Anti-GDF15 antibodies and drug regimens for cancer treatment

Abstract

The present invention relates to an anti-GDF15 antibody and a drug regimen for the treatment of cancer in human patients using the anti-GDF15 antibody. [Solution] The inventors have identified the mechanism by which GDF-15 blocks the attachment and entry of primarily T lymphocytes into tissues. Therefore, the present invention establishes a novel treatment approach that enables the treatment of cancer in human patients by promoting the entry of effector T cells into tumor tissue through the blockade of GDF-15 using the antibody of the present invention.

Description

[Technical Field]

[0001] The present invention relates to an anti-GDF15 antibody and a drug regimen for the treatment of cancer in human subjects with cancer using the anti-GDF15 antibody. [Background technology]

[0002] GDF-15 is a diverse member of the TGF-beta superfamily whose functions in appetite regulation, metabolism, cell and tissue survival, and immune tolerance have been described. GDF-15 is produced as a homodimer that matures into a 25 kDa (2 × 112 aa) dimeric GDF-15 and as a proprotein that is cleaved into a 2 × 18 kDa (2 × 167 aa) propeptide present in tissues (Tsai 2018).

[0003] To date, two main categories of GDF-15 activity have been described. The first category concerns metabolic effects, namely, GDF-15 induces anorexia by mediating cachexia through changes in food intake behavior (Johnen 2007). This effect is mediated by a brainstem-specific receptor named GFRAL, described in late 2017 (Emmerson 2017). In contrast, the second category concerns immunomodulatory effects, namely, 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 diseases, and tumor evasion. GDF-15 inhibits leukocyte integrin activation, thereby preventing their infiltration (Kempf 2011).

[0004] In recent years, there has been growing evidence that GDF-15 plays an essential immunomodulatory role in physiological and pathophysiological situations, and particularly in cancer. For cancer cells, it would naturally be very appealing to utilize and "hijack" such immune cell evasion mechanisms to block the entry of immune cells into the tumor microenvironment, thereby preventing the immune system from eliminating cancer cells. Consistent with this, numerous publications have recently emerged indicating that high serum levels of GDF-15 in various cancer types correlate with shorter overall survival, and that GDF-15 is an independent factor for patient survival in various tumor types (Wischhusen et al., 2020).

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

[0006] Additionally, two proprietary analyses involving two different academic melanoma research groups suggest that GDF-15 levels also appear to correlate with the response to PD-1 antagonists.

[0007] As indicated, cancerous tissue, normal organ tissue in difficult conditions, and the placenta are known to overexpress GDF-15, most likely in all cases, to prevent excessive immune cell infiltration into their respective tissues. Therefore, the inventors hypothesized that GDF-15 produced by the above-mentioned tissues substantially reduces vascular T cell adhesion and endothelial migration, thereby preventing T cell entry into or immediate access to their respective tissues.

[0008] While anti-GDF-15 antibodies generally exhibit a benign and well-tolerated safety profile in animal models, this mode of action naturally presents various potential risks when intended to provide a suitable drug regimen for human treatment.

[0009] A reasonable combination partner for anti-GDF-15 antibodies would be T-cell activating compounds, such as anti-PD-1 / PD-L1 checkpoint inhibitors. The efficacy of such compounds may be substantially enhanced. However, their toxicity may also be potentially enhanced when combined with certain drug regimens for anti-GDF-15 neutralizing antibodies.

[0010] A second potential area of ​​challenge is the physiological role of GDF-15 in organ protection for organs in distress. When GDF-15 is suppressed using anti-GDF-15 antibodies and organ distress occurs (e.g., myocardial infarction, infection, or other major organ injury), excessive organ infiltration and undesirable tissue damage / destruction by immune cells may occur.

[0011] A third potential challenge is the scarcity of findings made in individual mouse knockout models for GDF-15 (Wischhusen 2020).

[0012] Furthermore, two important mechanisms supporting the cytotoxic effects of antibody drugs against tumor cells are antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Both mechanisms can cause undesirable effects in endogenous healthy cells, leading to the expression of antibody targets or nonspecific binding to antibodies.

[0013] In this regard, ADCC is a crucial mechanism for killing target cancer cells, based on the binding of certain antibody drugs to human FcγRIIIa receptors on immune cells (mostly natural killer cells), resulting in the activation of bound killer cells. Thus, recognition and binding of target antigens on tumor cells, as well as bridging between target cells and immune cells by antibodies, are essential for ADCC induction. The cross-linking of target and immune cells leads to the activation of the ADCC MOA (mechanism of action) pathway and ultimately to cell lysis.

[0014] It is even more important to provide a stable formulation of the antibody that can be safely administered to patients and meets all criteria for long-term stability. [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Tsai, Vicky WW, Yasmin Husaini, Amanda Sainsbury, David A. Brown, and Samuel N. Breit. 2018. “The MIC-1 / GDF15-GFRAL Pathway in Energy Homeostasis: Implications for Obesity, Cachexia, and Other Associated Diseases.” Cell Metabolism 28 (3): pp. 353–68, https: / / doi.org / 10.1016 / j.cmet.2018.07.018. [Non-Patent Document 2] Johnen H, Lin S, Kuffner T, et al., "Tumor-induced anorexia and weight loss are mediated by the TGF-β superfamily cytokine MIC-1." Nat Med. 2007;13(11):1333~1340, doi:10.1038 / nm1677 [Non-Patent Document 3] Emmerson, Paul J., Feng Wang, Yong Du, Qian Liu, Richard T. Pickard, Malgorzata D. Gonciarz, Tamer Coskun et al., 2017. "The Metabolic Effects of GDF15 Are Mediated by the Orphan Receptor GFRAL." Nature Medicine 23 (10): pp. 1215–19, https: / / doi.org / 10.1038 / nm.4393. [Non-Patent Document 4] Tong S, Marjono B, Brown DA et al., "Serum concentrations of macrophage inhibitory cytokine 1 (MIC 1) as a predictor of miscarriage." Lancet. 2004;363(9403):129-130, doi:10.1016 / S0140-6736(03)15265-8 [Non-Patent Document 5] Chung HK, Kim JT, Kim HW et al., "GDF15 deficiency exacerbates chronic alcohol- and carbon tetrachloride-induced liver injury." Sci Rep. 2017;7(1):1~13, doi:10.1038 / s41598-017-17574-w [Non-Patent Document 6] Abulizi P, Loganathan N, Zhao D, et al., "Growth Differentiation Factor-15 Deficiency Augments Inflammatory Response and Exacerbates Septic Heart and Renal Injury Induced by Lipopolysaccharide" Sci Rep. 2017;7(1):pp.1-10 [Non-Patent Document 7] Kempf, Tibor, Alexander Zarbock, Christian Widera, Stefan Butz, Anika Stadtmann, Jan Rossaint, Matteo Bolomini-Vittori et al. 2011. "GDF-15 Is an Inhibitor of Leukocyte Integrin Activation Required for Survival after Myocardial Infarction in Mice." Nature Medicine 17 (5): pp. 581–88, https: / / doi.org / 10.1038 / nm.2354. [Non-Patent Document 8] Wischhusen J, Melero I, and Fridman W. "GDF-15: From Biomarker to Novel Targetable Immune Checkpoint." Front. Immunol. Accepted April 23, 2020. doi: 10.3389 / fimmu.2020.00951. [Non-Patent Document 9] Welsh, John B., Lisa M. Sapinoso, Suzanne G. Kern, David A. Brown, Tao Liu, Asne R. Bauskin, Robyn L. Ward, et al. 2003. "Large-Scale Delineation of Secreted Protein Biomarkers Overexpressed in Cancer Tissue and Serum." Proceedings of the National Academy of Sciences 100 (6): pp. 3410–15, https: / / doi.org / 10.1073 / PNAS.0530278100. [Non-Patent Document 10] 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 [Non-Patent Document 11] Bottner, Martina, Martin Laaff, Birgit Schechinger, Gudrun Rappold, Klaus Unsicker, and Clemens Suter-Crazzolara. 1999. "Characterization of the Rat, Mouse, and Human Genes of Growth / Differentiation Factor-15 / Macrophage Inhibiting Cytokine-1 (GDF-15 / MIC-1)." Gene 237 (1): pages 105~11 [Non-Patent Document 12] Wang M, Yao LC, Cheng M, Cai D, Martinek J, Pan CX et al., "Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy." FASEB J. 2018;32(3):1537~49 [Non-Patent Document 13] Selby, Mark J., John J. Engelhardt, Robert J. Johnston, Li-Sheng Lu, Minhua Han, Kent Thudium, Dapeng Yao et al., 2016. "Preclinical Development of Ipilimumab and Nivolumab Combination Immunotherapy: Mouse Tumor Models, In Vitro Functional Studies, and Cynomolgus Macaque Toxicology.", Edited by Aamir Ahmad, PLOS ONE 11 (9): e0161779. https: / / doi.org / 10.1371 / journal.pone.0161779. [Non-Patent Document 14] van den Boorn, Jasper G. and Gunther Hartmann. 2013. "Turning Tumors into Vaccines: Co-Opting the Innate Immune System." Immunity 39 (1): pp. 27-37, https: / / doi.org / 10.1016 / j.immuni.2013.07.011. [Non-Patent Document 15] Vaupel, Peter. 2004. "Tumor Microenvironmental Physiology and Its Implications for Radiation Oncology." Seminars in Radiation Oncology 14 (3): pp. 198-206, https: / / doi.org / 10.1016 / j.semradonc.2004.04.008. [Non-Patent Document 16] Eisenhauer EA, Therasse P, Bogaerts J et al., "New Response Evaluation Criteria in Solid Tumours: Revised RECIST Guideline (Version 1.1)." Eur J Cancer. 2009 Jan;45(2):pp. 228-247 [Non-Patent Document 17] Hodi FS, Ballinger M, Lyons B et al., "Immune-Modified Response Evaluation Criteria In Solid Tumors (imRECIST): Refining Guidelines to Assess the Clinical Benefit of Cancer Immunotherapy." J Clin Oncol. 2018 Mar 20;36(9):pp. 850-858 [Summary of the Invention] [Problem to be Solved by the Invention]

[0016] An object of the present invention is to overcome the unmet clinical need for providing safe and effective compositions for use in the therapeutic treatment of human patients.

[0017] The present invention further provides safe and stable formulations for said antibodies. [Means for Solving the Problem]

[0018] Based on large-scale experimental studies, the inventors of the present application have surprisingly found that neither antibody-dependent cell-mediated cytotoxicity (ADCC) nor complement-dependent cytotoxicity (CDC) substantially contributes to the anti-cancer effect of the anti-GDF15 antibody of the present invention.

[0019] GDF-15 blocks the attachment and entry of T lymphocytes into tissues. Using the anti-GDF15 antibody of the present invention, which blocks GDF-15, a treatment approach has been established that promotes the entry of effector T cells into cancer tissue without any contribution of ADCC or CDC to the anticancer effect. This should substantially increase the efficacy of any T cell activator, such as a checkpoint inhibitor, and thus should enable the provision of effective immunotherapy, either alone or in combination with a checkpoint inhibitor.

[0020] Simultaneously, the inventors' discovery that neither ADCC nor CDC contributes to the anti-cancer effect makes it possible to use an anti-GDF-15 antibody that effectively treats cancer without inducing ADCC and / or CDC, thereby increasing overall safety.

[0021] Therefore, the anti-GDF-15 antibody of the present invention is particularly safe, thus remarkably combining complete efficacy against cancer with safety.

[0022] Furthermore, the applicant also provides the first drug regimen of an anti-GDF15 antibody that enables favorable treatment of human patients.

[0023] Therefore, the present invention provides the following preferred embodiments:

[0024] Item 1: A composition comprising an anti-GDF-15 antibody for use in a method for treating cancer and / or cancer cachexia in human patients.

[0025] Item 2 The composition according to Item 1, wherein the anti-GDF-15 antibody is administered in a dose of 0.3, 1.0, 3.0, 10.0, or 20.0 mg / kg, preferably 3, 10, or 20 mg / kg, more preferably 10 mg / kg, and in a drug regimen of at least one administration cycle, wherein the cycle is for a period of 2 weeks, and the dose is administered at least once in each of the at least one cycle.

[0026] Item 3 The composition according to Item 1, wherein the anti-GDF-15 antibody is administered in a dose of 10 to 20 mg / kg, preferably 20 mg / kg, and in a drug regimen of at least one administration cycle, the cycle having a duration of 4 weeks, and the dose is administered at least once in each of the at least one cycle.

[0027] Item 4 The composition according to Item 1, wherein the anti-GDF-15 antibody is administered in a dose of 10-20 mg / kg and in a drug regimen of at least one administration cycle, the cycle having a duration of 3 weeks, and the dose is administered at least once in each of the at least one cycle.

[0028] Item 5: The composition according to any one of items 1 to 4, wherein the antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0029] Item 6: The composition according to any one of items 1 to 5, wherein the anti-GDF-15 antibody is an IgG4 isotype antibody.

[0030] Item 7 The composition according to Item 6, wherein the antibody contains a hinge-stabilizing mutation.

[0031] Item 8: The composition according to Item 7, wherein the hinge-stabilizing mutation is the S228P mutation.

[0032] Item 9 The composition according to any one of items 1 to 8, wherein the anti-GDF-15 antibody comprises a heavy chain variable domain including 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 including 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.

[0033] Item 10 The composition according to any one of items 1 to 9, wherein the heavy chain variable domain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable domain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 7 or the amino acid sequence shown in SEQ ID NO: 7.

[0034] Item 11 The composition according to any one of items 1 to 10, wherein the heavy chain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: 8, and the light chain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 9 or the amino acid sequence shown in SEQ ID NO: 9.

[0035] Item 12 The composition according to any one of items 1 to 11, wherein the anti-GDF-15 antibody can be obtained by expression in CHO cells.

[0036] Item 13 The composition according to any one of items 1 to 12, wherein the concentration of GDF-15 in the patient's serum / plasma is less than 10 ng / mL at the end of the administration cycle.

[0037] Item 14 The composition according to Item 13, wherein the concentration of GDF-15 in the patient's serum is less than 2 ng / mL at the end of the administration cycle.

[0038] Item 15: The composition according to Item 13, wherein the concentration of GDF-15 in the patient's serum is less than 0.5 ng / mL at the end of the administration cycle.

[0039] Item 16 The composition according to any one of items 1 to 15, wherein the administration cycle is for a period of 3 weeks, and the dose is administered at least once in each of the at least one cycle.

[0040] Item 17 The composition according to any one of items 1 to 15, wherein the administration cycle is for a period of 4 weeks, and the dose is administered at least once in each of the at least one cycle.

[0041] Item 18 The composition according to any one of items 1 to 15, wherein the drug regimen comprises multiple cycles and optionally up to 52 administration cycles.

[0042] Item 19 The composition according to Item 18, wherein the drug regimen consists of 26 to 52 administration cycles.

[0043] Item 20 The composition according to Item 16, wherein the drug regimen consists of up to 34 administration cycles.

[0044] Item 21 The composition according to Item 20, wherein the drug regimen consists of 17 to 34 administration cycles.

[0045] Item 22 The composition according to Item 17, wherein the drug regimen comprises up to 26 administration cycles.

[0046] Item 23 The composition according to Item 22, wherein the drug regimen consists of 12 to 26 administration cycles.

[0047] Item 24 The composition according to any one of items 1 to 23, wherein the anti-GDF-15 antibody is administered in combination with a checkpoint inhibitor.

[0048] Item 25 The composition according to Item 24, wherein the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CD40 antibody.

[0049] Item 26 The composition according to item 24 or 25, wherein the checkpoint inhibitor is administered in the same drug regimen as the GDF-15 antibody.

[0050] Item 27 The composition according to any one of items 24 to 26, wherein the checkpoint inhibitor is administered prior to the administration of the GDF-15 antibody.

[0051] Item 28 The composition according to Item 27, wherein the checkpoint inhibitor is administered within 120 minutes prior to the administration of the GDF-15 antibody.

[0052] Item 29 The composition according to Item 28, wherein the checkpoint inhibitor is administered within 30 minutes prior to the administration of the GDF-15 antibody.

[0053] Item 30 The composition according to any one of items 1 to 29, wherein the cancer is selected from the group consisting of all brain cancers (including gliomas), all cancers of the nervous system, melanoma, lung cancer, liver cancer, endometrial cancer, cervical cancer, ovarian cancer, breast cancer, renal cell carcinoma, bone and soft tissue tumors (including, for example, Ewing's sarcoma), non-small cell lung cancer and small cell lung cancer, lip cancer, nasopharyngeal cancer, laryngeal cancer, pharyngeal cancer, any type of head and neck cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, bladder cancer, any type of urothelial carcinoma, testicular cancer, thyroid cancer, kidney cancer, gallbladder and common bile duct cancer, multiple myeloma, esophageal cancer, gastrointestinal tumors including stomach, colorectal, rectal and anal cancers, pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, and carcinomas of unknown primary origin, and any other type of human cancer, and the use is optionally and for the treatment of cancer cachexia.

[0054] Item 31 The composition according to any one of items 1 to 30, wherein the dose of the anti-GDF-15 antibody is administered intravenously.

[0055] Item 32. An anti-GDF-15 antibody, wherein the antibody is an IgG4 isotype antibody having a hinge-stabilizing mutation, and the heavy chain variable domain of the antibody contains an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable domain of the antibody contains an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 7 or the amino acid sequence shown in SEQ ID NO: 7.

[0056] Item 33 The anti-GDF-15 antibody according to Item 32, wherein the heavy chain of the antibody comprises an amino acid sequence having at least 90% identity, preferably at least 95% identity, and more preferably at least 98% identity with respect to the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: 8, and the light chain of the antibody comprises an amino acid sequence having at least 90% identity, preferably at least 95% identity, and more preferably at least 98% identity with respect to the amino acid sequence represented by SEQ ID NO: 9 or the amino acid sequence shown in SEQ ID NO: 9.

[0057] Item 34. An anti-GDF-15 antibody as described in item 32 or 33, wherein the hinge-stabilizing mutation is the S228P mutation.

[0058] Item 35 An antibody that does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC), as described in any one of items 32-34.

[0059] Item 36: An antibody described in any one of items 32-35, which can be obtained by expression in CHO cells.

[0060] An antibody according to any one of items 32 to 36, comprising a heavy chain variable domain including 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 including 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.

[0061] A formulation comprising an anti-GDF-15 antibody described in any one of items 32 to 37, or a composition described in any one of items 1 to 31, wherein the formulation comprises 10 to 50 mg / ml of the anti-GDF-15 antibody.

[0062] Item 39: The preparation described in Item 38, comprising histidine / histidine HCl, sucrose, arginine-HCl, and polysorbate at a pH of 5-6.

[0063] A formulation according to item 38 or 39, comprising 10-50 mg / ml of CTL-002, 10-50 mg / ml of histidine / histidine HCl, 100-200 mM of sucrose, 20-80 mM of arginine-HCl, and 0.01-0.05% w / v of polysorbate 20 or polysorbate 80 at a pH of 5.0-6.0, preferably pH 5.3-5.7.

[0064] Item 41: A preparation according to any one of items 38 to 40, containing 25 mg / ml of CTL-002, 20 mM histidine / histidine HCl, 150 mM sucrose, 50 mM arginine-HCl, and 0.02% w / v polysorbate 20 at pH 5.5.

[0065] The preparation described in item 41, comprising 25 mg / ml of CTL-002, 20 mM histidine / histidine HCl, 150 mM sucrose, 50 mM arginine-HCl, and 0.02% w / v polysorbate 20 at pH 5.5. [Brief explanation of the drawing]

[0066] [Figure 1] Treatment of GDF-15-producing tumors with anti-GDF-15 antibody B1-23 (a mouse precursor of CTL-002) substantially improves the response to anti-PD1 antibody. While anti-GDF-15 antibody B1-23 alone had only minimal effect, its combination with anti-PD-1 substantially reversed the impairment caused by anti-PD-1 treatment. [Figure 2] Baseline GDF-15 serum levels correlate with response to anti-PD1 / -L1 treatment. (A+B) Study in patients with advanced melanoma. Baseline GDF-15 levels are associated with (A) response outcomes and (B) overall survival in patients with advanced melanoma. (C+D) Baseline GDF-15 levels are associated with (C) response outcomes and (D) overall survival. [Figure 3] Affinity determination of the human antibody CTL-002 (=H1L5 IgG4). Binding kinetics were analyzed using Biacore T3000. Different GDF-15 concentrations were applied to a flow cell with CTL-002. The association and dissociation phases were recorded to calculate the antibody dissociation constant. Kinetic data were evaluated by global fitting using the software BIAevaluation 4.1. One of three representative measurements is shown. [Figure 4]GDF-15 has an IC50 of 7–14 ng / ml in an in vitro flow adhesion assay. HUVECs are cultured for 3 days before overnight activation. T cells are purified from healthy donors and purified on the day of the experiment. A physiological flow is generated through a mounted HUVEC channel slide using a softened pump. Primary T cells are pre-treated for 20 minutes with a dose titration of 0.4–100 ng / ml of GDF-15. The HUVEC monolayer is equilibrated for 20 minutes with a wash buffer containing 1 μM CXCL12, then the pre-treated T cells are perfused for 6 minutes, followed by a 40-minute co-culture step with the wash buffer. Individual images are recorded every 30 seconds in one fixed field of view, and adhesion events are recorded as the total number of cells per unit field of view. Due to technical and biological variability in the assay, the IC50 is typically 7–14 ng / ml. [Table 1] [Figure 5-1] CTL-002, along with EC50 at 690-725 ng / ml, restores GDF-15 adhesion inhibition in a concentration-dependent manner. HUVECs are cultured for 3 days before overnight activation. T cells are purified from healthy donors and purified on the day of the experiment. 50 ng / ml of GDF-15 is conjugated by pre-incubating with different concentrations of CTL-002 for 20 minutes. Primary T cells and HUVEC monolayers are pre-treated with the CTL-002 / GDF-15 complex for 20 minutes. After CTL-002 / GDF-15 incubation, the HUVEC monolayer is perfused with a wash buffer containing 1 μM CXCL12 for 5 minutes, followed by 15 minutes of quiescence. After washing, the pre-treated T cells are perfused for 6 minutes, followed by 50 minutes of washing. The first time point was recorded at 10 minutes, and then individual images were recorded every 30 seconds in one fixed field of view, recording adhesion events as the total number of cells per unit field of view for a total of 50 minutes. The EC50 was determined by generating a plot of AUC values. This is shown for three individual donors. Due to technical and biological variability in the assay, the IC50 is typically 690–725 ng / ml. [Figure 5-2]CTL-002, along with EC50 at 690-725 ng / ml, restores GDF-15 adhesion inhibition in a concentration-dependent manner. HUVECs are cultured for 3 days before overnight activation. T cells are purified from healthy donors and purified on the day of the experiment. 50 ng / ml of GDF-15 is conjugated by pre-incubating with different concentrations of CTL-002 for 20 minutes. Primary T cells and HUVEC monolayers are pre-treated with the CTL-002 / GDF-15 complex for 20 minutes. After CTL-002 / GDF-15 incubation, the HUVEC monolayer is perfused with a wash buffer containing 1 μM CXCL12 for 5 minutes, followed by 15 minutes of quiescence. After washing, the pre-treated T cells are perfused for 6 minutes, followed by 50 minutes of washing. The first time point was recorded at 10 minutes, and then individual images were recorded every 30 seconds in one fixed field of view, recording adhesion events as the total number of cells per unit field of view for a total of 50 minutes. The EC50 was determined by generating a plot of AUC values. This is shown for three individual donors. Due to technical and biological variability in the assay, the IC50 is typically 690–725 ng / ml. [Figure 6] Animals treated with CTL-002 show higher immune cell infiltration enriched with CD3+ cells. [Figure 7] MC38-hGDF-15 exhibits proliferative advantages only in immunocompetent mice. [Figure 8A] GDF-15 expression interferes with successful anti-PD-1 therapy. MC38-blank tumors responded to anti-PD-1 treatment (Figure 8A), while GDF-15-secreting tumors showed a significantly impaired response to anti-PD-1 (Figure 8B). [Figure 8B] GDF-15 expression interferes with successful anti-PD-1 therapy. MC38-blank tumors responded to anti-PD-1 treatment (Figure 8A), while GDF-15-secreting tumors showed a significantly impaired response to anti-PD-1 (Figure 8B). [Figure 9A]Treatment of hyper-GDF-15 tumors with anti-GDF-15 antibodies and anti-PD-1 antibodies can re-establish the success of checkpoint inhibitor therapy. Anti-GDF-15 antibody B1-23 had only minimal effect when used alone (solid line, Figure 9A), while its combination with anti-PD-1 (dashed line, Figure 9B) was able to partially reverse the impairment of anti-PD-1 treatment. [Figure 9B] Treatment of hyper-GDF-15 tumors with anti-GDF-15 antibodies and anti-PD-1 antibodies can re-establish the success of checkpoint inhibitor therapy. Anti-GDF-15 antibody B1-23 had only minimal effect when used alone (solid line, Figure 9A), while its combination with anti-PD-1 (dashed line, Figure 9B) was able to partially reverse the impairment of anti-PD-1 treatment. [Figure 10] The combination of B1-23 with anti-CD40 / poly(IC:LC) immunotherapy eradicated MC38 expressing human GDF-15, in contrast to anti-CD40 / poly(IC:LC) alone. [Figure 11] The combination of B1-23 with anti-CD40 / poly(IC:LC) immunotherapy eradicated MC38, in contrast to anti-CD40 / poly(IC:LC) alone. [Figure 12] ADCC induction in UACC-257 was investigated using test antibody CTL-001, variant CTL-001 IgG1* PG LALA, and CTL-002. Target cells were incubated with effector cells and different concentrations of test or control antibodies in triple incubators at 37°C for 6 hours. Luminescence signals were then measured. The induction factor of the luminescence signal or the resulting relative light units (RLU) were plotted against the antibody concentration. [Figure 13]The binding of human complement protein C1q to various concentrations of CTL-001, different isotype variants of CTL-001, CTL-002, and the control antibody rituximab was investigated. The interaction between C1q protein and therapeutic antibodies was determined using an ELISA-based approach with 10 μg / ml human C1q protein and an HRP-conjugated anti-C1q antibody for detection. Here, C1q binding was obtained after incubation of CTL-001 and different isotype variants with pre-coated human GDF-15 protein. The experiment was performed in triplicate, and the average absorption (A450nm) was plotted against antibody concentration. [Figure 14] Complement-dependent cytotoxicity (CDC) induced by test antibodies and different control antibodies against GDF-15-expressing cells. Cells were incubated with the indicated concentrations of test or control antibodies in the presence of 10% serum containing active rabbit complement protein. Cell viability was determined by adding Alamar Blue Viability dye. Mean fluorescence intensity (MFI) was measured after 6 hours and 24 hours of incubation at 37°C and 5% CO2, respectively. Calculated viability after 6h[A] and 24h[B] incubations is shown. The plotted values ​​are always the highest antibody concentration used. [Figure 15] Serum concentration-time profiles of CTL-001 and CTL-002 in cynomolgus monkeys. [Figure 16] Observed CTL-002 PK and total GDF-15 NHP DRF studies. Note: The PK of CTL-002 is linear in the range of 1–100 mg / kg and is approximately dose-proportional. The plateau in GDF-15 capture at both doses of 10 and 100 mg / kg indicates that all available GDF-15 is captured, and increasing the dose simply increases the duration of complete target capture. [Figure 17]Observed CTL-002 PK and total GDF-15 - NHP 4wk GLP toxicity study. Observed values ​​(symbols) for individual animals in the 4wk GLP toxicity study. Solid line - PK-PD model fitted to observed data: Data shown as black circles are included in the PK-PD model, and data shown as crosses are outliers and excluded from the PK-PD modeling. [Figure 18] Predicted human CTL-002 PK (serum concentration). [Figure 19] Predicted human free and total GDF-15 (serum concentration). Predicted suppression of GDF-15 in systemic circulation. Three baseline levels of serum GDF-15 are considered: 0.5 ng / mL (mean level in healthy subjects and approximately 15th percentile in the cancer patient cohort), 2 ng / mL (median level in the cancer patient cohort; 50th percentile), and 10 ng / mL (98th percentile in the cancer patient cohort). [Figure 20] Predicted suppression of GDF-15 in tumor microangiologies. Assumption: Three baseline levels of systemic GDF-15, 0.5, 2, and 10 ng / mL, result in tumor microangiological GDF-15 concentrations of 0.5, 25, and 161 ng / mL, respectively. [Figure 21] Predicted inhibition of serum GDF-15 at planned clinical doses and within the baseline level range. Normal serum levels of GDF-15 (0.5 ng / mL) in healthy subjects are indicated by a dashed line. [Figure 22] Sequences of the CTL-002 binding region in various species. The sequences of the CTL-002 binding region are shown for humans, cynomolgus monkeys, mice, and rats (first four rows from top to bottom). [Figure 23] GDF-15 serum levels in female monkeys after the first dose of CTL-002. [Figure 24]Sequence liability map of the H1L5 light chain. The sequence map graphically represents the locations of all identified sequence liabilities in the H1L5 light chain. In addition to the locations of domain boundaries and CDRs, the type of liability is detected at a given location relative to the overall sequence. The types of liabilities are indicated as follows: (I) Asn N-linked glycosylation, (II) Ser / Thr O-linked glycosylation, (III) Asn deamide, (IV) Asp isomerization / fragmentation, (V) pyroglutamic acid, (VI) C-terminal lysate, (VII) Met / Trp oxidation, (VIII) free thiol. [Figure 25] Sequence liability map of the H1L5 heavy chain. The sequence map graphically represents the locations of all identified sequence liabilities in the H1L5 heavy chain. In addition to the locations of domain boundaries and CDRs, the type of liability is detected at a given location relative to the overall sequence. The types of liabilities are indicated as follows: (I) Asn N-linked glycosylation, (II) Ser / Thr O-linked glycosylation, (III) Asn deamide, (IV) Asp isomerization / fragmentation, (V) pyroglutamic acid, (VI) C-terminal Lys, (VII) Met / Trp oxidation, (VIII) free thiol. [Modes for carrying out the invention]

[0067] Unless otherwise defined herein, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art in the fields of gene therapy, immunology, biochemistry, genetics, and molecular biology.

[0068] All methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0069] In this specification, each occurrence of terms such as "comprising" or "comprises" may be optionally replaced with "consisting of" or "consists of."

[0070] This invention provides an anti-GDF15 antibody that can be used in the treatment of cancer in human patients.

[0071] In one embodiment of the present invention, the anti-GDF15 antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC reporter assay for determining whether the antibody induces antibody-dependent cell-mediated cytotoxicity is not particularly limited and may be any ADCC reporter assay known in the art. An exemplary ADCC reporter assay may be the ADCC Reporter Bioassay, Core Kit (Technical Manual TM383, Promega Corporation) and may be performed according to the manufacturer's protocol. In this regard, the test and control antibodies may be applied to target cells at different concentrations and incubated with FcγRIIIa-expressing effector cells for 6 hours at 37°C. A luciferase substrate may then be added, and the luminescence signal may be determined with a luminescence reader after incubation at RT for 30 minutes.

[0072] GDF-15 can be measured by ELISA. ELISAs that can be used to measure GDF-15 include, but are not limited to, the Quantikine ELISA, immunoradiometric assays, Luminex® sandwich assays, and electrochemiluminescence sandwich assays from R&D Systems, and the ELECSYS® GDF15 assay (Roche Diagnostics), for example, 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 assays mentioned are based on the immunosandwich principle, which uses monoclonal or polyclonal antibodies to capture and quantify GDF-15. Depending on the reagents used and their combinations, free GDF-15 or total GDF-15 (free GDF-15 and GDF-15 bound to CTL-002) is measured.

[0073] In all other preferred embodiments of the present invention, cancer is a “solid tumor.” A “solid tumor” is cancer that forms one or more solid tumors. Such solid tumors that form solid tumors are generally known in the art. The term “solid tumor” encompasses both the primary tumor formed by cancer and any possible secondary tumors, also known as metastases. Preferred solid tumors treated according to the present invention are selected from the group consisting of melanoma, colorectal cancer, prostate cancer, head and neck cancer, urothelial carcinoma, gastric cancer, pancreatic cancer, liver cancer, testicular cancer, ovarian cancer, endometrial cancer, cervical cancer, brain cancer, breast cancer, gastric cancer, renal cell carcinoma, Ewing's sarcoma, non-small cell lung cancer and small cell lung cancer, and carcinoma of unknown primary origin; preferably selected from the group consisting of melanoma, colorectal cancer, prostate cancer, head and neck cancer, urothelial carcinoma, gastric cancer, pancreatic cancer, liver cancer, testicular cancer, ovarian cancer, endometrial cancer and cervical cancer; more preferably selected from the group consisting of melanoma, colorectal cancer, prostate cancer, head and neck cancer, urothelial carcinoma and gastric cancer; and most preferably selected from the group consisting of melanoma, colorectal cancer and prostate cancer.

[0074] In this specification, the terms "CTL-002," "CTL-001 IgG4*," "CTL-001 IgG4," and "H1L5 IgG4*" are used synonymously. They refer to antibodies having the heavy chain amino acid sequence of SEQ ID NO: 8 and the light chain amino acid sequence of SEQ ID NO: 9.

[0075] In all other preferred embodiments of the present invention, 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").

[0076] Provision of stable formulations Therapeutic proteins are complex and highly heterogeneous due to post-translational modifications (PTMs) and chemical modifications. These modifications include glycosylation, deamide, oxidation, and N- and C-terminal variations. Modifications that result in associated product-related variants are classified by regulatory authorities as critical quality attributes (CQAs). CQAs are given narrow tolerance criteria, and their variations are monitored by appropriate qualitative and quantitative methods. Providing stable antibody formulations is therefore often not at all direct.

[0077] In the first step of approaching the goal of providing a stable formulation for the antibody of the present invention, the applicant undertook to determine which parts and sequences of the antibody pose a potential risk in future formulation efforts. To do so, in silico determination was performed. The humanized anti-GDF-15 antibody H1L5 was screened using an in silico manufacturability assessment tool. As described in detail in the following examples, the amino acid sequence of H1L5, consisting of a full-length kappa isotype light chain and a full-length IgG1 heavy chain, was screened for sequence motifs, features of numerous potential development challenges, and aggregation risks. The applicant found that H1L5 has potential CDR deamide and oxidation sites that need to be further evaluated. The antibody also has other potential stability challenges in the form of C-terminal clipping, in addition to potential oxidation and acid-instability sites. Thus, it was clear that the antibody of the present disclosure is potentially not easily stabilized.

[0078] As evident from the experimental data from the initial rounds, several risk factors were identified that could potentially destabilize the antibody during further formulation efforts.

[0079] Therefore, in the second step, as described elsewhere in this specification, the antibody H1L5 was modified to have an IgG4 backbone and was then designated CTL-002. Along with the IgG4 backbone, three of the identified risk factors mentioned above are eliminable, namely, 1) K448 of IgG1 is deleted. 2) The N at position 204 of IgG1 is replaced by D in the IgG4 antibody. 3) The sulfur (S) at position 132 of IgG1 is replaced by cin (C) in the IgG4 antibody.

[0080] The transition from IgG1 to IgG4 thus eliminated three potential risk factors for providing a stable antibody formulation.

[0081] Based on the changes and further stability studies described below, the applicant provides a stable antibody formulation. The formulation preferably comprises histidine / histidine HCl, arginine-HCl, polysorbate, and sucrose at a pH of 5-6. Further preferred formulations are described in the embodiments and claims above. [Examples]

[0082] Drug substances (DS) CTL-002 is a humanized, hinge-stabilized IgG4 monoclonal antibody that targets growth and differentiation factor-15 (GDF-15), and relates to the antibody of the present invention.

[0083] In the example liquid formulation of the CTL-002 drug substance, the CTL-002 antibody is given 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 pH 5.5.

[0084] Manufacturing & Management The CTL-002 drug substance may be produced in CHO cells, for example, CHOK1SV GS KO® cells. The downstream process comprises two chromatographic steps: one protein A-based affinity chromatography (e.g., MabSelect SuRe), followed by anion exchange membrane chromatography (e.g., Sartobind Q).

[0085] Virus inactivation can be achieved, for example, by Triton-X 100 treatment.

[0086] Analytical testing is routinely performed during manufacturing and before final shipment.

[0087] stability Drug manufacturing stability research is currently underway as follows: A pilot non-cGMP (batch #DPS026) representative stability study to provide representative stability data for setting the provisional shelf life of a GMP batch of drug products, for a maximum of 3 years under intended long-term storage conditions at +2 to 8°C, as well as for 12 months at +25°C and 6 months at +40°C. • GMP (Batch # F19235) stability studies to confirm the stability of the proposed IMP, for a maximum of 3 years under intended long-term storage conditions at +2 to 8°C, as well as for 12 months at +25°C and 6 months at +40°C.

[0088] storage CTL-002 drug product vials must be stored in their original secondary packaging at +2 to 8°C in a safe environment, protected from light and separated from other pharmaceuticals or investigational products. The product should not be frozen.

[0089] Preparation & Administration To prepare CTL-002 for intravenous administration, add the CTL-002 solution to an infusion bag containing 0.9% NaCl. The CTL-002 solution for infusion may be administered using an IV bag made of polyethylene (PVC, DEHP, and latex-free) or polyvinyl chloride (latex-free) and an infusion line made of PE (PVC, DEHP, and latex-free) or PVC (DEHP and latex-free) material. The use of a 0.2 μm in-line filter (positively charged / uncharged PES membrane) is mandatory.

[0090] Once prepared, the CTL-002 solution in the infusion bag may be used immediately or administered at ambient temperature. The infusion bag may be stored at room temperature for up to 6 hours and at +2 to 8°C for up to 24 hours, but should be used within 24 hours of preparation.

[0091] Nonclinical pharmacology The inventors identified the mechanism by which GDF-15 blocks the adhesion and entry of primarily CD8+-T lymphocytes into tissues. Using CTL-002, which blocks GDF-15, a novel therapeutic approach was established to promote the entry of effector T cells into tumor tissue. This can substantially enhance the effectiveness of any T cell activator, such as a checkpoint inhibitor.

[0092] In particular, in flow adhesion assays, different immunocell subsets pretreated with + / - GDF-15 were perfused onto layers of activated endothelial cells or recombinant adhesion molecules. Adhesion and migration processes were monitored by live imaging microscopy. T cell adhesion to the endothelial cell layer was significantly impaired by the addition of GDF-15. CD8+ T cells were most affected among the T cell subsets, but adhesion of other immunocells was not reduced. The inhibitory effect of GDF-15 on CD8+ T cell adhesion was equivalent to the potent blockade of LFA-1 by TS1 / 18 antibody and was rescueable with the anti-GDF-15 antibody CTL-002 at an EC50 of approximately 700 ng / ml.

[0093] This initial finding was further validated by data from relevant animal models, in which the anti-GDF-15 antibody CTL-002 or a mouse surrogate induced a strong increase in tumor-infiltrating lymphocyte counts and enhanced the response to T cell activation therapy. Neutralization of GDF-15 with CTL-002 in HV18-MK melanoma-bearing humanized mice resulted in a strong increase in tumor-infiltrating leukocyte counts. Subset analysis revealed excessive proportional enrichment of T cells, particularly CD8+-T cells (see Figure 6).

[0094] Furthermore, syngeneic mouse tumor models containing genetically modified mouse colon tumor MC38 cells expressing human GDF-15 (MC38-GDF15) showed an increased response to anti-PD-1 or anti-CD40 / poly(IC:LC) combination therapy, which would otherwise be reduced. No adverse effects were observed in any of the animals (Figures 1, 13, and 14).

[0095] Therefore, CTL-002 was developed to neutralize the pathological effects mediated by GDF-15. The biological activity of the GDF-15 adhesion and elution processes was monitored by live-cell imaging microscopy in an in vitro flow adhesion system with primary immune cells, and key parameters for inhibiting the GDF-15 effect by CTL-002 were determined in this system.

[0096] Furthermore, secondary pharmacological studies investigated the ability of CTL-002 to induce off-target binding, in addition to on-target and off-tissue binding, as well as CDC and ADCC. Safety pharmacological evaluations were included in standard repeated dose-toxicity studies.

[0097] Overall, these studies provide a thorough characterization of the mechanism of action of CTL-002, as well as well-supported evidence for its clinical laboratory findings and GDF-15 elevation in the tumor microenvironment in patients with cancer.

[0098] Binding of the drug CTL-002 to target GDF-15 As shown in Table 1 (Table 2), GDF-15 and CTL-002 form the main complex in solution consisting of two CTL-002 antibodies and two dimeric GDF-15 molecules. Other complexes appeared less favorable, and only one additional complex of three CTL-002 and three GDF-15 molecules was reliably detected. This complex was maximal during equimolar incubation of CTL-002 and GDF-15, but was still below 8% and decreased when the ratio was changed in either direction. In excess antibodies of more than 3 molars, all GDF-15 complexed with CTL-002 molecules, and no tendency for high molecular weight aggregates was observed.

[0099] [Table 2]

[0100] The affinity of CTL-002 for recombinant human GDF-15 was determined by measuring surface plasmon resonance on a Biacore T3000. Additionally, the affinity for cynomolgus monkey, rat, and mouse GDF-15 was measured (see Table 2 (Table 3)).

[0101] 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 antibody (Jackson, order # 109-005-008, lot # 111148) was covalently immobilized on a Biacore CM5 chip (GE Healthcare, order # 61144275, lot # 10236645) by EDC / NHS chemistry. To dynamically characterize the antigen-antibody interaction, pulses of increasing GDF-15 concentrations (e.g., 156.3 pM, 312 pM, 625 pM, 1,250 pM) were injected at a flow rate of 30 μl / min. After each measurement cycle (8 minutes of association, followed by 30 minutes of dissociation), the antibody-antigen complexes were separated by surface regeneration with 10 mM glycine-HCl at pH 2.0. The association and dissociation phases were recorded to calculate the dissociation constant of CTL-002 and evaluated by global fitting using the BIAevaluation 4.1 software. These antigen concentrations were taken into account for global fit analysis only, which allowed for analysis following either a Langmuir 1:1 binding model or a 1:1 binding model with a drifting baseline.

[0102] K about human GDF-15 D The values ​​are shown in Table 2 (Table 3).

[0103] [Table 3]

[0104] Table 3 (Table 4) provides an overview and comparison of the three antibodies used in a series of nonclinical studies.

[0105] [Table 4A]

[0106] [Table 4B]

[0107] In summary, CTL-002 is a humanized IgG4 antibody derived from mouse antibody B1-23, which exhibits high specificity for human GDF-15. CTL-002 binds to human, cynomolgus monkey, and rat GDF-15 at picomolar affinities (38.3, 108, and 449 pM, respectively), and to mouse GDF-15 at a low nanomolar affinity (9.76 nM). CTL-002 binds to a discontinuous conformational epitope at the carboxyl terminus of mature GDF-15, specifically recognizing the physiological dimeric conformation.

[0108] In vitro biological activity of CTL-002 in GDF-15-mediated inhibition of T cell adhesion Two proprietary analyses further indicated that GDF-15 levels also appear to correlate with non-response to PD-1 antagonists. This again aligns with the concept that GDF-15 acts as an immune and T-cell repellent, keeping CD8+ / CD4+ T cells outside the tumor and preventing PD-1 antagonist-based activity (Figure 2).

[0109] A flow adhesion assay system was used to mimic the dynamics of immune cells in vascular walls separating them from tumor tissue, and to analyze the effect of CTL-002 on neutralizing GDF-15. To evaluate the efficacy 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 (Figure 4).

[0110] 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 T cell flow adhesion assay. As a result, a mean concentration of CTL-002 of 707 ± 17 ng / ml was effective in increasing T cell adhesion by 50%, as shown in different donors (see Figure 5).

[0111] In vivo pharmacology Among all TGF-beta superfamily members, the orthologous GDF-15 molecule exhibits the lowest sequence conservation. While mature rat, mouse, and human TGF-beta-1 and BMP-2 proteins have 99–100% sequence identity between humans and mice, homology for GDF-15 is lower than 70% (Bottner 1999). Biological differences between different species cannot therefore be ruled out. Mouse GDF-15 shows a considerably low 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 mouse homologs. Pharmacodynamic effects were investigated according to two different in vivo approaches.

[0112] Firstly, a humanized mouse model was used, in which immunodeficient mice were transplanted with human umbilical cord blood-derived CD34+ hematopoietic stem cells.

[0113] Three months after reconstitution, these mice developed a functional human-like immune system containing all major human immune cell subsets (Wang 2018). These mice were then inoculated with HV-18MK, a patient-derived human melanoma cell line that has been shown to secrete high levels of GDF-15. Mice were treated twice weekly with isotype controls or CTL-002 starting at 3 days post-treatment, and tumors were harvested at 4 weeks and analyzed for immune cell infiltration by flow cytometry.

[0114] Tumor size was not affected, but the CTL-002 treatment group showed a 9-fold increase in human tumor-infiltrating CD45+ cells (Figure 6). Within the infiltrating cell population, T cells were enriched 4-fold. In follow-up studies analyzing the infiltrating immune cell population in more detail, an increase in CD45+ infiltration and enrichment of CD3+ were confirmed, although less evident, with a 3-fold increase in CD45+ and a 4-fold enrichment of CD3+ T cells.

[0115] As a model for the second experiment, a genetically modified mouse cell line overexpressing human GDF-15 was generated to test the therapeutic efficacy of anti-GDF-15 antibodies.

[0116] In this regard, MC38 colon adenocarcinoma cells are preferred mouse tumor cells for analyzing immune checkpoint blocker activity (Selby 2016), and were used to generate in vivo data to support the development of approved anti-PD-1 antibodies. Overexpression was performed by stable transfection and did not affect in vitro proliferation compared to control-treated MC38 cells.

[0117] Age-matched immunocompetent C57BL / 6 and immunodeficient NCI nu / nu mice were subcutaneously injected with 5 × 10⁶ cells. 5 Transgenic derivative MC38 expressing MC38 blank colon tumor cells or recombinant human GDF-15. GDF-15 The suspension was inoculated. In contrast to in vitro proliferation and in vivo proliferation in immunodeficient NCI nu / nu mice, GDF-15 expression was shown to mediate a proliferation advantage in immunocompetent C57BL / 6 mice, supporting the concept that GDF-15 interferes with the tumor host immune system (Figure 7).

[0118] Furthermore, human GDF-15 expression transformed anti-PD-1 responsive MC38 colon cancer tumors into anti-PD-1 resistant tumors. This was partially reversed by anti-GDF-15(B1-23) / anti-PD-1 combination treatment, but not by anti-GDF-15 monotherapy, and was partially reversed by anti-PD-1 alone. (Data are also shown in Figure 1)

[0119] Similarly, monotherapy using anti-GDF-15 antibodies showed only minimal improvement, while GDF-15 secretory tumors (solid line, Figure 9B) showed better survival when treated with a combination of anti-GDF-15 B1-23 and anti-PD-1 (Figure 9). This combination treatment was non-significantly better than anti-PD-1 monotherapy (Figure 8).

[0120] Since anti-GDF-15 treatment has been suggested to increase T cell infiltration, other cancer immunotherapies that rely on the presence of T cells in tumors should benefit from neutralizing GDF-15.

[0121] Another immunotherapy previously shown to be dependent on the T cell immune response, anti-CD40 and poly(IC:LC) tumor treatment (van den Boorn 2013), was tested in a mouse model with MC38 cells expressing human GDF-15. The combination treatment using GDF-15 neutralization and anti-CD40 / poly(IC:LC) resulted in complete tumor rejection in 8 out of 10 animals, while only 3 out of 10 tumors were eliminated by anti-CD40 / poly(IC:LC) treatment alone (Figure 10).

[0122] Mouse GDF-15 could not be detected in wild-type MC38 cells, which may have been due to an inadequate analytical assay. However, a similar experiment comparing anti-GDF-15 treatment combined with anti-CD40 / poly(IC:LC) with isotype control antibodies was performed using MC38 cells that had not been manipulated to secrete human GDF-15. Similar to experiments using genetically modified MC38 cells, anti-GDF-15 improved the efficacy of anti-CD40 / poly(IC:LC) treatment, albeit to a lower degree (Figure 11).

[0123] Secondary pharmacology (ADCC, CDC) GDF-15 is a soluble factor that is provisionally present on cells during their maturation. The ability of CTL-002 to bind to cell surface-associated GDF-15 in vitro implies that CTL-002 may potentially mediate cell and complement-mediated cytotoxicity against healthy tissues possessing associated GDF-15.

[0124] ADCC induction was analyzed using an ADCC reporter assay (Promega). Test and control antibodies were applied to target cells at different concentrations and incubated with FcγRIIIa-expressing effector cells for 6 hours at 37°C. Luciferase substrate was then added, and after 30 minutes of incubation in RT, the luminescence signal was determined using a luminescence reader.

[0125] As a result, CTL-002 did not induce any measurable ADCC, but the positive control trastuzumab was found to induce it in both cell lines as expected (Figure 12).

[0126] Additionally, the ability of CTL-002 to mediate CDC binding was also analyzed by measuring C1q binding using ELISA and by a cell reporter assay. As a result, CTL-002 did not bind to human complement protein C1q, in contrast to the control antibody rituximab (Figure 13).

[0127] In the second approach, the ability to mediate CDC was tested in cell assays. CDC was analyzed in Raji cells and GDF-15-expressing UACC-257 cells using rituximab as a positive control. Anti-CD55 and anti-CD59 antibodies were selected as positive controls because neutralization of complement inhibitory molecules was sufficient to induce CDC, but antibodies that directly induce CDC in UACC-257 were not available. As a result, CTL-002 did not induce any CDC in combination with anti-CD55 and anti-CD59 antibodies, but high levels of the target protein GDF-15 were found to be detectable by flow cytometry (Figure 14).

[0128] In conclusion, experimental data surprisingly revealed that the CTL-002 antibody does not induce either ADCC or CDC.

[0129] Pharmacokinetics and metabolism of products in animals The pharmacokinetics of CTL-002 were analyzed in non-human primates, and a PK model of CTL-002 was generated to predict its pharmacokinetics in humans. Pharmacodynamic data were generated in the same study (and subsequent repeated dose studies in monkeys) by measuring the inhibition of GDF-15 in serum. These datasets were combined into a PK / PD model and used to predict the pharmacodynamic activity of CTL-002 in humans.

[0130] Data from the PK / PD model also provided important information for estimating a safe starting dose in the first-in-human study of CTL-002.

[0131] absorption A preliminary study was conducted in cynomolgus monkeys to compare the pharmacokinetics of IgG1 and IgG4-based anti-GDF-15 antibodies (CTL-001 and CTL-002, respectively). In this study, one male and one female monkey were each given a single intravenous injection of either CTL-001 or CTL-002 at a dose of 25 mg / kg. Blood samples were collected over 60 days.

[0132] The serum concentration-time profile is shown in Figure 15, and the key pharmacokinetic parameters are summarized in Table 4 (Table 5).

[0133] [Table 5]

[0134] In summary, CTL-002 (IgG4 isotype) showed a slightly longer half-life and a slightly higher AUC compared to CTL-001 (IgG1 isotype) in the tested monkeys, and no adverse effects were observed in either animal.

[0135] Dose-response information and modeling of human therapeutic doses A two-compartment population pharmacokinetic (PK) model describing systemic exposure to CTL-002 has been constructed from non-human primate (NHP) PK data after a single dose, including trough concentrations prior to the third weekly dose.

[0136] Accumulation of inactive GDF-15 was observed after administration of CTL-002, and the PK model was extended to include a binding PD model describing the suppression of GDF-15 (Figures 16 and 17). This model describes the observed single-dose PK-PD data in NHP and predicts the observed data up to 14 days after repeated weekly administrations (two doses) in a 4-week GLP toxicity study. There is no evidence to suggest a significant saturable component in the clearance of CTL-002, and maximum target capture may be achieved at doses ≥ 10 mg / kg / wk.

[0137] It should be noted that the observed IgG clearance for CTL-002 in NHP is typical for human IgG-like molecules in NHP. A possible explanation for the observed excessive increase in exposure and loss of GDF-15 capture in some animals after repeated administration of CTL-002 is an ADA response, which has not been experimentally confirmed (unavailability of ADA detection assays in this species).

[0138] The NHP PK-PD model was allometrically scaled to predict human PK in CTL-002 using powers of 0.75 for clearance, 0.67 for intercompartmental exchange, and 1.0 for volume. Target binding to NHP and human GDF-15 was also included in the model to predict the degree and duration of target suppression (Table 5 (Table 6)).

[0139] [Table 6]

[0140] Taking these assumptions into account, human CTL-002 exposure was predicted for different dosing regimens (Figure 18), and the resulting safety limits for Cmax and AUC were compared to the exposure achieved at 100 mg / kg / Q1wk in a 4wk GLP toxicity study (Table 6 (Table 7)).

[0141] [Table 7]

[0142] Observed baseline levels of GDF-15 in the target patient population were analyzed in a cohort of 34 patients who were previously treated, refractory, or relapsed after anti-PD-1 antibody treatment. In this cohort, baseline GDF-15 levels ranged from 0.35 to 12 ng / mL. The degree and duration of systemic GDF-15 suppression appeared to depend on baseline levels of GDF-15, with higher rates of GDF-15 production requiring higher doses to achieve suppression (Figure 19).

[0143] The estimated FIH dose is a conservative approach because, while it suppresses GDF-15 levels below physiological levels over an extended period, this has not yet been achieved over the full duration of treatment, even in patients with lower GDF-15 levels. This was done at the request of the consulted distributor (PEI, Germany) for the FIH dose. The relationship between baseline GDF-15 levels and the degree and duration of targeted suppression will be explored in a Phase I clinical trial.

[0144] The PK-PD model described above was developed to describe the suppression of systemic GDF-15 at various levels of baseline GDF-15. However, the desired target is the tumor microenvironment. If the tumor is a major source of the increase in systemic GDF-15, the amount of GDF-15 in the tumor vascular system should also be considered. Consequently, an estimate of GDF-15 suppression in the tumor microenvironment was included as an extension to the human PK-PD model.

[0145] The serum half-life of GDF-15 in humans is predicted to be 18 minutes (allometric scaling from NHP). Based on this efflux rate, GDF-15 needs to be produced at a rate of 1.67 mg / day to produce a stable serum GDF-15 concentration of 10 ng / mL. Due to a tumor size of 36 g and an average blood flow velocity of 0.2 mL / min / g in the tumor tissue (range 0.01–2 mL / min / g - Vaupel 2004), the resulting GDF-15 homodimer concentration in the tumor vascular system is 161 ng / mL (6.5 nM), which is approximately 16 times higher than the systemic GDF-15 concentration.

[0146] Based on these assumptions, predictions for tumor GDF-15 suppression in the tumor microvascular system are shown in Figure 20 for various levels of tumor GDF-15. It should be emphasized that the predicted tumor GDF-15 is intrinsically dependent on tumor blood flow velocity.

[0147] In patients with a baseline serum GDF-15 level of 2 ng / mL, tumor microangiological GDF-15 concentrations are expected to be suppressed to <0.5 ng / mL (mean level in healthy individuals) for approximately 5 days at a proposed starting dose of 0.3 mg / kg. The expected duration of suppression is much shorter than in patients with higher serum baseline levels of GDF-15 (Table 7 (Table 8)).

[0148] Note: The suppression of sGDF-15 in the tumor stromal space outside the vascular system is incorporated into the PK-PD model because it requires additional assumptions regarding CTL-002 permeation into the tumor environment and GDF-15 levels in the stromal space.

[0149] [Table 8]

[0150] Furthermore, as observed in patients with progressive melanoma (10 ng / ml), the proposed starting dose of CTL-002 at 0.3 mg / kg is at the upper end of the range of GDF-15 baseline levels. max This only lowers serum GDF-15 to a level slightly below the normal level (0.5 ng / ml), and as a result, its endogenous function should not be impaired (Figure 21).

[0151] In conclusion, CTL-002 is described by a linear PK model. In other words, CTL-002 does not exhibit saturable target-mediated kinetic behavior, as is sometimes observed with IgG-like molecules targeted to membrane receptors.

[0152] In a 4-week NHP GLP toxicity study, CTL-002 was shown to be safe and well-tolerated at doses that provided sufficient exposure limits for clinical trials.

[0153] The proposed initiation dose of 0.3 mg / kg / Q2wk for first-in-hives (FIH) is predicted to give a Cmax at the end of a 1-hour infusion of 6 μg / mL, which is 683 times lower than the maximum plasma concentration (Cmax) of CTL-002 exposure at the no-observed-effect-level (NOEL) in NHP. This dose may only achieve transient suppression of GDF-15 in the tumor microenvironment and is considered to be the estimated minimum pharmacokinetic level (MABEL).

[0154] Whole serum GDF-15 has been shown to be a useful biomarker for GDF-15 targeted engagement in NHP, and CTL-002 doses ≥ 10 mg / kg are associated with maintenance of GDF-15 capture (and presumably, GDF-15 suppression). Therefore, whole human GDF-15 may be a potential clinical biomarker for GDF-15 targeted engagement, and FIH clinical studies will be designed to explore both maximal suppression of GDF-15 over a limited period and continuous suppression of GDF-15 throughout each dosing cycle.

[0155] toxicology CTL-002 is being tested for the treatment of patients with advanced cancer.

[0156] To identify suitable species for nonclinical studies, GDF-15 sequence homology was compared across species. Sequence homology from humans to cynomolgus monkeys, mice, and rats was 94.6%, 67.9%, and 66.1%, respectively.

[0157] CTL-002 binds to a nonlinear conformational epitope within GDF-15, which is illustrated as two boxes in Figure 22. The sequence of the binding region of CTL-002 is shown for cynomolgus monkeys, humans, mice, and rats (first four rows from top to bottom).

[0158] The cynomolgus macaque exhibits 100% sequence homology to the human CTL-002-binding epitope within GDF-15. It is therefore considered a suitable species for toxicity testing. The binding affinities of CTL-002 to human and cynomolgus macaque GDF-15 are 38.3 pM and 108 pM, respectively.

[0159] A dose-response finding (DRF) study of CTL-002 was conducted in rats because the product was expected to be pharmacologically active enough to achieve sustained complete target inhibition at reasonable intravenous dose levels (rat GDF-15 binding affinity: 449 pM). However, the PK / PD data obtained in the study indicated that CTL-002 could not saturate GDF-15 binding, even at the highest dose level of 100 mg / kg. Therefore, pivotal toxicology was evaluated only in cynomolgus monkeys in a 4-week study with weekly intravenous administration of CTL-002. No toxicity was observed up to the highest tested dose of 100 mg / kg.

[0160] Tissue cross-reactivity studies conforming to Good Laboratory Practice (GLP) standards were performed using human and cynomolgus monkey tissues. Staining with CTL-002 in the examined tissue panel was limited to the cytoplasm of the trophoblast layer in human and monkey placentas, which was consistent with the reported expression of its target protein, GDF-15, in the placenta. No unexpected cross-reactivity was observed.

[0161] Toxicology: Non-Pivotal Research A non-GLP single-dose dose discovery study was conducted in cynomolgus monkeys. The primary objective of this study was to support dose selection for subsequent GLP-compliant 28-day repeated dose toxicity studies. Furthermore, the pharmacokinetics of CTL-002 at various dose levels were characterized.

[0162] The drug was administered to one male and one female animal per dose level (0.1; 1; 10 or 100 mg / kg) via a single 30-minute intravenous infusion, and the pharmacokinetic profile was recorded over 14 weeks. In addition to pharmacokinetic analysis, the development of anti-drug antibodies was evaluated before administration, as well as at 6 and 12 weeks after administration. Serum levels of GDF-15 and CTL-002 were quantified, and pharmacokinetic data were calculated separately for total CTL-002 (total PK) and free CTL-002 (free PK).

[0163] As indicators of toxicity, mortality and clinical signs were checked daily. Body weight and food and water consumption were analyzed weekly, and clinical chemistry, including cytokines, was assessed several times during the study, in addition to hematology including body temperature, ECG, blood pressure, and coagulation.

[0164] None of the animals died prematurely, and there were no signs of CTL-002-related toxicity at any of the tested dose levels.

[0165] A single infusion of 0.1, 1, 10, or 100 mg / kg of CTL-002 on day 1 of the study led to a dose-related increase in total GDF-15 serum levels in all males and females. Target saturation with CTL-002 at dose levels of 10 and 100 mg / kg was indicated by overlapping GDF-15 curves at these two dose levels.

[0166] Measurements of total and free CTL-002 in serum samples obtained by day 43 of the study (groups 1 and 2; treated with 0.1 or 1 mg / kg of CTL-002) or day 99 of the study (groups 3 and 4; treated with 10 or 100 mg / kg of CTL-002) revealed the animals' dose-related exposure to CTL-002.

[0167] Key pharmacokinetic data are given in Table 8 (Table 9) as the average for one male and one female animal per group.

[0168] [Table 9]

[0169] Based on the results of this study, dose levels of 10, 30, and 100 mg / kg were recommended for pivotal 4-week repeated dose-toxicity studies.

[0170] Toxicology: Research on Pivotella An additional pivotal study was a 4-week repeated dose-toxicity study of CTL-002 in cynomolgus monkeys (age: 3-4 years) with a 4-week recovery period.

[0171] In this study, CTL-002 was administered once a week, i.e., on days 1, 8, 15, 22, and 29 of the study, by intravenous infusion over 30 minutes. The recovery period was completed on day 58. Dose levels of 0; 10; 30 or 100 mg / kg were administered to 3 male and 3 female monkeys per group, as well as to 2 male and 2 female recovered animals in the control and high-dose groups.

[0172] None of the animals died prematurely, nor did slaughter become necessary, and no signs of local intolerance related to the test items were observed.

[0173] No test-item-related effects were observed in any animal at any dose level for behavior and appearance, body weight and 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, visual and auditory function, organ weight, or bone marrow:red blood cell ratio. No macroscopic organ changes were observed in any of the animals examined at any of the tested dose levels.

[0174] Histopathological diagnosis did not reveal any local or systemic lesions related to the test items. No test item-related changes were observed during or at the end of the 4-week untreated recovery period.

[0175] Based on the above results, the no-effect level (NOEL) was 100 mg of CTL-002 / kg, administered via repeated 30-minute intravenous infusions once a week for four weeks, i.e., five doses per animal.

[0176] The Cmax levels and AUC area for all CTL-002 revealed roughly linear dose-related systemic exposure in animals. No sex-specific differences were observed. Accumulation of all CTL-002 over time was observed, ranging from approximately 2 to 6 times. The calculated mean terminal-phase serum elimination half-life (t) of all CTL-002 was also observed. 1 / 2 ) ranged from 119 to 651 hours.

[0177] Key pharmacokinetic data for male and female animals after the first (days 1-8) and fourth (days 22-29) administrations of CTL-002 are shown in Table 9 (Table 10).

[0178] [Table 10]

[0179] Following CTL-002 infusion, serum GDF-15 levels in all animals increased 100-1000-fold at all dose levels and remained elevated throughout the entire dosing interval. The increase was similar across dose groups, as indicated in Figure 23, which shows GDF-15 levels in female monkeys throughout the first week after dosing as an example.

[0180] Therefore, it can be concluded that complete targeted inhibition was achieved in pivotal monkey studies at all dose levels and throughout the entire dosing interval.

[0181] Overall research 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 objectives of the study are to (a) demonstrate the safety of CTL-002 and the CTL-002 + anti-PD1 / PD-L1 combination, and (b) demonstrate that patients who have relapsed or are refractory to anti-PD1 / PD-L1 therapy due to elevated GDF-15 respond again and exhibit tumor shrinkage when administered with the CTL-002 + anti-PD1 / PD-L1 combination.

[0182] Part A (Dosage escalation) At least 21 subjects in the "3+3" cohort will receive escalating doses of CTL-002 administered IV as monotherapy and in combination with an anti-PD-1 checkpoint inhibitor in subjects with advanced-stage solid tumors that have relapsed or become refractory to prior anti-PD-1 / PD-L1 therapy and have exhausted or are no longer eligible for all available approved standard treatments. The "backfill cohort" will supplement with additional patients at the highest dose level.

[0183] Part B (Enlarged) To further evaluate the safety and efficacy of CTL-002 as monotherapy (one monotherapy cohort to be explored) or in combination with an anti-PD-1 checkpoint inhibitor (up to four combination cohorts to be explored) in defined tumor entities that have relapsed or become refractory to prior anti-PD-1 / PD-L1 therapy, and to confirm RP2D, the study will consist of up to five expanded cohorts of up to 25 subjects per cohort. The dedicated monotherapy cohort will help establish the safety profile of CTL-002 in prolonged monotherapy (without anti-PD-1 / PD-L1) at doses considered therapeutically effective. Enrollment in all five cohorts may be conducted in parallel.

[0184] Treatment period Part A (Dosage escalation) This study uses a standard "3+3" dose escalation design, where 3 to 6 subjects per cohort are enrolled at their respective assigned dose levels, depending on the occurrence of DLTs.

[0185] The planned dose of CTL-002 to be tested is 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

[0186] The starting dose of 0.3 mg / kg for Cohort 1 is fixed. The doses explored in Cohorts 2–5 (outlined above) may be modified by the Safety Review Committee (SRC) based on the data that emerges (i.e., available safety, PK / pharmacodynamic, and other biomarker data).

[0187] The DLT observation period is the first two treatment cycles (i.e., the first four weeks) for each treatment cohort. All treatment cycles are initially defined as having a duration of two weeks. CTL-002 is administered once every two weeks as an IV infusion. Subjects are initially given one dose of CTL-002 as monotherapy over one cycle, followed by a combination of CTL-002 and a defined checkpoint inhibitor over one cycle, with the defined checkpoint inhibitor administered at a dose of 240 mg once every two weeks via IV.

[0188] For the combination, CTL-002 and the defined checkpoint inhibitor are administered concomitantly on the same day, with CTL-002 administered first for the first combination infusion, followed by a 30-minute observation period to assess safety, and then the defined checkpoint inhibitor infusion. The observation period may be modified (i.e., shortened or lengthened) based on the safety data that emerges.

[0189] The first two treatment cycles (i.e., the first four weeks) represent the DLT observation period.

[0190] Subsequently, the subject will continue to receive the combined treatment until the treatment progresses or until they withdraw from the study for any other reason (e.g., toxicity or consent to withdraw).

[0191] Additional, intermediate dose cohorts may be explored based on emerging data and at the request of the Safety Review Committee (SRC). The maximum dose of CTL-002 tested in this study will not exceed 20 mg / kg.

[0192] All subjects will be hospitalized overnight for safety observation purposes and to enable logistical collection (e.g., PK) at the sampling time, after being administered a first dose of CTL-002 and a first combined dose of CTL-002 and a defined checkpoint inhibitor.

[0193] Intrapatient dose escalation in expanded treatment If, upon completion of Cohort 2 and evaluation by SRC, any Cohort 1 subject is still receiving the 0.3 mg / kg dose, the subject may be increased to the Cohort 2 dose of 1.0 mg / kg.

[0194] If, upon completion of Cohort 3 and evaluation by SRC, any Cohort 1 or 2 subject is still receiving the 1.0 mg / kg dose, the subject may be increased to the Cohort 3 dose of 3.0 mg / kg.

[0195] Note: Any subject still receiving treatment at 0.3 mg / kg must be treated at 1.0 mg / kg before proceeding to 3.0 mg / kg, in agreement with the Sponsor Medical Monitor.

[0196] The maximum target dose can be increased to 3.0 mg / kg through dose escalation within the target range.

[0197] In addition to available safety and PK / pharmacodynamic data, preliminary efficacy data will provide information for determining the MTD / dose, which will be further explored in Part B of the study.

[0198] MTD is defined as the highest dose level of CTL-002 in which one or fewer of the six subjects experienced a DLT during the first two treatment cycles (i.e., the first four weeks in which CTL-002 is administered as monotherapy [weeks 1 and 2] and in combination with a defined checkpoint inhibitor [weeks 3 and 4]).

[0199] Additionally, for cohorts 3–5, in the absence of any DLTs, three additional subjects may be added to each of these cohorts (up to a total of six subjects per cohort) to enhance the understanding of PK and pharmacodynamic data. This will be done while dose escalation is ongoing. These additional “supplement” subjects will be given a combination treatment of CTL-002 and a defined checkpoint inhibitor once every two weeks starting from day 1 of cycle 1, with CTL-002 always administered first and the defined checkpoint inhibitor administered afterward, as outlined above.

[0200] For subjects in Part A (excluding supplemental subjects), three sequential tumor biopsies will be taken; one biopsy at baseline, a second biopsy prior to the initiation of combination therapy (2 weeks later), and a third biopsy after the first cycle of combination therapy (at the end of treatment, or, if combination treatment is continued, at the end of cycle 2 / beginning of cycle 3).

[0201] For replacement patients, only two biopsies are required: one at baseline and the second after 4 weeks of combined treatment (at the end of treatment, or, if combined treatment is continued, at the end of cycle 2 / start of cycle 3).

[0202] These biopsies are mandatory to assess immune cell infiltration in the tumor. If a biopsy is not possible for safety reasons, this must be discussed with the medical supervisor.

[0203] In addition to monotherapy (1 cycle), treatment with CTL-002 in combination with a checkpoint inhibitor (nivolumab) has been safely tolerated up to CTL-002 dose level 5 (20 mg / kg). No DLTs or grade 4 adverse events have occurred in any treated patients. Combination treatments can be initiated simultaneously, as demonstrated by so-called supplementation cohorts with immediate combinations of CTL-002 and anti-PD1 / PD-L1 treatment.

[0204] Biomarker analysis consistently shows tumor-selective influx of CD8+ and CD4+ cells from DL1-4. Preliminary analysis indicates increased T cell proliferation in tumors in several patients, as demonstrated by CD3 / ki-67+ staining. Tumors with "cold tumors," characterized by significantly low CD8 and CD4 counts at baseline, transform into inflammatory tumors (hot tumors) by increasing CD8 and CD4 counts.

[0205] The first tumor shrinkage has been observed at various dose levels. Most notably, in a patient with carcinoma of unknown primary origin (squamous cell type) treated in a dose-level 3 supplementation cohort who had relapsed under prior nivolumab treatment and only maintained a slowly progressive disease (within the range of stable disease according to RECIST) when escalated to ipilimumab + nivolumab, the patient has so far achieved a -49% tumor shrinkage, equivalent to confirmed partial remission. Treatment is ongoing. Another patient with hepatocellular carcinoma at dose level 4 has so far shown a -11% tumor shrinkage, and treatment is ongoing.

[0206] Part B (Enlarged) In Part B of the study, up to six cohorts (with a maximum of 20 subjects per cohort) may be enrolled, each enrolling subjects with a specific tumor type.

[0207] A dedicated CTL-002 monotherapy cohort may be prepared in this expanded part of the study to explore the safety profile of CTL-002 administered as monotherapy (for example, in subjects with advanced-stage melanoma). Additionally, mandatory sequential tumor biopsies are required for this monotherapy cohort to broaden the understanding of the pharmacodynamic effects of CTL-002 in tumor tissue.

[0208] In this monotherapy cohort, two consecutive tumor biopsies are mandatory; one biopsy is taken at baseline, and the second biopsy is taken two weeks later (at the end of cycle 1 / beginning of cycle 2). All subjects will be treated until disease progression.

[0209] Next, up to five additional expanded cohorts of the defined tumor population may be treated with a combination of CTL-002 and a defined checkpoint inhibitor. Tumor indications consist of tumor types for which PD-1 / PD-L1 treatment is approved and subjects who have relapsed / progressed in or after anti-PD-1 / PD-L1 treatment. Enrollment in expanded cohorts may be carried out in parallel. All subjects will be treated until progression.

[0210] For safety observation purposes and to enable logistical collection at the sampling time (i.e., PK sampling), all subjects were admitted overnight either after receiving a first dose of CTL-002 (monotherapy cohort) or after receiving a first combination dose of CTL-002 and a defined checkpoint inhibitor (combination therapy cohort).

[0211] Research evaluation Tumor biopsy The timing of the biopsy is as follows: • Part A (all patients except those receiving supplemental treatment): Three sequential tumor biopsies are mandatory; one biopsy at baseline, a second prior to the initiation of combination therapy (2 weeks later), and a third biopsy after the first cycle of combination treatment (at the end of the treatment visit, or, if combination treatment continues, at the end of cycle 2 / start of cycle 3). These biopsies are mandatory to assess immune cell infiltration in the tumor. • Part A (Supplementary): Two consecutive tumor biopsies are required; one biopsy at baseline, and the second biopsy 4 weeks later (at the end of the treatment visit, or, if combined treatment continues, at the end of cycle 2 / start of cycle 3). • Part B (individuals enrolled in the monotherapy cohort): Two consecutive tumor biopsies are mandatory; one biopsy is performed at baseline, and the second biopsy is performed after the first monotherapy cycle (i.e., 2 weeks).

[0212] Where possible, there should be lesions suitable for sequential biopsies, or adjacent lesions, but these lesions should not be the only target lesions to be radiologically evaluated during the course of the study.

[0213] Biomarkers may be analyzed from biopsy tumor tissue samples. Additional immune cell markers and / or tumor markers specific to any of the tumor types may be included.

[0214] The biopsied tumor tissue is 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 and different lymphocytes (e.g., CD4+ and CD8+ T cells, B cells, NK cells), before and after treatment with CTL-002 or a defined checkpoint inhibitor, by histology. Furthermore, the expression of the CTL-002 drug target, GDF-15 protein, and mRNA is determined.

[0215] Tumor lesions For subjects in Part A of the study, target skin lesions selected for RECIST evaluation will be measured by caliper and photographed. Additionally, the number of skin lesions will be recorded. For clinical measurement of skin lesions in Part A, documentation by color photography of lesions (including size measurement) and caliper measurement will be performed at baseline within 7 days prior to CTL-002 injection, every 4 weeks during extended treatment, at the end of treatment visits, and during follow-up.

[0216] Safety evaluation The safety and tolerability of CTL-002 monotherapy and intravenous infusion of CTL-002 in combination with defined checkpoint inhibitors are evaluated by the occurrence of adverse events (AEs - all AEs are evaluated according to NCI CTCAE v5.0), serious adverse event emergencies (SAEs), drug-induced thromboembolic (DLTs), and the use of concomitant medications. Safety evaluations include physical examination, including ECG, neurological examination to rule out motor neuropathy, ECOG performance status, vital signs, and laboratory samples (evaluation of hematology, clinical chemistry, coagulation, thyroid function (thyroid-stimulating hormone [TSH] and free T3), cytokines, hemoglobin A1c [HbA1c], N-terminal B-type natriuretic peptide [NT proBNP], and urinalysis).

[0217] In addition to screening, participants will be evaluated for safety during the procedure up to their safety follow-up visit. Subsequently, study-related safety will be further assessed during follow-up visits at 12 months (Part A) and 24 months (Part B) after the procedure.

[0218] Vital signs Vital signs, including systolic and diastolic blood pressure (sitting), heart rate, body temperature, respiratory rate, and oxygen saturation, should be assessed. Additional vital sign measurements may be performed if clinically justified.

[0219] Physical and neurological examination A physical examination is performed during screening, which includes examination of the head, eyes, ears, nose, throat, neck, cardiovascular system, chest / lungs, abdomen (including liver and spleen size), limbs, skin, and lymph nodes, as well as a brief neurological examination to assess motor neuropathy.

[0220] The timing of additional physical examination evaluations is outlined below, as well as in the Statements of Analysis (SoA) in Tables 5, 6, and 7.

[0221] Performance Status Performance status is assessed at the time of screening according to the following ECOG criteria: • 0 = Fully active, able to perform all pre-illness activities without restriction. • 1 = Physically limited in strenuous activity, but able to walk and perform light or sedentary tasks (e.g., light housework, office work). • 2 = Able to walk and perform self-care, but unable to perform any occupational activities. Able to function at approximately 50% of the level of activity during wakefulness. • 3 = Only limited self-care is possible; more than 50% of the time while awake, it is restricted to being in bed or a chair. • 4 = Completely incapacitated, unable to perform self-care, completely confined to bed or chair. ·5=Death.

[0222] Monitoring of cardiac function Participants will undergo thorough monitoring for cardiovascular accidents, and protective measures will be taken to exclude those at risk from participating in the study.

[0223] At baseline, participants undergo ECG, echocardiography (or MUGA if echocardiography is not possible), and N-terminal pro-β natriuretic peptide level testing (NT-proBNP, heart failure screening). NT-proBNP testing is repeated every two weeks for three months, and thereafter monthly or whenever there is any suspicion of any type of cardiac / vascular injury (again in combination with ECG and echocardiography).

[0224] A single 12-lead ECG is performed.

[0225] All ECG monitoring is performed locally at the researcher's facility.

[0226] The subject should be relaxed and in a supine or semi-supine position at least 5 minutes before recording the ECG.

[0227] If deemed clinically justifiable, additional ECG testing may be performed at the researcher's discretion.

[0228] Clinical Laboratory Evaluation Samples for the laboratory tests listed below shall be collected. All tests shall be performed locally.

[0229] Prior to administration of CTL-002 / PD-1 / PD-L1, the results of hematology / coagulation and clinical chemistry must be available, evaluated by the investigator or an authorized designee, and must be considered acceptable.

[0230] Clinically significant abnormal tests must be repeated to confirm the nature and degree of the abnormality. Appropriate auxiliary studies shall be initiated when necessary. If the abnormality does not resolve or cannot be explained by events or conditions unrelated to the study medicinal product or its administration, the medical monitor must be consulted.

[0231] Within the context of the disease under study, the clinical significance of any abnormal test must be determined by the investigator, which includes significant shifts from baseline within the normal range that the investigator considers clinically important.

[0232] [Table 11]

[0233] Pharmacokinetics The PK of CTL-002 administered as monotherapy and / or in combination with a defined checkpoint inhibitor is measured from blood samples collected at the start of treatment and at various subsequent time points (Part A). Additional PK data may be evaluated in the expansion cohort (Part B).

[0234] Blood samples are collected at the start of treatment and at various subsequent time points to determine whether antibodies directed against CTL-002 have potentially developed.

[0235] Systemic Cytokine / Chemokine Monitoring (Pharmacodynamics) Serum samples are collected for the measurement of cytokines, chemokines, and other circulating biomarkers to assess safety in addition to pharmacodynamic effects. The 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).

[0236] exploratory evaluation Serum biomarker testing of specimens (retained aliquots) specifically collected during this clinical trial for future biomedical research may be performed to identify serum factors (e.g., metabolites, soluble growth factors, cytokines, chemokines, etc.) important for anti-GDF-15 (CTL-002) therapy. Retrospective biomarker studies may be performed with appropriate biostatistical design and analysis, and the PK / pharmacodynamic results may be compared with previously evaluated biomarkers or clinical outcomes.

[0237] Effectiveness evaluation: Imaging evaluation (localized examination) Tumor response will be assessed according to institutional standards using the imRECIST criteria in addition to RECIST V1.1. For the purposes of this study, subjects should be assessed at screening for a baseline scan, reassessed every 8 weeks from the start of cycle 3 and / or the end of treatment visits, and then response assessments may be performed after this point in accordance with local institutional guidelines until the end of efficacy and survival follow-up.

[0238] All lesions identified at screening / baseline must be consistently tracked using a unique lesion number assigned at screening / baseline and documented in the target file and eCRF.

[0239] For each individual subject, the same method of evaluation (imaging modality, e.g., MRI, CT) must be used to characterize each identified and reported lesion at baseline and throughout all follow-up examinations. If there are changes in modality, the study site may be asked to explain the reason for the changes in the eCRF. Changes in modality may be considered a deviation from the protocol.

[0240] Each effectiveness point / visit can be completed within a ±7 day window.

[0241] Image reading by the reading center is performed post-test, in addition to localized reading by researchers during the test.

[0242] Definitions of progressive diseases according to RECIST V1.1 and imRECIST: RECIST assessments are used to identify subjects with potential disease progression (Eisenhaur et al., 2009). The date of initial potential progression by RECIST scanning is defined as the date of immuno-undetermined progressive disease (iUPD), as defined by the modified RECIST V1.1 or imRECIST criteria for immuno-based therapies (Hodi et al., 2018). Subjects with a stable iUPD date continue to participate in the study as planned and are reassessed for progression 4–8 weeks after the initial assessment. If the confirmatory assessment supports PD, the date of disease progression is the iUPD date. If the confirmatory assessment does not support PD, the subject does not have disease progression, and the iUPD date is ignored; such subjects remain in the study as planned and continue with subsequent imaging assessments as planned in the protocol.

[0243] Antitumor activity is assessed according to investigator assessment using immune response criteria from RECIST V1.1 and imRECIST, as described below. • Contrast-enhanced CT scan, MRI, or positron emission tomography (PET-CT) of the chest, abdomen, and pelvis. • Disease response and disease progression are evaluated using RECIST and imRECIST criteria in this study. • Subjects with symptomatic or untreated brain and / or leptomeningeal metastases that require current therapy are not eligible for the study. Brain imaging must be no older than 12 weeks. Results with abnormal / unexpected findings on brain MRI should be discussed with the medical monitor as part of the screening process. • The same method of assessment and the same technique should be used to characterize each identified and reported lesion at baseline and during follow-up, respectively. If there is a change in modality, the study site may be required to explain the reason for the change in the eCRF. A change in modality may be considered a protocol deviation.

[0244] Results The study was initiated in December 2020, and the first patient was enrolled on December 9, 2020. Cohorts 1 to 4 have been completed without dose-limiting toxicity (DLT), and dose escalation continues. Note: provisional data.

[0245] Patients and CTL-002 treatment: This interim report includes demographic and preliminary safety data for the first 16 patients treated in the CTL-002-001 trial.

[0246] [Table 12]

[0247] Preliminary safety and tolerability of CTL-002: In addition to monotherapy, combination therapy with nivolumab has shown excellent tolerability. No dose-limiting toxicities (DLTs) have occurred, and no problematic safety events have been observed to date. Overall, a total of 111 adverse events have been reported for dose levels 1–5. Three of the AEs were classified as SAEs and at least potentially related to CTL-002 (two resulting from prolonged hospitalization, and one explicitly stated as a significant medical event according to investigator assessment; all were Common Terminology Criteria for Adverse Events (CTCAE) grades 1–2).

[0248] No CTCAE grades of 4 or higher were observed.

[0249] In summary, the side effect profile is very mild.

[0250] Biomarker strategies and analysis This clinical trial explores serum and tissue-based biomarkers. In addition to classical immune system activation markers, such as serum cytokines, specific analyses are performed to evaluate the immunomodulatory effects of GDF-15 in the tumor microenvironment. Among other parameters, baseline GDF-15 levels, intratumoral GDF-15 levels, as well as the number and profile of tumor-infiltrating leukocytes, were analyzed prior to and under GDF-15 neutralization with CTL-002. Substantial tumor-selective GDF-15 expression was confirmed in most tumors analyzed. Primarily CD8 expression was observed under CTL-002 administration. + and CD4 + Selective influx of T cells into the tumor and tumor stroma was observed in the majority of patients, with effects seen from dose level 1 onwards. Preliminary analysis indicates increased T cell proliferation in tumors in several patients, as demonstrated by CD3 / ki-67+ staining. Tumors with "cold tumors," characterized by significantly low CD8 and CD4 counts at baseline, transform into inflammatory tumors by increasing CD8 and CD4 counts.

[0251] Furthermore, some tumors showed upregulation of reactive PD-L1, which is an indirect sign of IFN-gamma release.

[0252] [Table 13]

[0253] Preliminary response evaluation Initial tumor shrinkage was observed at various dose levels. Most notably, in a patient with carcinoma of unknown primary origin (squamous cell type) treated in a dose-level 3 supplementation cohort, who had relapsed under prior nivolumab treatment and only maintained a slowly progressive disease (within the range of stable disease according to RECIST) when escalated to ipilimumab + nivolumab, the patient has so far achieved a -49% tumor shrinkage, equivalent to confirmed partial remission. Treatment is ongoing. Another patient with hepatocellular carcinoma at dose level 4 has so far shown a -11% tumor shrinkage, and treatment is ongoing.

[0254] conclusion Recent preclinical data from us and others indicate that GDF-15 powerfully (1) prevents T cell infiltration into the tumor microenvironment (TME) and (2) suppresses a robust immune response within the TME through other mechanisms. Therefore, GDF-15 plays a key role in the effective suppression of antitumor immune responses.

[0255] The GDFATHER (GDF-15 Antibody-mediated Effector Cell Relocation) Phase 1 trial will explore the safety, pharmacokinetic (PK) and pathogenesis (PD) of the GDF-15 neutralizing antibody CTL-002 as monotherapy and in combination with checkpoint inhibitors (CPIs) in a patient population with relapsed / refractory disease. Anticachexic effects will also be investigated.

[0256] Dose levels 1-5 were safely completed without dose-limiting trials (DLTs) with excellent tolerability.

[0257] Preliminary pharmacodynamic analyses from sequential tumor biopsies (dose levels 1-4) indicate a CTL-002-mediated selective T cell shift into the tumor microenvironment.

[0258] The preferred dose and dosing regimen for the antibody of the present invention, including CTL-002, is 3, 10, or 20 mg / kg / Q2wk; a more preferred dose and dosing regimen is 10 mg / kg / Q2wk. These dose levels are expected to be highly effective, as reflected by the favorable clinical effects observed above in patients at dose levels 3 and 4. In addition, the selection of dose and dosing regimens is based on a large-scale study conducted by the inventors, which includes pharmacodynamic modeling and PK / pharmacodynamic data obtained from Part A indicating complete GDF-15 neutralization at this dose in patients with the full range of baseline GDF-15 serum levels. Consideration of all treatment doses and their adverse events, observed PK / pharmacodynamics to date, and the thorough pharmacodynamic modeling performed indicates complete GDF-15 suppression in the tumor microenvironment of CTL-002 at a dose of 10 mg / kg, even at elevated baseline serum GDF-15 concentrations of up to 10 ng / ml in serum, corresponding to approximately 160 ng / ml of GDF-15 in immediate tumor proximity. No problematic safety events have been observed at this dose, no DLTs have occurred, and the safety limit remains >10 times lower compared to NOAEL in non-human primates (NHP). Therefore, the preferred dose indicated above is expected to be particularly effective and safe.

[0259] The observations indicated above regarding efficacy, safety, and PK / pharmacodynamic data also indicate that it is advantageous to administer the anti-GDF-15 antibody at a preferred dose of 10–20 mg / kg, more preferably 20 mg / kg, and in a dosing regimen of at least one administration cycle, where the cycle is 4 weeks long, and where the dose is administered at least once (i.e., preferably once) in each of the at least one cycle. Although this dosing regimen has a longer administration cycle of 4 weeks, the preferred dose of 10–20 mg / kg, more preferably 20 mg / kg, allows for obtaining a favorable safety and efficacy profile similar to the preferred 10 mg / kg / Q2wk regimen, and is compatible with the observed PK / pharmacodynamic profile.

[0260] Similarly, the observations indicated above regarding efficacy, safety, and PK / pharmacodynamic data also indicate that it is advantageous to administer the anti-GDF-15 antibody at a preferred dose of 10–20 mg / kg and in a dosing regimen of at least one administration cycle, where the cycle is 3 weeks long, and where the dose is administered at least once (i.e., preferably once) in each of the at least one cycle. Although this dosing regimen has a longer 3-week dosing cycle, the preferred dose of 10–20 mg / kg allows for obtaining a favorable safety and efficacy profile similar to the preferred 10 mg / kg / Q2wk regimen, and is compatible with the observed PK / pharmacodynamic profile.

[0261] The above findings indicate that treatment with the anti-GDF-15 antibody according to the present invention can provide significant clinical benefits for cancer patients. Importantly, this benefit is observed even in patients who were previously refractory to some of the most advanced treatment options, such as therapy with PD-1 / PD-L1 axis antagonists (e.g., nivolumab).

[0262] In silico determination of potential stability risks in antibodies with the IgG1 backbone "H1L5". In the first step of approaching the goal of providing a stable formulation for the antibody of the present invention, the applicant undertook to determine which parts and sequences of the antibody pose a potential risk in future formulation efforts. To do this, in silico determination was performed. The humanized anti-GDF-15 antibody H1L5 was screened using an in silico manufacturability assessment tool. The amino acid sequence of H1L5, consisting of a full-length kappa isotype light chain and a full-length IgG1 heavy chain, was screened for sequence motifs, features of numerous potential development challenges, and aggregation risk. H1L5 was shown to have potential CDR deamide and oxidation sites that could benefit from in vitro evaluation. The antibody also has other challenges in the form of C-terminal clipping, in addition to potential oxidation and acid instability sites.

[0263] Therapeutic proteins are complex and highly heterogeneous due to post-translational modifications (PTMs) and chemical modifications. These modifications include glycosylation, deamide, oxidation, and N- and C-terminal variations. Modifications that result in associated product-related variants are classified as critical quality characteristics (CQAs) by regulatory authorities. CQAs are given narrow tolerance criteria, and their variations are monitored by appropriate qualitative and quantitative methods.

[0264] Modifications can be attributed to the host cell system, manufacturing method, and storage conditions. These may relate to either the chemical stability of the molecule or the intrinsic physical stability of its aggregation potential. Aggregation is a challenge that has such potential impact on the safety, quality, and efficacy that one or more CQAs generally define for this purpose.

[0265] Protein aggregation is a common problem encountered during biopharmaceutical development. It can occur at several different stages of manufacturing, such as fermentation, purification, formulation, and storage. The potential impact of aggregation extends not only to the manufacturing process but also to the target product profile, delivery, and, essentially, patient safety.

[0266] Aggregation depends on the protein itself (endogenous aggregation tendency) as well as environmental factors such as pH, concentration, buffers, excipients, and shear force. However, the fundamental differences in why one antibody aggregates during a process or manufacturing while others do not are encoded in the amino acid sequence of the antibodies and their endogenous aggregation tendency. Aggregation poses a risk to the safety, quality, and efficacy of the antibody.

[0267] Asparagine deamide is a non-enzymatic reaction that produces a heterogeneous mixture of asparagine, iso-aspartic acid, and aspartic acid over time at the site of influence. Deamide is caused by the hydrolysis of the amide groups on the side chains of asparagine and glutamine. While glutamine deamide can occur in therapeutic proteins, the focus of manufacturability is on asparagine deamide. Three main factors influence the deamide rate of peptides: pH, temperature, and primary sequence. The secondary and tertiary structures of proteins can significantly alter the deamide rate. In addition to causing charge heterogeneity, asparagine deamide can affect protein function when it occurs at binding interfaces, such as antibody CDRs. Deamide has also been reported to cause aggregation.

[0268] Aspartate isomerization is a non-enzymatic interconversion of aspartic acid and iso-aspartic acid residues. The peptide bond at the C-terminus of aspartic acid can be susceptible to fragmentation under acidic conditions. As these reactions proceed through intermediates similar to those in the asparagine deamide reaction, the rates of aspartic acid isomerization and fragmentation are influenced by pH, temperature, and primary sequence. Aspartic acid isomerization can affect protein function if it occurs at binding interfaces, such as antibody CDRs. Isomerization can also cause charge heterogeneity and result in fragmentation caused by cleavage of the peptide backbone. Fragmentation reactions mainly occur below pH 5, and the Asp-Pro peptide bond is less stable than other peptide bonds. Aspartic acid isomerization has the potential to increase immunogenicity, and the risk is further increased as fragmentation promotes the formation of aggregates.

[0269] C-terminal lysine processing is a modification in antibodies and other proteins that occurs during bioprocessing, apparently due to the action of basic carboxypeptidases. Since C-terminal lysine processing can result in species with 2, 1, or 0 lysines, it is a major source of charge and mass heterogeneity in antibody products.

[0270] The isoelectric point (pI) of a protein is the pH at which the protein has a net charge of 0. The isoelectric point depends on the number and type of charged residues in the protein, their spatial arrangement, and the degree of solvent accessibility. Predicting the isoelectric point from the amino acid sequence assumes a denatured protein. While it is known that predicted and measured isoelectric points can differ, a relationship may be observed between the two values. When a protein solution has a pH equal to the protein's pI, the repulsive electrostatic force between charges on the protein molecule is minimized. The lack of repulsive electrostatic force can increase the risk of hydrophobic surface patches forming aggregation hotspots.

[0271] N- and O-glycosylation are post-translational modifications that appear in therapeutic proteins, such as antibodies, blood factors, EPOs, hormones, and interferons. Carbohydrate attachment to amino acid residues occurs at the side-chain nitrogen atom of asparagine in N-glycosylation, and at the side-chain oxygen atoms of serine and threonine in O-linked glycosylation. Some immunoglobulin V- genes contain asparagine residues in their CDRs, which can result in the formation of N-glycosylation motifs during selection, and approximately 20% of all antibodies are glycosylated in the variable region in vivo. Proper glycosylation is important not only for folding but also for stability, solubility, potency, pharmacokinetics, and immunogenicity. Unintended glycan structures in or near the binding interface, for example in the CDR, can occlude the binding region or introduce steric hindrance, thereby reducing binding affinity. Glycan structures can vary in branching and composition, thereby introducing further heterogeneity that may require characterization and control.

[0272] Oxidation: Several amino acids are susceptible to damage from oxidation caused by reactive oxygen species (ROS), including histidine, methionine, cysteine, tyrosine, and tryptophan. Oxidation is generally divided into two categories: site-directed metal-catalyzed oxidation and non-site-directed oxidation. Methionine, and to a lesser extent tryptophan, are more susceptible to non-site-directed oxidation. Methionine is primarily sensitive to free ROS, while tryptophan is more sensitive to photo-induced oxidation. The degree of sensitivity is partly determined by the solvent accessibility of the side chain; buried residues are less sensitive or take longer to react.

[0273] Pyroglutamate formation is a modification that occurs in proteins with an N-terminal glutamine or glutamate residue, where the side chain cyclizes with the N-terminal amine group to form a five-membered ring structure. Since many antibody light and heavy chains have an N-terminal glutamine or glutamate residue, pyroglutamate formation is a common modification, especially for sequences with an N-terminal glutamine. N-terminal cyclization causes mass and charge heterogeneity that needs to be controlled and monitored. Pyroglutamate formation is commonly found in antibodies with an N-terminal glutamine. While the conversion from glutamate to pyroglutamate is unlikely to pose a safety risk, the N-terminus in antibodies is proximal to the CDR, and charge fluctuations can affect binding affinity.

[0274] Abbreviation CDR Complementarity Determination Region Crystallizable fragments of Fc antibodies FR Framework Domain FRx Framework Domain x (FR1, FR2, FR3, FR4) Hx CDR x, heavy chain (H1, H2, H3) H:Ala11 Heavy chain alanine at position 11 IgG immunoglobulin G antibody Lx CDR x, light chain (L1, L2, L3) L:Ala11 Light chain alanine at position 11 pI isoelectric point VH variable domain, heavy chain VL (Variable Domain), Light Chain

[0275] result Array Liability Map The two sequence maps shown in Figures 24 and 25 provide a graphical representation of the locations of all identified sequence liabilities. In these maps, the domain boundaries and CDR locations are indicated in relation to the overall sequence. Schemes are used to indicate the type of liability detected at a given location. Asparagine residues that are predicted to be potentially involved in both deamidation and N-glycosylation are indicated using both schemes. All indicated residues in both Figures 24 and 25 represent potential challenges for the development of stable formulations for the antibodies of this disclosure.

[0276] This section focuses on the anticipated aggregation and most important developmental challenges in H1L5.

[0277] The results of the antibody aggregation risk prediction are shown in Table 13 (Table 14).

[0278] Potential development challenges are summarized in Table 14 (Table 15), which summarizes the areas that may pose particular problems for formulation design during development.

[0279] [Table 14]

[0280] [Table 15]

[0281] Clearly, there were several risk factors that could potentially destabilize the antibody during further formulation efforts.

[0282] In the second step, as described elsewhere in this specification, the antibody H1L5 was modified to form an IgG4 skeleton and subsequently designated as CTL-002. Along with the IgG4 skeleton, three of the identified risk factors mentioned above are eliminable, namely, 1) K448 of IgG1 is deleted. 2) The N at position 204 of IgG1 is replaced by D in the IgG4 antibody. 3) The sulfur (S) at position 132 of IgG1 is replaced by cin (C) in the IgG4 antibody.

[0283] The transition from IgG1 to IgG4 thus eliminated three potential risk factors for providing a stable antibody formulation.

[0284] Thermal and colloidal stability testing Based on the findings obtained from the in silico experiments performed above, the inventors have determined the first basic formulation that may be suitable for stabilizing specific antibody structures that have the challenges found in the in silico experiments performed above.

[0285] While it was clearly preferable to have a stable liquid formulation, it was decided that a potential lyophilized formulation should also be included, considering the potential stability issues associated with this antibody in a liquid environment. Based on this, - Four liquid formulations - One lyophilized preparation - Two different pH points, both selected to be much lower than the isoelectric point of the antibody. - Two different buffer systems - Several different excipients at different concentrations It was decided that it was necessary to test it.

[0286] The pH point was selected after particular consideration of determining the pI of the antibody, as well as after considering the potentially best environment for reducing aggregation and increasing the repulsive electrostatic force between protein molecules for the specific antibodies of this disclosure.

[0287] This led to the following research design: pH and ionic strength screening were performed on a microliter scale using selective analytical methods. Thermal and colloidal stability were studied using dynamic light scattering analysis, intrinsic fluorescence, light scattering analysis, and trace calorimetry, respectively.

[0288] [Table 16]

[0289] [Table 17]

[0290] material The drug substance (DS) for CTL-002 was processed and supplied by Lonza Biologics Plc, Slough, UK. The DS batches were transported and stored at 2-8°C from the day of arrival until the material was aliquoted, and used for different studies.

[0291] [Table 18]

[0292] [Table 19]

[0293] [Table 20]

[0294] [Table 21]

[0295] method Preparation of test materials DS was subjected to buffer replacement in formulations F2 and F3 using a centrifugal concentrator. Protein concentration was monitored at the end of processing.

[0296] In all formulations, the protein concentration was adjusted by adding a specific formulation buffer.

[0297] result thermal stability The thermal structural stability of a protein can be evaluated not only by the temperature at which the protein aggregates (aggregation onset temperature (Tagg)), but also by the temperature at which the protein unfolds from its native (folded) state to a denatured (unfolded) state. The midpoint of the unfolding transition, defined as the temperature at which equal populations of folded and unfolded proteins exist in solution, is called the melting temperature (Tm) when evaluated by traditional DSC measurements and the unfolding temperature (Tunfold) when evaluated by endogenous fluorescence. Unfolding of IgG molecules exhibits two or three transitions, reflecting the unfolding of both Fab and Fc(CH2 and CH3) fragments.

[0298] The onset of aggregation was determined by light scattering at approximately 61–68°C for five samples. The Tagg values ​​can be ranked as follows: F5 > F1–F4 > F3 > F2

[0299] The unfolding temperature was observed at approximately 64–68°C, and the melting temperature was observed at approximately 65–69°C. Both methods gave similar formulation rankings: F5 > F1- F3 > F4 > F2.

[0300] Overall, thermal stability was higher at pH 6.0 than at pH 5.5. NaCl decreased it, while sodium citrate buffer improved it.

[0301] [Table 22]

[0302] Colloidal Stability Both a certain dissociation coefficient (kD) and the osmotic second virial coefficient (A2) are indicators of colloidal stability that measure interactions resulting from non-covalent forces between different molecules in solution. High values of kD and A2 indicate a strong net repulsive interaction, while low values indicate net attractive forces. While it is possible to distinguish between net attractive forces and net repulsive forces by the sign of A2, this is not possible for kD. A formulation with good average colloidal stability has an A2 value higher than 1.10 -4 mol.ml.g 2 .

[0303] Negative dissociation constants have been measured under all tested conditions, which correlates with weak negative or neutral osmotic second virial coefficient values, reflecting a tendency for weak attractive protein-protein interactions.

[0304] kD values can be ranked as follows: F2 > F4 > F3 > F5 > F1.

[0305] Colloidal stability is therefore improved by reducing the pH value to 5.5.

[0306] Increasing ionic strength with NaCl, arginine-HCl or sodium citrate also improves colloidal stability.

[0307] [Table 23]

[0308] Therefore, regarding which pH should be selected for the final formulation, the results of the determination of colloidal and thermal stability points point in different directions. A pH of 6.0 provides improved thermal stability, but colloidal stability may be worse at this pH compared to pH 5.5.

[0309] If selecting one pH over others negatively impacts one of two properties (thermal stability and colloidal stability, respectively), and this negative impact cannot be overcome by carefully selecting other components of the formulation, it may potentially have a negative impact on the stability of the final formulation.

[0310] Early-stage formulation development After reconsidering the results obtained from experiments conducted to date, we decided to continue using both the liquid and lyophilized formulations.

[0311] In this further preparation for formulation, the inventors ultimately arrived at three formulations (two liquids and one lyophilized), which were tested under specific stress conditions. The target concentration for the antibody was set at 25 mg / ml.

[0312] As defined elsewhere in this specification, the antibody to be stabilized in this case is CTL-002.

[0313] [Table 24]

[0314] [Table 25A]

[0315] [Table 25B]

[0316] [Table 26A]

[0317] [Table 26B]

[0318] material The bulk purified drug substance (BPDS) of CTL-002 was supplied at a concentration of approximately 25 g / L.

[0319] [Table 27A]

[0320] [Table 27B]

[0321] [Table 28]

[0322] [Table 29]

[0323] All formulation samples were labeled and stored at 5±3°C until distribution for stability studies.

[0324] The liquid formulation test samples were subjected to shaking stress and low-temperature conditions for approximately 5 days at room temperature in a horizontal position within a reciprocating (horizontal) shaker at a target speed of 200 rpm.

[0325] The liquid formulation test samples were subjected to five freeze / thaw cycles from -65°C or below to room temperature in a vertical position.

[0326] The lyophilized formulation vials were reconstituted using 2.3 mL of purified water. The volume required for reconstitution was calculated taking into account the volume change due to solids. During reconstitution, the vials were gently agitated to ensure completion, and were then used for further analysis.

[0327] result Formulation after compounding The pH, protein concentration, and gravimetric osmolality were determined for the prepared solutions of the liquids after preparation and filtration (F1, F2, and F4) and for the lyophilized (F3) formulation. The results are shown in Table 20 (Table 30). For completeness, protein concentrations determined by UV spectrophotometer (A280) at the initial point in time during the short-term stability study are also included. • All results are close to the target pH value. The prepared solution was colorless (BY7) after preparation and filtration and contained virtually no visible particles.

[0328] [Table 30]

[0329] Freeze-thaw and shaking research 5 freeze-thaw cycles (-65 o All formulations except F3, subjected to shaking stress (from C to RT) or at ambient temperature and low temperatures, showed no relevant changes in any analytical method compared to the initial unstressed sample, indicating that the formulations effectively stabilize the CTL-002 molecule under both freeze-thaw and shaking stress.

[0330] No significant differences in aggregation and fragmentation were observed for all liquid formulations by SE-HPLC. Furthermore, no significant chemical degradation was observed by iCE, and the overall number of visible and subvisible particles was low under shaking and F / T stress. The data suggested that both surfactants, polysorbate 20 and 80, protected the formulations from shaking, freeze, and thaw stress.

[0331] Short-term stability study Overall, pH and protein concentrations remained stable in all four tested formulations over short-term stability tests of up to 8 weeks. Stability tests revealed a low initial number of subvisible particles for all formulations. Lyophilized formulation F3 demonstrated a trend towards higher levels of subvisible particles compared to liquid formulations F1, F2, and F4, which is intrinsic to lyophilized cake reconstitution. No significant changes in the number of subvisible particles were detected in any of the tested formulations after 8 weeks of storage under any of the tested storage conditions. Furthermore, all samples contained virtually no visible particles at T0 in evaluations during visual inspection using black and white backgrounds. Only formulation F4 demonstrated an increase in the number of visible particles, with a small number of particles and white fibers observed after 8 weeks of storage at 25°C. This increase, however, was not observed at 40°C. Overall, the levels of visible particles remained unchanged over short-term stability studies.

[0332] Lyophilized formulation F3 demonstrated high stability. No changes in purity were observed under 8 weeks of stability stress under all tested storage conditions, as measured by SE-HPLC, icIEF, RP-HPLC, and CE-SDS. However, the results for liquid formulations other than formulation F1, in particular, indicated that antibodies can be suitably stabilized in liquid formulations as well.

[0333] The CTL-002 molecule exhibited a low tendency for aggregation and fragmentation in all three tested liquid formulations when stressed at 40°C. Besides aggregation, monomer loss due to fragmentation was more pronounced in formulation F1 than in formulations F2 and F4. After 8 weeks of storage at 40°C, 1.2% aggregates and 0.3% degradation products were measured in F1 (pH 5.5), while formulations F2 (pH 6.4) and F4 (pH 6.0) contained approximately 1.0–0.9% aggregates and 0.1–0.2% degradation products. The formation of high and low molecular weight species is therefore considered pH-dependent.

[0334] The low solution turbidity, low levels of subvisible particles, and absence of visible particles measured in all tested formulations throughout the stability study highlighted the low tendency to aggregate. Furthermore, neither the tendency to aggregate nor the tendency to fragment was detected by tip-based CE-SDS, and no changes were observed under normal or reducing conditions.

[0335] The chemical purity of CTL-002 was altered by thermal stress at 40°C and, to a lower extent, 25°C, as measured by iciEF. The loss in the main peak purity was more significant in formulations F2 and F4 than in formulation F1, which was mainly attributed to the formation of acidic species. Only formulation F1 at pH 5.5 showed additional significant incorporation of basic species.

[0336] RP-HPLC analysis under non-reducing conditions showed a loss in the main peak at 40°C, followed by a post-peak increase in species, for all liquid formulations, but no change was observed when RP-HPLC was used under reducing conditions. The changes in RP-HPLC were smaller in formulation F1 than in formulations F2 and F4 in icIEF. Formulation F1, with a lower pH of 5.5, demonstrated overall higher chemical stability than formulations F2 and F4.

[0337] No significant changes in polysorbate content were observed across all formulations throughout the short-term stability studies. Both surfactants, polysorbate 20 and polysorbate 80, are suitable for the CTL-002 formulation.

[0338] Conclusion: • The lyophilized formulation F3 remained stable throughout the entire short-term stability study. All liquid formulations were stable for 8 weeks of storage at 5°C, in addition to freeze / thaw and shaking stress. Formulations F2 and F4 demonstrated generally similar stability profiles under the tested stress load conditions. The formulation F1 with the lowest pH showed a greater tendency towards aggregation and fragmentation under accelerated storage conditions at 40°C. Formulation F1, with the lowest pH, demonstrated higher chemical stability than formulations F2 and F4, primarily at 40°C and to a lower degree at 25°C by icIEF, where the tendency towards acidic species increased under stress loading conditions, and at 40°C by RPHPLC, where species increased after the peak.

[0339] Based on the combined results of all experimental data collected up to this point for this formulation project, the following liquid formulations were provided for CTL-002: 25 mg / mL CTL-002, 20 mM histidine / histidine HCl, 150 mM sucrose, 50 mM arginine-HCl, 0.02% w / v polysorbate 20, pH 5.5

[0340] At this stage, the data (see above) appeared somewhat contradictory regarding chemical stability and physical stability, making it extremely difficult to make a decision. However, the inventors, taking all the data obtained up to this point into consideration, found that chemical stability was particularly important in the context of stabilization efforts for this antibody.

[0341] Long-term data Two long-term stability studies were conducted using the above formulation for the IgG4 antibody CTL-002.

[0342] Stability samples for product CTL-002 (250 mg / 10 mL) stored under long-term storage conditions of inverted and upright at 5°C ± 3°C were tested.

[0343] After 18 months under long-term storage conditions of inverted and upright storage at 5℃±3℃, product CTL-002 demonstrates stability in the following manner: SE-HPLC (main peak, fragments, aggregates) and CE-DSD (reduced LC+HC, unreduced intact IgG) This does not show decomposition.

[0344] Only a slight shift from the main peak towards acidic species is indicated by the icIEF results. A decrease in polysorbate content may also be detected.

[0345] As the results show, the antibody was stabilized to a high degree, not only with respect to the chemical stability parameters determined during this study, but also, quite surprisingly, with respect to its aggregation properties.

[0346] array Sequence ID No. 1 (peptide sequence of the heavy chain CDR1 region of monoclonal anti-human GDF-15 antibody): GFSLSTSGMG Sequence ID No. 2 (peptide sequence of the heavy chain CDR2 region of monoclonal anti-human GDF-15 antibody): IYWDDDK Sequence ID No. 3 (peptide sequence of the heavy chain CDR3 region of monoclonal anti-human GDF-15 antibody): ARSSYGAMDY Sequence ID No. 4 (light chain CDR1 region peptide sequence of monoclonal anti-human GDF-15 antibody): QNVGTN Peptide sequence of the light chain CDR2 region of monoclonal anti-human GDF-15 antibody: SAS Sequence ID No. 5 (light chain CDR3 region peptide sequence of monoclonal anti-human GDF-15 antibody): QQYNNFPYT Sequence ID 6 (heavy chain variable domain of monoclonal anti-human GDF-15 antibody):

[0347] [ka]

[0348] Sequence ID 7 (Light chain variable domain of monoclonal anti-human GDF-15 antibody):

[0349] [ka]

[0350] Sequence ID 8 (heavy chain of monoclonal anti-human GDF-15 antibody CTL-002 lacking a leader peptide sequence):

[0351] [ka]

[0352] Sequence ID 9 (light chain of monoclonal anti-human GDF-15 antibody CTL-002 lacking a leader peptide sequence):

[0353] [ka]

[0354] Sequence ID No. 10 (Heavy chain variable domain of anti-human GDF-15 antibody H1L5):

[0355] [ka]

[0356] Sequence ID 11 (Light chain variable domain of anti-human GDF-15 antibody H1L5):

[0357] [ka]

[0358] [References] TIFF2026143722000041.tif242162TIFF2026143722000042.tif97162 [Industrial applicability]

[0359] Anti-GDF-15 antibodies are usable in methods for treating cancer in human patients and can be industrially manufactured and sold as products for use and by the methods described in this document, in accordance with known standards for the manufacture of pharmaceutical products. Therefore, the present invention is industrially applicable.

Claims

1. A composition comprising an anti-GDF-15 antibody for use in methods for treating cancer and / or cancer cachexia in human patients.

2. The composition according to claim 1, wherein the anti-GDF-15 antibody is administered in a dose of 0.3, 1.0, 3.0, 10.0, or 20.0 mg / kg, preferably 3, 10, or 20 mg / kg, more preferably 10 mg / kg, and in a drug regimen of at least one administration cycle, wherein the cycle is for a period of two weeks, and the dose is administered at least once in each of the at least one cycle.

3. The composition according to claim 1, wherein the anti-GDF-15 antibody is administered in a dose of 10 to 20 mg / kg, preferably 20 mg / kg, and in a drug regimen of at least one administration cycle, the cycle having a duration of 4 weeks, and the dose is administered at least once in each of the at least one cycle.

4. The composition according to claim 1, wherein the anti-GDF-15 antibody is administered in a dose of 10 to 20 mg / kg and in a drug regimen of at least one administration cycle, the cycle having a duration of 3 weeks, and the dose is administered at least once in each of the at least one cycle.

5. The composition according to any one of claims 1 to 4, wherein the antibody does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

6. The composition according to any one of claims 1 to 5, wherein the anti-GDF-15 antibody is an IgG4 isotype antibody.

7. The composition according to claim 6, wherein the antibody comprises a hinge-stabilizing mutation.

8. The composition according to claim 7, wherein the hinge-stabilizing mutation is the S228P mutation.

9. The composition according to any one of claims 1 to 8, wherein the anti-GDF-15 antibody comprises a heavy chain variable domain including 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 including 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.

10. The composition according to any one of claims 1 to 9, wherein the heavy chain variable domain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable domain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 7 or the amino acid sequence shown in SEQ ID NO:

7.

11. The composition according to any one of claims 1 to 10, wherein the heavy chain of the antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: 8, and the light chain of the antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence represented by SEQ ID NO: 9 or the amino acid sequence shown in SEQ ID NO:

9.

12. The composition according to any one of claims 1 to 11, wherein the anti-GDF-15 antibody can be obtained by expression in CHO cells.

13. The composition according to any one of claims 1 to 12, wherein the concentration of GDF-15 in the patient's serum / plasma is less than 10 ng / mL at the end of the administration cycle.

14. The composition according to claim 13, wherein the concentration of GDF-15 in the patient's serum is less than 2 ng / mL at the end of the administration cycle.

15. The composition according to claim 13, wherein the concentration of GDF-15 in the patient's serum is lower than 0.5 ng / mL at the end of the administration cycle.

16. The composition according to any one of claims 1 to 15, wherein the cycle is for a period of three weeks, and the dose is administered at least once in each of the at least one cycle.

17. The composition according to any one of claims 1 to 15, wherein the cycle is for a period of 4 weeks, and the dose is administered at least once in each of the at least one cycle.

18. The composition according to any one of claims 1 to 15, wherein the drug regimen comprises multiple cycles and optionally up to 52 administration cycles.

19. The composition according to claim 18, wherein the drug regimen consists of 26 to 52 administration cycles.

20. The composition according to claim 16, wherein the drug regimen consists of a maximum of 34 administration cycles.

21. The composition according to claim 20, wherein the drug regimen consists of 17 to 34 administration cycles.

22. The composition according to claim 17, wherein the drug regimen consists of a maximum of 24 administration cycles.

23. The composition according to claim 22, wherein the drug regimen consists of 12 to 24 administration cycles.

24. The composition according to any one of claims 1 to 23, wherein the anti-GDF-15 antibody is administered in combination with a checkpoint inhibitor.

25. The composition according to claim 24, wherein the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CD40 antibody.

26. The composition according to claim 24 or 25, wherein the checkpoint inhibitor is administered in the same drug regimen as the GDF-15 antibody.

27. The composition according to any one of claims 24 to 26, wherein the checkpoint inhibitor is administered prior to the administration of the GDF-15 antibody.

28. The composition according to claim 27, wherein the checkpoint inhibitor is administered within 120 minutes prior to the administration of the GDF-15 antibody.

29. The composition according to claim 28, wherein the checkpoint inhibitor is administered within 30 minutes prior to the administration of the GDF-15 antibody.

30. The composition according to any one of claims 1 to 29, wherein the cancer is selected from the group consisting of brain cancer including glioma, nervous system cancer, melanoma, lung cancer, head and neck cancer, urothelial carcinoma, liver cancer, endometrial cancer, cervical cancer, gastric cancer, renal cell carcinoma, Ewing's sarcoma, non-small cell lung cancer and small cell lung cancer, lip cancer, liver cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, bladder cancer, cervical cancer, endometrial cancer, testicular cancer, thyroid cancer, kidney cancer, gallbladder cancer, multiple myeloma, gastrointestinal tumors including nasopharyngeal cancer, laryngeal cancer, pharyngeal cancer, esophageal cancer, gastric cancer and colorectal cancer, pancreatic cancer, prostate cancer, ovarian cancer, breast cancer, and carcinoma of unknown primary origin, and the use is optional and for the treatment of cancer cachexia.

31. The composition according to any one of claims 1 to 30, wherein the dose of the anti-GDF-15 antibody is administered intravenously.

32. An anti-GDF-15 antibody, wherein the antibody is an IgG4 isotype antibody having a hinge-stabilizing mutation, the heavy chain variable domain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 6, and the light chain variable domain of the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by SEQ ID NO: 7 or the amino acid sequence shown in SEQ ID NO:

7.

33. The anti-GDF-15 antibody according to claim 32, wherein the heavy chain of the antibody comprises an amino acid sequence having at least 90% identity, preferably at least 95% identity, and more preferably at least 98% identity with respect to the amino acid sequence represented by SEQ ID NO: 8 or the amino acid sequence shown in SEQ ID NO: 8, and the light chain of the antibody comprises an amino acid sequence having at least 90% identity, preferably at least 95% identity, and more preferably at least 98% identity with respect to the amino acid sequence represented by SEQ ID NO: 9 or the amino acid sequence shown in SEQ ID NO:

9.

34. The anti-GDF-15 antibody according to claim 32 or 33, wherein the hinge-stabilizing mutation is the S228P mutation.

35. An antibody according to any one of claims 32 to 34, which does not induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

36. The antibody according to any one of claims 32 to 35, which can be obtained by expression in CHO cells.

37. The antibody according to any one of claims 32 to 36, comprising a heavy chain variable domain including 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 including 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.

38. A formulation comprising an anti-GDF-15 antibody according to any one of claims 32 to 37, or a composition according to any one of claims 1 to 31, wherein the formulation comprises 10 to 50 mg / ml of the anti-GDF-15 antibody.

39. The formulation according to claim 38, comprising histidine / histidineHCl, sucrose, arginine-HCl, and polysorbate at a pH of 5 to 6.

40. The formulation according to claim 38 or 39, comprising 10-50 mg / ml of CTL-002, 10-50 mg / ml of histidine / histidineHCl, 100-200 mM of sucrose, 20-80 mM of arginine-HCl, and 0.01-0.05% w / v of polysorbate 20 or polysorbate 80 at a pH of 5.0-6.0, preferably pH 5.3-5.

7.

41. A formulation according to any one of claims 38 to 40, comprising 25 mg / ml of CTL-002, 20 mM histidine / histidine HCl, 150 mM sucrose, 50 mM arginine-HCl, and 0.02% w / v polysorbate 20 at pH 5.

5.

42. The formulation according to claim 41, comprising 25 mg / ml of CTL-002, 20 mM histidine / histidine HCl, 150 mM sucrose, 50 mM arginine-HCl, and 0.02% w / v polysorbate 20 at pH 5.5.

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