomodulatory role in physiological and pathophysiological situations, particularly in cancer. It would naturally be very attractive for cancer cells to exploit and "hijack" such immune cell evasion mechanisms, blocking immune cell entry into the tumor microenvironment and thereby preventing the immune system from eliminating cancer cells. Consistent with this, numerous publications have recently shown that high GDF-15 serum levels correlate with shorter overall survival in various cancer types, and that GDF-15 is an independent factor in patient survival within various tumor types (Wischhusen et al., 2020).

[0005] Elevated GDF-15 levels have been frequently reported in cancer patients: a microarray-based study comparing 150 carcinomas from 10 anatomical sites with 46 normal tissues showed the highest levels of tumor-associated (over)expression of GDF-15 (Welsh, 2003), and several studies have correlated GDF-15 serum levels with poorer response / worse prognosis in cancer.

[0006] Furthermore, two independent analyses from two different academic melanoma research groups have shown that pretreatment GDF-15 levels also appear to correlate with response to PD-1 antagonists.

[0007] As shown, cancer tissues, afflicted normal organ tissues, and the placenta are known to overexpress GDF-15, presumably to prevent excessive immune cell infiltration into the respective tissues. Therefore, the inventors hypothesized that GDF-15 produced by the above tissues would actually substantially reduce vascular T cell adhesion and endothelial transmigration, preventing T cell entry into or in the immediate vicinity of the respective tissues.

[0008] While anti-GDF-15 antibodies generally exhibit benign and well-tolerated safety profiles in animal models, this mechanism of action naturally carries a variety of potential risks when aiming to provide appropriate dosing regimens for human treatment.

[0009] Rational combination partners for anti-GDF-15 antibodies would be T cell-activating compounds, such as anti-PD-1 / PD-L1 checkpoint inhibitors, or cellular T cell therapies, such as CAR-T or TCR-T therapies. The efficacy of such compounds and treatments can be substantially enhanced by increasing the intratumoral presence of anti-tumor T cells. However, when combined with certain dosing regimens of anti-GDF-15 neutralizing antibodies, their toxicity can also potentially be enhanced.

[0010] WO 2022 / 101263 describes anti-GDF-15 antibodies and dosing regimens for the treatment of cancer.

[0011] A second area of ​​potential interest is the physiological role of GDF-15 in organ protection for distressed organs. If GDF-15 is inhibited using anti-GDF-15 antibodies and organ distress (e.g., myocardial infarction, infection, or other significant organ damage) occurs, organ infiltration by excessive immune cells and unwelcome tissue dysfunction / destruction may occur.

[0012] A third area of ​​potential interest is the rare finding in individual mouse knockout models of GDF-15 (Wischhusen, 2020).

[0013] A further challenge in the field of cancer treatment is the identification of cancer subtypes that are particularly responsive to respective cancer treatments. Although the average therapeutic effect of a particular cancer treatment on cancer is only moderate, certain subtypes of each cancer and respective subgroups of cancer patients may be particularly responsive to treatment and therefore may be oriented toward a more favorable clinical outcome than other patients. Indeed, growing evidence suggests that for many types of cancer, there exist distinct cancer subtypes (especially molecular and / or histological subtypes) with distinct prognostic and therapeutic implications. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] WO 2022 / 101263 [Patent Document 2] WO 2017 / 055613 [Non-patent literature]

[0015] [Non-Patent Document 1] Tumeh et al., Nature., 2014 Nov 27;515(7528):568-71 [Non-patent document 2] Yarchoan et al., JCI Insight., 2019 Mar 21;4(6):e126908 [Non-patent document 3] Chen et al., Exp Hematol Oncol., August 3, 2020;9:17. doi: 10.1186 / s40164-020-00173-3. eCollection 2020 [Non-patent document 4] Wollert KC, Kempf T, Giannitsis Eら, An Automated Assay for Growth Differentiation Factor 15. J Appl Lab Med An AACC Publ., 2018;1(5): pp. 510~521. doi:10.1373 / jalm.2016.022376

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[0016] These considerations apply particularly to targeted cancer therapies, i.e., therapies that rely on the inhibition of specific target proteins. Because treatment with anti-GDF-15 antibodies is a targeted therapy, there is a need for the identification of cancer types and subtypes that are particularly responsive to treatment with anti-GDF-15 antibodies.

[0017] Similarly, targeting treatment to patient groups that are particularly responsive to therapy remains a challenge in the field of cancer therapy. Generally, characterization of patient groups can be based on various factors, such as the patient's treatment history or the presence of biological markers in cancer tissue. In this regard, immune checkpoint molecules or PD-L1 have previously been investigated as potential markers for cancer treatment using anti-PD1 or anti-PD-L1 antibodies. However, there is a particular need for identifying suitable biomarkers for combination treatment using anti-GDF-15 antibodies and immune checkpoint inhibitors, such as anti-PD1 or anti-PD-L1 antibodies. For example, such combination treatment can be successfully used even in patients who are refractory to monotherapy with anti-PD1 or anti-PD-L1 antibodies or combination therapy with anti-PD1 or anti-PD-L1 antibodies and other anti-cancer drugs. Therefore, such combination therapy is expected to be clinically different from monotherapy with anti-PD1 or anti-PD-L1 antibodies or combination therapy with anti-PD1 or anti-PD-L1 antibodies and other anti-cancer drugs, which are associated with different biomarkers. As a result, there remains a need in the art to enable the identification / enrichment of specific patient populations that are particularly responsive to cancer therapy, and to enable the treatment of these patient populations. [Means for solving the problem]

[0018] To meet the above needs, the present invention provides treatments for specific patient subgroups that are particularly responsive to treatment using checkpoint inhibitors in combination with anti-GDF-15 antibodies.

[0019] In a further aspect, the present invention also provides safe and effective dosing regimens of anti-GDF-15 antibodies in the therapeutic treatment of such patient subpopulations.

[0020] According to the present invention, these patient subgroups would particularly benefit from treatment with anti-GDF-15 antibodies.

[0021] PD-L1 was originally described as a marker to enrich for patients who respond to aPD-1 / -L1 monotherapy. The inventors have now found, inter alia, that patients who have previously relapsed or are refractory to anti-PD-1 / L-1 therapy and who have tumors that express PD-L1 at baseline (i.e., at the time treatment with an anti-GDF-15 antibody is initiated) and / or who have evidence of inflammatory tumors at baseline (e.g., as reflected by a high Tumor Inflammation Score (TIS) and a specific minimum number of CD3 or CD8 cell infiltrates) are more likely to have a particularly favorable clinical outcome (e.g., treatment response) when treated with an anti-GDF-15 antibody in combination therapy with an anti-PD1 / anti-PD-L1 antibody, such as nivolumab, compared to patients whose tumors do not have these characteristics.

[0022] It has previously been reported that baseline samples from anti-PD1 / anti-PD-L1 (monotherapy) responders showed higher numbers of CD8-, PD-1-, and PD-L1-expressing cells in the tumor (Tumeh et al., Nature. 2014 Nov. 27;515(7528):568-71). Furthermore, it has been reported that TIS correlates with the expression of PD-L1, also known as CD274 (Danaher et al., 2018).

[0023] However, the patients in our study had progressive disease when treated with anti-PD1 or anti-PD-L1 antibodies. Under these circumstances, where treatment with anti-PD1 or anti-PD-L1 antibodies has already proven to fail in the monotherapy setting, it was highly unexpected that patients with cancers with high TIS, specific initial CD3 or CD8 cell infiltration counts, and PD-L1 expression would particularly benefit from combination therapy with an anti-GDF-15 antibody and a checkpoint blockade, e.g., an anti-PD1 / anti-PD-L1 antibody such as nivolumab. Rather, it was anticipated that the clinical outcome of immunotherapy combining an anti-GDF-15 antibody with a checkpoint inhibitor would be governed by completely different biomarkers.

[0024] Furthermore, although inhibition of GDF-15 with anti-GDF-15 antibodies helps increase the percentage of CD3+ and CD8+ T cells in cancers, the inventors have unexpectedly found that the pre-existence of CD3+ and CD8+ cells and PD-L1 in cancers, as well as a high TIS score, are still beneficial to the clinical outcome of treatment, as previously described, for example, in WO 2017 / 055613. For example, in some embodiments of the present invention, baseline infiltration demonstrates a residual anti-tumor immune response, which can then be boosted by anti-GDF-15 and kept active by combination therapy with a checkpoint inhibitor, such as anti-GDF-15 and anti-PD-1 / -L1, ultimately resulting in tumor regression.

[0025] The selection of patient groups with more favorable clinical outcomes according to the present invention is expected to be a useful and effective selection criterion in clinical practice, since only a subset of patients will have cancers with such characteristics. For example, while PD-L1 can be expressed in many different cancer types, only a subset of cancer patients for each cancer type expresses PD-L1 (see Yarchoan et al., JCI Insight., 2019 Mar. 21;4(6):e126908 and Chen et al., Exp Hematol Oncol., 2020 Aug. 3;9:17. doi: 10.1186 / s40164-020-00173-3. eCollection 2020).

[0026] Thus, the present invention makes it possible to select and selectively treat patients in clinical situations that would be particularly favorable when treated with a combination therapy of an anti-GDF-15 antibody and a checkpoint blockade, e.g., an anti-PD1 / anti-PD-L1 antibody such as nivolumab.

[0027] According to the present invention, analysis of clinical data revealed, inter alia, PD-L1 TPS as a positive enrichment factor for clinical response. Furthermore, the clinical data also revealed that patient selection by PD-L1 TPS alone or in combination with any of CD8 cell density, CD3 cell density, or TIS enriches for clinical responders.

[0028] Therefore, the present invention provides the following preferred embodiments.

[0029] Item 1. A pharmaceutical composition comprising a combination of an anti-GDF-15 antibody and at least one checkpoint inhibitor for use in a method of treating cancer in a human patient having a PD-L1-positive cancer and / or having a cancer involving an inflammatory tumor.

[0030] Item 2. The pharmaceutical composition for the use as defined in item 1, wherein the cancer is PD-L1 positive as determinable by immunohistochemistry (IHC) assay.

[0031] Item 3. The pharmaceutical composition for use as defined in item 2, wherein the IHC assay uses an anti-PD-L1 antibody as a primary antibody to determine the percentage of PD-L1 positive cancer cells.

[0032] Item 4. The pharmaceutical composition for the use defined in any one of items 2 to 3, wherein the PD-L1 positive cancer has a percentage of cancer cells that show staining for the presence of PD-L1 on the cell surface in an immunohistochemistry (IHC) assay of at least 1.

[0033] Item 5. The pharmaceutical composition for use as defined in item 2, wherein the IHC assay uses an anti-PD-L1 antibody as the primary antibody to determine the total percentage of PD-L1-positive cancer cells and PD-L1-positive cancer-infiltrating cells.

[0034] Item 6. The pharmaceutical composition for use as defined in any one of items 1 to 5, wherein the inflammatory tumor is a T-cell inflammatory tumor.

[0035] Item 7. The pharmaceutical composition for use as defined in any one of items 1 to 6, wherein the inflammatory tumor is a tumor containing CD3+ cells.

[0036] Item 8. The pharmaceutical composition for use as defined in item 7, wherein the inflammatory tumor contains CD3+ cells that can be determined by an immunohistochemistry (IHC) assay, and the IHC assay uses an anti-CD3 antibody as the primary antibody.

[0037] Item 9 Inflammatory tumors have >300 cells / mm in immunohistochemical tissue sections with a thickness of 4 μm, as determined by immunohistochemistry (IHC) assay. 2 9. The pharmaceutical composition for use as defined in item 8, wherein the tumor has a mean CD3+ cell density of

[0038] Item 10. The pharmaceutical composition for use as defined in any one of items 1 to 9, wherein the inflammatory tumor is a tumor containing CD8+ cells.

[0039] Item 11. The pharmaceutical composition for use as defined in item 10, wherein the inflammatory tumor contains CD8+ cells that can be determined by an immunohistochemistry (IHC) assay, and the IHC assay uses an anti-CD8 antibody as the primary antibody.

[0040] Item 12 Inflammatory tumors have >300 cells / mm in immunohistochemical tissue sections with a thickness of 4 μm, as determined by immunohistochemistry (IHC) assay. 2 12. The pharmaceutical composition for use as defined in item 11, wherein the tumor has a mean CD8+ cell density of

[0041] Item 13. The pharmaceutical composition for use as defined in any one of items 6 to 12, wherein the T cell inflammatory tumor is inflammatory as indicated by its tumor inflammation signature (TIS).

[0042] Item 14. The pharmaceutical composition for the use as defined in item 13, wherein the TIS is based on the expression of at least one, and preferably all, of the marker genes selected from the group consisting of PSMB10, HLA-DQA1, HLA-DRB1, CMKLR1, HLA-E, NKG7, CD8A, CCL5, CXCL9, CD27, CXCR6, IDO1, STAT1, TIGIT, LAG3, CD274, PDCD1LG2, and CD276.

[0043] Item 15 TIS

[0044]

number

[0045] [where x i is the log2-transformed normalized expression level of the i-th gene, and w i is a predefined mass greater than zero] 15. The pharmaceutical composition for use as defined in item 14, wherein the linear combination of all of said marker genes is calculated as:

[0046] Item 16. A pharmaceutical composition for use as defined in Item 15, having a TIS of higher than 7.5.

[0047] Item 17. The pharmaceutical composition for use as defined in any one of items 1 to 16, wherein the human patient is an anti-PD-1 and / or anti-PD-L1 relapsed and / or refractory patient.

[0048] Item 18. The pharmaceutical composition for use as defined in any one of items 1 to 17, wherein the checkpoint inhibitor is selected from one or more of the group consisting of an anti-PD-1 antibody or a PD-1-binding fragment thereof, an anti-PD-L1 antibody or a PD-L1-binding fragment thereof, an anti-CD40 antibody or a CD40-binding fragment thereof, an anti-LAG-3 antibody or a LAG-3-binding fragment thereof, an anti-TIM-3 antibody or a TIM-3-binding fragment thereof, an anti-TIGIT antibody or a TIGIT-binding fragment thereof, and an anti-CTLA4 antibody or a CTLA4-binding fragment thereof.

[0049] Item 19. The pharmaceutical composition for use as defined in Item 18, wherein the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or a PD-1-binding fragment thereof and an anti-PD-L1 antibody or a PD-L1-binding fragment thereof.

[0050] Item 20. The pharmaceutical composition for use as defined in any one of items 1 to 19, wherein the cancer is a solid tumor.

[0051] Item 21. The pharmaceutical composition for use as defined in any one of items 1 to 20, wherein the cancer is selected from the group consisting of colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate adenocarcinoma, pancreatic cancer, uterine cancer, cervical cancer, thyroid cancer, cutaneous squamous cell carcinoma, mesothelioma, carcinoma of unknown primary (CUP), and breast cancer.

[0052] Item 22. The pharmaceutical composition for use as defined in any one of Items 1 to 21, wherein the anti-GDF-15 antibody comprises a heavy chain variable domain comprising a CDR1 region represented by the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 region represented by the amino acid sequence set forth in SEQ ID NO: 2, and a CDR3 region represented by the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable domain comprising a CDR1 region represented by the amino acid sequence set forth in SEQ ID NO: 4, a CDR2 region represented by the amino acid sequence ser-ala-ser, and a CDR3 region represented by the amino acid sequence set forth in SEQ ID NO: 5.

[0053] Item 23. The pharmaceutical composition for use as defined in any one of items 1 to 22, wherein the anti-GDF-15 antibody has a heavy chain variable domain comprising an amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence set forth in SEQ ID NO: 6, and a light chain variable domain comprising an amino acid sequence represented by SEQ ID NO: 7 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence set forth in SEQ ID NO: 7.

[0054] Item 24. The pharmaceutical composition for use as defined in any one of items 1 to 23, wherein the anti-GDF-15 antibody has a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence set forth in SEQ ID NO: 8, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 9 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence set forth in SEQ ID NO: 9.

[0055] Item 25. The pharmaceutical composition for use as defined in any one of items 1 to 24, wherein the anti-GDF-15 antibody has a heavy chain comprising the amino acid sequence represented by SEQ ID NO:8 and a light chain comprising the amino acid sequence represented by SEQ ID NO:9.

[0056] Item 26. The anti-GDF-15 antibody is administered to a human patient, preferably i) administered at a dose of 3 to 20 mg / kg, preferably at a dose of 10 mg / kg, in a dosing regimen of at least one administration cycle, the cycle being 2 weeks in duration, said dose being administered at least once during each of the at least one cycle; ii) at a dose of 3 to 20 mg / kg, preferably at a dose of 10 mg / kg, administered in a dosing regimen of at least one administration cycle, the cycle being 3 weeks in duration, said dose being administered at least once during each of the at least one cycle; or iii) administered at a dose of 3 to 20 mg / kg, preferably at a dose of 20 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 4 weeks in duration, and said dose is administered at least once during each of the at least one cycle; the cancer is selected from the group consisting of colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate adenocarcinoma, pancreatic cancer, uterine cancer, cervical cancer, thyroid cancer, cutaneous squamous cell carcinoma, mesothelioma, cancer of unknown primary (CUP), and breast cancer; 26. A pharmaceutical composition for the use as defined in any one of items 1 to 25.

[0057] Item 27. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is colorectal cancer.

[0058] Item 28. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is bladder cancer.

[0059] Item 29. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is cancer of unknown primary (CUP).

[0060] Item 30. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is mesothelioma.

[0061] Item 31. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is cutaneous squamous cell carcinoma.

[0062] Item 32. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is non-small cell lung cancer.

[0063] Item 33. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is hepatocellular carcinoma.

[0064] Item 34. The pharmaceutical composition for use as defined in any one of items 1 to 26, wherein the cancer is melanoma.

[0065] Item 35. The pharmaceutical composition for use as defined in any one of items 1 to 34, wherein the use is also a use for the treatment of cancer cachexia.

[0066] Item 36. The pharmaceutical composition for use as defined in any one of items 1 to 35, wherein the anti-GDF-15 antibody is administered at a dose of 3 to 20 mg / kg, preferably 20 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 4 weeks in duration, and wherein said dose is administered at least once during each of the at least one cycle.

[0067] Item 37. The pharmaceutical composition for use as defined in any one of items 1 to 36, wherein the anti-GDF-15 antibody is administered at a dose of 3 to 20 mg / kg, preferably 10 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 3 weeks in duration, and wherein said dose is administered at least once during each of the at least one cycle.

[0068] Item 38. The pharmaceutical composition for use as defined in any one of items 1 to 37, wherein the anti-GDF-15 antibody is administered at a dose of 3 to 20 mg / kg, preferably 10 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 2 weeks in duration, and wherein said dose is administered at least once during each of the at least one cycle.

[0069] Item 39. The pharmaceutical composition for use as defined in any one of items 1 to 38, wherein said anti-GDF-15 antibody is obtainable by expression in CHO cells.

[0070] Item 40. The pharmaceutical composition for use as defined in any one of items 1 to 39, wherein the checkpoint inhibitor is administered in the same dosing regimen as the anti-GDF-15 antibody.

[0071] Item 41. The pharmaceutical composition for use as defined in any one of items 1 to 40, wherein the checkpoint inhibitor is administered prior to administration of the anti-GDF-15 antibody, preferably within 120 minutes prior to administration of the anti-GDF-15 antibody, more preferably within 30 minutes prior to administration of the anti-GDF-15 antibody.

[0072] Item 42. The pharmaceutical composition for use as defined in any one of items 1 to 41, wherein the dose of anti-GDF-15 antibody is administered intravenously.

[0073] Item 43 (i) analyzing whether the cancer is a PD-L1 positive cancer and / or whether the cancer includes an inflammatory tumor; (ii) predicting the patient's clinical outcome for the treatment based on the analysis; 1. A method for predicting the clinical outcome of a human cancer patient on treatment comprising a combination of an anti-GDF-15 antibody and at least one checkpoint inhibitor, comprising:

[0074] Item 44 (a) analyzing whether the cancer is a PD-L1 positive cancer and / or whether the cancer includes an inflammatory tumor; (b) predicting the patient's clinical outcome for the treatment based on the analysis; and (c) treating the human patient with a combination of an anti-GDF-15 antibody and at least one checkpoint inhibitor; 10. A method of treating a human patient diagnosed with cancer, comprising:

[0075] Item 45. The method of Item 43 or 44, wherein the clinical outcome comprises response, complete response, partial response, stable disease, progressive disease, and / or survival of the human cancer patient.

[0076] Item 46. The method of any one of items 43 to 45, wherein the patient is predicted to show improved clinical outcome if the cancer is PD-L1 positive.

[0077] Item 47 The method of Item 46, wherein the PD-L1 positive cancer is as defined in Items 2 to 5.

[0078] Item 48. The method of any one of items 43 to 47, wherein the patient is predicted to show improved clinical outcome when the cancer is an inflammatory tumor.

[0079] Item 49. The method of item 48, wherein the inflammatory tumor is as defined in items 6 to 16.

[0080] Item 50. The method of any one of items 43 to 49, wherein the checkpoint inhibitor is as defined in item 18 or 19.

[0081] Item 51. The method of any one of Items 43 to 50, wherein the cancer is selected from the cancers defined in Item 20 or 21.

[0082] Item 52. The method of any one of items 43 to 51, wherein the human patient is an anti-PD-1 and / or anti-PD-L1 relapsed and / or refractory patient.

[0083] Item 53. An anti-GDF-15 antibody for use in a method of treating cancer in a human patient, wherein the anti-GDF-15 antibody is administered in combination with at least one checkpoint inhibitor, and the human patient has a PD-L1-positive cancer and / or has a cancer comprising an inflammatory tumor.

[0084] Item 54. The anti-GDF-15 antibody for use as defined in Item 53, wherein the patient has a cancer as defined in any one of Items 2 to 16, 19 to 21, and 26 to 34.

[0085] Item 55. An anti-GDF-15 antibody for use as defined in item 53 or 54, wherein the patient is as defined in item 17.

[0086] Item 56. The anti-GDF-15 antibody for use as defined in any one of items 53 to 55, wherein the checkpoint inhibitor is as defined in any one of items 18 to 19.

[0087] Item 57. An anti-GDF-15 antibody for use as defined in any one of items 53 to 56, wherein the anti-GDF-15 antibody is as defined in any one of items 22 to 25 and 39.

[0088] Item 58. The anti-GDF-15 antibody for use as defined in any one of items 53 to 57, wherein the anti-GDF-15 antibody is administered as defined in any one of items 26, 36 to 38, and 42.

[0089] Item 59. The anti-GDF-15 antibody for use as defined in any one of items 53 to 58, wherein the checkpoint inhibitor is administered as defined in any one of items 40 to 41.

[0090] Item 60. An anti-GDF-15 antibody for use as defined in any one of items 53 to 59, wherein the use is also for the treatment of cancer cachexia.

[0091] Item 61. The pharmaceutical composition for use as defined in any one of items 1 to 42 or the anti-GDF-15 antibody for use as defined in any one of items 53 to 60, wherein the PD-L1 positive cancer has a percentage of cancer cells that stain for the presence of PD-L1 on the cell surface in an immunohistochemistry (IHC) assay of at least 1, preferably at least 2, more preferably at least 5.

[0092] Item 62. The pharmaceutical composition or anti-GDF-15 antibody for use as defined in Item 61, wherein the cancer is bladder cancer.

[0093] In accordance with the present invention, it will be understood that reference to "at least one checkpoint inhibitor" means that the indicated checkpoint inhibitor is required, and that additional checkpoint inhibitors may or may not be included, i.e., are optional. For example, in instances where the present invention refers to at least one checkpoint inhibitor and the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or a PD-1-binding fragment thereof and an anti-PD-L1 antibody or a PD-L1-binding fragment thereof, it will be understood that this means that a checkpoint inhibitor selected from the group consisting of an anti-PD-1 antibody or a PD-1-binding fragment thereof and an anti-PD-L1 antibody or a PD-L1-binding fragment thereof is required, and that additional checkpoint inhibitors (e.g., an anti-CD40 antibody or a CD40-binding fragment thereof, an anti-LAG-3 antibody or a LAG-3-binding fragment thereof, an anti-TIM-3 antibody or a TIM-3-binding fragment thereof, an anti-TIGIT antibody or a TIGIT-binding fragment thereof, and an anti-CTLA4 antibody or a CTLA4-binding fragment thereof) may or may not be included, i.e., are optional. [Brief explanation of the drawings]

[0094] [Figure 1](A) This figure shows that treatment of GDF-15-producing tumors with anti-mGDF-15 antibodies substantially improves the response to anti-PD-1 antibodies. While anti-mGDF-15 antibodies alone had a moderate effect, combination with anti-PD-1 antibodies could substantially improve the therapeutic effect of anti-PD-1 treatment. Female albino C57B1 / 6 mice were injected with 1×10 Panc02-Luc cells into the pancreatic tail. On day 5, animals were randomized by bioluminescence imaging and treated twice weekly with vehicle / anti-mGDF-15 / anti-PD-1 / anti-mGDF-15 + anti-PD-1. (B) This figure shows that treatment of GDF-15-producing tumors with anti-mGDF-15 antibodies substantially improves the response to anti-PD-1 antibodies. While anti-mGDF-15 antibodies alone had a moderate effect, combination with anti-PD-1 antibodies could substantially improve the therapeutic effect of anti-PD-1 treatment. Tumor burden was assessed by bioluminescence in vivo imaging at least once a week. Tumor growth was assessed by correlating measurements on day 35 with the signal during randomization. Tumor growth was compared using the Mann-Whitney test and corrected for multiple comparisons according to Bonferroni-Holm. (C) This figure shows that treatment of GDF-15-producing tumors with anti-mGDF-15 antibody substantially improves the response to anti-PD-1 antibody. While anti-mGDF-15 antibody alone had a moderate effect, combination with anti-PD-1 could substantially improve the therapeutic effect of anti-PD-1 treatment. Tumor burden was assessed by bioluminescence in vivo imaging at least once a week. Tumor growth was assessed by correlating measurements on day 35 with the signal during randomization. Tumor growth was compared using the Mann-Whitney test and corrected for multiple comparisons according to Bonferroni-Holm. [Figure 2] Figure 1 shows that animals treated with CTL-002 exhibit higher immune cell infiltration enriched for CD3+ cells. The syngeneic mouse models MBT-2 (left panel) and Pan02 (right panel) show higher immune cell infiltration in animals treated with aGDF-15. [Figure 3]Figure 1 shows the sequences of the binding region of CTL-002 in various species. The sequences of the binding region of CTL-002 are shown for human, cynomolgus monkey, mouse, and rat (first four columns, top to bottom). [Figure 4] FIG. 1 shows GDF-15 serum levels in female monkeys after the first dose of CTL-002. [Figure 5] Figure 1 shows predicted GDF-15 suppression in tumor microvasculature considering various dosing intervals. Predicted suppression of GDF-15 in tumor microvasculature. Assumptions: three systemic GDF-15 baseline levels (0.5, 2, and 10 ng / mL), three different CTL-002 concentrations (3, 10, and 20 mg / kg), and three different CTL-002 dosing intervals (2, 3, and 4 weeks). [Figure 6] Figure 1 shows a visualization of the correlation between clinical response and PD-L1 TPS and TIS. Baseline biopsies from clinical responders 1-02-006, 1-03-004, 1-03-002, and long-term SD 2-01-003 showed PD-L1 TPS>1 and tumor inflammation score (TIS)>7.5, suggesting the predictive value of PD-L1 TPS and TIS for response to CTL-002 / nivolumab in anti-PD-1 / PD-L1 refractory patients. Furthermore, 6 of 19 patients showed PD-L1 TPS>1, of which 50% (3 of 6) showed a PR. [Figure 7](A) Correlation of PD-L1 TPS versus CD8+ cell count. Baseline biopsies from clinical responders 1-02-006, 1-03-004, 1-03-002, and long-term SD 2-01-003 demonstrated a PD-L1 TPS >1 and a CD8+ T cell density >300 cells / mm², suggesting the predictive value of PD-L1 TPS for CTL-002 / nivolumab response and CD8+ T cell density in anti-PD-1 / PD-L1 refractory patients. Furthermore, 6 of 19 patients demonstrated a PD-L1 TPS >1, of which 50% (3 of 6) demonstrated a PR. Furthermore, 5 of 19 patients demonstrated a PD-L1 TPS >1 and a CD8+ cell density >300 cells / mm², of which 60% (3 of 5) demonstrated a PR. (B) Correlation of PD-L1 TPS versus CD8+ cell count. An updated version of (A), including additional patients, notably clinical responders 1-02-036 and 1-06-005, also demonstrates PD-L1 TPS>1 and CD8+ T cell density>300 cells / mm2. Furthermore, 27 of 60 patients demonstrate PD-L1 TPS>1, of which 18.5% (5 of 27) demonstrate PR. Furthermore, 15 of 60 patients demonstrate PD-L1 TPS>1 and CD8+ T cell density>300 cells / mm2, of which 33.3% (5 of 15) demonstrate PR. [Figure 8] Figure 1 shows the correlation between PD-L1 TPS and CD3 cell count. Baseline biopsies from clinical responders 1-02-006, 1-03-004, 1-03-002, and long-term SD 2-01-003 demonstrated a PD-L1 TPS >1 and a CD3+ T-cell density >300 cells / mm2, suggesting the predictive value of PD-L1 TPS for CTL-002 / nivolumab response and CD3+ T-cell density in anti-PD-1 / PD-L1 refractory patients. Furthermore, 6 of 18 patients demonstrated a PD-L1 TPS >1, with 50% (3 of 6) of these patients experiencing a PR. [Figure 9]Figure 1 shows the fold change in CD8+ and CD3+ ki67+ cells compared to baseline (Bsl) in treated human patients on days 14 and 28. Immune cell analysis in biopsies from patients at baseline (bsl), day 14, and day 28 revealed an increase in CD8+ T cells (upper panel) and an expansion of CD3+ ki67+ T cells (lower panel) under treatment. DETAILED DESCRIPTION OF THE INVENTION

[0095] Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the present invention.

[0096] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0097] All references mentioned herein are incorporated by reference in their entirety for all purposes. Patent applications are referred to herein by using their application and / or publication numbers. Non-patent literature mentioned herein may be cited either as a full reference or in an abbreviated form (e.g., Danaher et al., 2018) followed by the full reference in the "References" section.

[0098] As used herein, each occurrence of the terms "comprising" or "comprises" or the like may optionally be replaced with "consisting of" or "consists of."

[0099] The present invention provides anti-GDF15 antibodies that can be used in the treatment of cancer in human patients.

[0100] GDF-15 can be measured by ELISA. ELISAs that can be used to measure GDF-15 include, but are not limited to, R&D Systems' Quantikine ELISA, immunoradiometric assays, luminex™ sandwich assays, and electrochemiluminescence sandwich assays, such as the ELECSYS® GDF15 assay (Roche Diagnostics) (summarized by Wollert et al. (Wollert KC, Kempf T, Giannitsis E, et al., An Automated Assay for Growth Differentiation Factor 15. J Appl Lab Med An AACC Publ., 2018;1(5):510-521. doi:10.1373 / jalm.2016.022376)). All of the assays mentioned are based on the immunosandwich principle, using monoclonal or polyclonal antibodies to capture and quantify GDF-15. Depending on the reagents and their combination used, free GDF-15 or total GDF-15 (free GDF-15 and GDF-15 bound to CTL-002) is measured.

[0101] The term "PD-L1-positive cancer" as used herein relates to a cancer that has been detected as PD-L1-positive. It will be understood that the term "PD-L1-positive cancer" refers to a cancer that is PD-L1-positive at the time an anti-GDF-15 antibody is first administered. In other words, the cancer is a PD-L1-positive cancer, as can be assessed by analysis of a baseline biopsy from a human patient. This distinguishes from, but does not exclude, the further possibility that changes in PD-L1 expression, such as upregulation of PD-L1 expression, may occur during the course of anti-GDF-15 antibody administration. However, the "PD-L1-positive cancer" referred to herein distinguishes from and excludes a cancer that was not PD-L1-positive at the time an anti-GDF-15 antibody was first administered, but that only subsequently became PD-L1-positive due to upregulation of PD-L1 expression during the course of anti-GDF-15 antibody administration. The detection method used to determine whether a cancer is PD-L1-positive is not particularly limited. Any method that allows for direct / indirect detection of the presence of PD-L1 on the cell surface of cells in cancer can be used in the context of the present invention. Direct methods for detecting PD-L1 can be based on assays for detecting PD-L1 at the protein level. An exemplary method for direct detection of PD-L1 uses immunohistochemistry to stain cells that have PD-L1 on their cell surface. Indirect methods for detecting PD-L1 can be based on assays that aim to detect PD-L1 expression at the nucleotide level, for example, using gene expression data. In a preferred embodiment of a direct method for detecting PD-L1, an immunohistochemical (IHC) assay is used to determine whether a human patient's cancer is a PD-L1-positive cancer. Those skilled in the art are aware of IHC assays that allow for the determination of the proportion of PD-L1-positive cells in a cancer sample based on general knowledge. In this regard, those skilled in the art also recognize that immunohistochemical assays of cancer tissues to detect the presence / proportion of PD-L1-positive cells are mandatory for some therapeutic agents in order to meet the requirements of drug authorities (e.g., FDA) for treatment with checkpoint inhibitors.Therefore, those skilled in the art can easily recognize, using common general knowledge, assays that allow for determining whether a cancer is positive for PD-L1. Generally, IHC assays can be based on an anti-PD-L1 antibody as the primary antibody, which is capable of binding to the PD-L1 protein located on the surface of cells in the cancer. Further analysis allows for the determination of the tumor proportion score (TPS) of the cancer, which is the percentage of cancer cells that stain for the presence of PD-L1 on the cell surface. This percentage of cancer cells can be determined by counting the number of all cancer cells in a cancer sample that stain for the presence of PD-L1 on their cell surface, dividing this number by the total number of cancer cells in the sample, and multiplying the resulting quotient by 100. Alternatively, the combined positive score (CPS) of PD-L1 can be determined, which is the combined percentage of cancer cells and cancer-infiltrating cells (e.g., cancer-infiltrating immune cells) that stain for the presence of PD-L1 on their cell surface. This total percentage of cancer cells and cancer-infiltrating cells can be determined by counting the sum of the number of all cancer cells and cancer-infiltrating cells in a cancer sample that stain for the presence of PD-L1 on their cell surface, dividing that number by the sum of the total number of cancer cells and cancer-infiltrating cells in the sample, and multiplying the resulting quotient by 100. Preferably, a "PD-L1 positive cancer" has a percentage of cancer cells that stain for the presence of PD-L1 on their cell surface (also known as TPS) of at least 1.

[0102] An exemplary method for determining PD-L1 TPS is as follows.

[0103] For test interpretation and scoring, please refer to the manufacturer's interpretation guide: "VENTANA PD-L1 (SP263) Assay Staining of Non-Small Cell Lung Cancer Interpretation Guide," version 1015317EN. Briefly, the percentage of PD-L1-positive tumor cells (TPS) can be determined in an immunohistochemistry (IHC) assay procedure based on formalin-fixed, paraffin-embedded (FFPE) sections using an anti-PD-L1 monoclonal antibody assay, such as the SP263 monoclonal antibody assay (Ventana, catalog number 790-4905). For anti-PD-L1 monoclonal antibody assays, tissue sections can be deparaffinized and heat-pretreated prior to antibody incubation to expose all target antigens. Anti-PD-L1 antibody binding to PD-L1-positive cancer cells was then visualized using a secondary HRP enzyme-conjugated antibody (e.g., Ventana optiView Universal DAB Detection Kit, catalog number 760-700). The specific antibody-enzyme complex is visualized based on the precipitating enzyme reaction product. A CE-IVD-approved predictive assay (741-4905) can be used to determine PD-L1 TPS. Preferably, a defined cutoff of ≥1% membrane-stained tumor cells is used to distinguish between negative and positive cancer tissue (e.g., NSCLC tissue) specimens. Positive cases can be further subdivided by applying additional cutoffs of ≥5% and ≥10%. Exemplary slide interpretation and cutoff determination can be performed as described in more detail in the official scoring guideline "VENTANA PD-L1 (SP263) Assay fur Non-Small Cell Lung Cancer Interpretationsanleitung, 2016," version 1015317EN.

[0104] For scoring, the percentage of PD-L1 positive tumor cells was determined for each staining intensity (0, 1, 2, 3), and the total percentage of stained tumor cells was calculated by summing the percentages at intensities 1, 2, and 3.

[0105] As used herein, the term "inflammatory tumor" refers to an inflammatory tumor phenotype. Those skilled in the art will recognize assays to determine whether a tumor exhibits an inflammatory tumor phenotype using common general knowledge.

[0106] As an exemplary method, the question of whether a tumor is an inflammatory tumor may be assessed based on gene expression analysis using a subset of genes related to antigen presentation, chemokine expression, cytotoxic activity, and adaptive immune resistance, which is translated into the tumor inflammatory signature (TIS) of the tumor. The subset of genes used to determine the TIS is not particularly limited and may be related to at least one gene selected from the group consisting of PSMB10, HLA-DQA1, HLA-DRB1, CMKLR1, HLA-E, NKG7, CD8A, CCL5, CXCL9, CD27, CXCR6, IDO1, STAT1, TIGIT, LAG3, CD274, PDCD1LG2, and CD276. In a preferred embodiment, the subset of genes used to determine the TIS consists of all of the above-mentioned genes. The TIS score may be determined as a linear combination of the 18 genes listed above, as described, for example, in Ayers M, Lunceford J, Nebozhyn M, Murphy E, Loboda A, Kaufman DR, Albright A, Cheng JD, Kang SP, Shankaran V, Piha-Paul SA, Yearley J, Seiwert TY, Ribas A, McClanahan TK. IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest. 2017 Aug 1;127(8):2930-2940. doi: 10.1172 / JCI91190. Epub 2017 Jun 26. PMID: 28650338; PMCID: PMC5531419.

[0107]

number

[0108] [where x i is the log2-transformed normalized expression (e.g., mRNA expression) level of the i-th gene, and w i is a predefined mass greater than zero].

[0109] Alternatively or additionally, the question of whether a tumor is an inflammatory tumor can be assessed by assessing whether the tumor is a tumor containing CD8+ or CD3+ cells. Preferably, the inflammatory tumor contains CD8+ or CD3+ cells, which can be determined by immunohistochemistry (IHC) assay, preferably based on anti-CD8 or anti-CD3 antibodies as primary antibodies. More preferably, this means that the inflammatory tumor contains >300 cells / mm2 in an immunohistochemistry tissue section having a thickness of, for example, 4 μm, as determined by immunohistochemistry (IHC) assay. 2 It can be assessed whether tumors have a mean CD8+ or CD3+ cell density of 100 or more.

[0110] An exemplary immunohistochemistry (IHC) assay for CD8+ cells is as follows: A primary (monoclonal) anti-CD8 antibody (e.g., monoclonal mouse anti-human CD8, clone C8 / 144B) can be applied to formalin-fixed, paraffin-embedded (FFPE) cancer tissue sections (e.g., 4 μm thick) in a multiplex IHC assay procedure. To expose all target antigens, the tissue sections are deparaffinized and heat-pretreated prior to antibody incubation. Binding of specific antibodies (e.g., anti-CD8 antibodies) can be visualized by applying one or more linker antibodies, e.g., hydroxyquinoxaline (HQ)- or nitropyrazole (NP)-conjugated, followed by a secondary horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-enzyme-conjugated antibody, thereby enabling amplification of the resulting signal. The enzyme coupled to the secondary antibody catalyzes a chromogenic precipitate-forming reaction at the binding site of the primary antibody. Each step is followed by incubation. After each incubation step, the automated slide stainer (e.g., the VENTANA DISCOVERY ULTRA automated slide stainer) can wash the sections to stop each reaction and remove unbound material. If applicable, before the application of the next primary antibody, the previous reagent is removed by heat incubation ("stripping"), leaving only insoluble precipitates on the slide. Quantitative evaluation of stained tissue sections can be performed using appropriate digital image analysis software (e.g., Visiopharm Digital Image Analysis Software VIS 2019.02 (TID 001673)). Stained slides can be scanned at 20x magnification using, for example, an Aperio ScanScope XT (Leica) before image analysis. The final workflow not only allows for the detection of positive staining for a single biomarker (e.g., CD8), but also allows for the identification of tumor, normal tissue, and peritumoral and intratumoral tissue compartments.To achieve this, additional guidance staining with tumor-specific markers panCK (solid tumors) or SOX10 (malignant melanoma) can be used, allowing for the alignment of serial sections to generate virtual overlay images and the transfer of information from one slide to another. The final readout is an area measurement (mm) per tissue section. 2 ), cell count per cell type per compartment (e.g., CD8 cell count), density calculation (cell count / mm 2 ), and distance measurements for combinations of biologically relevant cell types.

[0111] CD3+ T cell density can be determined, for example, by immunohistochemistry (IHC) assay using an anti-CD3 antibody, for example, using the ULTIVUE® Multiplex Immunofluorescence Assay (UltiMapper 1 / 0 T-act Panel CD3, Granzyme B, Ki67, and panCK / SOX10) in 4 μm thick FFPE tissue sections using the UltiMapper® I / O T-act kit by ULTIVUE (p / n ULT20110), stained with Leica Bond RX, digitized as full slide images using a ZEISS Axioscan Z1, and subsequently digitally analyzed in Visiopharm.

[0112] The term "clinical outcome" as used herein has the ordinary meaning understood by those skilled in the art. The term "clinical outcome" refers to an event or endpoint reached in medical care. Clinical outcome can be described using classical means for evaluating the efficacy of treatment in a clinical perspective. Exemplary means reflecting clinical outcome are overall survival (OS) and progression-free survival (PFS). Furthermore, clinical outcome can also include initial efficacy endpoints such as response rate, complete response, partial response, stable disease, survival, or progressive disease.

[0113] In a preferred embodiment according to all other embodiments of the present invention, the cancer is a "solid cancer." A "solid cancer" is a cancer that forms one or more solid tumors. Such solid cancers that form solid tumors are generally known in the art. The term "solid cancer" encompasses both the primary tumor formed by the cancer and potential secondary tumors, also known as metastases. "Solid cancer" encompasses all non-hematological cancers, including, but not limited to, colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate adenocarcinoma, pancreatic cancer, uterine cancer, cervical cancer, thyroid cancer, cutaneous squamous cell carcinoma, mesothelioma, carcinoma of unknown primary site (CUP), and breast cancer.

[0114] The terms "CTL-002," "CTL-001 IgG4*," "CTL-001 IgG4," and "H1L5 IgG4*," as well as visgromab, referred to herein, are used interchangeably and refer to antibodies having a heavy chain amino acid sequence of SEQ ID NO: 8 and a light chain amino acid sequence of SEQ ID NO: 9. It will be understood that any reference to the amino acid sequences of the antibodies of the invention is meant to encompass post-translational modifications of these sequences that occur in mammalian cells, such as CHO cells, including, but not limited to, N-glycosylation, O-glycosylation, deamidation, Asp isomerization / fragmentation, formation of pyroglutamic acid, removal of C-terminal lysine, and Met / Trp oxidation.

[0115] In a preferred embodiment according to all other embodiments of the present invention, the GDF-15 is human GDF-15 (also referred to herein as "hGDF-15") and the anti-GDF-15 antibody is an anti-human GDF-15 antibody (also referred to herein as "anti-hGDF-15 antibody").

[0116] It will be appreciated that the antibodies according to the present invention can be administered in the form of pharmaceutical compositions, which are prepared by using pharmaceutically acceptable ingredients, such as carriers, excipients, or stabilizers, in a manner that allows them to be stored and administered appropriately.

[0117] Such pharmaceutically acceptable ingredients are non-toxic in the amounts used when the pharmaceutical composition is administered to a human patient. The pharmaceutically acceptable ingredients added to a pharmaceutical composition can depend on the route of administration.

[0118] Generally, pharmaceutically acceptable ingredients used in connection with the present invention are used in accordance with knowledge available in the art, e.g., from Remington's Pharmaceutical Sciences, edited by A.R. Gennaro, 20th Edition, 2000, Williams & Wilkins, PA, USA.

[0119] The invention is illustrated by the following non-limiting examples and reference examples. [Example]

[0120] ((1) Reference Example) Drug substance (DS) CTL-002 is a humanized, hinge-stabilized IgG4 monoclonal antibody that targets growth differentiation factor-15 (GDF-15) and relates to the antibodies of the present invention.

[0121] In an exemplary liquid formulation of the CTL-002 drug substance, the CTL-002 antibody is presented at a concentration of approximately 25 mg / mL and further comprises 20 mM histidine / histidine HCl, 150 mM sucrose, 50 mM arginine-HCl, and 0.02% w / v polysorbate 20, at a pH of 5.5.

[0122] Manufacturing and Control CTL-002 drug substance can be manufactured in CHO cells, such as CHOK1SV GS KO™ cells. Downstream processing involves two chromatography steps: one Protein A-based affinity chromatography (e.g., MabSelect SuRe), followed by anion exchange membrane chromatography (e.g., Sartobind Q).

[0123] Viral inactivation is achieved, for example, by Triton-X 100 treatment.

[0124] Analytical testing is performed routinely in-process and for final release.

[0125] stability Stability studies of the drug product are currently underway as follows: A pilot non-cGMP (Batch No. DPS026) representative stability study for the intended long-term storage conditions of (up to) 3 years at +2-8°C, as well as 12 months at +25°C and 6 months at +40°C to provide representative stability data for establishing tentative shelf-life of drug product GMP batches. GMP (Batch No. F19235) stability studies to confirm the stability of the proposed IMP under the intended long-term storage conditions of +2-8°C for up to 3 years, as well as 12 months at +25°C and 6 months at +40°C.

[0126] storage CTL-002 drug product vials must be stored at +2-8°C in a certified environment in their original secondary packaging, protected from light and separated from other pharmaceutical or investigational products. The product should not be frozen.

[0127] Preparation and Administration To prepare CTL-002 for intravenous administration, add the CTL-002 solution to an infusion bag containing 0.9% NaCl. CTL-002 solution for infusion can be administered using polyethylene (PVC-, DEHP-, and latex-free) or polyvinyl chloride (latex-free) IV bags and infusion lines made of PE (PVC-, DEHP-, and latex-free) or PVC (DEHP- and latex-free) materials. The use of a 0.2 μm in-line filter (positively / uncharged PES membrane) is essential.

[0128] After compounding, the CTL-002 solution for infusion in the infusion bag can be used immediately and administered at room temperature. The infusion bag can be stored at room temperature for up to 6 hours and at +2-8°C for up to 24 hours, but should be used within 24 hours of preparation.

[0129] Nonclinical Pharmacology The present inventors have identified the mechanism by which GDF-15 primarily blocks the adhesion and transgression of CD8+ T lymphocytes into tissues. Because CTL-002 blocks GDF-15, a novel treatment approach has been established that facilitates the entry of effector T cells into tumor tissues. This may substantially enhance the efficacy of any T cell activator, such as a checkpoint inhibitor.

[0130] Specifically, in a flow adhesion assay, various immune cell subsets pretreated with + / - GDF-15 were perfused over an activated layer of endothelial cells or recombinant adhesion molecules. The adhesion and transmigration processes were monitored by live-imaging microscopy. T cell adhesion to the endothelial cell layer was significantly impaired by the addition of GDF-15. Among T cell subsets, CD8+ T cells were most affected, whereas adhesion of other immune cells was not impaired. The inhibitory effect of GDF-15 on CD8+ T cell adhesion was comparable to the potent blockade of LFA-1 by the TS1 / 18 antibody and could be rescued by the anti-GDF-15 antibody CTL-002 with an EC50 of approximately 700 ng / ml.

[0131] This initial finding was further substantiated with data from related animal models, in which the anti-GDF-15 antibody CTL-002 or a mouse surrogate induced a strong increase in the number of tumor-infiltrating lymphocytes and enhanced the response to T cell-activating therapy. Neutralization of GDF-15 with CTL-002 in humanized mice bearing HV18-MK melanoma resulted in a strong increase in the number of tumor-infiltrating leukocytes. Subset analysis revealed a disproportionately high enrichment of T cells, particularly CD8+ T cells (see Figure 6 in WO 2022 / 101263). In line with this, neutralization of GDF-15 in the syngeneic mouse models MBT-2 and Pan02 resulted in a strong increase in T cell infiltration (Figure 2).

[0132] The ability of GDF-15 to enhance responses to checkpoint blockade was confirmed in orthotopic Pan02 tumors, where the combination of aGDF-15 and aPD-1 doubled the number of tumor-free animals compared with aPD-1 monotherapy (Figure 1). Furthermore, a syngeneic mouse tumor model (MC38-GDF15) harboring genetically engineered mouse colon tumor MC38 cells expressing human GDF-15 showed an increased response to anti-PD-1 or anti-CD40 / poly(IC:LC) combination therapy, compared with the otherwise reduced response. No adverse effects were observed in any of the animals (Figures 9-11 of WO 2022 / 101263).

[0133] Therefore, CTL-002 was developed to neutralize the pathological effects mediated by GDF-15. The biological activity of GDF-15 in the adhesion and transmigration processes was monitored by live-cell imaging microscopy in an in vitro flow adhesion system using primary immune cells, and the key parameters of the inhibition of GDF-15 effects by CTL-002 were determined in this system.

[0134] Additionally, secondary pharmacology studies examined the ability of CTL-002 to induce CDC and ADCC, on-target / off-tissue binding, and off-target binding. Safety pharmacology evaluations included standard repeat-dose toxicity studies.

[0135] Overall, these studies provide a thorough characterization of the mechanism of action of CTL-002 and a well-supported rationale for its clinical testing in patients with cancer and elevated GDF-15 in the tumor microenvironment.

[0136] Binding of the drug CTL-002 to the target GDF-15 As shown in Table 1, GDF-15 and CTL-002 formed a primary complex of two CTL-002 antibodies and two dimeric GDF-15 molecules in solution. Other complexes appeared less favorable, with only one additional complex of three CTL-002 and three GDF-15 molecules reliably detected. This complex was maximal during equimolar incubations of CTL-002 and GDF-15, but was still less than 8% and decreased when the ratio was changed in either direction. Above a 3 molar excess of antibody, all GDF-15 was complexed by CTL-002 molecules, and no tendency toward the formation of high-molecular-weight aggregates was observed.

[0137] [Table 1]

[0138] The affinity of CTL-002 for recombinant human GDF-15 was determined by surface plasmon resonance measurements on a Biacore T3000. Additionally, the affinity for cynomolgus monkey, rat, and mouse GDF-15 was measured (see Table 2).

[0139] All experiments were performed in 10 mM HEPES buffer, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.05% Tween. Anti-human IgG Fc-specific antibodies (Jackson, order number 109-005-008, lot number 111148) were covalently immobilized onto Biacore CM5 chips (GE Healthcare, order number 61144275, lot number 10236645) using EDC / NHS chemistry. To characterize the kinetics of antigen-antibody interactions, pulses of increasing GDF-15 concentrations (e.g., 156.3 pM, 312 pM, 625 pM, and 1,250 pM) were injected at a flow rate of 30 μl / min. After each measurement cycle (8 min association, followed by 30 min dissociation), the antibody-antigen complex was separated by regeneration of the surface with 10 mM glycine-HCl, pH 2.0. For the calculation of the dissociation constant of CTL-002, the association and dissociation phases were recorded and evaluated by global fitting using the software BIAevaluation 4.1. Only for the global fitting analysis were these antigen concentrations considered, which allowed analysis according to the Langmuir 1:1 binding model or 1:1 binding with a drifting baseline.

[0140] Human GDF-15 K D The values ​​are shown in Table 2.

[0141] [Table 2]

[0142] A summary and comparison of the three antibodies used in the panel of non-clinical studies is given in Table 3.

[0143] [Table 3A]

[0144] [Table 3B]

[0145] [Table 3C]

[0146] In summary, CTL-002 is a humanized IgG4 antibody with high specificity for human GDF-15, derived from the murine antibody B1-23. CTL-002 binds to human, cynomolgus monkey, and rat GDF-15 with picomolar affinity (38.3, 108, and 449 pM, respectively) and to murine GDF-15 with low nanomolar affinity (9.76 nM). CTL-002 binds to a discontinuous conformational epitope at the carboxy terminus of mature GDF-15 and specifically recognizes the physiological dimeric conformation.

[0147] In vitro biological activity of CTL-002 on GDF-15-mediated inhibition of T cell adhesion A flow adhesion assay system was used to mimic the dynamics of the blood vessel wall that separates immune cells from tumor tissue and to analyze the effect of GDF-15 neutralization by CTL-002. To evaluate the potency of GDF-15 adhesion inhibition and the sensitivity of the system, the IC50 of GDF-15 in the assay system was determined to be 13.8±2.3 ng / ml, as shown in Figure 4 of WO 2022 / 101263.

[0148] To evaluate the efficacy of CTL-002 in preventing GDF-15-mediated inhibition of T cell adhesion, the EC50 of CTL-002 was determined in a flow adhesion assay using T cells. As a result, an average concentration of 707±17 ng / ml of CTL-002 was effective in increasing T cell adhesion by 50% across various donors. This is shown in Figure 5 of WO 2022 / 101263.

[0149] In vivo pharmacology Among all TGF-beta superfamily members, the orthologous GDF-15 molecule shows the lowest sequence conservation. While mature rat, mouse, and human TGF-beta 1 and BMP-2 proteins share 99-100% sequence identity between humans and mice, the homology for GDF-15 is less than 70% (Bottner, 1999). Therefore, biological differences between different species cannot be excluded. Mouse GDF-15 shows a lower sequence identity of 67.9% with its human counterpart, which is also reflected in the fact that anti-GDF-15 antibodies show lower affinity for the mouse homolog. Pharmacodynamic effects were investigated using two different in vivo approaches.

[0150] First, we used a humanized mouse model in which immunodeficient mice were transplanted with CD34+ hematopoietic stem cells derived from human umbilical cord blood.

[0151] Three months after reconstitution, these mice developed functional human-like immune systems containing all major human immune cell subsets (Wang, 2018). These mice were then inoculated with a patient-derived human melanoma cell line, HV-18MK, which has been shown to secrete high levels of GDF-15. Starting three days later, mice were treated twice weekly with an isotype control or CTL-002. After four weeks, tumors were harvested and analyzed for immune cell infiltration by flow cytometry.

[0152] Although tumor size was unaffected, the CTL-002-treated group showed a nine-fold increase in human tumor-infiltrating CD45+ cells. This is shown in Figure 6 of WO 2022 / 101263. Within the infiltrating cell population, T cells were four-fold enriched. A follow-up study analyzing the infiltrating immune cell population in more detail confirmed an increased infiltration of CD45+ cells and enrichment of CD3+ cells, but this was less pronounced, with a three-fold increase in CD45+ cells and a four-fold enrichment of CD3+ T cells. Similarly, the syngeneic mouse models MBT-2 (Figure 2, left panel) and Pan02 (Figure 2, right panel) show higher immune cell infiltration in animals treated with aGDF-15.

[0153] As a second experimental model, a genetically modified mouse cell line overexpressing human GDF-15 was generated to test the therapeutic efficacy of anti-GDF-15 antibodies.

[0154] In this regard, MC38 colon adenocarcinoma cells are the preferred murine tumor cell line for analyzing the activity of immune checkpoint blockade agents (Selby, 2016) and were used to generate in vivo data to support the development of approved anti-PD-1 antibodies. Overexpression was implemented by stable transfection and did not affect in vitro proliferation compared to control-treated MC38 cells.

[0155] Age-matched immunocompetent C57BL / 6 and immunodeficient NCI nu / nu mice were treated with 5 × 10 5 MC38 blank colon tumor cells or transgenic derivative MC38 expressing recombinant human GDF-15 GDF-15 In contrast to in vitro and in vivo growth in immunodeficient NCI nu / nu mice, it has previously been shown that GDF-15 expression mediated a growth advantage in immunocompetent C57BL / 6 mice, supporting the idea that GDF-15 interferes with the tumor host's immune system (see Figure 7 in WO 2022 / 101263).

[0156] Furthermore, expression of human GDF-15 rendered anti-PD-1-responsive MC38 colon cancer tumors anti-PD-1-resistant, which was partially reversed by anti-GDF-15 (B1-23) / anti-PD-1 combination treatment but not by anti-GDF-15 monotherapy and partially by anti-PD-1 alone.

[0157] Similarly, tumors secreting GDF-15 (solid line, Figure 9B of WO 2022 / 101263) showed better survival when treated with a combination of anti-GDF-15 B1-23 and anti-PD-1 (Figure 9 of WO 2022 / 101263), while monotherapy with an anti-GDF-15 antibody showed only minimal improvement. This combination treatment was non-significantly better than anti-PD-1 monotherapy (Figure 8 of WO 2022 / 101263).

[0158] Because CTL-002 and its derivatives have only nanomolar affinity for mouse GDF-15, a surrogate antibody, anti-mGDF-15 (having the light chain variable region domain amino acid sequence of SEQ ID NO: 19 and the heavy chain variable region domain amino acid sequence of SEQ ID NO: 20), with picomolar affinity for mouse GDF-15, was used in another syngeneic mouse model. To monitor tumor growth, luciferase-expressing mouse pancreatic adenocarcinoma Panc02 cells, which secrete GDF-15, were implanted into the mouse pancreas. Implantation of the cell line into the corresponding tissue allows tumor development to be assayed in a relevant environment that more closely mimics the disease process in humans. Five days later, mice were randomized into four groups and treated twice weekly with isotype antibodies, anti-PD-1, anti-GDF-15, or anti-PD-1, or a combination of anti-GDF-15 and anti-PD-1. Tumor size was measured periodically by luciferase imaging. The relative results at day 35 are summarized in Figure 1, a waterfall plot ranking the tumor incidence of each animal within a group from baseline, and a bar graph showing the relative mean tumor size change from baseline (Figure 1). In contrast to the vehicle group, in which 1 of 12 animals showed spontaneous tumor shrinkage for unknown reasons, tumor shrinkage was observed in only 5 of 12 animals in the combination treatment group and, to a lesser extent, in 3 of 12 animals in the anti-PD-1 monotherapy group (Figure 1).

[0159] Anti-GDF-15 treatment is suggested to increase T cell infiltration, so other cancer immunotherapies that rely on the presence of T cells in the tumor should benefit from neutralization of GDF-15.

[0160] Another immunotherapy previously shown to rely on T cell immune responses, anti-CD40 and poly(IC:LC) tumor treatment (van den Boorn, 2013), was previously tested in a mouse model bearing MC38 cells expressing human GDF-15. Combined treatment with GDF-15 neutralization and anti-CD40 / poly(IC:LC) resulted in complete tumor rejection in 8 of 10 animals, whereas only 3 of 10 tumors were eliminated by anti-CD40 / poly(IC:LC) treatment alone (Figure 10 in WO 2022 / 101263).

[0161] Although mouse GDF-15 was undetectable in wild-type MC38 cells, which may be due to a poor analytical assay, a similar experiment comparing an isotype control antibody with anti-GDF-15 treatment in combination with anti-CD40 / poly(IC:LC) was performed using MC38 cells that had not been engineered to secrete human GDF-15. Similar to the experiment with genetically modified MC38, anti-GDF-15 was able to improve the efficacy of anti-CD40 / poly(IC:LC) treatment, but to a lesser extent (Figure 11 of WO 2022 / 101263).

[0162] In a 4-week GLP toxicology study in NHPs, CTL-002 has been shown to be safe and well-tolerated at doses that provide an adequate margin of exposure for clinical trials.

[0163] The proposed FIH starting dose of 0.3 mg / kg / Q2wk was designed to achieve a maximum plasma concentration (Cmax) at the end of a 1-hour 6 μg / mL infusion, which is 683-fold lower than the Cmax exposure to CTL-002 at the no-observed-effect level (NOEL) in NHPs. This dose may achieve only transient suppression of GDF-15 in the tumor microenvironment and is considered the minimum acceptable biological effect level (MABEL).

[0164] Serum total GDF-15 has been shown to be a useful biomarker of GDF-15 target engagement in NHPs, with CTL-002 doses ≥ 10 mg / kg being associated with sustained GDF-15 capture (and, by interference, GDF-15 suppression). Thus, total human GDF-15 may be a potential clinical biomarker of GDF-15 target engagement, and the FIH clinical study is designed to explore both limited-duration maximal suppression of GDF-15 and continuous suppression of GDF-15 throughout each dosing cycle.

[0165] toxicology CTL-002 is being tested for the treatment of patients with advanced cancer.

[0166] To identify relevant species for preclinical testing, the sequence homology of GDF-15 was compared across species. The sequence homology from human to cynomolgus monkey, mouse, and rat is 94.6%, 67.9%, and 66.1%, respectively.

[0167] CTL-002 binds to a nonlinear conformational epitope within GDF-15, illustrated as two boxes in Figure 3. The sequences of the binding region of CTL-002 are shown for cynomolgus monkey, human, mouse, and rat (first four columns, top to bottom).

[0168] Cynomolgus monkeys show 100% sequence homology with the human CTL-002 binding epitope within GDF-15, and are therefore considered a relevant species for toxicity studies. The binding affinity of CTL-002 to human and cynomolgus GDF-15 is 38.3 pM and 108 pM, respectively.

[0169] A dose-response discovery (DRF) study of CTL-002 was conducted in rats because the product was expected to be sufficiently pharmacologically active to achieve sustained and complete target inhibition at reasonable intravenous dose levels (binding affinity for rat GDF-15: 449 pM). However, the PK / PD data obtained in the study showed that CTL-002 was unable to saturate GDF-15 binding, even at the highest dose level of 100 mg / kg. Therefore, central toxicology was evaluated in cynomolgus monkeys only in a 4-week study using weekly intravenous administration of CTL-002. No toxicity was observed up to the highest tested dose of 100 mg / kg.

[0170] A tissue cross-reactivity study was conducted using human and cynomolgus monkey tissues in accordance with Good Laboratory Practice (GLP). CTL-002 staining in the tissue panel examined was restricted to the cytoplasm of trophoblast cells in human and monkey placenta, consistent with the reported expression of its target protein, GDF-15, in the placenta. No unexpected cross-reactivity was observed.

[0171] Toxicology: Non-central research A non-GLP single-dose dose-ranging study was conducted in cynomolgus monkeys. The primary objective of this study was to support dose selection for a subsequent GLP-compliant 28-day repeat-dose toxicity study. Additionally, the pharmacokinetics of CTL-002 at various dose levels was characterized.

[0172] One male and one female animal per dose level (0.1, 1, 10, or 100 mg / kg) was dosed via a single 30-minute intravenous infusion, and pharmacokinetic profiles were recorded over 14 weeks. In addition to pharmacokinetic analysis, the development of anti-drug antibodies was assessed pre-dose and 6 and 12 weeks after dosing. Serum levels of GDF-15 and CTL-002 were quantified, and pharmacokinetic data were calculated separately as total CTL-002 (PK Total) and free CTL-002 (PK Free).

[0173] Mortality and clinical signs were monitored daily as indicators of toxicity. Body weight and food and water consumption were analyzed weekly, and temperature, ECG, blood pressure, hematology including coagulation, and clinical chemistry including cytokines were assessed at several times during the study.

[0174] No animals died during the study, and there were no signs of CTL-002-related toxicity at any of the dose levels tested.

[0175] A single infusion of 0.1, 1, 10, or 100 mg / kg of CTL-002 on study day 1 resulted in a dose-related increase in total GDF-15 serum levels in all males and females. Target saturation by CTL-002 at the 10 and 100 mg / kg dose levels was demonstrated by overlaying the GDF-15 curves at these two dose levels.

[0176] Measurement of total and free CTL-002 in serum samples obtained by Study Day 43 (Groups 1 and 2, treatment with 0.1 or 1 mg / kg CTL-002) or Study Day 99 (Groups 3 and 4, treatment with 10 or 100 mg / kg CTL-002) revealed dose-related exposure of animals to CTL-002.

[0177] The primary pharmacokinetic data are given in Table 4 as the mean of one male and one female animal per group.

[0178] [Table 4]

[0179] Based on the results of this study, dose levels of 10, 30, and 100 mg / kg were recommended for a pivotal 4-week repeat-dose toxicity study.

[0180] Toxicology: central research A further pivotal study was a 4-week repeat-dose toxicity study of CTL-002 in cynomolgus monkeys (age: 3-4 years) with a 4-week recovery period.

[0181] In this study, CTL-002 was administered by 30-minute intravenous infusion once weekly on study days 1, 8, 15, 22, and 29. The recovery period ended on day 58. Dose levels of 0, 10, 30, or 100 mg / kg were administered to three male and three female monkeys per group plus two male and two female recovery animals in the control and high-dose groups.

[0182] No animals died or had to be sacrificed during the study, and no test-related signs of local intolerance were noted.

[0183] No study-related effects were noted in any animal at any dose level on behavior and appearance, body weight and body weight gain, food and water consumption, electrocardiogram parameters and heart rate, circulatory function, hematology including coagulation parameters, D-dimer levels, clinical chemistry parameters, cytokine levels, urinary parameters, ophthalmological and auditory function, organ mass, and bone marrow:erythroid ratio. No macroscopic organ changes were noted in any animal examined at any dose level tested.

[0184] Histopathology did not reveal any test article-related local or systemic lesions. No test article-related changes were noted during or at the end of the 4-week treatment-free recovery period.

[0185] Based on the above results, the no observed effect level (NOEL) was 100 mg CTL-002 / kg by intravenous 30-minute infusion repeated once weekly for 4 weeks, ie, 5 doses per animal.

[0186] Cmax levels and AUC areas of total CTL-002 revealed a roughly linear, dose-related systemic exposure of the animals. No gender-specific differences were noted. Accumulation of total CTL-002 over time was noted and ranged from approximately 2-fold to 6-fold. The calculated mean terminal serum elimination half-life (t 1 / 2 ) ranged from 119 to 651 hours.

[0187] The primary pharmacokinetic data for male and female animals after the first (days 1-8) and fourth (days 22-29) doses of CTL-002 are given in Table 5.

[0188] [Table 5]

[0189] After infusion of CTL-002, GDF-15 serum levels increased 100- to 1000-fold in all animals at all dose levels and remained elevated throughout the dosing interval. This increase was comparable across dose groups, as shown in Figure 4, which illustrates GDF-15 levels in female monkeys throughout the first week after dosing.

[0190] It can therefore be concluded that in the central monkey study, complete target inhibition was obtained at all dose levels and throughout the dosing interval.

[0191] Overall study design A Phase 1, multicenter, first-in-human (FIH), open-label study consisting of Part A (dose escalation) followed by Part B (expansion) will be conducted using the CTL-002 antibody. The primary intent of the study is to (a) demonstrate the safety of CTL-002 and the combination of CTL-002 plus anti-PD1 / PD-L1, and (b) demonstrate that patients who have relapsed after / are refractory to anti-PD1 / PD-L1 treatment due to elevated GDF-15 will again respond and show tumor regression when administered the combination of CTL-002 plus anti-PD1 / PD-L1.

[0192] Part A (dose escalation) At least 21 subjects will receive escalating doses of CTL-002 IV in the "3+3" cohort, both as monotherapy and in combination with an anti-PD-1 checkpoint inhibitor, given to subjects with advanced solid tumors that have relapsed after or are refractory to prior anti-PD-1 / PD-L1 therapy and who have exhausted or are no longer eligible for all available approved standard treatments. A "backfill cohort" will recruit additional patients up to the highest dose level.

[0193] Part B (enlarged) The safety and efficacy of CTL-002 will be further evaluated as monotherapy (exploring one monotherapy cohort) or in combination with anti-PD-1 checkpoint inhibitors (exploring up to eight combination cohorts) to confirm RP2D in defined tumor entities that have relapsed after or are refractory to prior anti-PD-1 / PD-L1 therapy, consisting of up to eight expansion cohorts of up to 25 subjects / cohort. A dedicated monotherapy cohort will serve to establish the safety profile of CTL-002 in long-term monotherapy at a dose considered therapeutic (without the addition of anti-PD-1 / PD-L1). Enrollment into all eight cohorts may occur in parallel.

[0194] Treatment period Part A (dose escalation) The study will use a standard "3+3" dose escalation design, enrolling 3-6 subjects / cohorts at each assigned dose level, depending on the occurrence of DLTs.

[0195] The planned doses of CTL-002 to be tested are outlined below: Cohort 1: 0.3 mg / kg Cohort 2: 1.0 mg / kg Cohort 3: 3.0 mg / kg Cohort 4: 10 mg / kg Cohort 5: 20 mg / kg

[0196] The starting dose of 0.3 mg / kg will be fixed for Cohort 1. The doses explored in Cohorts 2-5 (outlined above) may be modified by the Safety Research Committee (SRC) based on emerging data (i.e., available safety, PK / pharmacodynamic, and other biomarker data).

[0197] The DLT observation period is the first two treatment cycles (i.e., the first 4 weeks) for each dosing cohort. All treatment cycles are initially defined as a 2-week period. CTL-002 is administered as an IV infusion once every two weeks. Subjects will initially receive one dose of CTL-002 as monotherapy for one cycle, followed by one cycle of CTL-002 combined with a defined checkpoint inhibitor, which is administered at a dose of 240 mg IV once every two weeks.

[0198] The combination involves giving CTL-002 and the defined checkpoint inhibitor simultaneously on the same day, with CTL-002 administered first, and the initial combination infusion will have a 30-minute observation period to assess safety, followed by the infusion of the defined checkpoint inhibitor. The observation period may be modified (i.e., shortened or extended) based on emerging safety data.

[0199] The first two treatment cycles (ie, the first 4 weeks) represent the DLT observation period.

[0200] Thereafter, subjects continue combination treatment until progression or withdrawal from the study for any other reason (eg, toxicity or subject withdraws consent).

[0201] Based on emerging data and Safety Research Committee (SRC) requirements, additional intermediate dose cohorts may be explored. The maximum dose of CTL-002 tested in this study will not exceed 20 mg / kg.

[0202] For safety observation purposes and to allow for logistical collection of sampling time points (e.g., PK), all subjects will be hospitalized overnight after receiving their first dose of CTL-002 and their first combination dose of CTL-002 and defined checkpoint inhibitor.

[0203] Intrapatient dose escalation during extended treatment Upon completion of Cohort 2 and follow-up by SRC, any Cohort 1 subject still receiving 0.3 mg / kg treatment may be increased to the Cohort 2 dose of 1.0 mg / kg.

[0204] Upon completion of Cohort 3 and follow-up by SRC, any Cohort 1 or 2 subject still receiving 1.0 mg / kg treatment may be escalated to the Cohort 3 dose of 3.0 mg / kg.

[0205] Note: Any subjects still receiving 0.3 mg / kg treatment must be treated at 1.0 mg / kg before progressing to 3.0 mg / kg per Sponsor Medical Monitor.

[0206] The maximum dose a subject can be increased to through intra-dose escalation is 3.0 mg / kg.

[0207] Available safety, PK / pharmacodynamic, and preliminary efficacy data will inform decisions regarding the MTD / dose to further explore in Part B of the study.

[0208] The MTD is defined as the highest dose level of CTL-002 at which 1 in 6 subjects or less experience a DLT during the first two treatment cycles (i.e., the first 4 weeks when CTL-002 is given as monotherapy [weeks 1 and 2] and in combination with a defined checkpoint inhibitor [weeks 3 and 4]).

[0209] Additionally, in Cohorts 3-5, in the absence of any DLTs, three more subjects may be recruited in each of these cohorts (up to a total of six subjects / cohort) to increase understanding of PK and pharmacodynamic data. This will occur while dose escalation continues. These additional "backfill" subjects will receive the combination treatment of CTL-002 and the defined checkpoint inhibitor once every two weeks, starting on Day 1 of Cycle 1, as outlined above, with CTL-002 always administered first, followed by the defined checkpoint inhibitor.

[0210] Part A subjects (non-backfill subjects) will have three serial tumor biopsies taken: one at baseline, a second biopsy before the start of combination therapy (2 weeks later), and a third biopsy after the first cycle of combination therapy (either at the end of treatment or, if combination treatment is continued, at the end of cycle 2 / beginning of cycle 3).

[0211] Backfill patients are required to undergo only two biopsies: one at baseline and a second after 4 weeks of combination treatment (either at the end of treatment or, if combination treatment is continued, at the end of cycle 2 / beginning of cycle 3).

[0212] These biopsies are mandatory to assess immune cell infiltration in the tumor. If a biopsy cannot be taken for safety reasons, this must be discussed with the medical monitor.

[0213] Treatment with CTL-002 in monotherapy (1 cycle) and in combination with a checkpoint inhibitor (nivolumab) has been safely tolerated up to dose level 5 (20 mg / kg) of CTL-002. No DLTs or grade 4 adverse events occurred in any treated patient. Combination treatment can be initiated simultaneously, as demonstrated by the so-called backfill cohort with immediate combination of CTL-002 and anti-PD1 / PD-L1 treatment.

[0214] Consistent with preclinical data on increased immune infiltration by aGDF-15, biomarker analysis demonstrates a consistent tumor-selective influx of CD8+ cells from DL1-5. Preliminary analysis indicates that in some patients, T cell proliferation is increased in the tumor, as demonstrated by CD3 / ki-67+ staining. Some tumors that are "cold tumors," characterized by low CD8 and CD4 counts at baseline, can be converted to hot tumors by increasing CD8 and CD4 counts.

[0215] Tumor shrinkage was observed at various dose levels. Data are available for 24 evaluable subjects (Part A: 24 patients / Part B: 0 patients). All subjects had been treated in the heavily pretreated setting (average 4.4 prior lines of therapy) and had relapsed / refractory to previous anti-PD-1 / PD-L1 therapy.

[0216] A clear and durable clinical benefit was observed in six subjects in this heavily pretreated, end-line tumor cohort. Three subjects experienced a confirmed partial response (PR), currently lasting up to 10 months or longer (one with unknown primary carcinoma at dose level [DL] 3 and one with mesothelioma at DL 5), and a third subject experienced a confirmed PR with durable, long-term tumor regression (hepatocellular carcinoma at DL 4). Three additional subjects experienced long-term disease stabilization, two of which lasted longer than 6 months (one with NSCLC and two with melanoma). A third subject had disease stabilization for 5.5 months before experiencing local progression of one liver lesion compressing a blood vessel. At the investigator's discretion, study treatment was continued and local irradiation was added to the site (study discontinuation). Shortly thereafter, the subject achieved a deep confirmed partial response and demonstrated an abscopal response in unirradiated lesions (under continued study treatment with CTL-002 and nivolumab with sponsor permission). Two other subjects had minor tumor regression in isolated lesions.

[0217] In summary, in dose escalation (Part A), 6 subjects were treated with an optional extension treatment phase for up to 10 months, and 2 were treated even beyond 1 year, and showed durable tumor control, including durable confirmed partial responses.

[0218] Part B (enlarged) Part B of the study may enroll up to eight cohorts (up to 25 subjects / cohort), each enrolling subjects with a specific tumor type.

[0219] To explore the safety profile of CTL-002 given as monotherapy (e.g., in subjects with advanced melanoma), a dedicated CTL-002 monotherapy cohort may be established in the expansion part of this study. Furthermore, this monotherapy cohort will require mandatory serial tumor biopsies to broaden our understanding of the pharmacodynamic effects of CTL-002 in tumor tissue.

[0220] In this monotherapy cohort, two serial tumor biopsies are mandatory: one biopsy taken at baseline and a second biopsy taken two weeks later (end of cycle 1 / beginning of cycle 2). All subjects will be treated until progression.

[0221] Up to eight other expansion cohorts of defined tumor populations may then be treated with the combination of CTL-002 and the defined checkpoint inhibitors. Tumor indications consist of tumor types approved for PD-1 / PD-L1 treatment and subjects who have relapsed / progressed on or after anti-PD-1 / PD-L1 treatment. Enrollment into expansion cohorts may occur in parallel. All subjects will be treated until progression.

[0222] For safety observation purposes and to allow for logistical collection of sampling time points (i.e., PK sampling), all subjects will be hospitalized overnight after receiving their first dose of CTL-002 (monotherapy cohort) or their first combination dose of CTL-002 and a defined checkpoint inhibitor (combination therapy cohort).

[0223] Research evaluation Tumor biopsy The timing of biopsy is as follows: Part A (all subjects except backfill subjects): Three serial tumor biopsies are required: one at baseline, a second biopsy before the start of combination therapy (after 2 weeks), and a third biopsy after the first cycle of combination treatment (either at the end-of-treatment visit or, if combination treatment is continued, at the end of cycle 2 / beginning of cycle 3). These biopsies are required to assess immune cell infiltration in the tumor. Part A (eligible for backfill): Two serial tumor biopsies are required: one at baseline and a second biopsy 4 weeks later (either at the end-of-treatment visit or, if combination treatment is continued, at the end of cycle 2 / beginning of cycle 3). Part B (subjects enrolled in the monotherapy cohort): Two serial tumor biopsies are required: one at baseline and a second biopsy after the first monotherapy cycle (i.e., 2 weeks).

[0224] There should be a lesion at or near which serial biopsies are amenable, if possible, but this lesion should not be the only target lesion evaluated radiologically during the course of the study.

[0225] Biomarkers may be analyzed from biopsied tumor tissue samples and may include additional immune cell markers and / or tumor markers specific to any of the tumor types.

[0226] Biopsied tumor tissues will be fixed in formalin and embedded in paraffin (FFPE) to determine treatment-induced changes in the number, frequency, and spatial location of infiltrating immune cells, including but not limited to leukocytes, various lymphocytes (e.g., CD4+ and CD8+ T cells, B cells, NK cells), and expression of the CTL-002 drug target, GDF-15 protein and mRNA, by histology before and after treatment with CTL-002 or in combination with defined checkpoint inhibitors.

[0227] Tumor lesions For subjects in Part A of the study, target skin lesions selected for RECIST assessment will be measured with calipers and photographed. In addition, the number of skin lesions will be recorded. Clinical measurements of skin lesions in Part A will include color photographic documentation (including size measurements) and caliper measurements of lesions at baseline within -7 days prior to CTL-002 infusion, every 4 weeks during extension treatment, at the end-of-treatment visit, and during follow-up.

[0228] Safety evaluation Safety and tolerability of IV infusion of CTL-002 monotherapy and in combination with defined checkpoint inhibitors will be assessed by incidence of AEs (all AEs will be assessed according to NCI CTCAE v5.0), SAEs, DLTs, and concomitant medication use. Safety assessments will include ECG, physical examination including neurological examination to rule out motor neuropathy, ECOG performance status, vital signs, and clinical laboratory samples (hematology, clinical chemistry, coagulation, thyroid function (thyroid-stimulating hormone [TSH] and free T3), cytokines, hemoglobin A1c assessment [HbA1c], N-terminal B-type natriuretic peptide [NT-proBNP], and urinalysis).

[0229] Subjects will be assessed for safety at screening and during treatment until safety follow-up visits, after which study-related safety will be further captured during 12-month (Part A) / 24-month (Part B) post-treatment follow-up.

[0230] vital signs Vital signs should be assessed, including systolic and diastolic BP (sitting), pulse rate, temperature, respiratory rate, and oxygen saturation. Additional vital sign measurements may be taken if clinically warranted.

[0231] Physical and neurological examination A physical examination will be performed at screening and will include an examination of the head, eyes, ears, nose, pharynx, neck, cardiovascular, chest / lungs, abdomen (including liver and spleen size), extremities, skin, and lymph nodes, as well as a brief neurological examination to assess for motor neuropathy.

[0232] Performance Status Performance status will be assessed at screening according to the following ECOG criteria: · 0 = fully active, able to perform all pre-disease activities without limitation. 1 = Physically strenuous activity is limited, but the patient is able to walk and perform tasks of a light or sedentary nature (e.g., light housework, office work). 2 = Able to walk and take care of themselves, but unable to perform any occupational activities. Active more than 50% of waking hours. 3 = Able to perform only limited self-care and remains in bed or chair for more than 50% of waking hours. · 4 = Totally incapacitated, unable to perform self-care, completely confined to bed or chair. ·5=Death.

[0233] Cardiac function monitoring Subjects are undergoing intensive monitoring for cardiac / vascular AEs, and safeguards are in place to exclude at-risk subjects from study participation.

[0234] At baseline, subjects undergo ECG, echocardiography (or MUGA if ECHO is not available), and N-terminal pro-b-type natriuretic peptide (Peptid) level testing (NT-proBNP, heart failure screening). NT-proBNP testing is repeated every 2 weeks for 3 months, then monthly thereafter, or if there is any suspicion of any kind of cardiac / vascular damage (in which case it is again combined with ECG and echocardiography).

[0235] A single 12-lead ECG is performed.

[0236] All ECG monitoring will be performed on-site at the study site.

[0237] The subject should be relaxed and in a recumbent or semi-recumbent position for at least 5 minutes before the ECG is recorded.

[0238] If clinically justified, additional ECG testing may be performed at the investigator's discretion.

[0239] Clinical Laboratory Evaluation Collect samples for the laboratory tests listed below. All testing will be done on-site.

[0240] Hematology / coagulation and clinical chemistry results must be available, reviewed, and deemed acceptable by the investigator or authorized designee prior to administration of CTL-002 / PD-1 / PD-L1.

[0241] Clinically significant abnormal tests should be repeated to confirm the nature and magnitude of the abnormality. Appropriate ancillary investigations should be initiated, if necessary. If the abnormality fails to resolve or cannot be explained by events or conditions unrelated to the study drug or its administration, the medical monitor should be consulted.

[0242] The clinical significance of abnormal test values ​​must be determined by the investigator in the context of the disease under study, and includes a significant shift from a baseline within the normal range that the investigator considers to be clinically important.

[0243] [Table 6]

[0244] Pharmacokinetics The PK of CTL-002 given as monotherapy and / or in combination with defined checkpoint inhibitors will be measured from blood samples taken at the start of treatment and at various subsequent time points (Part A). Additional PK data may be evaluated in an expansion group (Part B).

[0245] Blood samples will be taken at the start of treatment and at various subsequent time points to determine whether antibodies directed against CTL-002 may have developed.

[0246] Systemic cytokine / chemokine monitoring (pharmacodynamics) Serum samples will be collected for measurement of cytokines, chemokines, and other circulating biomarkers to assess pharmacodynamic efficacy and safety. Cytokines and chemokines analyzed may include, but are not limited to, tumor necrosis factor alpha (TNF-α), interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, CXCL9 (monokine induced by gamma [MIG]), and CXCL10 (IP-10).

[0247] Exploratory evaluation During this clinical trial, serum biomarker testing may be performed on samples (retention aliquots) specifically collected for future biomedical studies to identify serum factors (e.g., but not limited to, metabolites, soluble growth factors, cytokines, chemokines) important to anti-GDF-15 (CTL-002) treatment. Retrospective biomarker studies will be performed using appropriate biostatistical design and analysis to compare with PK / pharmacodynamic results, previously evaluated biomarkers, or clinical outcomes.

[0248] Efficacy evaluation: Imaging evaluation (on-site test) Tumor response will be assessed using RECIST V1.1 and imRECIST criteria according to institutional standards. For the purposes of this study, subjects will be evaluated for a baseline scan at screening and reassessed every 8 weeks starting at cycle 3 and / or end-of-treatment visits; after this time, response assessments may be performed as per local institutional guidelines until the end of efficacy and survival follow-up.

[0249] All lesions identified at screening / baseline must be tracked at all times using the unique lesion number assigned at screening / baseline and documented in the subject file and eCRF.

[0250] The same assessment method (imaging modality, e.g., MRI, CT) must be used for each subject at baseline and during all follow-up examinations to characterize each identified and reported lesion. If there is a change in modality, the study site may be asked to explain the reason for the change in the eCRF. A change in modality may be considered a protocol deviation.

[0251] Each efficacy time point / visit may be completed up to a ±7 day window.

[0252] In addition to on-site reading by investigators during the trial, centralized reading of images will be performed after the trial by a reading center.

[0253] Definition of progressive disease according to RECIST V1.1 and iRECIST: RECIST assessment is used to identify subjects with potential disease progression (Eisenhaur et al., 2009). As defined by the modified RECIST V1.1 criteria for immune-based therapeutics, or iRECIST criteria (Seymour et al., 2017), the date of first potential progression by RECIST scan is defined as the immune-unconfirmed progressive disease (iUPD) date. Subjects who have an iUPD date and are stable will continue to participate in the study as planned and will be re-evaluated for progression 4-8 weeks after their initial evaluation. If the confirmatory assessment supports PD, the date of disease progression will be the iUPD date. If the confirmatory assessment does not support PD, the subject does not have disease progression, and the iUPD date will be ignored. Such subjects will remain in the study as planned and continue with the next imaging evaluation scheduled by the protocol.

[0254] Antitumor activity will be assessed by investigator assessment using immune response criteria according to RECIST V1.1 and iRECIST, as described below. · Contrast CT scan, MRI, or positron emission tomography-computed tomography (PET-CT) of the chest, abdomen, and pelvis. Disease response and disease progression will be assessed in this study using RECIST and iRECIST criteria. Subjects with brain and / or leptomeningeal metastases that are symptomatic, untreated, or requiring current therapy are not eligible for the study. Brain imaging must not be older than 12 weeks. Abnormal / unexpected findings on brain MRI should be discussed with the medical monitor as part of the screening process. The same assessment method and the same techniques should be used at baseline and during follow-up to characterize each identified and reported lesion. If there is a change in modality, the study site may be asked to explain the reason for the change in the eCRF. A change in modality may be considered a protocol deviation.

[0255] result The study began in December 2020, and the first patient enrolled on December 9, 2020. Cohorts 1-5 have been completed without dose-limiting toxicities (DLTs), and Phase 1 (Part A) of the study is complete. Note: Interim data.

[0256] Patients and CTL-002 Treatment: This interim report includes demographic and preliminary safety data for all 25 patients treated in Phase 1 (Part A) of the CTL-002-001 clinical trial.

[0257] [Table 7]

[0258] Preliminary safety and tolerability of CTL-002: To date, monotherapy and combination with nivolumab have been well tolerated. No dose-limiting toxicities (DLTs) or grade 4 or 5 TEAEs have occurred, and no concerning safety events have been observed to date. Twenty-five subjects received at least one dose of visgromab (CTL-002) in Part A (Phase 1) of the clinical trial CTL-002-001. Adverse events were coded according to the Medical Dictionary for Regenerative Medicine (MedDRA).

[0259] No DLTs or ≥ Grade 4 AEs were reported in all subjects treated in Part A of the study.

[0260] No significant safety findings arose from this clinical trial during the reporting period. Treatment has not been associated with any adverse events of major clinical concern to date and has been well tolerated in all subjects treated to date in the context of the treatment of advanced stage cancer. As of the interim report (cutoff date February 10, 2022), 12 SAEs have been reported in 8 subjects (32.0%) to date in Study CTL-002-001. A total of 4 of these SAEs were assessed by the investigators and / or sponsor as possibly related to CTL-002.

[0261] No grade 4 (life-threatening) or grade 5 (fatal) AEs related to CTL-002 were reported during the reporting period.

[0262] Grade 3 AEs reported in multiple patients were increased GGT and anemia (2 subjects each; transfusions were not permitted during the DLT period). No subjects discontinued the study prematurely due to AEs.

[0263] Biomarker Strategy and Analysis This clinical trial explores serum- and tissue-based biomarkers. Apart from classical immune system activation markers such as serum cytokines, specific analyses will be performed to evaluate the immunomodulatory effects of GDF-15 in the tumor microenvironment. Among other parameters, baseline GDF-15 levels, intratumoral GDF-15 levels, and the number and profile of tumor-infiltrating leukocytes will be analyzed before and during GDF-15 neutralization with CTL-002. Substantial tumor-selective GDF-15 expression was confirmed for most tumors analyzed. Under CTL-002 treatment, primarily CD8 + and CD4 + Selective influx of T cells into the tumor and tumor stroma was observed in the majority of patients, with efficacy seen from dose level 1 onward. Preliminary analyses indicate that in some patients, T cell proliferation is increased in the tumor, as evidenced by CD3 / ki-67+ staining. Some tumors, which are "cold tumors" at baseline, characterized by low CD8 and CD4 counts, can be converted to hot tumors by increasing the CD8 and CD4 counts.

[0264] Furthermore, some tumors showed reactive PD-L1 upregulation, an indirect sign of IFN-gamma release and an active immune response against tumor cells.

[0265] [Table 8]

[0266] Precedent response evaluation A clear and durable clinical benefit was observed in six subjects in this heavily pretreated, end-line tumor cohort. Three subjects experienced a confirmed partial response (PR), two of which are now durable for more than 10 months (one with unknown primary carcinoma at dose level [DL] 3 and one with mesothelioma at DL 5), and a third with hepatocellular carcinoma with durable, long-term tumor regression. Three additional subjects experienced long-term disease stabilization, two of which lasted for more than 6 months (one with NSCLC and two with melanoma). A third subject had disease stabilization for 5.5 months before experiencing local progression with one liver lesion compressing a blood vessel. At the investigator's discretion, study treatment was continued and local irradiation was added to the site (study discontinuation). Shortly thereafter, the subject achieved a deep, confirmed partial response, with abscopal response observed in unirradiated lesions (under continued study treatment with CTL-002 and nivolumab with sponsor permission). Two other subjects had minor tumor regression in isolated lesions.

[0267] In summary, in dose escalation (Part A), 6 subjects were treated with an optional extension treatment phase for up to 11 months and demonstrated durable tumor control, including durable confirmed partial responses.

[0268] conclusion Recent preclinical data from the present inventors and others indicate that GDF-15 potently (1) prevents T cell infiltration into the tumor microenvironment (TME) and (2) also suppresses potent immune responses by other mechanisms within the TME. Thus, GDF-15 plays a key role in suppressing effective antitumor immune responses.

[0269] The GDFATHER (GDF-15 Antibody-Mediated Effector Cell Repositioning) Phase 1 / 2 trial will explore the safety, PK, and PD, as well as the upfront antitumor activity of the GDF-15 neutralizing antibody CTL-002, as monotherapy and in combination with checkpoint inhibitors (CPIs) in a CPI relapsed / refractory patient population. Anti-cachexia effects will also be investigated.

[0270] Dose levels 1 to 5 have been safely completed with good tolerability and no DLTs, and the Phase 2 portion has begun.

[0271] Pharmacodynamic analysis from serial tumor biopsies (dose levels 1-5) demonstrates CTL-002-mediated selective T cell shift into the tumor microenvironment in the majority of samples examined.

[0272] The preferred dose and dosing regimen for antibodies of the invention, including CTL-002, is 3, 10, or 20 mg / kg / Q2wk, with 10 mg / kg / Q2wk being a more preferred dose and dosing regimen. These dose levels are expected to be highly effective, as reflected by the favorable clinical effects observed in patients to date. Furthermore, the selection of the dose and dosing regimen is based on extensive research conducted by the inventors, including pharmacological modeling and PK / pharmacodynamic data obtained from Part A, which demonstrates complete GDF-15 neutralization at this dose in patients with a full range of baseline GDF-15 serum levels. A review of all treatment doses and their adverse events, the PK / pharmacodynamics observed to date, and the extensive pharmacological modeling exercises performed indicates complete GDF-15 suppression within the tumor vasculature at 10 mg / kg CTL-002, even at elevated baseline serum concentrations of up to 10 ng / ml GDF-15 in serum (corresponding to approximately 160 ng / ml GDF-15 in the immediate vicinity of the tumor). No concerning safety events have been observed to date at this dose, no DLTs have occurred at this dose, and there is still a >10-fold safety margin compared to the NOAEL in non-human primates (NHPs). Therefore, the preferred dose indicated above is expected to be particularly effective and safe.

[0273] The above-described observations regarding efficacy, safety, and PK / pharmacodynamic data also indicate that anti-GDF-15 antibodies can be advantageously administered at a preferred dose of 10-20 mg / kg, more preferably 20 mg / kg, in at least one dosing cycle, where the cycle comprises a 3-week period for the 10 mg / kg dose and a 4-week period for the 20 mg / kg dose, with said doses administered at least once (i.e., preferably once) in each of the at least one cycle. Although this dosing regimen has a longer dosing cycle of 3 or 4 weeks, the preferred dose of 10-20 mg / kg, more preferably 20 mg / kg, allows for a similar advantageous safety and efficacy profile to the preferred 10 mg / kg / Q2wk regimen. This dosing regimen is compatible with the observed PK / pharmacodynamic profiles for 3, 10, and 20 mg / kg q2w, q3w, and q4w dosing, as well as the population PK / PD modeling approach used to model serum total CTL-002 and total GDF-15 in combination with a previously developed tumor model of GDF-15 to predict systemic and tumor levels of serum free GDF-15. The dosing expected to maintain free GDF-15 in the tumor below the nominal healthy value of 0.5 ng / mL depends on baseline GDF-15, with higher baseline levels requiring higher doses. These data support a dosing regimen of 10 mg / kg at least every 2 or 3 weeks or 20 mg / kg at least every 4 weeks to maintain free GDF-15 in the tumor vasculature below the nominal healthy value of 0.5 ng / mL (Figure 5).

[0274] The above findings indicate that treatment with anti-GDF-15 antibodies according to the invention can provide substantial clinical benefit in cancer patients with a very low associated risk profile. Importantly, this benefit is observed in patients who were previously refractory to some of the most cutting-edge treatment options, such as treatment with PD-1 / PD-L1 axis antagonists (e.g., nivolumab).

[0275] ((2) Example 1) In light of the advantageous findings made in the foregoing Reference Examples, the present inventors sought to identify and further analyze cancers that would be particularly responsive to treatment with anti-GDF-15 antibodies and aPD-1 / -L1 antibodies together. Based on their further analysis, the following cancer subgroups are predicted to be particularly responsive to treatment:

[0276] In colorectal cancer (CRC), 13 selected immune-related analyses were performed in silico using mRNA expression data from numerous colorectal cancer patients. All calculations and correlation analyses were performed using the statistical software environment R. In these analyses, GDF-15 mRNA expression was correlated with 13 immune signatures, including mRNA expression of genes related to CD8+ cell infiltration, T cell dysfunction, T cell inflammatory tumors, interferon gamma, inflammation, immune prediction score, cytolytic activity, cytotoxic T lymphocytes, and T cell elimination, as well as mRNA expression of genes encoding PD-1 and PD-L1, and marker genes for CD8 cells and cytotoxic T lymphocytes.

[0277] Strikingly, in all 13 immune-related analyses, a significant inverse correlation with GDF-15 mRNA expression was observed in CRC patients. Furthermore, a clear decrease in putative infiltrating CD8+ T cells could be observed from lower to higher GDF15 expression.

[0278] Similarly, for BLCA, BRCA, PAAD, LUAD, KIRC, CESC, and TGCT, an inverse correlation with GDF-15 mRNA expression could be observed in at least 10 of the 13 selected immune-related analyses.

[0279] These findings are consistent with the potent T cell deletion caused by GDF-15 in patients with CRC, BLCA, BRCA, PAAD, LUAD, KIRC, CESC, and TGCT.

[0280] Therefore, treatment with anti-GDF-15 antibodies according to the invention is expected to be particularly effective in at least CRC, BLCA, BRCA, PAAD, LUAD, KIRC, CESC, and TGCT patients due to the restoration of tumor infiltration by T cells.

[0281] ((3) Example 2) The present inventors sought to identify and further analyze cancer patients across a variety of different cancer types who would be particularly responsive to combination treatments comprising an anti-GDF-15 antibody and a checkpoint inhibitor. Based on these further experiments, the present inventors surprisingly found that the following patients are predicted to be particularly responsive to combination treatments:

[0282] In this regard, patients' baseline biopsies are analyzed for PD-L1 TPS and tumor inflammation score (TIS) and T cell infiltration counts.

[0283] PD-L1 TPS method For test interpretation and scoring, please refer to the manufacturer's interpretation guide: "VENTANA PD-L1 (SP263) Assay Staining of Non-Small Cell Lung Cancer Interpretation Guide", version 1015317EN.

[0284] Briefly, the percentage of PD-L1-positive tumor cells (TPS) was determined in an immunohistochemistry (IHC) assay procedure on formalin-fixed, paraffin-embedded (FFPE) sections using an anti-PD-L1 (SP263) monoclonal antibody (Ventana, catalog no. 790-4905). To expose all target antigens, tissue sections were deparaffinized and heat-pretreated prior to antibody incubation. Anti-PD-L1 antibody binding to PD-L1-positive cancer cells was then visualized using a secondary HRP enzyme-conjugated antibody (Ventana optiView Universal DAB Detection Kit, catalog no. 760-700). The specific antibody-enzyme complex was visualized based on the precipitating enzyme reaction product. To determine PD-L1 TPS, a CE-IVD-licensed predictive assay (741-4905) was used, utilizing a defined cutoff of ≥1% membrane-stained tumor cells to distinguish between negative and positive cancer tissue (e.g., NSCLC tissue) specimens. Because increased overall survival after treatment with nivolumab (OPDIVO) was observed in patients with higher PD-L1 expression levels, positive cases were further subdivided by applying additional cutoffs of ≥5% and ≥10%. Slide interpretation and cutoff determination are described in more detail in the official scoring guideline "VENTANA PD-L1 (SP263) Assay fur Non-Small Cell Lung Cancer Interpretationsanleitung 2016," version 1015317EN.

[0285] For scoring, the percentage of PD-L1 positive tumor cells was determined for each staining intensity (0, 1, 2, 3), and the total percentage of stained tumor cells was calculated by summing the percentages at intensities 1, 2, and 3.

[0286] Analysis of clinical data revealed PD-L1 TPS as a positive enrichment factor for clinical response (see Figure 6, Figure 7A / B, Figure 8).

[0287] Tumor Inflammation Score (TIS) Methodology: The TIS includes 18 functional genes known to be associated with response to PD-1 / PD-L1 inhibitor pathway blockade (PSMB10, HLA-DQA1, HLA-DRB1, CMKLR1, HLA-E, NKG7, CD8A, CCL5, CXCL9, CD27, CXCR6, IDO1, STAT1, TIGIT, LAG3, CD274, PDCD1LG2, and CD276), as well as IFN-γ-responsive genes associated with antigen presentation, chemokine expression, cytotoxic activity, and adaptive immune resistance genes (see Danaher, Patrick et al.; Ayers M et al.). The TIS method may be performed as briefly described below. First, RNA extracts from formalin-fixed, paraffin-embedded tumor blocks were hybridized with the NanoString® PanCancer IO360™ CodeSet using nCounter® technology. Next, to eliminate assay technical variability, the raw data for each sample and gene were normalized to the internal ERCC control. Counts were then normalized to the geometric mean of endogenous housekeeping genes, followed by log2 transformation. Finally, a gene expression signature, including TIS, was calculated as a weighted linear average of the constituent genes.

[0288] Based on analysis of baseline biopsies from patients, PD-L1 and TIS are expected to show predictive value for clinical response to combination treatment including anti-GDF-15 Ab (e.g., CTL-002) and nivolumab, especially in patients who are refractory to anti-PD-1 / PD-L1 (see clinical responders 1-02-006, 1-03-004, 1-03-002, and long-term SD 2-01-003 with PD-L1 TPS>1 and tumor inflammation score (TIS)>7.5 in Figure 6).

[0289] These findings by the inventors are surprising because the present combination treatment can be successfully used even in patients who are refractory to monotherapy with an anti-PD1 or anti-PD-L1 antibody or combination therapy with an anti-PD1 or anti-PD-L1 antibody and other anti-cancer agents. Therefore, such combination therapy is clinically different from monotherapy with an anti-PD1 or anti-PD-L1 antibody or combination therapy with an anti-PD1 or anti-PD-L1 antibody and other anti-cancer agents, and therefore, the combination treatment of the present invention was expected to be associated with different biomarkers. Based solely on the experimental results provided by the inventors, it was surprisingly found that the combination treatment of the present invention achieves particularly beneficial treatment outcomes in patients with PD-L1-positive cancers or inflammatory tumors as indicated by TIS, which subsequently confirmed the failure of previous anti-PD1 / PD-L1 treatment in the final line of treatment.

[0290] CD8+ T cell density (cell number / mm) by immunohistochemistry (IHC) 2Determination of CD8+ antigens: The CD8 protein is a glycoprotein that is primarily expressed on the surface of CTLs, thereby serving as a general marker for this specific cell population. The function of CD8+ CTLs is to eliminate cancer cells or virus-damaged or infected cells by recognizing antigen fragments presented on MHC class I on these cells. Activated CTLs release cytokines and cytotoxic enzymes, such as granzyme B and perforin, which lead to the death of cancerous or infected cells. The primary (monoclonal) antibodies (monoclonal mouse anti-human CD8, clone C8 / 144B) applied in the multiplex IHC assay procedure described herein bound to their respective antigens in FFPE tissue sections (thickness: 4 μm). To expose all target antigens, tissue sections were deparaffinized and heat-pretreated prior to antibody incubation. Specific antibody binding can be visualized by applying one or more linker antibodies, most often hydroxyquinoxaline (HQ)- or nitropyrazole (NP)-conjugated, followed by a secondary horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-enzyme-conjugated antibody, thus allowing amplification of the resulting signal. The enzyme coupled to the secondary antibody catalyzed a chromogenic precipitate-forming reaction at the actual primary antibody binding site. Each incubation step was followed by an incubation. After each incubation step, the VENTANA DISCOVERY ULTRA automated slide stainer washed the sections to terminate the respective reaction and remove any unbound material that would interfere with the desired reaction in the subsequent step. Before applying the next primary antibody, the previous reagents were removed by heat incubation ("stripping"), leaving only the insoluble precipitate on the slide. Quantitative evaluation of stained slides was performed using Visiopharm digital image analysis software VIS 2019.02 (TID 001673). Stained slides were scanned using an Aperio ScanScope XT (Leica) at 20x magnification prior to image analysis.The final workflow not only allows for the detection of positive staining for a single biomarker, but also distinguishes between tumor, normal tissue, and peritumoral and intratumoral tissue compartments. To achieve this, additional guidance staining with tumor-specific markers panCK (solid tumors) or SOX10 (malignant melanoma) is introduced, allowing for the alignment of serial sections to generate virtual overlay images and the transfer of information from one slide to another. The final readout is an area measurement (mm) per tissue compartment. 2 ), cell number per cell type per compartment, density calculation (cells / mm 2 ), and distance measurements for biologically relevant cell type combinations.

[0291] Determination of CD3+ T cell density (cells / mm2) by IHC: CD3+ T cell density was determined in 4 μm thick FFPE tissue sections using the UltiMapper® I / O T-act kit by ULTIVUE® (p / n ULT20110) in an ULTIVUE® multiplex immunofluorescence assay (UltiMapper 1 / 0 T-act panel CD3, granzyme B, Ki67, and panCK / SOX10), stained with Leica Bond RX, digitized as full slide images using a ZEISS Axioscan Z1, and subsequently digitally analyzed in Visiopharm.

[0292] Baseline biopsies from clinical responders 1-02-006, 1-03-004, 1-03-002, 1-02-036, and 1-06-005, and long-term SD 2-01-003, showed PD-L1 TPS >1 and >300 cells / mm 2Figures 7A and 7B show CD8+ T cell densities in the 100% TPS population (Figure 7A). Therefore, CD8+ T cell density, as well as TPS, is expected to have predictive value for the treatment of human patients with anti-GDF-15 antibodies such as CTL-002 in combination with anti-PD-1 or anti-PD-L1 antibodies, such as nivolumab, when administered to anti-PD-1 and / or anti-PD-L1 refractory patients. The same applies to CD3+ T cell density (Figure 8).

[0293] In patients treated with anti-GDF15, an average 2- to 4-fold change in CD8+ cell influx compared to baseline (Bsl) was observed at days 14 and 28, indicating that anti-GDF15 antibodies increase the percentage of CD8+ cells in the tumor compared to baseline (Figure 9, upper panel). Similarly, immune cell analysis of biopsies from patients' baseline (Bsl), days 14, and 28 revealed increased proliferation of CD3+Ki67+ T cells under treatment (Figure 9, lower panel).

[0294] In summary, analysis of clinical data revealed, among other things, PD-L1 TPS as a positive enrichment factor for clinical response (see Figure 6, Figure 7A / B, Figure 8).

[0295] Furthermore, clinical data also demonstrated that patient selection by PD-L1 TPS alone or in combination with any of CD8 cell density, CD3 cell density, or TIS enriched for clinical responders.

[0296] For example, of 14 bladder cancer patients, 2 patients showed PD-L1 TPS > 5, and both patients showed PR. Similarly, of 14 bladder cancer patients, 7 patients showed PD-L1 TPS > 1, and 28.6% (2 of 7) of them showed PR.

[0297] Furthermore, of 14 bladder cancer patients, 2 patients showed PD-L1 TPS>5 and CD8 cell density>300 cells / mm2, and both patients showed PR.Similarly, of 14 bladder cancer patients, 3 patients showed PD-L1 TPS>1 and CD8 cell density>300 cells / mm2, and 66.7% (2 of 3) of them showed PR.

[0298] array SEQ ID NO: 1 (heavy chain CDR1 region peptide sequence of monoclonal anti-human GDF-15 antibody): GFSLSTSGMG

[0299] SEQ ID NO: 2 (heavy chain CDR2 region peptide sequence of monoclonal anti-human GDF-15 antibody): IYWDDDK

[0300] SEQ ID NO: 3 (heavy chain CDR3 region peptide sequence of monoclonal anti-human GDF-15 antibody): ARSSYGAMDY

[0301] SEQ ID NO: 4 (light chain CDR1 region peptide sequence of monoclonal anti-human GDF-15 antibody): QNVGTN

[0302] Peptide sequence of the light chain CDR2 region of the monoclonal anti-human GDF-15 antibody: SAS

[0303] SEQ ID NO: 5 (light chain CDR3 region peptide sequence of monoclonal anti-human GDF-15 antibody): QQYNNFPYT

[0304] SEQ ID NO: 6 (heavy chain variable domain of monoclonal anti-human GDF-15 antibody):

[0305] [ka]

[0306] SEQ ID NO: 7 (light chain variable domain of monoclonal anti-human GDF-15 antibody):

[0307] [ka]

[0308] SEQ ID NO: 8 (heavy chain of monoclonal anti-human GDF-15 antibody CTL-002 without leader peptide sequence):

[0309] [ka]

[0310] SEQ ID NO: 9 (light chain of monoclonal anti-human GDF-15 antibody CTL-002 without leader peptide sequence):

[0311] [ka]

[0312] SEQ ID NO: 10 (heavy chain variable domain of anti-human GDF-15 antibody H1L5):

[0313] [ka]

[0314] SEQ ID NO: 11 (light chain variable domain of anti-human GDF-15 antibody H1L5):

[0315] [ka]

[0316] Discontinuous epitope (EVQVTMCIGACPSQFR (SEQ ID NO: 12) --- 38 amino acids --- TDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 13))

[0317] The sequence shown in Figure 3: GDF-15 human: SEQ ID NO: 14 GDF-15 macfa: SEQ ID NO: 15 GDF-15 mouse: SEQ ID NO: 16 GDF-15 Rat: SEQ ID NO: 17 Partial consensus CTL-002 epitope (b): SEQ ID NO: 18

[0318] SEQ ID NO: 19 (light chain variable region domain of anti-mGDF-15 antibody)

[0319] [ka]

[0320] SEQ ID NO: 20 (heavy chain variable region domain of anti-mGDF-15 antibody)

[0321] [ka]

[0322] (References) TIFF2025531837000021.tif212160TIFF2025531837000022.tif234160TIFF2025531837000023.tif169160 [Industrial Applicability]

[0323] The anti-GDF-15 antibodies used in the method for treating cancer in human patients can be industrially manufactured and sold as products for specified methods and uses according to known standards for the manufacture of pharmaceutical products. Thus, the present invention is industrially applicable.

Claims

1. A pharmaceutical composition comprising a combination of an anti-GDF-15 antibody and at least one checkpoint inhibitor for use in a method of treating cancer in a human patient having a PD-L1-positive cancer and / or having a cancer involving an inflammatory tumor.

2. 2. The pharmaceutical composition for use as defined in claim 1, wherein the cancer is PD-L1 positive as determinable by an immunohistochemistry (IHC) assay.

3. 3. The pharmaceutical composition for use as defined in claim 2, wherein the IHC assay uses an anti-PD-L1 antibody as the primary antibody to determine the percentage of PD-L1 positive cancer cells.

4. 4. The pharmaceutical composition for use as defined in claim 2 or 3, wherein the PD-L1 positive cancer has a percentage of cancer cells that show staining for the presence of PD-L1 on the cell surface in an immunohistochemistry (IHC) assay of at least 1.

5. 3. The pharmaceutical composition for use as defined in claim 2, wherein the IHC assay uses an anti-PD-L1 antibody as the primary antibody to determine the total percentage of PD-L1 positive cancer cells and PD-L1 positive cancer infiltrating cells.

6. 6. The pharmaceutical composition for use as defined in any one of claims 1 to 5, wherein the inflammatory tumor is a T-cell inflammatory tumor.

7. 7. The pharmaceutical composition for use as defined in any one of claims 1 to 6, wherein the inflammatory tumor is a tumor containing CD3+ cells.

8. 8. The pharmaceutical composition for use as defined in claim 7, wherein the inflammatory tumor contains CD3+ cells that can be determined by an immunohistochemistry (IHC) assay, and the IHC assay uses an anti-CD3 antibody as the primary antibody.

9. Inflammatory tumors were assessed by immunohistochemistry (IHC) assay to have >300 cells / mm in immunohistochemistry tissue sections with a thickness of 4 μm. 2 9. The pharmaceutical composition for use as defined in claim 8, wherein the tumor has an average CD3+ cell density of 0.1 to 0.5%.

10. 10. The pharmaceutical composition for use as defined in any one of claims 1 to 9, wherein the inflammatory tumor is a tumor containing CD8+ cells.

11. 11. The pharmaceutical composition for use as defined in claim 10, wherein the inflammatory tumor contains CD8+ cells that can be determined by an immunohistochemistry (IHC) assay, and the IHC assay uses an anti-CD8 antibody as the primary antibody.

12. Inflammatory tumors were assessed by immunohistochemistry (IHC) assay to have >300 cells / mm in immunohistochemistry tissue sections with a thickness of 4 μm. 2 12. The pharmaceutical composition for use as defined in claim 11, wherein the tumor has an average CD8+ cell density of 0.05 to 0.

15.

13. 13. The pharmaceutical composition for use as defined in any one of claims 6 to 12, wherein the T cell inflammatory tumor is inflammatory as indicated by its tumor inflammation signature (TIS).

14. 14. A pharmaceutical composition for use as defined in claim 13, wherein the TIS is based on the expression of at least one, preferably all, of the marker genes selected from the group consisting of PSMB10, HLA-DQA1, HLA-DRB1, CMKLR1, HLA-E, NKG7, CD8A, CCL5, CXCL9, CD27, CXCR6, IDO1, STAT1, TIGIT, LAG3, CD274, PDCD1LG2, and CD276.

15. TIS, [Equation 1] [where x i is the log2-transformed normalized expression level of the i-th gene, and w i is a predefined mass greater than zero] 15. The pharmaceutical composition for use as defined in claim 14, wherein the marker gene expression level is a linear combination of all of the marker genes calculated as:

16. 16. A pharmaceutical composition for use as defined in claim 15, having a TIS higher than 7.

5.

17. 17. The pharmaceutical composition for use as defined in any one of claims 1 to 16, wherein the human patient is an anti-PD-1 and / or anti-PD-L1 relapsed and / or refractory patient.

18. 18. The pharmaceutical composition for use as defined in any one of claims 1 to 17, wherein the checkpoint inhibitor is selected from one or more of the group consisting of an anti-PD-1 antibody or a PD-1-binding fragment thereof, an anti-PD-L1 antibody or a PD-L1-binding fragment thereof, an anti-CD40 antibody or a CD40-binding fragment thereof, an anti-LAG-3 antibody or a LAG-3-binding fragment thereof, an anti-TIM-3 antibody or a TIM-3-binding fragment thereof, an anti-TIGIT antibody or a TIGIT-binding fragment thereof, and an anti-CTLA4 antibody or a CTLA4-binding fragment thereof.

19. 19. The pharmaceutical composition for use as defined in claim 18, wherein the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody or a PD-1-binding fragment thereof and an anti-PD-L1 antibody or a PD-L1-binding fragment thereof.

20. 20. The pharmaceutical composition for use as defined in any one of claims 1 to 19, wherein the cancer is a solid tumor.

21. 21. The pharmaceutical composition for use as defined in any one of claims 1 to 20, wherein the cancer is selected from the group consisting of colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate adenocarcinoma, pancreatic cancer, uterine cancer, cervical cancer, thyroid cancer, cutaneous squamous cell carcinoma, mesothelioma, carcinoma of unknown primary (CUP), and breast cancer.

22. 22. The pharmaceutical composition for use as defined in any one of claims 1 to 21, wherein the anti-GDF-15 antibody comprises a heavy chain variable domain comprising a CDR1 region represented by the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region represented by the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region represented by the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain comprising a CDR1 region represented by the amino acid sequence shown in SEQ ID NO: 4, a CDR2 region represented by the amino acid sequence ser-ala-ser, and a CDR3 region represented by the amino acid sequence shown in SEQ ID NO:

5.

23. 23. The pharmaceutical composition for use as defined in any one of claims 1 to 22, wherein the anti-GDF-15 antibody has a heavy chain variable domain comprising the amino acid sequence represented by SEQ ID NO: 6 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence shown in SEQ ID NO: 6, and a light chain variable domain comprising the amino acid sequence represented by SEQ ID NO: 7 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence shown in SEQ ID NO:

7.

24. 24. The pharmaceutical composition for use as defined in any one of claims 1 to 23, wherein the anti-GDF-15 antibody has a heavy chain comprising an amino acid sequence represented by SEQ ID NO: 8 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence shown in SEQ ID NO: 8, and a light chain comprising an amino acid sequence represented by SEQ ID NO: 9 or an amino acid sequence having at least 90% identity, preferably at least 95% identity, more preferably at least 98% identity with the amino acid sequence shown in SEQ ID NO:

9.

25. 25. The pharmaceutical composition for use as defined in any one of claims 1 to 24, wherein the anti-GDF-15 antibody has a heavy chain comprising the amino acid sequence represented by SEQ ID NO: 8 and a light chain comprising the amino acid sequence represented by SEQ ID NO:

9.

26. The anti-GDF-15 antibody is administered to a human patient, preferably i) administered at a dose of 3 to 20 mg / kg, preferably at a dose of 10 mg / kg, in a dosing regimen of at least one administration cycle, the cycle being of 2 weeks duration, said dose being administered at least once during each of the at least one cycle; ii) administered at a dose of 3 to 20 mg / kg, preferably at a dose of 10 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 3 weeks in duration, and wherein said dose is administered at least once during each of the at least one cycle; or iii) administered at a dose of 3 to 20 mg / kg, preferably at a dose of 20 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 4 weeks in duration, and said dose is administered at least once during each of the at least one cycle; the cancer is selected from the group consisting of colorectal cancer, gastric cancer, bladder cancer, melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate adenocarcinoma, pancreatic cancer, uterine cancer, cervical cancer, thyroid cancer, cutaneous squamous cell carcinoma, mesothelioma, cancer of unknown primary (CUP), and breast cancer; 26. A pharmaceutical composition for use as defined in any one of claims 1 to 25.

27. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is colorectal cancer.

28. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is bladder cancer.

29. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is cancer of unknown primary (CUP).

30. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is mesothelioma.

31. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is cutaneous squamous cell carcinoma.

32. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is non-small cell lung cancer.

33. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is hepatocellular carcinoma.

34. 27. The pharmaceutical composition for use as defined in any one of claims 1 to 26, wherein the cancer is melanoma.

35. 35. The pharmaceutical composition for use as defined in any one of claims 1 to 34, wherein the use is also for the treatment of cancer cachexia.

36. 36. The pharmaceutical composition for use as defined in any one of claims 1 to 35, wherein the anti-GDF-15 antibody is administered at a dose of 3 to 20 mg / kg, preferably 20 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 4 weeks in duration, and wherein said dose is administered at least once during each of the at least one cycle.

37. 37. The pharmaceutical composition for use as defined in any one of claims 1 to 36, wherein the anti-GDF-15 antibody is administered at a dose of 3 to 20 mg / kg, preferably 10 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is 3 weeks in duration, and wherein said dose is administered at least once during each of the at least one cycle.

38. 38. The pharmaceutical composition for use as defined in any one of claims 1 to 37, wherein the anti-GDF-15 antibody is administered at a dose of 3 to 20 mg / kg, preferably 10 mg / kg, in a dosing regimen of at least one administration cycle, wherein the cycle is of two weeks duration, and wherein said dose is administered at least once during each of the at least one cycle.

39. 39. The pharmaceutical composition for use as defined in any one of claims 1 to 38, wherein said anti-GDF-15 antibody is obtainable by expression in CHO cells.

40. 40. The pharmaceutical composition for use as defined in any one of claims 1 to 39, wherein said checkpoint inhibitor is administered in the same dosage regimen as the anti-GDF-15 antibody.

41. 41. The pharmaceutical composition for use as defined in any one of claims 1 to 40, wherein the checkpoint inhibitor is administered prior to administration of the anti-GDF-15 antibody, preferably within 120 minutes prior to administration of the anti-GDF-15 antibody, more preferably within 30 minutes prior to administration of the anti-GDF-15 antibody.

42. 42. The pharmaceutical composition for use as defined in any one of claims 1 to 41, wherein the dose of anti-GDF-15 antibody is administered intravenously.

43. (i) analyzing whether the cancer is a PD-L1 positive cancer and / or whether the cancer includes an inflammatory tumor; (ii) predicting the patient's clinical outcome for the treatment based on the analysis; 1. A method for predicting the clinical outcome of a human cancer patient on treatment comprising a combination of an anti-GDF-15 antibody and at least one checkpoint inhibitor, comprising:

44. (a) analyzing whether the cancer is a PD-L1 positive cancer and / or whether the cancer includes an inflammatory tumor; (b) predicting the patient's clinical outcome for the treatment based on the analysis; and (c) treating the human patient with a combination of an anti-GDF-15 antibody and at least one checkpoint inhibitor; 10. A method of treating a human patient diagnosed with cancer, comprising:

45. 45. The method of claim 43 or 44, wherein the clinical outcome comprises response, complete response, partial response, stable disease, progressive disease, and / or survival of the human cancer patient.

46. 46. ​​The method of any one of claims 43 to 45, wherein the patient is predicted to have an improved clinical outcome if the cancer is PD-L1 positive.

47. 47. The method of claim 46, wherein the PD-L1 positive cancer is as defined in claims 2 to 5.

48. 48. The method of any one of claims 43 to 47, wherein the patient is predicted to have an improved clinical outcome when the cancer is an inflammatory tumor.

49. 49. The method of claim 48, wherein the inflammatory tumor is as defined in claims 6 to 16.

50. 50. The method of any one of claims 43 to 49, wherein the checkpoint inhibitor is as defined in claim 18 or 19.

51. 51. The method of any one of claims 43 to 50, wherein the cancer is selected from the cancers defined in claim 20 or 21.

52. 52. The method of any one of claims 43 to 51, wherein the human patient is an anti-PD-1 and / or anti-PD-L1 relapsed and / or refractory patient.

53. 1. An anti-GDF-15 antibody for use in a method of treating cancer in a human patient, wherein the anti-GDF-15 antibody is administered in combination with at least one checkpoint inhibitor, and the human patient has a PD-L1-positive cancer and / or has a cancer comprising an inflammatory tumor.

54. 54. An anti-GDF-15 antibody for use as defined in claim 53, wherein the patient has a cancer as defined in any one of claims 2 to 16, 19 to 21, and 26 to 34.

55. 55. An anti-GDF-15 antibody for use as defined in claim 53 or 54, wherein the patient is as defined in claim 17.

56. 56. An anti-GDF-15 antibody for use as defined in any one of claims 53 to 55, wherein the checkpoint inhibitor is as defined in claim 18 or 19.

57. 57. An anti-GDF-15 antibody for use as defined in any one of claims 53 to 56, wherein the anti-GDF-15 antibody is as defined in any one of claims 22 to 25 and 39.

58. 58. An anti-GDF-15 antibody for use as defined in any one of claims 53 to 57, wherein the anti-GDF-15 antibody is administered as defined in any one of claims 26, 36 to 38, and 42.

59. 59. An anti-GDF-15 antibody for use as defined in any one of claims 53 to 58, wherein the checkpoint inhibitor is administered as defined in claim 40 or 41.

60. 60. The anti-GDF-15 antibody for use as defined in any one of claims 53 to 59, wherein the use is also for the treatment of cancer cachexia.

61. 61. The pharmaceutical composition for use as defined in any one of claims 1 to 42 or the anti-GDF-15 antibody for use as defined in any one of claims 53 to 60, wherein the PD-L1 positive cancer has a percentage of cancer cells that stain for the presence of PD-L1 on the cell surface in an immunohistochemistry (IHC) assay of at least 1, preferably at least 2, more preferably at least 5.

62. 62. The pharmaceutical composition or anti-GDF-15 antibody for use as defined in claim 61, wherein the cancer is bladder cancer.

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