Medical uses and administration planning of CCR8 antibodies

Abstract

The present invention relates to medical uses comprising administration of an anti-human CCR8 antibody in a specifically defined dosage regimen in monotherapy or in combination therapy with an anti-PD (L) 1 antibody. Dosing regimens have been developed against the anti-human / cynomolgus monkey CCR8 antibody TPP-23411, but they can also be used for other antibodies having similar properties to TPP-23411. The medical use or dose regime may include a stratified step of selecting a patient with an increased probability of success of treatment. The suggested biomarkers are: a) a tumor proportion score or a combined positive score as a measure of PD (L) 1 expression; b) analyzing inflammatory cytokines in a blood, plasma or serum sample, and c) prior treatment of cancer with an anti-PD-(L) 1 antibody for at least 6 months. Furthermore, provided are medical uses and methods of treatment based on the anti-human CCR8 antibodies, comprising administering Zr-89 labeled anti-CD8 microantibodies to determine the abundance and / or distribution of CD8 cells by PET scanning, for stratification or for monitoring the success of treatment or disease progression. The invention also provides a method for reliably determining the anti-CCR8 antibody in the plasma of cynomolgus monkeys or human. Finally, an anti-murine CCR8 replacement antibody that mimics the unusual half-life of TPP-23411 is disclosed.

Description

Technical Field

[0001] The present invention relates to medical uses comprising administering anti-human CCR8 antibodies in a specifically defined dosage regime in monotherapy or in combination therapy with anti-PD-(L)1 antibodies. Dosing regimens have been developed for the anti-human / cynomolgus CCR8 antibody TPP-23411, but they can also be used for other antibodies with similar properties to TPP-23411. In certain embodiments, medical uses or treatment methods based on anti-human CCR8 antibodies include a stratification step to select patients with an increased probability of treatment success. The proposed biomarkers are:

[0002] a. Tumor Proportion Score or Combined Positive Score as a measure of PD-(L)1 expression;

[0003] b. analyzing inflammatory cytokines selected from the group consisting of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-13 and TNF-α in a blood, plasma or serum sample, and

[0004] c. Prior treatment for cancer with anti-PD-(L)1 antibodies for at least 6 months.

[0005] Furthermore, according to the present invention, medical uses and methods of treatment based on anti-human CCR8 antibodies are provided, comprising administering Zr-89 labeled anti-CD8 minibodies to determine the abundance and / or distribution of CD8 cells by PET scanning for stratification or for monitoring treatment success or disease progression. According to the present invention, a method for reliably determining anti-anti-CCR8 antibodies in cynomolgus monkey or human plasma is also provided. Finally, an anti-mouse CCR8 surrogate antibody is disclosed that mimics the unusual half-life of TPP-23411. Background Art

[0006] In a monotherapy setting or in combination with immune checkpoint inhibitors, targeting regulatory T cells (Treg) is an attractive method to enhance anti-tumor immune responses because Tregs suppress the anti-tumor immune function of cytotoxic T cells and contribute to the immunosuppressive tumor microenvironment (TME). However, peripheral Tregs are physiologically indispensable for maintaining immune tolerance. Therefore, systemic depletion of Tregs may not only enhance anti-tumor immune responses, but also induce strong and undesirable autoimmunity. In essence, the key issue of customizing Treg-targeted cancer immunotherapy is to ensure the specific depletion of tumor-infiltrating Tregs without affecting peripheral Tregs.

[0007] In preclinical models, several Treg depletion methods have been shown to reduce tumor burden and enhance anti-tumor immune responses. However, most of these methods target surface receptors that are not specific for tumor-infiltrating Tregs, such as CD25 or CCR4, and are therefore associated with a number of side effects. Therefore, there is a great need for safe and effective medical uses to deplete tumor-infiltrating Tregs while sparing peripheral Tregs and effector T cells.

[0008] Compared with peripheral Treg, CC motif chemokine receptor 8 (CCR8) was identified as one of the receptors most differentially and specifically expressed on tumor-infiltrating Treg. CCR8 has 4 natural ligands: CCL1, CCL8, CCL16 and CCL18; wherein CCL1 specifically binds to CCR8. Neither gene knockout nor functional blockade of CCR8 significantly affects the tumor infiltration, activation or inhibition ability of CCR8+Treg (Campbell, Joseph R., et al. "Fc-optimized Anti-CCR8 antibody depletes regulatory T cells in human tumor models." Cancer Research 81.11 (2021): 2983-2994). This suggests that CCR8 and other chemokine receptors play an insignificant role in activating tumor homing of Treg. Therefore, depletion of tumor-infiltrating CCR8+ Tregs, rather than blocking CCR8 function, is the key to specific immunotherapy with pan-tumor potential (Whiteside, Sarah K., et al. "CCR8 marks highly suppressive Treg cells within tumours but is dispensable for their accumulation and suppressive function." Immunology 163.4(2021):512-520).

[0009] TPP-23411 is a new Treg depleting antibody that specifically depletes tumor-infiltrating Tregs while sparing peripheral Tregs and effector T cells because of the highly tumor-specific expression profile of its target CCR8. It was first described in U.S. Application Serial No. 17 / 358,841, filed on June 25, 2021, PCT Application No. PCT / EP2021 / 067504, PCT Application No. PCT / EP2021 / 067578, PCT Application No. PCT / EP2021 / 067574, PCT Application No. PCT / EP2021 / 067579, and PCT Application No. PCT / EP2021 / 067580. The entire contents of each of these documents are incorporated herein, particularly the description of the specific properties of TPP-23411 and the techniques used to analyze these properties.

[0010] TPP-23411 is a fully human IgG antibody and was generated using a phage display method using a chemically synthesized peptide containing the sulfated N-terminus of human or cynomolgus monkey CCR8 as an epitope. The corresponding sequences characterizing TPP-23411 are provided as SEQ ID NO: 1 to SEQ ID NO: 18, see also the "Brief Description of Sequence IDs" section herein.

[0011] TPP-23411 was shown to bind highly specifically to both human and cynomolgus CCR8 expressed by CHO cells, with respective affinities being of the same order of magnitude, e.g., low-digit nanomolar range. TPP-23411 does not bind to CCR4, the closest paralogue of CCR8.

[0012] TPP-23411 is a low / non-internalizing antibody, as demonstrated by human cells expressing endogenous CCR8. It is hypothesized that this property prolongs the presentation of TPP-23411 to effector cells and may therefore improve the efficacy of ADCC- and ADCP-based Treg depletion.

[0013] In cynomolgus monkeys and humans, TPP-23411 is characterized by relatively high clearance, see, e.g., Examples 12 or 15 herein.

[0014] An antibody with similar properties to TPP-23411 is the following antibody:

[0015] a. Characterized by a KD for binding to CHO cells transfected with human CCR8 that is of the same order of magnitude as the KD for binding to CHO cells transfected with human CCR8 by TPP-23411.

[0016] b. Antibodies that induce ADCC and ADCP

[0017] o Preferably, wherein the antibody binds to human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) that is in the same order of magnitude as TPP-23411 binding to human Fcγ receptor IIIA variant V176 (CD16a), and

[0018] o Preferably, wherein the antibody binds to human FcγRIIa (CD32a) with a dissociation constant (KD) that is in the same order of magnitude as the binding of TPP 23411 to human FcγRIIa (CD32a);

[0019] o Preferably, wherein the antibody is afucosylated;

[0020] c. Preferably, the antibody is characterized by a half-life in humans of <14 days, preferably <10 days, most preferably <7 days.

[0021] TPP-23411 is preferably non-fucosylated and induces ADCC and ADCP. Therefore, after binding Treg, TPP-23411 recruits respective effector cells (ADCC is NK cells, ADCP is macrophages) via FC receptor (FcR) interactions, so that these effector cells can deplete Treg expressing CCR8. In fact, TPP-23411 triggers effective and dose-dependent depletion of human primary CCR8+Treg or HEK293 target cells expressing epitopic human CCR8 by engaging human NK92V cells or human primary M2c macrophages as effector cells.

[0022] TPP-23411 does not block or neutralize CCL1-induced β-arrestin signaling.

[0023] In preclinical experiments, the TPP-23411 surrogate antibody was found to show significant efficacy in syngeneic tumor models, either alone or in combination with PD-(L)1 inhibitors.

[0024] CCR8 antibodies can be combined with PD-(L)1 inhibitors or other checkpoint inhibitors.

[0025] Pembrolizumab (KEYTRUDA) is an effective humanized IgG4mAb with a high specificity for binding to the PD-1 receptor, thereby inhibiting its interaction with PD-L1 and PD-L2. Based on preclinical in vitro data, pembrolizumab has high affinity and effective receptor blocking activity for PD-1. Pembrolizumab has an acceptable preclinical safety profile and is in clinical development as an intravenous (IV) immunotherapy for advanced malignancies. Pembrolizumab is indicated for the treatment of patients across multiple cancer indications. The dosage form and strength of pembrolizumab are solutions for injection provided in single-dose vials of 100mg / 4mL (25mg / mL) solution. Pembrolizumab can be administered, for example, at a dose of 200mg once every three weeks, or at a dose of 400mg once every six weeks. Therapeutic studies in mouse models have confirmed that, whether as a monotherapy or in combination with other treatment modalities, the administration of antibodies that block the interaction of PD-1 / PD-L1 enhances the infiltration of tumor-specific CD8+T cells and ultimately leads to tumor rejection.

[0026] Nivolumab (OPDIVO) is another PD-1 blocking antibody indicated for the treatment of patients across multiple cancer indications. The dosage form and strength is a solution for injection provided in a single-dose vial of 10 mg / mL (4 mL, 10 mL). Nivolumab can be administered by intravenous infusion after dilution, for example at a dose of 240 mg every two weeks, 360 mg every 3 weeks, or 480 mg every 4 weeks.

[0027] Atezolizumab (TECENTRIQ) is another PD-L1 blocking antibody and is also indicated for treating patients across multiple cancer indications. The dosage form and strength is a solution for injection provided in a single-dose vial of 840 mg / 14 mL (60 mg / mL) or 1200 mg / 20 mL) (60 mg / mL). Atezolizumab can be administered by intravenous infusion after dilution, for example, at a dose of 840 mg every two weeks, 1200 mg every 3 weeks, or 1680 mg every 4 weeks.

[0028] Zimberelimab (Arcus Biosciences) is a monoclonal antibody that binds to PD-1 and restores the anti-tumor activity of T cells. Zimberelimab is undergoing clinical studies for various cancer indications, such as for the treatment of first-line metastatic non-small cell lung cancer, for example in combination with domvanalimab (anti-TIGIT monoclonal antibody) and etrumadenant (dual A2a / A2b adenosine antagonist). Zimberelimab can be administered by intravenous infusion after dilution, for example at a dose of 360 mg every 3 weeks.

[0029] Toripalimab is a recombinant humanized PD-1 monoclonal antibody being developed by Shanghai Junshi Bioscience Co., Ltd. It binds to PD-1 and prevents PD-1 from binding to PD-L1 and PD-L2 for the treatment of various cancers. The approved dose of Toripalimab is 3 mg / kg every two weeks, intravenously (IV) infused.

[0030] Durvalumab (IMFINZI) is a PD-L1 blocking antibody designated for various cancer types. The dosage form and strength are solutions for injection provided in single-dose vials of 500 mg / 10 mL or 120 mg / 2.4 mL (50 mg / mL each). Durvalumab can be administered by intravenous infusion after dilution as part of a combination regimen, for example at a dose of 10 mg / kg every two weeks or 1500 mg every 3 weeks.

[0031] Other PD-(L)1 inhibitors and their dosing regimens (approved or in clinical investigation) are known in the art and can be used in the provided medical uses and treatment methods.

[0032] Technical issues

[0033] If the antibody cannot be properly tested in the relevant model species, it is challenging to come up with an appropriate dosing regimen. TPP-23411 cross-reacts with cynomolgus monkeys but not with the mouse CCR8 orthologue. Therefore, established models for finding the optimal dosing regimen cannot be applied to find appropriate solutions for dosing of monotherapy and combination therapy in human patients.

[0034] In addition, when characterizing TPP-23411, the inventors observed abnormal PK / PD behavior and increased clearance of the antibody in cynomolgus monkeys. Based on preliminary translation alpharmacokinetic estimations described herein, TPP-23411 is characterized by a half-life of about 4 days (the half-life of a typical antibody is 21 days). Therefore, this clearance behavior deviates not only from the predicted properties of TPP-23411, but also from the common half-life of other antibodies for medical use, and complicates the identification of safe and effective dosing regimens for treating patients.

[0035] In order to find a suitable regimen for dosing in human patients, the inventors had to come up with an anti-mouse CCR8 surrogate antibody that could be used to mimic the short half-life of TPP-23411. The anti-mouse CCR8 surrogate antibody is TPP-29338 provided herein.

[0036] In addition, it is necessary to determine and quantify anti-anti-CCR8 antibodies in cynomolgus monkey or human plasma or serum in order to propose a suitable dosage regime and for quality control. The medical use of TPP-23411 as provided herein is characterized by a specific dosage regime that ensures superior efficacy while meeting the necessary safety requirements. The successful mode of action of the dosing regime of the present invention is demonstrated in Example 24. In addition, the dosing regime according to the present invention also provides convenience in operation and administration, thereby reducing dosing errors while improving the quality of life and compliance of patients.

[0037] Furthermore, administration of Treg depleting agents may be associated with significant side effects or may not be effective in certain patient populations. When testing dosing regimes according to the present invention, the inventors proposed certain (pre-)dosing regimens that were found to prevent the adverse reactions observed when anti-CCR8 antibodies were administered intravenously, see Example 23. In order to identify those patients who may benefit from anti-CCR8 antibody treatment and in order to identify those patients for whom potential side effects are acceptable after a benefit-risk assessment, a stratification procedure is provided herein.

[0038] Finally, the inventors have found that IHC staining to determine the amount of T cells in tumor biopsies as a biomarker for monitoring stratification may lack robustness, for example if the T cell distribution is not normally distributed. Therefore, the inventors propose herein to apply a specific PET-based method to track the recruitment of T cells after administration of anti-human-CCR8 antibodies. Background Art

[0040] Several companies have started or announced plans to start clinical studies to administer compounds targeting CCR8. The various dosing regimes provided are distinct from the dosing regimens / medical uses of the methods of treatment of the invention described herein, at least in that the compounds targeting CCR8 are different from TPP-23411, and also differ in a number of other respects.

[0041] Jounce and Gilead have developed the anti-CCR8 antibody JTX-1811 / GS-1811 (see WO2021 / 163064A1), and Gilead has announced the initiation of a "Phase 1 study evaluating the safety and tolerability of GS-1811, a non-fucosylated anti-CCR8 monoclonal antibody, as monotherapy and in combination with pembrolizumab in adults with advanced solid tumors" (NCT05007782). During the dose escalation period, participants received GS-1811 at increasing dose levels for up to 12 months to determine the maximum tolerated dose (MTD) and / or the recommended Phase 2 dose.

[0042] Shionogi has developed the anti-CCR8 antibody S-531011 (see WO2020 / 138489A1) and has initiated a "Phase 1b / 2, multicenter, open-label study of S-531011 as monotherapy and in combination with immune checkpoint inhibitors in participants with locally advanced or metastatic solid tumors" (NCT05101070). Participants will receive escalating doses of S-531011 by intravenous infusion for up to approximately 12 months. For the combination arm, participants will receive escalating doses of S-531011 in combination with pembrolizumab by intravenous infusion for up to approximately 12 months.

[0043] BMS has developed the anti-CCR8 antibody BMS-986340 (see WO2021 / 194942A1), and has launched a "Phase 1 / 2 study of BMS-986340 as a monotherapy and in combination with nivolumab in participants with advanced solid tumors" (NCT04895709) in May 2021. Preliminary completion is expected in March 2024. In the dose escalation phase, 4A19 was administered intravenously to subjects at fixed doses of 0.3, 1, 3, 10, 30, 100, 300 and 800 mg once every two weeks (Q2W). In Part IB, 4A19 was administered intravenously (IV) to subjects at the same fixed dose, in combination with nivolumab administered IV at a fixed dose of 480 mg approved by the FDA once every 4 weeks (Q4W).

[0044] The international phase application WO2022 / 00443A1, entitled "METHODS AND COMPOSITIONS FOR TARGETING TREGS USING CCR8 INHIBITORS", was filed on 2020-07-03 by Nanjing Immunophage Biotech Co., Ltd, and discloses small molecule CCR8 inhibitors that block the CCR8 / CCL1 axis, as demonstrated in a calcium mobilization assay. On November 15, 2021, Nanjing Immunophage has launched a "Phase 1 study to evaluate the safety, tolerability, pharmacokinetics and preliminary anti-tumor activity of orally administered IPG7236 as a single agent in patients with advanced solid tumors" (see NCT05142592). The IPG7236 drug is provided in an oral tablet dosage form, including two strengths: 25 mg and 100 mg, respectively.

[0045] LM-108 is a humanized monoclonal anti-CCR8 antibody developed by LaNova Medicines. In August 2022, LaNova Medicines announced the launch of a "Phase I / II, open-label, dose-escalation and dose-expansion clinical study to evaluate the safety, tolerability, pharmacokinetics and preliminary efficacy of LM-108 as a single agent or in combination with Toripalimab in advanced solid tumors" (NCT05518045).

[0046] U.S. Application No. 17 / 280,137 discloses Zr-89 labeled anti-CD8 miniantibodies for PET scanning, but does not disclose the use of this miniantibody as part of a medical use of anti-CCR8 antibodies, where this particular method is superior to conventional histopathological methods to reliably track the recruitment of T cells as a biomarker after administration of anti-human CCR8 antibodies.

[0047] Descriptions of anti-drug antibody assays are known in the art, see, for example, EP3105592B1 or Seaman, Michael S., et al. "Optimization and qualification of a functional anti-drug antibody assay for HIV-1bnAbs." Journal of immunological methods 479 (2020): 112736, but the inventors are not aware of specific methods for detecting and quantifying anti-CCR8 antibodies.

[0048] There are various anti-CCR8 mouse surrogate antibodies, such as those previously described by the inventors in U.S. application Ser. No. 17 / 358,841 and PCT application Nos. PCT / EP2021 / 067504, PCT / EP2021 / 067578, PCT / EP2021 / 067574, PCT / EP2021 / 067579, and PCT application No. PCT / EP2021 / 067580, but the inventors are not aware that any of these anti-CCR8 mouse surrogate antibodies may be suitable for mimicking the rapid clearance rate of TPP-23411.

[0049] Solution to the problem

[0050] Based on various experimental data (see Examples 1 to 16), the inventors could successfully identify the medical uses of anti-CCR8 antibodies, including specific administration regimens, as further illustrated in Example 17:

[0051] Provided is an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment, comprising intravenously administering to a patient in need thereof an anti-CCR8 antibody in a total amount of:

[0052] a. About 1, 2.5, 3, 10, 30, 50, 100, 125 or 250 mg once a week, or

[0053] b. About 16, 450, 500, 750, 1000 or 1500 mg once every three weeks.

[0054] Optionally, the medical use may further comprise intravenously administering to a patient in need thereof an anti-PD-(L)1 antibody in a total amount of:

[0055] i. about 200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0056] ii. about 400 mg once every six weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0057] iii. about 240 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0058] iv. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0059] v. about 480 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0060] vi. about 840 mg every two weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or

[0061] vii. about 1200 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is atezolizumab, or

[0062] viii. about 1680 mg every four weeks, preferably wherein the anti-PD-(L)1 antibody is ateliizumab, or

[0063] ix. about 360 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is sepalizumab, or

[0064] x. about 3 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is toripalimab, or

[0065] xi. about 10 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab, or

[0066] xii. About 1500 mg / kg every 3 weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab.

[0067] Also provided is an anti-human CCR8 antibody having ADCC activity and / or ADCP activity for use in a therapeutic method.

[0068] a. comprising intravenously administering to a patient in need thereof a total amount of 2.7 mg to 75 mg of an anti-CCR8 antibody once a week,

[0069] b. preferably further comprising intravenously administering to the patient an anti-PD-(L)1 antibody in a total amount of:

[0070] i. about 200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0071] ii. about 400 mg once every six weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0072] iii. about 240 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0073] iv. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0074] v. about 480 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0075] vi. about 840 mg every two weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or

[0076] vii. about 1200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is atezolizumab, or

[0077] viii. about 1680 mg every four weeks, preferably wherein the anti-PD-(L)1 antibody is ateliizumab, or

[0078] ix. about 360 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is sepalizumab, or

[0079] x. about 3 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is toripalimab, or

[0080] xi. about 10 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab, or

[0081] xii. About 1500 mg / kg every 3 weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab.

[0082] Further provided is an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method,

[0083] a. comprising intravenously administering to a patient in need thereof a total amount of 16 mg to 450 mg of an anti-CCR8 antibody once every three weeks,

[0084] b. preferably further comprising intravenously administering to the patient an anti-PD-(L)1 antibody in a total amount of:

[0085] i. about 200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0086] ii. about 400 mg once every six weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0087] iii. about 240 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0088] iv. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0089] v. about 480 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0090] vi. about 840 mg every two weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or

[0091] vii. about 1200 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is atezolizumab, or

[0092] viii. about 1680 mg every four weeks, preferably wherein the anti-PD-(L)1 antibody is ateliizumab, or

[0093] ix. about 360 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is sepalizumab, or

[0094] x. about 3 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is toripalimab, or

[0095] xi. about 10 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab, or

[0096] xii. About 1500 mg / kg every 3 weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab.

[0097] The specific administration regimen provided herein avoids unacceptable side effects, yet maintains sufficient doses of the anti-CCR8 antibody in the blood for optimal efficacy.

[0098] In certain embodiments, the medical use or treatment method based on anti-human CCR8 antibodies includes a stratification step to select patients with an increased probability of treatment success and / or an improved benefit-risk ratio. The proposed biomarkers are:

[0099] a. Tumor Proportion Score or Combined Positive Score as a measure of PD(L)1 expression;

[0100] b. analyzing blood, plasma or serum samples for inflammatory cytokines selected from the group consisting of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL 12p70, IL-13 and TNF-α, and

[0101] c. Prior treatment for cancer with anti-PD-(L)1 antibodies for at least 6 months.

[0102] Further, according to the present invention, medical uses and treatment methods based on anti-human CCR8 antibodies are provided, comprising administering Zr-89-labeled anti-CD8 miniantibodies to determine the abundance and / or distribution of CD8 cells by PET scanning for stratification or for monitoring treatment success or disease progression.

[0103] According to the present invention, there is also provided a method for reliably measuring anti-anti-CCR8 antibodies in cynomolgus monkey or human plasma using an anti-CCR8 antibody-based bridging ELISA method. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 : Overview of the dose escalation portion of the study plan.

[0106] Figure 2 : Dose-normalized plasma concentrations of TPP-23411 after intravenous and subcutaneous administration.

[0107] Figure 3 : Predicted human plasma c / t profile following a one-hour single intravenous infusion of 1 mg / kg TPP-23411.

[0108] Figure 4 : For QW, Q2W, and Q3W administration, c / t profiles were simulated following multiple one-hour intravenous infusions of 1 mg / kg TPP-23411.

[0109] Figure 5: In vivo, correlation between responses to anti-CCR8 antibody treatment and anti-PD-L1 antibody (A) or anti-PD-1 (B) treatment. (A)-(B) The in vivo efficacy of antibody treatment was evaluated in various syngeneic murine carcinoma models. The solid and dashed lines indicate the regression line and the associated 95% confidence interval, respectively.

[0110] Figure 6: Correlation between response to anti-mouse CCR8 antibody treatment and PD-L1 mRNA expression (A) and IFN-γ mRNA expression (B) in early untreated tumors. In various syngeneic murine carcinoma models, the in vivo efficacy of anti-CCR8 antibody treatment was evaluated for correlation with (A) baseline PD-L1 expression and (B) baseline IFN-γ expression. The solid and dashed lines indicate the regression line and the associated 95% confidence interval, respectively.

[0111] Figure 7 : Starting from 3 days after the first administration of 10 mg of anti-CCR8 antibody to human patients, the number of activated proliferative CD8+ T cells increased relative to the total number of CD3+ T cells. In particular, a continuous increase was observed starting from the second week of treatment, presumably due to the induction of immune activation in the tumor. The treatment ended 3 weeks after the last infusion. The ratio of the two cell types was set to 1 at the screening time point.

[0112] Figure 8 : Starting ~1 day after the first anti-CCR8 antibody administration (dose of 10 mg), the activated Tregs decreased relative to the total number of CD3+ T cells. The ratio of the two cell types was set to 1 at the screening time point.

[0113] Fig. 9 : The ratio of activated Tregs to the total number of activated proliferative CD8+ T cells. The ratio of the two cell types was set to 1 at the screening time point.

[0114] Fig.10 : When treated with 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody, Tregs in patient blood samples decreased. For each patient, the ratio C1D2 to C2D15 (activated Tregs / total CD3+T cells) obtained at the time point was divided by the ratio (activated Tregs / total CD3+T cells) obtained by the same patient at screening (Scr) (ie, before the administration of anti-CCR8 antibody). In order to obtain data for one box, all patients treated with the specified dose were grouped together. Even the lowest 1 mg group showed a reduction in the median activated Tregs to ~53% of the screening value.

[0115] Fig.11 : Boxplots of TNF-α levels measured in pg / μl in blood or serum samples collected 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8-antibody to human patients.

[0116] Fig.12 : Box plot of IFN-γ levels measured in pg / μl in blood or serum samples collected 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0117] Fig.13 : Box plot of IP10 (CXCL10) levels measured in pg / μl in blood or serum samples collected 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0118] Fig.14 : Box plot of IL8 levels measured in pg / μl in blood or serum samples collected 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0119] Fig.15: Box plot of IL6 levels measured in pg / μl in blood or serum samples collected 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0120] Fig.16 : Box plot of IL10 levels measured in pg / μl in blood or serum samples collected 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0121] A brief description of sequence ID

[0122] The sequence listing associated with this application is incorporated herein by reference in its entirety. The name of the text file containing the sequence listing is BHC221019_WO_ST26_20230903.xml. The size of the text file is 92 kilobytes and the text file was created on 03.09.2023.

[0123]

[0124]

[0125]

[0126]

[0127] definition

[0128] Unless otherwise defined, all scientific and technical terms used in the specification, drawings and claims have the common meaning as commonly understood by those of ordinary skill in the art. All publications, patent applications, patents and other references mentioned herein are incorporated by reference as a whole. In the event of a conflict, this specification (including definitions) shall prevail. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosures, the document with a later effective date shall prevail. When citing a database, valid data shall be the version number applicable to 06.05.2022 (if not otherwise specified). Materials, methods and embodiments are exemplary only and are not meant to be limiting. Unless otherwise stated, the following terms used in this document (including specification and claims) have the definitions given below.

[0129] As used herein, the expression "about" or "~" refers to a value within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. The term "about" is also used to indicate that the amount or value described can be a specified value or some other value that is roughly the same. The phrase is intended to express that similar values ​​promote equivalent results or effects as described herein. In this context, "about" can refer to a range of up to 10% above and / or below. Regardless of where the term "about" is specified for a certain assay or embodiment, the definition applies to the specific context.

[0130] If not otherwise defined, the term "about" means + / - 10% of the provided value.

[0131] The terms "comprising", "including", "containing", "having" and the like should be interpreted broadly or openly without limitation. The term comprising when used in this specification includes "consisting of".

[0132] Unless the context clearly dictates otherwise, singular forms such as "a," "an," or "the" include plural references. Thus, for example, reference to "a monoclonal antibody" includes a single monoclonal antibody as well as multiple monoclonal antibodies, whether the same or different. Similarly, reference to "a cell" includes a single cell as well as multiple cells.

[0133] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. The terms "at least one" and "at least one of" include, for example, one, two, three, four, five or more elements.

[0134] It should also be understood that slight variations above and below the stated ranges can be used to achieve substantially the same results as the values ​​within the ranges. In addition, unless otherwise stated, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values.

[0135] As used herein, the term "amino acid" or "amino acid residue" generally refers to naturally occurring amino acids. The single letter code used herein refers to the corresponding amino acid. As used herein, "charged amino acids" are amino acids with a negative or positive charge. "Negatively charged amino acids" are aspartic acid (D) and glutamic acid (E). "Positively charged amino acids" are arginine (R), lysine (K) and histidine (H). "Polar amino acids" are all amino acids that form hydrogen bonds as donors or acceptors. These are all charged amino acids and asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y) and cysteine ​​(C). "Polar uncharged amino acids" are asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y) and cysteine ​​(C). "Amphoteric amino acids" are tryptophan (W), tyrosine (Y) and methionine (M). "Aromatic amino acids" are phenylalanine (F), tyrosine (Y) and tryptophan (W). "Hydrophobic amino acids" are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and cysteine. "Small amino acids" are glycine (G), alanine (A), serine (S), proline (P), threonine (T), aspartic acid (D), and asparagine (N).

[0136] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0137] When a gene or protein from a particular species (eg mouse) is referred to generically, analogs from humans are also intended unless otherwise stated or clearly incompatible. This is particularly true in the context of biomarkers.

[0138] When applied to nucleic acid, polypeptide, protein or antibody, the term "isolated" means that the nucleic acid, polypeptide, protein or antibody is substantially free of other cellular components associated therewith in the native state. It is preferably in a homogeneous state. It can be in an anhydrous solution or an aqueous solution. Purity and homogeneity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Proteins, polypeptides or antibodies that are the main species present in the preparation are substantially purified. In particular, the isolated gene is separated from the open reading frame that flanks the gene and encodes the protein other than the target gene. However, the isolated polypeptide can be, for example, fixed on a bead or particle via a suitable linker.

[0139] The term "purified" means that the nucleic acid or protein produces essentially one band in an electrophoretic gel. In particular, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0140] As used herein, for example, when referring to synthetic nucleic acid molecules or synthetic genes or synthetic peptides, the term "synthetic" refers to nucleic acid molecules or polypeptide molecules produced by recombinant methods and / or by chemical synthesis methods. As used herein, produced by recombinant methods means by using recombinant DNA methods, referring to the use of well-known molecular biology methods to express proteins encoded by cloned DNA.

[0141] "Sulfation" is a post-translational modification in which a sulfate group is added to an amino acid such as a tyrosine residue of a polypeptide or protein. Tyrosine sulfation occurs in all multicellular organisms. Under physiological conditions, it is catalyzed by tyrosyl protein sulfotransferases (TPST) 1 and 2, which are Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to context-dependent tyrosine in the protein substrate. Synthetic sulfation of tyrosine can be performed using techniques known in the art, such as Bunschoten, Anton et al, "A general sequence independent solid phase method for the site specific synthesis of multiple sulfated-tyrosine containing peptides." Chemical Communications 21 (2009): 2999-3001. Sulfated polypeptides are polypeptides that contain at least one sulfate. Non-sulfated polypeptides are polypeptides that do not contain sulfates.

[0142] As used herein, the term "N-terminus" or "N-terminus" of a chemokine receptor refers to the N-terminal amino acids of a chemokine receptor comprising at least the TRD. When the polypeptide or protein comprises a signal peptide, the N-terminus may also refer to the N-terminal sequence after the natural cleavage site of the polypeptide or protein. According to some preferred embodiments, the N-terminus comprises the LID domain and the TRD domain of the chemokine receptor, but does not comprise the natural cysteine ​​between these two domains. Instead, the cysteine ​​may be removed or may be replaced by a different amino acid.

[0143] "Sequence identity" or "percent identity" is a number that describes how similar a query sequence is to a target sequence, or more precisely, how many characters in each sequence are identical after alignment. The most commonly used tool for calculating sequence identity is BLAST (Basic Local Alignment Search Tool, https: / / blast.ncbi.nlm.nih.gov / ), which compares pairs of sequences, searching for areas of local similarity. Suitable alignment methods are known in the art, such as the Needleman Wunsch algorithm for global-global alignments, using the BLOSUM62 matrix, a gap open penalty of 11, and a gap extension penalty of 1. The pairs of identical residues in the alignment can then be counted and then divided by the total length of the alignment (including internal and external gaps) to obtain a percent identity value.

[0144] For "percent similarity" or "sequence similarity" values, the same method as for percent identity values ​​can be used, except that instead of counting identical residue pairs, pairs of aligned residues have non-negative BLOSUM62 values ​​(ie, ≥ 0) are counted.

[0145] "CC chemokine receptors" (CCRs, also called beta chemokine receptors) are integral membrane proteins that specifically bind and respond to cytokines of the CC chemokine family. They represent a subfamily of chemokine receptors, a large family of G protein-linked receptors, and are referred to as seven-transmembrane (7-TM) proteins because they span the cell membrane seven times. The CC chemokine receptor subfamily includes CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and CCR10.

[0146] The term "CCR8" refers to CC chemokine receptor type 8. The CCR8 protein is encoded by the gene CCR8 (NCBI gene ID 1237). Synonyms of CCR8 are, in particular, CC-CKR-8, CCR-8, CDw198, CKRL1, CMKBR8, CMKBRL2, GPRCY6, CY6, TER1. The CCR8 protein includes human, murine, rat, rhesus monkey and other mammalian and non-mammalian homologs. The sequence of human CCR8 can be obtained via the UniProt identifier P51685 (CCR8_HUMAN), such as human isoforms P51685-1 or P51685-2 (UniProt, November 29, 2019). The sequence of murine CCR8 can be obtained via the UniProt identifier P56484 (CCR8_MOUSE). The sequence of rhesus macaque CCR8 can be obtained via UniProt identification code O97665 (CCR8_MACMU). Different species may have different isoforms and variants, and are all included by the term CCR8. Also included are CCR8 molecules before and after maturation, i.e., independent of the cleavage of one or more pro-domains. In addition, synthetic variants of CCR8 protein can be produced and are included in the term CCR8. In addition, protein CCR8 can be subjected to various modifications, such as synthetic or naturally occurring modifications, such as post-translational modifications. Recombinant human CCR8 is commercially available or can be prepared as known in the art. CCR8 is a receptor for the chemokine CCL1 / SCYA1 / I-309. Brington et al. reported the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) in chemokine receptor CCR8 for ligand binding and activation (Barington, Line, et al. "Role of conserved disulfide bridges and aromatic residues in extracellular loop 2 of chemokine receptor CCR8 for chemokine and small molecule binding." Journal of Biological Chemistry 291.31 (2016): 16208-16220). In addition, they found that two different aromatic residues in ECL-2, Tyr184 (Cys+1) and Tyr187 (Cys+4), are critical for the binding of CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding.

[0147] "Programmed death 1 (PD1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes, but is not limited to, human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number U64863 (November 29, 2019). "Programmed death ligand 1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion when bound to PD-1. As used herein, the term "PD-L1" includes, but is not limited to, human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7 (November 29, 2019).

[0148] The term "PD-(L)1" refers to PD-1 and / or PD-L1.

[0149] The "tumor proportion score" (TPS) is the percentage of viable tumor cells that show partial or complete membrane staining at any intensity. For example, if the TPS is ≥ 1%, the sample should be considered to have PD-L1 expression, and if the TPS is ≥ 50%, the sample should be considered to have high PD-L1 expression. For example, PD-L1 protein expression in NSCLC is often determined by using the tumor proportion score (TPS).

[0150] A "historical tumor proportion score" is a tumor proportion score obtained in preparation for or during monitoring of a previous (cancer) therapy or medical analysis, ie no sample obtained from a fresh biopsy was used in preparation for the anti-CCR8 antibody therapy.

[0151] The "combined positive score" (CPS) is the number of stained cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. For example, if the CPS is ≥1, the sample should be considered to have PD-L1 expression, and if the CPS is ≥10, it should be considered to have high PD-L1 expression. The FDA has approved the use of the PD-L1 IHC 22C3 pharmDx assay and the use of the VENTANA PD-L1 (SP263) assay to determine whether patients are eligible for the therapeutic antibody pembrolizumab.

[0152] A "historical combined positive score" is a combined positive score obtained in preparation or during monitoring of a previous (cancer) therapy or medical analysis, ie no sample obtained from a fresh biopsy was used in preparation of the anti-CCR8 antibody therapy.

[0153] The "PD L1 IHC 22C3 pharmDx assay" is a qualitative immunohistochemistry assay using monoclonal mouse anti-PD-L1 (Clone 22C3) intended for detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue using the EnVision FLEX visualization system on an Autostainer Link 48 (see, e.g., https: / / www.accessdata.fda.gov / cdrh_docs / pdf15 / p150013s001c.pdf).

[0154] The "VENTANA PD-L1 (SP263) assay" is another qualitative immunohistochemistry assay using rabbit monoclonal anti-PD-L1 (Clone SP142) and can be used in FFPE tissues stained using, for example, the OptiView DABIHC Dectection Kit and the OptiView Amplification Kit on a BenchMark ULTRA instrument (see, e.g., https: / / www.accessdata.fda.gov / cdrh_docs / pdf16 / p160046c.pdf).

[0155] The term "modulate" refers to any change in an existing process or behavior, such as blocking (antagonism) and induction (agonism). For example, modulation of G protein-independent signaling refers to any significant change in G protein-independent signaling.

[0156] The term "internalization" of an antibody, fragment or conjugate refers to the uptake of an antibody, fragment or conjugate into a cell. Preferably, the internalization of a cell line with endogenous target expression (e.g., human or mouse CCR8) is determined. Preferably, internalization is determined by measuring the total internalization fluorescence intensity of each cell, and quantified relative to an isotype control. In short, an antibody, fragment or conjugate and a matching isotype control are labeled with a dye, and the internalization fluorescence of the antibody, fragment or conjugate is measured and quantified relative to an isotype control.

[0157] A "non-internalizing antibody" is defined as an antibody that exhibits essentially the same internalization as a corresponding isotype control.

[0158] A "low internalization antibody" is defined as an antibody that exhibits internalization equal to or less than 10-fold that of an isotype control, preferably less than 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, or 1.1-fold that of an isotype control.

[0159] An "isotype control" is an antibody or fragment that does not bind the target, but is of the same class and type as the reference antibody or fragment that recognizes the target.

[0160] An antibody or fragment is said to be "cross-reactive" or "cross-reactive" if it binds antigens from two or more different species, for example with a KD value of 10-7 M or less, more preferably less than 10-8 M, even preferably in the range of 10-9 M to 10-11 M.

[0161] As used herein, the term "specific binding" with respect to an antibody refers to an antibody that recognizes a specific antigen, but does not substantially recognize or bind to other molecules in a sample: antibodies characterized by substantially non-specific binding would lack therapeutic applicability, such that these embodiments are excluded. However, as is known in the art, specific binding of an antibody or binding agent does not necessarily exclude antibodies or binding agents that bind to other antigens / target molecules. An antibody that specifically binds to an antigen from one species may also bind to antigens from one or more other species. This cross-species reactivity does not itself change the specific classification of the antibody.

[0162] In some cases, the term "specific binding" or "specifically binds" can be used to refer to the interaction of an antibody, protein or peptide with a second chemical substance, indicating that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical substance; for example, an antibody recognizes and binds to a specific protein structure, not proteins in general. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody.

[0163] In case of doubt, specific binding of an antibody or binding agent preferably describes binding of an antibody, antibody fragment or binding agent to its antigen / target with an affinity of at least 10 -7 M (as KD value; i.e. preferably has a KD value of less than 10 -7 M), wherein the antibody or binding agent has at least two-fold lower affinity for a non-specific antigen (which is an unintended antigen / target molecule or a closely related antigen / target molecule).

[0164] The term "affinity" is a term of art and describes the strength of binding between a binding agent, antibody or antibody fragment and a target. The "affinity" of an antibody and its fragments for a target can be determined using techniques well known in the art or described herein, such as by ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), flow cytometry or fluorescence polarization assay. Preferably, affinity is provided as a dissociation constant, KD.

[0165] The "dissociation constant" (KD) has molar units (M) and corresponds to the concentration of the binding agent / antibody at which half of the target protein is occupied at equilibrium. The smaller the dissociation constant, the higher the affinity between the binding agent or antibody and its target. According to the present invention, the antibody preferably has a KD of at least 10 -7 M (as KD value), more preferably at least 10 -8 M, even more preferably at 10 -9 M to 10 -11 The KD value may preferably be determined by surface plasmon resonance spectroscopy, for example as described elsewhere herein. If the assay conditions are found to affect the determined KD, the assay setting with the smallest standard deviation should be used.

[0166] "Half maximal effective concentration" (EC50) refers to the concentration of a drug, antibody, fragment, conjugate or molecule that induces a response halfway between baseline and maximum after a specific given time. In the case of antibody binding, the EC50 thus reflects the antibody concentration required for half maximal binding. If the inflection point can be determined by mathematical modeling (e.g., nonlinear regression) of the dose-response curve of the relationship between the concentration of the drug, antibody, fragment, conjugate or molecule and the signal for the described application, the EC50 can be determined. For example, if the dose-response curve follows a sigmoidal curve, the EC50 can be determined. When the response is an inhibitory effect, the EC50 is called the half maximal inhibitory concentration (IC50). The EC80 can be determined after making necessary changes.

[0167] The (effective) "half-life" of an antibody is the time it takes to reach half of its maximum concentration (Cmax) in plasma. On average, the serum half-life of IgG subclasses (IgG1, IgG2, and IgG4) is ~23 days, compared to 2-6 days for IgG3 and other Ig classes. Various methods are known in the art for analyzing antibody half-life, such as mass spectrometry or ELISA-based methods.

[0168] The term "antibody (Ab)" refers to an immunoglobulin molecule (e.g., not limited to human IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgE, IgA1, IgA2, mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgA, IgD, IgE or IgM, rat IgG1, IgG2a, IgG2b, IgG2c, IgA, IgD, IgE or IgM, rabbit IgA1, IgA2, IgA3, IgE, IgG, IgM, goat IgA, IgE, IgG1, IgG2, IgE, IgM or chicken IgY), which specifically binds to a specific antigen or reacts immunologically with it. Antibodies or antibody fragments include complementary determining regions (CDRs), also referred to as hypervariable regions, in the light chain variable domain and the heavy chain variable domain. The more highly conserved portion of the variable domain is called a framework (FR). As is known in the art, the amino acid positions / boundaries that depict the hypervariable regions of antibodies may vary, depending on the context and various definitions known in the art. As used herein, the numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al. The variable domains of natural heavy and light chains each contain four FR regions. The three CDRs in each chain are tightly bound together by the FR regions, and together with the CDRs of the other chain, contribute to the formation of the antigen binding site of the antibody, see Kabat EA, et al. "Sequences of Proteins of Immunological Interest (Natl. Inst. Health, Bethesda, MD), GPO Publ." No165-462 (1987). Unless otherwise expressly stated, the term antibody as used herein also refers to an antibody fragment. Depending on the respective context, the term antibody may also refer to any proteinaceous binding molecule with immunoglobulin-like functions.

[0169] The term "CDR" refers to the complementary determining region of an antibody. As known in the art, a complementary determining region (CDR) is a part of a variable chain in an antibody and a T cell receptor. A group of CDRs constitutes a paratope. CDRs are essential for the diversity of antigen specificity. There are three CDRs (CDR1, CDR2 and CDR3), which are arranged discontinuously in the amino acid sequence of the variable domain of an antigen receptor. Since antigen receptors are usually composed of two variable domains (on two different polypeptide chains, heavy and light chains), each antigen receptor usually has six CDRs that can contact the antigen together. The CDRs of the light chain are LCDR1, LCDR2 and LCDR3. The CDRs of the heavy chain are called HCDR1, HCDR2 and HCDR3. HCDR3 is the most variable complementarity determining region (see, e.g., Chothia, Cyrus, and Arthur M. Lesk. "Canonical structures for the hypervariable regions of immunoglobulins." Journal of molecular biology 196.4 (1987): 901-917.; Kabat, EA, et al. "Sequences of proteins of immunological interest. Bethesda, MD: US Department of Health and Human Services." Public Health Service, National Institutes of Health (1991): 103-511).

[0170] "Constant region" refers to the portion of an antibody molecule that confers effector function. The heavy chain constant region can be selected from any of the five isotypes: alpha (α), delta (δ), epsilon (ε), gamma (g) or mu (μ).

[0171] As used herein, the term "Fc domain", "Fc region" or "Fc portion" refers to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. For example, a human IgG heavy chain Fc region can extend from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain.

[0172] The antibodies or binding fragments according to the invention may have been modified to alter at least one constant region-mediated biological effector function. For example, in certain embodiments, the antibodies may be modified to reduce or enhance at least one constant region-mediated biological effector function relative to an unmodified antibody, such as reduced or increased binding to an Fc receptor (FcγR). FcγR binding may be reduced, for example, by mutating the immunoglobulin constant segment of the antibody at specific regions necessary for FcγR interaction (see, e.g., Canfield, Stephen M. and Sherie L. Morrison. "The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region." The Journal of experimental medicine 173.6 (1991): 1483-1491; and Lund, John, et al. "Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG." The Journal of Immunology 147.8 (1991): 2657-2662). FcγR binding may be enhanced, for example, by afucosylation. Reducing FcγR binding may also reduce other effector functions that rely on FcγR interaction, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity ("ADCC").

[0173] In addition, solving the interaction of Fc and FcRn allows to regulate the half-life of the antibody in vivo. The elimination of the interaction by, for example, introducing the mutation H435A results in an extremely short half-life, because the antibody is no longer protected from lysosomal degradation by FcRn recovery. In some preferred embodiments according to all aspects, the antibody according to the invention comprises the mutation H435A or has been engineered in other ways for reducing the half-life.

[0174] In contrast, antibodies containing the "YTE" mutation (M252Y / S254T / T256E) and / or equivalent mutations such as the "LS" mutation (M428L / N434S) have been shown to significantly extend half-life in preclinical species as well as in humans through more efficient recycling from endosomes (Dall'Acqua, William F., et al. "Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences." The Journal of Immunology 169.9 (2002): 5171-5180.; Zalevsky, Jonathan, et al. "Enhanced antibody half-life improves in vivo activity." Nature biotechnology 28.2 (2010): 157-159). In some preferred embodiments according to all aspects, the antibody according to the present invention comprises a YTE mutation (M252Y / S254T / T256E) and / or an equivalent mutation such as LS (M428L / N434S) or has been otherwise engineered for improving half-life. Suitable Fc engineering methods for extending half-life can be found in Haraya, Kenta, Tatsuhiko Tachibana, and Tomoyuki Igawa. "Improvement of pharmacokinetic properties of therapeutic antibodies by antibody engineering." Drug metabolism and pharmacokinetics 34.1 (2019): 25-41. and / or Lee, Chang-Han, et al. "An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence." Nature communications 10.1 (2019): 1-11, both of which are incorporated herein by reference.

[0175] "Non-fucosylated" antibodies are antibodies that are engineered so that the oligosaccharides in the Fc region of the antibody do not have any fucose units. Glycosylation of antibodies can change their function. For example, if the glycosylation at N297 in the CH2 domain of IgG is completely eliminated, the binding to FcγR is lost. However, adjusting the specific carbohydrate composition at N297 can have the opposite effect and enhance the ADCC activity of the antibody. In short, the affinity of the antibody to the activated FcγR depends on the composition of the oligosaccharides connected to N297 N. There are 32 different possible oligosaccharide combinations at this site. Naturally occurring human IgG and those produced by hybridomas or other common expression systems are usually composed of N-acetylglucosamine (GlcNAc) and three mannose residues that form the core carbohydrate. This core is connected to two additional GlcNAc groups to form biantennary branches. Adding galactose at each branch and adding sialic acid to the ends of these galactose molecules can occur. Fucose is usually part of the core GlcNAc. The fucose hinders the interaction of the antibody with FcγRIIIA by steric hindrance. Therefore, eliminating the fucose molecule while maintaining other forms of glycosylation at this site increases the binding of the antibody to the activated FcγRs and enhances its ability to induce ADCC and / or ADCP (Almagro, Juan C., et al. "Progress and challenges in the design and clinical development of antibodies for cancer therapy." Frontiers in immunology 8 (2018): 1751). The method for preparing a fucose-less antibody includes growing in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme necessary for fucosylation of polypeptides. Non-fucosylated antibodies are preferred in the present invention.

[0176] "Antibody-dependent cellular cytotoxicity" ("ADCC"), also known as "antibody-dependent cell-mediated cytotoxicity", is a cell-mediated immune defense mechanism in which immune cells actively lyse target cells whose membrane-surface antigens have been bound by specific antibodies. ADCC is mediated via the interaction of antibodies or fragments with FcγRIIIa. In humans, FcγRIII exists in two different forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). While FcγRIIIa is expressed as a transmembrane receptor on monocytes, neutrophils, mast cells, macrophages and natural killer cells, FcγRIIIb is only expressed on neutrophils. These receptors bind to the Fc portion of IgG antibodies and then activate antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

[0177] Different assay systems for determining ADCC induction in human subjects have been described in the literature and are suitable for characterizing the subject matter disclosed herein. For example, Yao-Te Hsieh et al. have studied different ADCC assay systems, namely assays based on (i) natural killer cells (FcγRIIIA+primary NK) from human donors, (ii) FcγRIIIA engineered NK-92 cells, and (iii) FcγRIIIA / NFAT-RE / Luc2 engineered Jurkat T cells (Hsieh, Yao-Te, et al. "Characterization of FcγRIIIA effector cells used in in vitro ADCC bioassay: comparison of primary NK cells with engineered NK-92and Jurkat T cells." Journal of Immunological Methods 441 (2017): 56-66, which is incorporated herein in its entirety; in particular, reference is made to the method descriptions of these assays). In short, all three effector cell systems differentially express FcγRIIIA and provide dose-dependent ADCC pathway activity, but only primary NK and engineered NK92 cells are able to induce ADCC-mediated cell lysis. For functional assessment of ADCC activity, primary NK or NK-92 (V-158) cells therefore better reflect the physiologically relevant ADCC mechanism of action. As an engineered cell line, NK-92 cells may show higher reproducibility than primary NK cells and are therefore the preferred assay system for determining ADCC responses in human subjects, such as in doubtful cases.

[0178] The antibody or antigen binding fragment that induces ADCC is an antibody that can induce a large amount of target cell lysis in the presence of NK effector cells. Preferably, ADCC induction results in at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% target cell lysis.

[0179] "Antibody-dependent cellular phagocytosis" ("ADCP") is a mechanism by which antibody-opsonized target cells activate FcγRs on the surface of macrophages to induce phagocytosis, leading to internalization and degradation of the target cells. For ADCP, binding to macrophages as effector cells typically occurs via the interaction of the antibody FC portion with FcγRIIa (CD32a) expressed by macrophages.

[0180] The antibody or antigen-binding fragment that induces ADCP is an antibody that can induce a large amount of phagocytosis of target cells in the presence of NK macrophages. Preferably, ADCP induction results in phagocytosis of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of target cells.

[0181] "Complement dependent cytotoxicity" ("CDC") is an effector function of IgG and IgM antibodies. When they bind to surface antigens on target cells (e.g., bacterial or virally infected cells), the classical complement pathway is triggered by binding of the protein C1q to these antibodies, leading to the formation of a membrane attack complex (MAC) and target cell lysis. The complement system is effectively activated by human IgG1, IgG3 and IgM antibodies, weakly activated by IgG2 antibodies, but not by IgG4 antibodies. It is one mechanism of action by which therapeutic antibodies - and also a specific embodiment of the antibodies according to the invention - can achieve an anti-tumor effect. Several laboratory methods exist for determining the efficacy of CDC and are known in the art.

[0182] The antibody or antigen binding fragment that induces CDC is an antibody that can cause a large amount of membrane attack complex formation and lysis of target cells. Preferably, CDC induction will result in at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% target cell lysis.

[0183] An antibody comprising an Fc region may or may not comprise modifications that promote association of the first and second subunits of the Fc domain.

[0184] Most preferably, induction of ADCC and ADCP results in at least 50% depletion of Tregs.

[0185] As used herein, the "fragment" of an antibody needs to substantially retain the affinity required for a full-length antibody. Therefore, a suitable fragment of an anti-human CCR8 antibody will retain the ability to bind to a target chemokine receptor, such as the ability to bind to a human CCR8 receptor. An antibody fragment comprises a portion of a full-length antibody, typically an antigen binding region or its variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, single-chain antibody molecules, bispecific antibodies and domain antibodies, see Holt, Lucy J., et al. "Domain antibodies: proteins for therapy." Trends in biotechnology 21.11 (2003): 484-490.

[0186] A "Fab fragment" contains the constant domain of the light chain and the first constant domain (CH2) of the heavy chain.

[0187] A "Fab' fragment" differs from a Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH2 domain including one or more cysteines from the antibody hinge region.

[0188] "F(ab') fragments" are produced by cleavage of disulfide bonds at the hinge cysteine ​​of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more quickly from the circulation of animals, and may have less nonspecific tissue binding than intact antibodies, see, for example, Wahl, Richard L., Charles W. Parker, and Gordon W. Philpott. "Improved radioimaging and tumor localization with monoclonal F(ab')2." Journal of nuclear medicine: official publication, Society of Nuclear Medicine 24.4 (1983): 316-325.

[0189] "Fv fragment" is the smallest fragment of an antibody containing a complete target recognition and binding site. This region consists of a dimer (VH / VL dimer) of a heavy chain and a light chain variable domain in tight non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH / VL dimer. Typically, six CDRs confer antigen binding specificity to antibodies. However, in some cases, even a single variable domain (or half of an Fv containing only three CDRs specific to a target) can have the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0190] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0191] "Single domain antibodies" are composed of a single VH or VL domain that exhibits sufficient affinity for a target. In a specific embodiment, a single domain antibody is a camelized antibody, see, e.g., Riechmann, Lutz, and Serge Muyldermans. "Single domain antibodies: comparison of camel VH and camelised human VH domains." Journal of immunological methods 231.1-2 (1999): 25-38.

[0192] "Minibodies" are a form of antibody that has a smaller molecular weight than full-length antibodies while maintaining bivalent binding properties to antigens. For example, minibodies can be bivalent homodimers in which each monomer has a single-chain variable fragment (scFv) connected to a human IgG1 CH3 domain via a modified IgG1 hinge sequence. Due to their smaller size, minibodies are cleared from the system more quickly and have enhanced permeability when targeting tumor tissue. With their powerful targeting capabilities combined with rapid clearance, minibodies are beneficial for diagnostic imaging and delivery of cytotoxic / radioactive payloads, for which extended circulation time may result in unfavorable patient dosing or dosimetry.

[0193] "Zr-89 labeled anti-CD8 miniantibodies" are miniantibodies that specifically bind to CD8 and are further labeled with Zr-89. Preferably, the Zr-89 labeled anti-CD8 miniantibodies bind to human CD8 glycoprotein with an EC50 of <1 nM. For example, the miniantibodies can be conjugated to the positron emitting radionuclide "zirconium-89" (89Zr; Tm 78.4 hours) via deferoxamine (Df) and radiolabeled. According to the most preferred embodiment, the Zr-89 labeled anti-CD8 miniantibody is the Zr-89 labeled anti-CD8 miniantibody described in U.S. Application No. 17 / 280,137. "Bispecific antibodies" are monoclonal antibodies that have binding specificity for at least two different epitopes on the same or different antigens. In the present disclosure, one of the binding specificities may be directed to a target chemokine receptor such as CCR8, and the other may be directed to any other antigen, such as but not limited to a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacteria-derived protein, or a bacterial surface protein. The bispecific antibody constructs according to the present invention also encompass multispecific antibody constructs comprising multiple binding domains / binding sites, such as trispecific antibody constructs, wherein the construct comprises three binding domains.

[0194] "Derivatized antibodies" are usually modified by glycosylation, acetylation, pegylation, phosphorylation, sulfation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, and bonding to cellular ligands or other proteins. Any of a variety of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, derivatives can contain one or more non-natural amino acids, for example using ambrx technology, see, for example, Wolfson, Wendy. "Amber codon flashing ambrx augments proteins with unnatural amino acids." Chemistry & biology 13.10 (2006): 1011-1012. The antibodies according to the present invention can be derivatized, for example, glycosylated or sulfated.

[0195] "Monoclonal antibody" is a substantially homogeneous population of antibodies that bind a specific antigen. Monoclonal immunoglobulins can be obtained by methods well known to those skilled in the art (see, e.g., Georges and Cesar Milstein. "Continuous cultures of fused cells secreting antibody of predefined specificity." Nature 256.5517 (1975): 495-497 and U.S. Patent No. 4,376,110). Immunoglobulins or immunoglobulin fragments with specific binding affinity can be separated, enriched or purified from prokaryotes or eukaryotes. Conventional methods known to those skilled in the art enable the production of immunoglobulins or immunoglobulin fragments and proteinaceous binding molecules with immunoglobulin-like functions in prokaryotes and eukaryotes. Antibodies according to the present invention are preferably monoclonal.

[0196] "Humanized antibodies" contain CDR regions derived from non-human species, such as mice, which are, for example, transplanted into V regions derived from human sequences together with any necessary framework back mutations. Therefore, in most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable regions are replaced by residues from the hypervariable regions of non-human species (donor antibodies) such as mice, rats, rabbits or non-human primates with desired specificity, affinity and capacity. See, for example, U.S. Patent Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205, each of which is incorporated herein by reference. In some cases, framework residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues not found in recipient antibodies or donor antibodies. These modifications are made in order to further improve antibody performance (e.g., to obtain desired affinity). Typically, a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions are framework regions of human immunoglobulin sequences. A humanized antibody optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones, Peter T., et al. "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321. 6069 (1986): 522-525.; Riechmann, Lutz, et al. "Reshaping human antibodies for therapy." Nature 332. 6162 (1988): 323-327.; and Presta, Leonard G. "Antibody engineering." Current Opinion in Structural Biology 2.4 (1992): 593-596, each of which is incorporated herein by reference.

[0197] Fully human antibodies (human antibodies) comprise human-derived CDRs, i.e. CDRs of human origin. Preferably, a fully human antibody according to the present invention is an antibody having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity with the closest human VH germline gene (e.g., sequences taken from the recommended list and analyzed in IMGT / Domain-gap-align). As accepted by the commonly used nomenclature system (e.g., the INN species subsystem) in effect as of 2017, a fully human antibody may contain a small amount of germline deviation compared to the closest human germline reference determined based on the IMGT database (http: / / www.imgt.org, November 29, 2019). For example, a fully human antibody according to the invention may comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14 or 15 germline deviations in the CDRs compared to the closest human germline reference. Fully human antibodies can be developed from human-derived B cells by combining cloning techniques with cell enrichment or immortalization steps.However, most fully human antibodies in clinical use are isolated from immunized mice transgenic for the human IgG locus or from complex combinatorial libraries by phage display (Brüggemann, Marianne, et al. "Human antibody production in transgenic animals." Archivum immunologiae ettherapiae experimentalis 63.2 (2015): 101-108.; Carter, Paul J. "Potent antibody therapeutics by design." Nature reviews immunology 6.5 (2006): 343-357.; Frenzel, André, Thomas Schirrmann, and Michael Hust. "Phage display-derived human antibodies in clinical development and therapy." MAbs. Vol. 8. No. 7. Taylor & Francis, 2016.; Nelson, Aaron L., Eugen Dhimolea, and Janice M. Reichert. "Development trends for human monoclonal antibody therapeutics." Nature reviews drug discovery 9.10(2010):767-774).

[0198] Several techniques can be used to produce fully human antibodies or antibodies containing human-derived CDRs (see WO2008112640). Cambridge Antibody Technologies (CAT) and Dyax have obtained antibody cDNA sequences from peripheral B cells obtained from immunized people, and designed phage display libraries for identifying human variable region sequences with specific specificity. In short, the antibody variable region sequence is fused with the gene III or gene VIII structure of M13 phage. These antibody variable region sequences are expressed as Fab or single-chain Fv (scFv) structures at the tip of the phage carrying the corresponding sequence. Using different degrees of antigen binding conditions (stringency) through multiple rounds of panning processes, phages expressing Fab or scFv structures specific to the target antigen can be selected and isolated. Then, the antibody variable region cDNA sequences of the selected phage can be elucidated using standard sequencing procedures. Then, these sequences can be used to reconstruct complete antibodies with desired isotypes using established antibody engineering techniques. The antibodies constructed according to this method are considered to be fully human antibodies (including CDRs). In order to improve the immunoreactivity (antigen binding affinity and specificity) of the selected antibody, an in vitro maturation process can be introduced, including the combined association of different heavy and light chains, deletion / addition / mutation at the CDR3 of the heavy and light chains (to simulate VJ and VDJ recombination), and random mutation (to simulate somatic hypermutation). An example of a "fully human" antibody produced by this method is the anti-tumor necrosis factor alpha antibody, Humira (adalimumab).

[0199] "Anti-drug antibodies" (ADA) are antibodies that bind to therapeutic antibodies and are produced by a subject or patient's immune response to the therapeutic antibodies. ADA can inactivate the therapeutic effect of the treatment and can potentially induce adverse effects.

[0200] The term "quantitative" means estimating or measuring the amount of a molecule, such as an antibody, at least in a semi-quantitative manner.

[0201] The term "polynucleotide" refers to a recombinant or synthetically produced polymerized deoxyribonucleotide or its analog, or a modified polynucleotide. The term encompasses double-stranded and single-stranded DNA or RNA. The polynucleotide can be integrated into, for example, a minicircle, a plasmid, a cosmid, a minichromosome or an artificial chromosome. The polynucleotide can be isolated or integrated into another nucleic acid molecule, for example, incorporated into an expression vector or a chromosome of a eukaryotic host cell.

[0202] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating a nucleic acid molecule connected thereto. The term further includes plasmids (non-viral) and viral vectors. Certain vectors are capable of directing the expression of nucleic acids or polynucleotides operably connected thereto. Such vectors are referred to herein as "expression vectors". Expression vectors for use by eukaryotic organisms can be constructed by inserting a polynucleotide sequence encoding at least one target protein (POI) into a suitable vector backbone. The vector backbone may include necessary elements to ensure the maintenance of the vector and to provide amplification in the host if necessary. For viral vectors, such as lentiviral vectors or retroviral vectors, more viral-specific elements such as structural elements or other elements may be required, and these elements are well known in the art. These elements may be provided, for example, in cis (on the same plasmid) or in trans (on different plasmids). Viral vectors may require helper viruses or packaging lines for large-scale transfection. The vector may contain more elements, such as enhancer elements (e.g., viral, eukaryotic), introns, and viral origins of plastid replication for replication in mammalian cells. According to the present invention, the expression vector generally has a promoter sequence that drives the expression of the POI. Expression of the POI and / or selectable marker protein can be constitutive or regulated (e.g., inducible by addition or removal of a small molecule inducer). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high-level expression of the POI in mammalian cells, such as regulatory elements, promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter Ad LP) or polyoma virus. For further description of viral regulatory elements and sequences thereof, see, e.g., US 5,168,062, US 4,510,245 and US 4,968,615.

[0203] As used herein, the term "linker" or "spacer" refers to any molecule capable of directly topologically linking two moieties. The moieties may be, among other things, polypeptides, proteins, antibodies, antibody fragments, cytotoxic moieties, binding moieties, moieties for detection such as fluorophores, moieties for fixation or recovery such as beads or magnetic beads, reactive moieties, or any other molecule. The two moieties may be of the same type or of different types. The linker may be part of the conjugate and may even contribute to its function. For example, for a conjugate comprising a polypeptide and biotin, there is about 1 / 4 N of the carboxyl group of the biotin and the first large amino acid of the peptide. The spacer of (~5 atoms) allows biotin to reach the (strept)avidin binding pocket. Various linkers are known in the art and can be selected based on the moieties that should be connected. Linker lengths are generally between 4 atoms and more than 200 atoms. Linkers with a length of more than 60 atoms generally comprise a population of compounds with an average length.

[0204] "Polypeptide linkers" can be connected by amide bonds or any other functional residues. The polypeptide linker can be connected to the N-terminus or C-terminus of the polypeptide, or can be connected via a reactive functional group or an amino acid side chain. The polypeptide can be coupled to, for example, biotin, proteins such as human serum albumin (HAS), carrier proteins such as keyhole limpet hemocyanin (KLH), ovalbumin (OVA) or bovine serum albumin (BSA), fluorescent dyes, short amino acid sequences such as Flag tags, HA tags, Myc tags or His tags, reactive tags such as maleimide, iodoacetamide, alkyl halides, 3-mercaptopropyl or 4-azidobutyric acid, or various other suitable parts. Suitable linkers, for example, non-limiting examples for conjugation of polypeptides include β-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)-acetic acid (AEA), 5-aminopentanoic acid (Ava), 6-aminohexanoic acid (Ahx), PEG2-spacer (8-amino-3,6-dioxaoctanoic acid), PEG3-spacer (12-amino-4,7,10-trioxadodecanoic acid), PEG4-spacer (15-amino-4,7,10,13-tetraoxapentadecanoic acid) and Ttds (trioxatridecanoic acid). In some cases, the linker can be derived from a reactive moiety, such as maleimide, iodoacetamide, alkyl halide, 3-mercaptopropyl or 4-azidobutyric acid. In some cases, the linker can include polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or a copolymer of polyethylene glycol or polypropylene glycol.

[0205] "Antibody linkers" are linkers that establish covalent bonds between different antibody moieties and include peptide linkers and non-proteinaceous polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol and polypropylene glycol.

[0206] "Treating" a disease in a subject or "treating" a subject having a disease means subjecting the subject to medical treatment, such as administration of a drug, such that at least one symptom of the disease is alleviated or its worsening is prevented.

[0207] The terms "prevent," "preventing," "preventing," and the like refer to reducing the probability of developing a disease, disorder or condition in a subject who does not have the disease, disorder or condition, but who is at risk of or susceptible to developing the disease, disorder or condition.

[0208] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount sufficient to achieve a specific biological result in a subject or to modulate or improve symptoms, or the time of symptom onset, in a subject, generally at least about 10%; generally at least about 20%, preferably at least about 30%, or preferably at least about 50%. The efficacy of using antibodies in cancer therapy can be evaluated based on changes in tumor burden. Tumor shrinkage (objective response) and the time to progression of the disease process are both important endpoints in cancer clinical trials. Standardized response criteria (known as RECIST (Response Evaluation Criteria in Solid Tumors)) were published in 2000. An updated version (RECIST 1.1) was published in 2009. The RECIST criteria are commonly used in clinical trials with objective response as the primary endpoint of the study, as well as in trials in which stable disease, tumor progression or progression time analysis are performed, because these outcome measures are based on an assessment of anatomical tumor burden and its changes during the trial. The effective amount for a particular subject may vary due to a variety of factors, such as the condition being treated, the overall health of the subject, the method, route and dose of administration, and the severity of side effects. When combined, the effective amount is the ratio of the components combined, and the effect is not limited to a single component alone.

[0209] If not defined otherwise, a Complete Response (CR) was defined as the disappearance of all target lesions. Any pathological lymph node (whether target or non-target) must achieve a reduction of <10 mm in the short axis. For a Partial Response (PR), the sum of the diameters of the target lesions must decrease by at least 30%, with the baseline sum as a reference. For Progressive Disease (PD), the sum of the diameters of the target lesions must increase by at least 20%, with the smallest sum on study as a reference (including the baseline sum if that was the smallest sum on study). In addition to a relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. In Stable Disease (SD), neither sufficient atrophy to qualify for PR nor sufficient increase to qualify for PD was observed, with the smallest sum of diameters on study as a reference.

[0210] Secondary outcome measures that can be used to determine the therapeutic benefit of the antibodies of the invention described herein include the following: "Objective Response Rate" (ORR) is defined as the percentage of subjects who achieve a complete response (CR) or a partial response (PR). "Progression Free Survival" (PFS) is defined as the time from the first date of administration of the antibody to disease progression or death (whichever occurs first). "Overall Survival" (OS) is defined as the length of time from the date of diagnosis or the start of disease treatment that the patient is diagnosed with the disease and is still alive. "Overall Response Duration" (Duration of Overall Response, DOR) is defined as the time from the participant's initial CR or PR to disease progression. "Depth of Response" (DpR) is defined as the percentage of tumor shrinkage observed at the maximum response point compared to the baseline tumor burden. The clinical endpoints of ORR and PFS can be determined based on the above-mentioned RECIST 1.1 criteria.

[0211] The aforementioned parameters for determining efficacy and benefit of a treatment must be adjusted when analyzing non-human subjects.

[0212] According to the present invention, typical "subjects" include human and non-human subjects. The subject can be a mammal, such as a mouse, rat, cat, dog, primate and / or human.

[0213] The term "patient" refers to a human subject suffering from a medical condition.

[0214] "Pharmaceutical compositions" (also referred to as "therapeutic formulations") of antibodies, fragments or conjugates can be prepared by mixing an antibody having the desired degree of purity with an optional physiologically acceptable carrier, excipient or stabilizer, for example, according to Remington's Pharmaceutical Sciences (18th edition; Mack Pub. Co.: Eaton, Pa., 1990), for example, in the form of a lyophilized dosage form or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as or polyethylene glycol (PEG).

[0215] "Host cell" is a cell that is used to receive, maintain, replicate and amplify the vector. Host cells can also be used to express polypeptides encoded by the vector, such as antibodies or fragments thereof. The nucleic acid contained in the vector is replicated when the host cell divides, thereby amplifying the nucleic acid. Preferred host cells are mammalian cells, such as CHO cells or HEK cells. Further preferred host cells are rat myeloma YB2 / 0 cells.

[0216] A "cell with endogenous target expression" is a cell that expresses the target protein at levels comparable to physiological or diseased conditions. Typically, cells that have been engineered for overexpression express the target protein at much higher levels.

[0217] As used herein, "lesion" refers to an area of ​​abnormal tissue. A lesion can be benign or malignant ("cancer lesion", also called "tumor lesion").

[0218] The terms "intra-tumoral," "intratumoral," "tumor-invasive," or "neoplastic" in the context of cells, structures, proteins, antibodies or markers refer to their localization within tumor tissue.

[0219] Cells that are "positive" or "+" for certain markers or proteins are cells characterized by expressing the marker or protein in large amounts. The expression of markers or proteins can be determined and quantified as known in the art, e.g. to define different cell populations. For the characterization of (immune) cell populations, marker expression can be determined by FACS or using any other technique described herein.

[0220] "Leukocytes" are immune cells that express CD45.

[0221] As used herein, "CD45+ cells" refer to all leukocytes. CD45 can be used as a marker to distinguish immune cells from non-immune cells. The term "lymphocyte" refers to all immature, mature, undifferentiated and differentiated white lymphocyte populations, including tissue-specific and specialized (specialized) species. By way of non-limiting example, it encompasses B cells, T cells, NKT cells and NK cells. In certain embodiments, lymphocytes include all B cell lineages, including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, Bl cells, B-2 cells and anergic AN1 / T3 cell populations.

[0222] "T cells" are immune cells expressing TCR α β, CD3 and CD8 or CD4. As used herein, the term includes naive T cells, CD4+T cells, CD8+T cells, regulatory T cells, memory T cells, activated T cells, anergic T cells, tolerant T cells, chimeric B cells and antigen-specific T cells, and other T cell populations known in the art. In certain embodiments, the presence of a T cell receptor (TCR) on the cell surface will distinguish T cells from other lymphocytes.

[0223] "CD8+T cells" (also known as "cytotoxic T cells", "TC", "cytotoxic T lymphocytes", "CTL", "T killer cells", "cytolytic T cells", "CD8+T cells" or "killer T cells") are T cells that express CD3, CD45 and CD8. CD8+T cells can kill cancer cells, infected cells (especially virus-infected) or otherwise damaged cells.

[0224] "CD4+T cells" (also known as "T helper cells", "Th cells") are immune cells that express CD3, CD4, and CD45. There are multiple subtypes of T helper cells, such as, but not limited to, Th1, Th2, and Th17. CD4+T cells help suppress or regulate the immune response. They are essential in B cell antibody class conversion, activation and growth of cytotoxic T cells, and maximizing the bactericidal activity of phagocytes such as macrophages.

[0225] As used herein, the term "Treg cells" (also referred to as "Tregs," "regulatory T cells," "T regulatory cells," "suppressive T cells") refers to immune cells that express CD3, CD4, CD45, and FoxP3, and further express high levels of CD25 and low levels of CD127. The identification of Treg cells can be performed as described elsewhere herein. Treg cells also typically express high levels of CTLA-4, GITR, and LAG-3. In the literature, Tregs have been further classified based on the memory marker CD45RO.

[0226] Under physiological conditions, Treg cells maintain immune tolerance. During an immune response, Treg cells stop T cell-mediated immunity and suppress autoreactive T cells that escape negative selection in the thymus. Treg cells can also suppress other types of immune cells, such as NK cells and B cells. Adaptive Treg cells (called Th3 or Tr1 cells) are thought to be generated during an immune response.

[0227] Treg cells further play an important role in immune escape by suppressing anti-tumor immunity, thereby providing an environment of immune tolerance. T cells that recognize cancer cells are often present in large numbers in tumors, but their cytotoxic functions are suppressed by nearby immunosuppressive cells. Tregs are abundant in many different cancers, highly enriched in the tumor microenvironment, and are well known for their role in tumor progression. "Activated Treg cells" express CD4, CD45, FoxP3, CD69, and CCR8, and further have high expression of CD25 and low expression of CD127. CD69 is a T cell activation marker.

[0228] "CCR8-positive regulatory T cells" or "CCR8+ regulatory T cells" are Tregs that express CCR8.

[0229] "CD4conv cells" are conventional CD4+, CD25- T cells.

[0230] "γδ T cells" are T cells that express a unique T cell receptor, TCRγδ, on their surface. γδ T cells also express CD3.

[0231] "B cells" are immune cells that express CD19, and mature B cells express CD20 and CD22. When activated by CD40, B cells undergo differentiation, in which somatic hypermutation and enhanced immunoglobulin class switching occur, producing mature B cells or plasma cells (capable of secreting antibodies). B cells participate in the humoral immunity of the adaptive immune system and are antigen presenting cells.

[0232] "Macrophages" are immune cells that express low CD14, high CD16, CD11b, CD68, CD163, and CD206. Macrophages engulf and digest cellular debris, foreign material, microorganisms, or cancer cells through phagocytosis. In addition to phagocytosis, macrophages also play an important role in innate immunity, and can also help initiate adaptive immunity by recruiting other immune cells. For example, macrophages are important as antigen presenters to T cells. Macrophages that promote inflammation are called M1 macrophages, while macrophages that reduce inflammation and promote tissue repair are called M2 macrophages.

[0233] As used herein, "M1 macrophages" are a subtype of macrophages that express ACOD 1. M1 macrophages have pro-inflammatory, bactericidal, and phagocytic functions.

[0234] As used herein, "M2 macrophages" are a subtype of macrophages that express MRC1 (CD206). M2 macrophages secrete anti-inflammatory interleukins, play a role in wound healing, and are essential for revascularization and epithelial regeneration. Tumor-associated macrophages are predominantly of the M2 phenotype and appear to actively promote tumor growth.

[0235] "Dendritic cells" (DC) are bone marrow-derived leukocytes and are the most effective antigen presenting cell type. DC is specialized for capturing and processing antigens, converting proteins into peptides that are presented on the major histocompatibility complex (MHC) molecules recognized by T cells. As defined herein, DC is characterized by the expression of CD1c, CD14, CD16, CD141, CD11c, and CD123. There are different dendritic cell subsets. In humans, DC1 is immunogenic, while DC2 cells are tolerogenic. Mature DC expresses CD83, while plasmacytoid DC expresses CD123.

[0236] "NK cells" (also called natural killer cells) are immune cells that express CD45, CD16, CD56, NKG2D, but are CD3 negative. NK cells do not require activation to kill cells that lack MHC class 1 "self" markers. NCR1 (also called CD335 or NKp46) is expressed on NK cells and NKT cell subtypes.

[0237] "Natural Killer T (NKT) cells" are a heterogeneous population of T cells that share properties of both T cells and natural killer cells.

[0238] "iNKT cells" (also called "invariant natural killer T cells") express the invariant αβTCR (Vα24-Jα18, CD24lo), CD44hi, NK1.1 (mouse), and NKG2D. The invariant TCR recognizes glycolipid antigens presented by the non-polymorphic MHC class I-like molecule CD1d. These cells can affect immune responses by rapidly producing large amounts of cytokines (i.e., IFNγ).

[0239] As known in the art, "effector cells" are immune cells that actively support an immune response after stimulation. As used herein, effector cells refer to immune cells that express Fcγ receptors and are therefore capable of mediating ADCC or ADCP. Non-limiting examples of effector cells are monocytes, neutrophils, mast cells, and preferably macrophages and natural killer cells.

[0240] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell surface receptor engineered to be expressed on immune effector cells and specifically binds to an antigen. CAR can be used as a therapy for adoptive cell transfer. Mononuclear cells are taken out from patients (blood, tumors or ascites) and modified so that they express receptors specific for a particular form of antigen. In certain embodiments, CAR is expressed with specificity for tumor-associated antigens. CAR may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. In some aspects, CAR comprises a fusion of a monoclonal antibody derived from a single-chain variable fragment (scFv), which is fused to the transmembrane and intracellular domains of CD3-ζ. The specificity of CAR design can be derived from a ligand (e.g., a peptide) of a receptor. In certain embodiments, CAR can be directed to target cancer by redirecting monocytes / macrophages expressing CARs specific for tumor-associated antigens.

[0241] Dosing regimens are abbreviations as known in the art, such as daily (QD), every 2 days (Q2D), or every 3 days (Q3D). Accordingly, "QW" means once a week, "Q2W" means once every two weeks, "Q3W" means once every three weeks, "Q4W" means once every four weeks, "Q5W" means once every five weeks, and "Q6W" means once every six weeks.

[0242] A "dosing cycle" or "treatment cycle" is a treatment period repeated according to a fixed schedule, followed by a rest period (no treatment). When this cycle is repeated multiple times according to a fixed schedule, it constitutes a course of treatment.

[0243] In pharmacology, "trough concentration," abbreviated "Ctrough," is the concentration achieved by a drug immediately before administration of the next dose.

[0244] "Cytokine release syndrome" (CRS) and when severe "cytokine storm" is a supraphysiological response that can occur in response to any immunotherapy and is a sequela of immune system activation associated with infusion reactions. The binding of mAbs results in the activation or engagement of endogenous or infused T cells and / or other immune effector cells, resulting in the rapid release of inflammatory cytokines from target immune cells into the circulation. CRS usually occurs within hours to days after the infusion of monoclonal antibodies. The incidence of CRS is relatively low for monoclonal antibodies, but for chimeric antigen receptor (CAR)-T and T cell engagers, the incidence of CRS is high, varying between 17% and 94%.

[0245] An intravenous line or "IV line" is a tube or cannula that can be used for intravenous infusions.

[0246] "PET scanning" is a diagnostic technique that can be used to observe the function and metabolism of human organs and tissues at the molecular level. For PET scanning, positron radiopharmaceuticals (e.g., 18F-FDG) can be injected into the human body. If FDG is used, because the metabolism of fluorodeoxyglucose (FDG) is similar to that of glucose, FDG will accumulate in cells that digest glucose. The uptake of radiopharmaceuticals by fast-growing tumor tissues is different. Because the positrons emitted by the decay of 18F annihilate with the electrons in the tissue, two γ-photons with the same energy are generated in opposite directions. For PET scanning, an array of detectors around the human body can detect two photons using coincidence measurement technology and determine the position information of the positrons. Then, a tomographic image of the positrons in the human body can be constructed by processing the position information using image reconstruction software. In some cases, immuno-PET can be used, in which a marker (e.g., 18F) is connected or associated with an antigen-binding construct. In such an embodiment, the distribution or abundance of the antigen-binding construct can be monitored, which will depend on the binding properties and distribution properties of the antigen-binding construct. For example, if a CD8-directed miniantibody or a Zr-89-labeled anti-CD8 miniantibody is used, a PET scan can be used to monitor the distribution and / or abundance of the CD8-directed miniantibody or the Zr-89-labeled anti-CD8 miniantibody, and thus monitor the distribution and / or abundance of CD8 molecules in the subject's system. PET systems are known in the art and include, for example, U.S. Patent Publication Nos. 20170357015, 20170153337, 20150196266, 20150087974, 20120318988, and 20090159804, each of which is incorporated herein by reference in its entirety for its description of PET, PET scanning, and its uses.

[0247] "Standardized uptake value", also called "standard uptake value" or "SUV" is a term in the field of nuclear medicine and is used in positron emission tomography (PET) and modern calibrated single photon emission tomography (SPECT) imaging for semi-quantitative analysis.

[0248] Computed tomography or "CT scan" (formerly known as computerized axial tomography or CAT scan) is a medical imaging technique used to obtain detailed internal images of the body. A CT scanner uses a rotating X-ray tube and an array of detectors placed in a gantry to measure the attenuation of X-rays by different tissues in the body. Multiple X-ray measurements taken from different angles are then processed on a computer using a tomographic reconstruction algorithm to produce tomographic (cross-sectional) images (virtual "slices") of the body.

[0249] The term "distribution", in the context of monitoring, detecting, comparing or observing the distribution of Zr-89-labeled anti-CD8 miniantibodies that have been administered to a subject, means a visual or mathematical image of the distribution of the Zr-89-labeled anti-CD8 miniantibodies relative to a full body or partial body scan of the subject, which image can be represented as a planar image (2-dimensional) or a computer-assisted three-dimensional representation (including holograms) and is in a format useful for an operator or clinician to observe the distribution of the Zr-89-labeled anti-CD8 miniantibodies at an independent tissue level and an independent tumor level.

[0250] "Antihistamines" are a class of pharmaceutical compounds designed to relieve various allergic reactions and symptoms by blocking the effects of histamine in the body. Examples include diphenhydramine, loratadine, cetirizine, fexofenadine, desloratadine, and levocetirizine.

[0251] "Diphenhydramine" (DPH) is an antihistamine and sedative known in the art, used primarily to treat symptoms of allergies, insomnia, and colds. It is also less commonly used for tremors and nausea in Parkinson's disease.

[0252] "Paracetamol" (acetaminophen[a] or parahydroxyacetanilide) is a non-opioid analgesic and antipyretic known in the art that is used to treat fever and mild to moderate pain. Common trade names include Tylenol and Panadol.

[0253] "Corticosteroids" are a class of synthetic or naturally occurring steroid hormones produced by the adrenal cortex that have potent anti-inflammatory, immunosuppressive and metabolic effects. Examples include prednisone, dexamethasone or methylprednisolone.

[0254] "Dexamethasone" is a glucocorticoid drug known in the art, used in particular to treat rheumatic problems, various skin diseases, severe allergies, asthma, chronic obstructive pulmonary disease, croup or brain swelling.

[0255] Implementation

[0256] Aspect 1

[0257] Medication regime

[0258] According to a first aspect of the present invention, there is provided an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment, comprising intravenously administering to a patient in need thereof an anti-CCR8 antibody in a total amount of:

[0259] a. 1 to 250 mg once a week, or

[0260] b. 16 to 1500 mg once every three weeks. More specifically, and as detailed in, for example, Example 17, an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment is provided, the method comprising intravenously administering to a patient in need thereof an anti-CCR8 antibody in a total amount of:

[0261] a. About 1, 2.5, 3, 10, 30, 50, 100, 125 or 250 mg once a week, or

[0262] b. About 16, 450, 500, 750, 1000, or 1500 mg once every three weeks.

[0263] For example, an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment is provided, comprising intravenously administering to a patient in need thereof a total amount of about 1, 2.5, 3, 10, 30, 50, 100, 125 or 250 mg once a week, preferably 10, 30, 50, 100, 125 or 250 mg of an anti-CCR8 antibody once a week. In one embodiment, the total amount of anti-CCR8 antibody is about 1 mg once a week. In another embodiment, the total amount of anti-CCR8 antibody is about 2.5 mg once a week. In another embodiment, the total amount of anti-CCR8 antibody is about 10 mg once a week. In a preferred embodiment, the total amount of anti-CCR8 antibody is about 30 mg once a week. In a further preferred embodiment, the total amount of anti-CCR8 antibody is about 50 mg once a week. In a further preferred embodiment, the total amount of anti-CCR8 antibody is about 100 mg once a week. In another preferred embodiment, the total amount of anti-CCR8 antibody is about 125 mg once a week. In a highly preferred embodiment, the total amount of anti-CCR8 antibody is about 250 mg once per week.

[0264] For example, provided is an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method, comprising intravenously administering to a patient in need thereof a total amount of about 500, 750, 1000 or 1500 mg of the anti-CCR8 antibody once every three weeks.

[0265] In a highly preferred embodiment, the total amount of anti-CCR8 antibody is about 500 mg once every three weeks. In another highly preferred embodiment, the total amount of anti-CCR8 antibody is about 750 mg once every three weeks. In a highly preferred embodiment, the total amount of anti-CCR8 antibody is about 1000 mg once every three weeks. In another preferred embodiment, the total amount of anti-CCR8 antibody is about 1500 mg once every three weeks.

[0266] For the anti-CCR8 antibody TPP-23411, which induces a large amount of ADCC and ADCP but is characterized by a relatively low half-life, the pharmacologically active / effective dose range for a 70 kg patient is 2.7 mg to 75 mg on a QW schedule and 16 mg to 450 mg on a Q3W schedule, see Example 16. Data demonstrating a successful mode of action are shown, for example, in Example 24, see Figure 7 , Figure 8 , Fig. 9 and Fig.10 .

[0267] Based on these findings, in a most preferred embodiment, an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method is provided, wherein the therapeutic method comprises intravenously administering a total amount of 2.7 mg to 75 mg of the anti-CCR8 antibody to a patient in need thereof once a week.

[0268] The proposed medical use for the QW dosing schedule is superior because the anti-CCR8 antibodies have pharmacologically relevant plasma exposure levels and also because medical use allows plasma C trough concentrations of the anti-CCR8 antibodies to be above the estimated EC80 value for CCR8+ cell killing, which the inventors derived from in vitro studies.

[0269] In another most preferred embodiment, an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment is provided, wherein the method comprises intravenously administering a total amount of 16 mg to 450 mg of the anti-CCR8 antibody to a patient in need thereof once every three weeks.

[0270] Medical use with a Q3W dosing schedule starts at a higher dose, but is still advantageous because it can be administered less frequently while still achieving the desired plasma exposure during the dosing interval to produce the desired pharmacological response (CCR8+Treg killing). The proposed Q3W dosing schedule also provides ease of dosing and coordination with other drug infusions.

[0271] As will be appreciated by those skilled in the art, the total amounts in the embodiments described herein are designed for patients with an average weight of 70 kg and can be adjusted proportionally based on the actual weight of the patient, ie, by using the appropriate mg / kg.

[0272] When referring to an anti-CCR8 antibody, the antibody is preferably TPP-23411, most preferably afucosylated TPP-23411.

[0273] In a most preferred embodiment, the anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to this aspect is

[0274] a. comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID Nos.: 2, 3, 4, 6, 7 and 8, and / or

[0275] b. comprising a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5, and / or

[0276] c. comprising a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17 and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0277] However, the anti-CCR8 antibody may also be an alternative anti-human CCR8 antibody, such as an antibody described herein, which induces ADCC and ADCP and has a half-life comparable to TPP-23411, i.e., an anti-human CCR8 antibody with a short half-life compared to other antibodies of the same IgG class. For example, the anti-human CCR8 antibody described herein may be characterized by a half-life in humans of <14 days, preferably <12 days, more preferably <10 days, and most preferably <7 days, such as 0-120 hours. Preferably, the anti-human CCR8 antibody is characterized by a half-life of <14 days, more preferably <10 days in humans, and most preferably <7 days in humans.

[0278] Preferably, the anti-CCR8 antibody is a human IgG1 antibody.

[0279] Preferably, the anti-CCR8 antibody is (further) a low internalizing antibody or a non-internalizing antibody.

[0280] In a preferred embodiment, the anti-CCR8 antibody is characterized in that the dissociation constant (KD) for binding to CHO cells transfected with human CCR8 is of the same order of magnitude as the KD of TPP-23411. As will be appreciated by those skilled in the art, the coefficient of difference in magnitude between the two values ​​is 10. In a most preferred embodiment according to this aspect, the anti-CCR8 antibody is

[0281] a. characterized in that the dissociation constant (KD) for binding to CHO cells transfected with human CCR8 is of the same order of magnitude as the KD for binding to CHO cells transfected with human CCR8 by TPP-23411,

[0282] b. Antibodies that induce ADCC and ADCP

[0283] o Preferably, wherein the antibody binds to human Fcγ receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) that is in the same order of magnitude as TPP-23411 binding to human Fcγ receptor IIIA variant V176 (CD16a), and

[0284] o Preferably, wherein the antibody binds to human FcγRIIA (CD32a) with a dissociation constant (KD) that is in the same order of magnitude as TPP-23411 binding to human FcγRIIA (CD32a);

[0285] o Preferably, wherein the antibody is afucosylated; and

[0286] c. wherein the antibody is characterized by a half-life in humans of <14 days, preferably <10 days, most preferably <7 days.

[0287] For preparation of intravenous infusion, (a) the desired volume of anti-CCR8 antibody solution can be removed from the vial and transferred to an intravenous (IV) bag containing 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP. The diluted solution can be mixed by gentle inversion without shaking. The final concentration of the diluted solution can be, for example, between 1 mg / mL and 10 mg / mL.

[0288] Administration of the anti-CCR8 antibody (e.g., in a diluted solution) can be performed intravenously within 15 to 120 minutes, preferably within 30 to 60 minutes, and most preferably within 30, 45, 60, or 75 minutes. Administration of the diluted solution can be performed through an intravenous line, e.g., containing a sterile, non-pyrogenic, low protein binding 0.2 micron to 5 micron in-line or add-on filter. To account for differences in infusion pumps at different sites, a window between -5 min and +10 min is allowed (i.e., an infusion time of 30 min [-5 min / +10 min]).

[0289] The medical use according to the first aspect may further comprise intravenously administering to a patient in need thereof an anti-PD-(L)1 antibody in a total amount of:

[0290] i. about 200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0291] ii. about 400 mg once every six weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0292] iii. about 240 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0293] iv. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0294] v. about 480 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0295] vi. about 840 mg every two weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or

[0296] vii. about 1200 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is atezolizumab, or

[0297] viii. about 1680 mg every four weeks, preferably wherein the anti-PD-(L)1 antibody is ateliizumab, or

[0298] ix. about 360 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is sepalizumab, or

[0299] x. about 3 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is toripalimab, or

[0300] xi. about 10 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab, or

[0301] xii. About 1500 mg / kg every 3 weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab.

[0302] For example, preferably, the medical use according to the first aspect comprises intravenously administering to a patient in need thereof a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks of an anti-PD-(L)1 antibody, wherein the anti-PD-(L)1 antibody is pembrolizumab.

[0303] For example, preferably, the medical use according to the first aspect comprises intravenously administering to a patient in need thereof a total amount of about 240 mg once every two weeks, or about 360 mg once every three weeks, or about 480 mg once every four weeks of an anti-PD-(L)1 antibody, wherein the anti-PD-(L)1 antibody is nivolumab.

[0304] For example, preferably, the medical use according to the first aspect comprises intravenously administering to a patient in need thereof a total amount of about 840 mg every two weeks, or about 1200 mg every three weeks, or about 1680 mg every four weeks of an anti-PD-(L)1 antibody, wherein the anti-PD-(L)1 antibody is atezolizumab.

[0305] In another example, the medical use according to the first aspect comprises intravenously administering to a patient in need thereof a total amount of about 360 mg of an anti-PD-(L)1 antibody every three weeks, wherein the anti-PD-(L)1 antibody is sepalizumab.

[0306] In another example, the medical use according to the first aspect comprises intravenously administering to a patient in need thereof a total amount of about 3 mg / kg every two weeks of an anti-PD-(L)1 antibody, wherein the anti-PD-(L)1 antibody is toripalimab.

[0307] In another example, the medical use according to the first aspect comprises intravenously administering to a patient in need thereof a total amount of about 10 mg / kg every two weeks, or about 1500 mg every three weeks of an anti-PD-(L)1 antibody, wherein the anti-PD-(L)1 antibody is durvalumab.

[0308] In an alternative, the medical use according to the first aspect may comprise intravenously administering to a patient in need thereof an anti-PD-(L)1 antibody in a total amount of:

[0309] a. About 200 mg once every three weeks, or

[0310] b. About 480 mg once every four weeks, or

[0311] c. About 480 mg once every six weeks.

[0312] In this alternative, the anti-PD-(L)1 antibody is pembrolizumab, nivolumab, atezolizumab, avelumab, or durvalumab.

[0313] The use of a fixed dose of anti-PD-(L)1 antibody reduces dosing complexity and leads to a reduced likelihood of dosing errors. Although administration in a different order or even in a corresponding setting is feasible, the anti-PD-(L)1 antibody is preferably administered after the anti-CCR8 antibody.

[0314] In the most preferred embodiment of the present invention, an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method is provided.

[0315] a. comprising intravenous administration to a patient in need thereof in a total amount of 2.7 mg to 75 once a week

[0316] mg of anti-CCR8 antibody,

[0317] b. Preferably further comprising intravenously administering to the patient a total amount of an anti-PD-(L)1 antibody of

[0318] i. about 200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0319] ii. about 400 mg once every six weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0320] iii. about 240 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0321] iv. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0322] v. about 480 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0323] vi. about 840 mg every two weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or

[0324] vii. about 1200 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is atezolizumab, or

[0325] viii. about 1680 mg every four weeks, preferably wherein the anti-PD-(L)1 antibody is ateliizumab, or

[0326] ix. about 360 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is sepalizumab, or

[0327] x. about 3 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is toripalimab, or

[0328] xi. about 10 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab, or

[0329] xii. About 1500 mg / kg every 3 weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab.

[0330] In another most preferred embodiment, provided is an anti-human CCR8 antibody having DCC activity and DCP activity for use in a method of treatment.

[0331] a. comprising intravenously administering to a patient in need thereof a total amount of 16 mg to 450 mg of an anti-CCR8 antibody once every three weeks,

[0332] b. Preferably further comprising intravenously administering to the patient a total amount of an anti-PD-(L)1 antibody of

[0333] i. about 200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0334] ii. about 400 mg once every six weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or

[0335] iii. about 240 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0336] iv. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0337] v. about 480 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or

[0338] vi. about 840 mg every two weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or

[0339] vii. about 1200 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is atezolizumab, or

[0340] viii. about 1680 mg every four weeks, preferably wherein the anti-PD-(L)1 antibody is ateliizumab, or

[0341] ix. about 360 mg every three weeks, preferably wherein the anti-PD-(L)1 antibody is sepalizumab, or

[0342] x. about 3 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is toripalimab, or

[0343] xi. about 10 mg / kg every two weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab, or

[0344] xii. About 1500 mg / kg every 3 weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab.

[0345] For preparation of intravenous infusion, the desired amount of anti-PD-(L)1 antibody solution can be removed from the vial and transferred to an intravenous (IV) bag containing 0.9% Sodium Chloride Injection, USP or 5% Dextrose Injection, USP. The diluted solution can be mixed by gentle inversion without shaking. The final concentration of the diluted solution can be, for example, between 1 mg / mL and 10 mg / mL.

[0346] For example, intravenous administration of an anti-PD-(L)1 antibody can be performed as a 15 to 60 minute intravenous infusion and preferably a 30 minute intravenous infusion.

[0347] Administration of anti-PD-(L)1 antibodies can be performed through an intravenous line, for example, containing a sterile, non-pyrogenic, low protein binding 0.2 micron to 5 micron in-line or add-on filter. Taking into account the differences in infusion pumps at different sites, a window between -5 min and +10 min is allowed (i.e., an infusion time of 30 min [-5 min / +10 min]).

[0348] For example, intravenous administration of an anti-PD-(L)1 antibody can be performed using the same IV line previously used for intravenous administration of an anti-human CCR8 antibody. This setup is preferred and advantageous because the complexity of therapeutic administration is reduced and because it is highly convenient for both patients and medical professionals. Preferably, the IV line is flushed with saline prior to intravenous administration of the second antibody (i.e., anti-human PD-(L)1 antibody).

[0349] For example, the anti-human CCR8 antibody with ADCC activity and ADCP activity for use in the treatment method according to this aspect may include at least one 21-day administration cycle. Moreover, both the anti-CCR8 antibody and the anti-PD-(L)1 antibody may be administered on day 1 of the 21-day administration cycle.

[0350] If the patient tolerated all previous infusions (e.g., in the 1st, 2nd, 3rd, or 4th cycle) well, the PD-(L)1 antibody can be administered directly after the anti-CCR8 antibody without substantial delay, i.e., without pausing for 15-60 minutes. For example, in cases where the medical use includes at least two and preferably more dosing cycles, for the second, third, fourth, fifth, or any subsequent dosing cycle, the anti-CCR8 antibody and the anti-PD-(L)1 antibody can be administered directly after each other without substantial delay. This approach is superior because it is more time-efficient for patients and medical professionals.

[0351] In a preferred embodiment, the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks. For example, in one embodiment, the total amount of anti-CCR8 antibody is about 10 mg once a week, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0352] For example, in one embodiment, the total amount of anti-CCR8 antibody is about 30 mg once a week, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0353] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once a week, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0354] For example, in one embodiment, the total amount of anti-CCR8 antibody is about 100 mg once a week, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0355] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once a week, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0356] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg once a week, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0357] In a further highly preferred example, the total amount of anti-CCR8 antibody is about 500 mg once every three weeks, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks. In a highly preferred example, the total amount of anti-CCR8 antibody is about 750 mg once every three weeks, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0358] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg once every three weeks, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0359] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg once every three weeks, and the anti-PD-(L)1 antibody is pembrolizumab and is administered in a total amount of about 200 mg once every three weeks, or about 400 mg once every six weeks.

[0360] In another preferred embodiment, the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0361] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once a week, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0362] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once a week, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0363] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once a week, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0364] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once a week, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0365] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once a week, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0366] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg once a week, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0367] In a further highly preferred example, the total amount of the anti-CCR8 antibody is about 500 mg once every three weeks, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0368] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 750 mg once every three weeks, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0369] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg once every three weeks, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0370] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg once every three weeks, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg once every two weeks, about 360 mg once every three weeks, about 480 mg once every four weeks, or about 480 mg once every six weeks.

[0371] In a preferred embodiment, the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0372] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once a week, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0373] For example, in one embodiment, the total amount of anti-CCR8 antibody is about 30 mg once a week, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0374] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once a week, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0375] For example, in one embodiment, the total amount of anti-CCR8 antibody is about 100 mg once a week, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0376] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once a week, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0377] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg once a week, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0378] In a further highly preferred example, the total amount of the anti-CCR8 antibody is about 500 mg once every three weeks, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0379] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 750 mg once every three weeks, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0380] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg once every three weeks, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0381] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg once every three weeks, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg once every two weeks, about 1200 mg once every three weeks, or about 1680 mg once every four weeks.

[0382] In a preferred embodiment, the anti-PD-(L)1 antibody is sepalizumab and is administered in a total amount of about 360 mg once every three weeks.

[0383] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once a week, and the anti-PD-(L)1 antibody is sepalimumab and is administered in a total amount of about 360 mg once every three weeks.

[0384] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once a week, and the anti-PD-(L)1 antibody is sepalimumab and is administered in a total amount of about 360 mg once every three weeks.

[0385] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once a week, and the anti-PD-(L)1 antibody is sepalizumab and is administered in a total amount of about 360 mg once every three weeks.

[0386] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once a week, and the anti-PD-(L)1 antibody is sepalimumab and is administered in a total amount of about 360 mg once every three weeks.

[0387] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once a week, and the anti-PD-(L)1 antibody is sepalizumab and is administered in a total amount of about 360 mg once every three weeks.

[0388] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg once a week, and the anti-PD-(L)1 antibody is sepalizumab and is administered in a total amount of about 360 mg once every three weeks.

[0389] In a further highly preferred example, the total amount of the anti-CCR8 antibody is about 500 mg once every three weeks, and the anti-PD-(L)1 antibody is sepalizumab and is administered in a total amount of about 360 mg once every three weeks.

[0390] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 750 mg once every three weeks, and the anti-PD-(L)1 antibody is sepalizumab and is administered in a total amount of about 360 mg once every three weeks.

[0391] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg once every three weeks, and the anti-PD-(L)1 antibody is sepalimumab and is administered in a total amount of about 360 mg once every three weeks.

[0392] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg once every three weeks, and the anti-PD-(L)1 antibody is sepalizumab and is administered in a total amount of about 360 mg once every three weeks.

[0393] In a preferred embodiment, the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0394] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once a week, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0395] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once a week, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0396] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once a week, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0397] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once a week, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0398] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once a week, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0399] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg once a week, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0400] In a further highly preferred example, the total amount of the anti-CCR8 antibody is about 500 mg once every three weeks, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0401] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 750 mg once every three weeks, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0402] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg once every three weeks, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0403] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg once every three weeks, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg once every two weeks.

[0404] In a preferred embodiment, the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every three weeks.

[0405] For example, in one embodiment, the total amount of anti-CCR8 antibody is about 10 mg once a week, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every three weeks.

[0406] For example, in one embodiment, the total amount of anti-CCR8 antibody is about 30 mg once a week, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 30 mg / kg every two weeks, or about 1500 mg every three weeks.

[0407] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once a week, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every three weeks.

[0408] For example, in one embodiment, the total amount of anti-CCR8 antibody is about 100 mg once a week, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every three weeks.

[0409] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once a week, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every three weeks.

[0410] In a highly preferred example, the total amount of anti-CCR8 antibody is about 250 mg once a week, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every three weeks.

[0411] In a further highly preferred example, the total amount of the anti-CCR8 antibody is about 500 mg once every three weeks, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every 3 weeks.

[0412] In a highly preferred example, the total amount of anti-CCR8 antibody is about 750 mg once every three weeks, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every 3 weeks.

[0413] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg once every three weeks, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every 3 weeks.

[0414] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg once every three weeks, and the anti-PD-(L)1 antibody is durvalumab and is administered in a total amount of about 10 mg / kg every two weeks, or about 1500 mg every 3 weeks.

[0415] The medical use according to the first aspect preferably comprises at least one 21-day administration cycle. In some preferred embodiments of these embodiments, both the anti-CCR8 antibody and the anti-PD-(L)1 antibody are administered on day 1 of the 21-day administration cycle.

[0416] The dosing regimen according to the present invention is particularly suitable for using anti-CCR8 antibodies in methods of treating cancers such as non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), triple negative breast cancer (TNBC), melanoma, other types of skin cancer and cancer.

[0417] The dosing regimen according to the invention is particularly suitable for using anti-CCR8 antibodies in methods for treating patients suffering from tumors or diseases characterized by chemokine receptor positive cells, preferably CCR8 positive cells, such as CCR8 positive regulatory T cells.

[0418] For example, the method of treatment is a method of treating cancer, preferably wherein the cancer is non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), head and neck squamous cell carcinoma (HNSCC), melanoma, or a skin cancer other than melanoma.

[0419] For example, the treatment is non-small cell lung cancer (NSCLC). For example, the treatment is triple negative breast cancer (TNBC). For example, the treatment is head and neck squamous cell carcinoma (HNSCC). For example, the treatment is melanoma or skin cancer other than melanoma.

[0420] According to the present invention, it has been further interestingly discovered that certain side effects of anti-CCR8 antibody administration can be prevented by administering an effective dose of an antihistamine, acetaminophen, corticosteroids or a combination thereof. Thus, the method of treatment according to this aspect may further comprise administering an effective dose of an antihistamine, acetaminophen, corticosteroids or a combination thereof, preferably

[0421] a. at least 500 mg or at least 650 mg of paracetamol prior to administration of the anti-CCR8 antibody, and / or

[0422] b. at least 50 mg or at least 100 mg of diphenhydramine prior to administration of the anti-CCR8 antibody, and / or

[0423] c. At least 8 mg of dexamethasone prior to administration of the anti-CCR8 antibody.

[0424] For example, acetaminophen can be administered orally. For example, diphenhydramine can be administered orally.

[0425] Tumor proportion score / combined positive score

[0426] Depending on the type of cancer, the response rate to immune checkpoint inhibitors (ICI) varies significantly, ranging from less than 15% to more than 60%. A considerable proportion of "immune-sensitive" tumor types are difficult to treat from the beginning of ICI treatment (primary resistance), or eventually acquire resistance (secondary or acquired resistance) after initial clinical benefit. Treatment options after failure of frontline standard of care (SoC) and ICI (including combination with other anticancer agents) are still limited in most cases of advanced tumors. Therefore, there is a high medical need for these "post-ICI therapy" patients to determine treatments that overcome their malignant diseases' resistance to (combination) immunotherapy, because these patients have limited alternative treatment options.

[0427] The correlation between gene expression and in vivo efficacy of treatment with CCR8-depleting antibodies was investigated by whole transcriptome sequencing of untreated syngeneic mouse tumor models. The inventors found that baseline expression of PD-L1 correlated well with in vivo anti-tumor efficacy of CCR8 replacement antibodies in 21 tumor models, see Figure 5.

[0428] According to certain preferred embodiments of the first aspect, the method of treatment is a method of treating cancer, comprising the following steps:

[0429] a. analyzing the tumor proportion score or combined positive score as a measure of PD-(L)1 expression in a patient's cancer tissue sample, and

[0430] b. If the patient's tumor proportion score is ≥50% or the combined positive score is ≥10% or ≥1%, administer an anti-human CCR8 antibody to the patient.

[0431] These stratification steps can be applied to determine the likelihood that a patient will benefit from the administration of an anti-human CCR8 antibody. In particular, PD-L1 expression can be used as a stratification strategy for patient selection and as a qualifying criterion for treating patients. For example, in the monotherapy-mode of action (MoA) expansion group (Group 2A) of a clinical study, a historical PD-L1 score of ≥50% of the tumor proportion score (TPS) was used as a qualifying criterion. PD-L1 expression appears to have general applicability as a predictive biomarker, see also Figure 6.

[0432] According to certain preferred embodiments of these embodiments,

[0433] a. the cancer is non-small cell lung cancer (NSCLC), and the tumor proportion score is analyzed as a measure of PD-(L)1 expression in a cancer tissue sample of the patient, or

[0434] b. the cancer is triple negative breast cancer and the combined positive score is analyzed as a measure of PD-(L)1 expression in the patient's cancer tissue sample, or

[0435] c. The cancer is head and neck squamous cell carcinoma, and the combined positive score is analyzed as a measure of PD-(L)1 expression in the patient's cancer tissue sample.

[0436] Preferably, the tumor proportion score is analyzed using the PD-L1-antibody 22C3 pharmDx assay. The PD-L1 antibody 22C3 pharmDx assay provides reliable results and has been approved by the FDA. PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemistry assay using monoclonal mouse anti-PD-L1, clone 22C3, and can be used to detect PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) cancer tissues, for example, using the EnVision FLEX visualization system on an Autostainer Link 48.

[0437] In an alternative approach, the VENTANA PD-L1 (SP142) Assay can be used. The VENTANA PD-L1 (SP142) Assay is another qualitative immunohistochemistry assay using rabbit monoclonal anti-PD-L1 clone SP142 and can be used on BenchMark ULTRA instruments (e.g.) using the OptiView DAB IHC Dectection Kit and OptiView Amplification Kit to stain FFPE tissue.

[0438] The tumor proportion score was assessed with reference to the PD-L1 IHC 22C3 pharmDx Interpretation Manual-NSCLC (Agilent Dako), and the PD-L1 IHC 22C3pharmDx Interpretation Manual-Headand Neck Squamous Cell Carcinoma (Agilent Dako) was also used to assess CPS.

[0439] For some cancer patients, historical tumor proportion scores and / or historical combined positive scores may be obtained. According to the present invention, historical tumor proportion scores or historical combined positive scores may be used to determine the likelihood that a patient will benefit from administration of an anti-human CCR8 antibody. Using these historical scores, sufficiently reliable, rapid, and very convenient stratification decisions may be made.

[0440] In particular, provided is an anti-human CCR8 antibody with ADCC activity and ADCP activity for use in a therapeutic method as described in the first aspect, wherein the therapeutic method is a method for treating cancer, comprising administering the anti-human CCR8 antibody to the patient if the patient's historical tumor proportion score is ≥50% or the historical combined positive score is ≥10% or ≥1%.

[0441] According to certain preferred embodiments of these embodiments,

[0442] a. The cancer is non-small cell lung cancer (NSCLC), and the method of treatment comprises administering an anti-human CCR8 antibody to the patient if the patient's historical tumor proportion score is ≥ 50%, or

[0443] b. The cancer is triple negative breast cancer, and the treatment method comprises administering to the patient an anti-human CCR8 antibody if the patient's historical combined positive score is ≥ 10% or ≥ 1%, or

[0444] c. The cancer is head and neck squamous cell carcinoma, and the treatment method comprises administering an anti-human CCR8 antibody to the patient if the patient's historical combined positive score is ≥20% or ≥1%.

[0445] According to certain embodiments described in this section, the tumor proportion score is analyzed or obtained using an FDA-approved PD-L1 assay, such as the PD-L1 IHC22C3 pharmDx assay or the VENTANA PD-L1 (SP263) assay.

[0446] Cytokine Biomarkers

[0447] Cytokine release has been identified as an important biomarker in the context of anti-CCR8 antibodies. According to the present invention, provided is an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treating a patient, for example, without limitation, according to any aspect or embodiment described herein, comprising the following steps:

[0448] a. Optionally, analyzing the patient's blood, plasma or serum screening sample for at least one and preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inflammatory cytokines selected from the group consisting of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL 12p70, IL-13 and TNF-α,

[0449] b. administering to the patient an effective dose of an anti-human CCR8 antibody,

[0450] c. analyzing a patient's blood, plasma or serum sample for at least one and preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inflammatory cytokines,

[0451] wherein a blood, plasma or serum sample is drawn or collected after administering an effective dose of the anti-human CCR8 antibody according to step b),

[0452] d. Comparison of cytokine levels obtained according to step c)

[0453] i. with the cytokine levels obtained according to step a), or

[0454] ii. With reference value,

[0455] To identify safety-related events, or as a surrogate biomarker of Treg depletion, or as a biomarker of treatment success.

[0456] A screening sample is defined herein as a sample obtained from a patient prior to the actual dosing event, preferably from a patient who has not been previously treated with an anti-human CCR8 antibody having ADCC activity and ADCP activity.

[0457] In order to use the corresponding cytokine level as biomarker, the respective cytokine level can be compared, for example, with the cytokine level obtained before the first anti-CCR8 antibody administration in the same patient or with a different reference value obtained for the same patient or for a different subject population.

[0458] It is well known to those skilled in the art that such reference values ​​can be obtained by, for example, calculating the mean or median of cytokine levels obtained from multiple patients at a clear time point (e.g., not limited to, before anti-CCR8 antibody administration, 1-12 hours after anti-CCR8 antibody administration, 1-3 days after anti-CCR8 antibody administration, any time point measured according to Example 24, or any other appropriate time point thereafter). If the reference value is calculated using a time point after antibody administration, then those skilled in the art will also know that the antibody dose used to treat the subject to determine the reference value should be clearly defined and can be, for example, any dose described herein for anti-CCR8 antibody administration. It is not advantageous to have a clearly defined pg / μl for each cytokine herein, as those skilled in the art know how to calibrate the corresponding assay for biomarker assessment. In any case, a substantial increase in the biomarker is associated with Treg depletion and / or treatment success (compare Fig.10 and Figures 11 to 16 ).

[0459] For example, a biomarker of treatment success can be a biomarker used for monitoring, prediction or stratification. The term stratification refers to patient selection for treatment.

[0460] In a preferred embodiment, at least one and preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inflammatory cytokines selected from the group consisting of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-13 and TNF-α is IFN-γ or comprises IFN-γ.

[0461] In a preferred embodiment, the reference value is a value obtained from a sample of the same patient drawn / collected before the start of treatment. For example, a blood, plasma or serum screening sample for analyzing cytokine levels can be drawn / collected 60-15 minutes, preferably ~30 minutes before administering an effective dose of an anti-human CCR8 antibody.

[0462] Cytokines as safety biomarkers of anti-CCR8 antibodies

[0463] Briefly, cytokine release assays were performed using human whole blood (containing added soluble antibodies) and human peripheral blood mononuclear cells (PBMCs) (containing wet-coated antibodies) to investigate the potential of anti-CCR8 antibodies alone or in combination with pembrolizumab to activate the secretion of cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10 and TNFα were analyzed), see Example 14. Reference values ​​for comparison of cytokine levels can be obtained for specific anti-CCR8 antibodies at specific concentrations as defined in this example. In the alternative, these reference values ​​can be obtained based on alternative antibodies with known safety profiles, as also explained in this example.

[0464] In fact, incubation of whole blood with soluble anti-human CCR8 antibodies resulted in a dose-dependent release of IFN-γ, IL-1β, IL-6, TNFα, and IL-8. When compared to rituximab (anti-human CCR8 antibody), higher IFN-γ, IL-6, TNFα, and IL-8 cytokine levels were induced at a dose of 10 μg / mL, but at comparable levels at lower antibody doses tested. IL-1 was not induced by rituximab, but by anti-human CCR8 antibodies. Due to its robust dose response and highest sensitivity, IFN-γ was defined as the main cytokine.

[0465] Based on these findings, the inventors propose the tested cytokine release panel as a safety biomarker for anti-CCR8 antibody cancer therapy to reduce the potential risk of infusion-related reactions due to cytokine release (including cytokine release syndrome) and mitigate the risk of immune-related adverse events such as skin toxicity (i.e., rash) observed with other Treg-depleting agents.

[0466] Cytokines as biomarkers for monitoring anti-CCR8 antibodies

[0467] When fucosylated anti-CCR8 antibody variants or "silent" Fc variants (reduced binding to FcgR) were tested on two donors, those variants did not - or to a much lesser extent - induce IFN-γ, IL-1β, IL-6, TNFα, and IL-8 in a whole blood / soluble antibody cytokine release assay format, indicating that cytokine release is indicative of successful Treg depletion via ADCC and ADCP.

[0468] Since the expected mode of action of non-fucosylated anti-CCR8 antibodies relies on ADCC and ADCP, the inventors concluded that cytokine release can be used as a surrogate biomarker for anti-CCR8 antibody-induced Treg depletion and as a predictive / stratification biomarker for treatment success. In fact, Figure 6 (B) shows the correlation between IFN-γ levels and treatment response in various mouse models, see also human patients Fig.10 and Figure 11-16 comparison.

[0469] Based on these findings, in certain further embodiments, the anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method further comprises

[0470] e. If the cytokine level obtained according to step c):

[0471] i. with the cytokine levels obtained according to step a), or

[0472] ii. is increased relative to the reference value,

[0473] At least one further effective dose of an anti-human CCR8 antibody is then administered to the patient.

[0474] In the course of Examples 13.2 and 13.3, cytokine levels were assessed in cynomolgus monkeys.Examples 14 and 25 disclose cytokine release assays in human donors and increased cytokine release in human patients with increasing doses of anti-CCR8 antibodies.

[0475] The analysis of cytokine levels can be performed, for example, using sandwich-based immunoassay techniques, such as the "Meso Scale Discovery" (MSD-ECL) platform. The MSD-ECL platform uses electrochemiluminescent markers combined with detection antibodies. These markers produce light when stimulated by electricity in an appropriate chemical environment, which can be used to measure key proteins and molecules. The detection process is started at the electrode located at the bottom of the platform microplate, and only the markers near the electrode are excited and detected. The plate electrodes are energized so that the SULFO-TAG markers emit light, and these labels are electrochemiluminescent labels that can achieve ultrasensitive detection. Then, the light intensity is measured to quantify the analyte in the sample.

[0476] In other words, in principle, each well of the microplate to be used is pre-coated with a capture antibody (specific for each cytokine to be detected) at a spatially different point. The plasma sample is added to the well of the microplate, and the bound cytokines in the sample are detected using an anti-cytokine detection antibody labeled with MSD SULFO-TAG. The electrochemiluminescence principle is used: for reading, a voltage is applied to the plate electrodes, and the intensity of the emitted light allows the quantitative measurement of the corresponding cytokine present in the sample.

[0477] In an alternative embodiment, analysis of cytokine levels can also be performed using a "single molecule array" (Simoa TM ). Simoa is based on the separation of individual immune complexes on paramagnetic beads using standard ELISA reagents. The main difference between Simoa and conventional immunoassays is that single molecules can be captured in femtoliter-sized wells, allowing a "digital" readout of each individual bead to determine whether it has bound to the target analyte.

[0478] In certain preferred embodiments, the level of at least one and preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inflammatory cytokines selected from the group of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-13 and TNF-α can be comparable to the baseline level of the same cytokine obtained from the same patient at an earlier time point or before administration of the anti-CCR8 antibody.

[0479] In certain preferred embodiments, the level of at least one and preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inflammatory cytokines selected from the group consisting of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-13 and TNF-α can be comparable to a reference value for each cytokine. The reference value can be determined by a person skilled in the art and can be derived based on the concentration of the anti-CCR8 antibody previously administered, see Example 14. The reference value can be a general reference value or an individual patient-specific reference value obtained from a sample drawn before treatment.

[0480] In certain embodiments, blood, plasma or serum samples for analysis of cytokine levels are drawn on the same day after administration of an effective dose of an anti-human CCR8 antibody, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours after administration of an effective dose of an anti-human CCR8 antibody. Blood samples drawn on the same day may be convenient, particularly if the patient can be treated in an outpatient setting.

[0481] In certain embodiments, blood, plasma or serum samples for analysis of cytokine levels are drawn 1-24 hours, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after administration of an effective dose of an anti-human CCR8 antibody.

[0482] According to certain preferred embodiments, the blood, plasma or serum sample for analyzing cytokine levels according to step c) is drawn 1-24 hours, 24-48 hours, 2-7 days, 7-14 days, 14-28 days or more than 28 days after administering an effective dose of anti-human CCR8 antibody according to step b).

[0483] According to certain preferred embodiments, the blood, plasma or serum sample for analyzing cytokine levels according to step c) is drawn within the first three days after administration of an effective dose of anti-human CCR8 antibody. Drawing samples within the first three days after administration of an effective dose of anti-human CCR8 antibody according to step b) is particularly suitable for monitoring safety-related events.

[0484] According to certain preferred embodiments, the blood, plasma or serum sample for analyzing cytokine levels according to step c) is drawn within 3-21 days after the administration of an effective dose of anti-human CCR8 antibody. Drawing samples within 3-21 days after the administration of an effective dose of anti-human CCR8 antibody according to step b) is particularly suitable for monitoring the success of treatment.

[0485] These embodiments are advantageous because they reduce the risk of infusion reactions and immune-related adverse events, and / or improve efficacy / treatment success by aiding in the identification of patient populations most likely to benefit from anti-CCR8 antibody treatment.

[0486] Preferably, the fold change in cytokine levels during treatment can be measured by an immuno-based assay compared to a baseline serum sample as a reference value.

[0487] Responsiveness Tregs are known to promote tumor growth by inhibiting the function of cytotoxic T cells and promote an immunosuppressive tumor microenvironment (TME) via various mechanisms. Accordingly, Tregs have also been identified as one of the key resistance mechanisms to ICIs in many tumor types. In addition, established PD-1 / PD-L1 inhibitors may not only induce the recovery of PD-1-positive dysfunctional cytotoxic T cells, but also enhance the proliferation and inhibitory activity of PD-1-positive Tregs. Therefore, a higher abundance of PD-1+Tregs with upregulated PD-1 expression was detected in patients whose tumors did not respond to ICIs. These observations support targeting Tregs as an attractive approach to enhance anti-tumor immune responses in monotherapy and in combination with ICIs.

[0488] This preclinical rationale also suggests that treatment with TPP-23411 may yield an innovative and effective therapy to counteract immune suppressive pathways in tumors by overcoming resistance to PD-(L)1 inhibitors, thereby potentially facilitating the failure of ICIs. after Improving the efficacy of established PD-(L)1 inhibitors in a combination setting together with anti-PD-(L)1 inhibitors in patients.

[0489] Non-responders are patients who have not received treatment with anti-PD-(L)1 antibodies for at least 6 months. This is due to the fact that the effect of anti-PD-(L)1 antibody treatment is monitored and if no response is seen, treatment is stopped. Therefore, responders are patients who have received treatment with anti-PD-(L)1 antibodies for at least 6 months.

[0490] According to a further aspect of the present invention, provided is an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method, wherein the therapeutic method is a method for treating cancer, comprising

[0491] a. stratify patients based on prior treatment of cancer with anti-PD-(L)1 antibodies for at least 6 months, and

[0492] b. If the patient has been previously treated with anti-PD-(L)1 antibody for at least 6 months, administer anti-human CCR8 antibody to the patient.

[0493] In some embodiments, the anti-human CCR8 antibody is an anti-human CCR8 antibody for use in a method of treatment according to the first aspect.

[0494] Aspect 2

[0495] Mini-antibodies

[0496] The inventors have demonstrated that anti-mouse CCR8 surrogate antibodies deplete tumor-infiltrating CCR8+Treg cells via ADCC and ADCP mechanisms and induce robust in vivo single-agent tumor growth inhibition in immunogenic murine tumor models. Furthermore, ex vivo analysis showed that these anti-tumor effects were associated not only with efficient depletion of CCR8+Tregs, but also with a substantial increase in CD8+T cells within the tumor microenvironment.

[0497] Thus, in U.S. Application No. 17 / 358,841, filed on June 25, 2021, PCT Application No. PCT / EP2021 / 067504, PCT Application No. PCT / EP2021 / 067578, PCT Application No. PCT / EP2021 / 067574, PCT Application No. PCT / EP2021 / 067579, and PCT Application No. PCT / EP2021 / 067580, the inventors have previously suggested that molecules that bind to T cell markers are used in a method for diagnosing / stratifying a subject having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the method comprising

[0498] a. Determining the level of T cell marker expression in the tumor (sample),

[0499] b. comparing the level of T cell marker expression to a reference sample or value, and

[0500] c. If the level of the T cell marker is higher than or equal to the reference sample or value, the subject is diagnosed / stratified as having a tumor that is sensitive to treatment with an anti-CCR8 antibody.

[0501] Since there is a strong interest in determining the levels of suitable T-cell markers in a highly reliable manner in order to analyze the number of T cells in tumor biopsy samples, the inventors have now developed a method that uses PET technology instead of biopsy tissue for evaluation, thereby obtaining a more comprehensive, reliable and less error-prone picture to evaluate treatment success or to decide whether a patient is eligible for treatment with (further doses of) anti-CCR8 antibodies.

[0502] According to this aspect of the present invention, provided is an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer, comprising the following steps:

[0503] a. administering to a subject a Zr-89 labeled anti-CD8 minibody,

[0504] b. performing at least one PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 miniantibody in the subject, thereby generating a first image of the subject,

[0505] c. determining the abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancer lesions of the subject based on the first subject image, and

[0506] d. If the first subject image indicates that the amount and / or distribution of Zr-89 labeled anti-CD8 miniantibodies in any of the one or more cancer lesions indicates a substantial likelihood that the subject will benefit from administration of an anti-human CCR8 antibody, administering an effective dose of an anti-human CCR8 antibody to the subject.

[0507] Minibodies can be bivalent homodimers in which each monomer has a single chain variable fragment (scFv) connected to a human IgG1 CH3 domain via a modified IgG1 hinge sequence. Minibodies preferably lack an Fc receptor interacting domain and have a smaller size than full monoclonal antibodies.

[0508] Preferably, the Zr-89 labeled anti-CD8 miniantibody binds to human CD8 glycoprotein with an EC50 of <1 nM. For example, the miniantibody can be conjugated via deferoxamine (Df) and radiolabeled with a positron emitting radionuclide zirconium-89 (89Zr; Tm 78.4 hours). According to a most preferred embodiment, the Zr-89 labeled anti-CD8 miniantibody is a Zr-89 labeled anti-CD8 miniantibody described in U.S. Application No. 17 / 280,137.

[0509] 89Zr-Df-crefmirlimab uptake in tumor lesions is related to CD8 expression in these lesions, and can be used according to the present invention to monitor the influx of T cells into tumor lesions after the first dose of anti-human CCR8 antibodies are administered. This influx records the successful activation of the immune system by anti-human CCR8 antibodies, and can be used as, for example, an alternative biomarker for treatment success, as a monitoring biomarker, or as a prediction / stratification biomarker. More particularly, the abundance increase and / or distribution change of Zr-Df-crefmirlimab indicate that the abundance of T cells is significantly higher or flows into at least one tumor lesion or tumor microenvironment in large quantities, indicating that the subject benefits from the substantial possibility of administering anti-human CCR8 antibodies.

[0510] Zr-89 labeled anti-CD8 miniantibodies or pharmaceutical preparations thereof can be prepared by conjugating the miniantibody with deferoxamine to form a Df-miniantibody; radiolabeling the Df-miniantibody with 89Zr to form a radiolabeled miniantibody; purifying the radiolabeled miniantibody; and mixing the radiolabeled miniantibody with a cold miniantibody to form a diagnostic composition, wherein the miniantibody and the cold miniantibody bind to the same epitope on CD8. A detailed description of the process can be found in U.S. Application No. 17 / 280,137.

[0511] Preferably, the abundance and / or distribution of the CD8 miniantibody in the subject is analyzed within 6 to 36 hours after administration of the CD8 miniantibody to the patient, such as 24 hours after administration of the CD8 miniantibody to the patient.

[0512] In some embodiments, the method of positron emission tomography ("PET scanning") comprises

[0513] a. administering to a subject a Zr-89 labeled anti-CD8 minibody,

[0514] b. Provide scintillator,

[0515] c. using a scintillator to detect a pair of photons generated by the Zr-89 labeled anti-CD8 minibody, and

[0516] d. using the detection of the pair of photons to locate the source of the Zr-89 labeled anti-CD8 minibody via a list of coincidence events processed by a processor configured to take the output from the scintillator and convert it into a list of coincidence events,

[0517] e. Each mm within the subject 3 About 300 to about 500 CD8-positive cells can be detected in tissues or cancer lesions.

[0518] In some embodiments, an MRI and / or CT scan may be performed to identify the location of the tumor and obtain more precise spatial information.

[0519] In some embodiments, the method comprises

[0520] a. Provides a distribution image of Zr-89 labeled anti-CD8 minibody,

[0521] b. providing a distribution image of the FDG marker via a second PET image of the subject,

[0522] c. Create a third PET image that contains the first PET image overlaid on the second PET image.

[0523] For example, methods for determining standard uptake values ​​include

[0524] a. applying Zr-89 labeled anti-CD8 miniantibodies to the subject,

[0525] b. determining r, where r is the concentration of radioactivity (kBq / ml) measured by a PET scanner within the target radiation area from the Zr-89 labeled anti-CD8 minibody,

[0526] c. Determine a', where a' is the decay-corrected amount of radiolabeled tracer injected (kBq),

[0527] d. Determine w, the subject's weight, and

[0528] e. Determine SUV as a result of r(a' / W).

[0529] For example, if the standardized uptake value (SUV) is >1, more preferably >2, >3 or >4, and most preferably >5, >6, >7 or >8, then the quantity of Zr-89 labeled anti-CD8 miniantibodies can be considered to indicate a substantial likelihood that the patient will benefit from administration of the anti-human CCR8 antibody.

[0530] In some embodiments, a method of analyzing an image comprises

[0531] a. Provide images,

[0532] b. defining a first region of interest (ROI) on the image by marking the image,

[0533] c. Determine the signal strength of the data points within the first ROI,

[0534] d. Determine the maximum signal intensity within the first ROI,

[0535] e. Determine the average value of the signal intensity within the first ROI,

[0536] f. adding the signal intensities within the first ROI to obtain a first summed signal level of the first ROI,

[0537] Wherein the first ROI represents data on the amount of Zr-89 labeled anti-CD8 minibody that has been administered to the subject.

[0538] In some preferred embodiments, the amount and / or distribution of Zr-89 labeled anti-CD8 minibody in any one or more cancer lesions is assessed relative to

[0539] i. the abundance and / or distribution of Zr-89 labeled anti-CD8 minibody in healthy tissues of patients, or

[0540] ii. One or more reference values ​​for the abundance and / or distribution of Zr-89 labeled anti-CD8 minibody.

[0541] According to some embodiments, provided is an anti-human CCR8 antibody having ADCC activity and / or ADCP activity for use in a therapeutic method, comprising the following steps:

[0542] a. administering to the subject a first dose of Zr-89 labeled anti-CD8 minibody,

[0543] b. performing a first PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 miniantibody in the subject, thereby generating a first image of the subject,

[0544] c. determining the abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancer lesions of the subject based on the first subject image,

[0545] d. administering to the subject an effective dose of an anti-human CCR8 antibody,

[0546] e. administering to the subject a second dose of Zr-89 labeled anti-CD8 minibody,

[0547] f. performing a second PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 miniantibody in the subject, thereby generating a second image of the subject,

[0548] g. determining a second abundance and / or distribution of the Zr-89 labeled anti-CD8 miniantibody in one or more cancer lesions of the subject based on the second subject image,

[0549] h. Compare the second subject image with the first subject image to evaluate whether the abundance of Zr-89 labeled anti-CD8 miniantibodies is substantially increased in one or more cancer lesions, or whether the distribution of Zr-89 labeled anti-CD8 miniantibodies is substantially changed, for monitoring disease progression or the success of anti-human CCR8 antibody treatment.

[0550] According to some of these embodiments, provided is an anti-human CCR8 antibody with ADCC activity and ADCP activity for use in a method for treating cancer, comprising the further step of administering to the patient at least one further effective dose of the anti-human CCR8 antibody if the abundance of the Zr-89-labeled anti-CD8 miniantibody is substantially increased or if the distribution of the Zr-89-labeled anti-CD8 miniantibody is substantially changed in one or more cancer lesions.

[0551] For example, Zr-89 labeled anti-CD8 minibodies can provide about 0.5 to 3.6 mCi of radioactivity.

[0552] For example, a PET scan can be performed about 6 hours to 36 hours after administration of a corresponding dose of Zr-89 labeled anti-CD8 minibody.

[0553] In some highly preferred embodiments, the Zr-89 labeled anti-CD8 minibody is 89Zr-Df-crefmirlimab.

[0554] In some highly preferred embodiments, the anti-human CCR8 antibody is an anti-human CCR8 antibody for use in a method of treating cancer according to another aspect disclosed herein.

[0555] In some highly preferred embodiments, the anti-human CCR8 antibody for use in the method of treating cancer is any one of TPP-23411, TPP-29338, TPP-27454, TPP-31741, TPP-31742, TPP-31743, TPP-31744.

[0556] The anti-CCR8 antibody according to this aspect is preferably an isolated anti-CCR8 antibody or antigen-binding fragment thereof comprising six CDR sequences, wherein each CDR sequence has at least 98% or 100% sequence identity to the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences:

[0557] a) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8,

[0558] b) SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44,

[0559] c) SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56

[0560] d) SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68,

[0561] e) SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80,

[0562] f) SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92.

[0563] Preferably, the antibody may further comprise

[0564] a. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and / or

[0565] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 5,

[0566] b. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 37, and / or

[0567] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 41,

[0568] c. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 49, and / or

[0569] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 53,

[0570] d. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 61, and / or

[0571] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 65,

[0572] e. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 73, and / or

[0573] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 77, or

[0574] f. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 85, and / or

[0575] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:89.

[0576] Preferably, the antibody may further comprise

[0577] a. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, and / or

[0578] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18,

[0579] b. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, and / or

[0580] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 48,

[0581] c. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 59, and / or

[0582] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60,

[0583] d. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 71, and / or

[0584] A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:72.

[0585] e. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 83, and / or

[0586] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 84,

[0587] f. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, and / or

[0588] A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:96.

[0589] Aspect 3

[0590] Plasma method

[0591] A method for measuring anti-CCR8 antibodies in cynomolgus monkey plasma is described herein, the method comprising the following steps:

[0592] a. diluting the plasma or serum sample with a buffer,

[0593] b. using immobilized biotinylated anti-human IgG-Fc antibody as a capture molecule to capture anti-CCR8 antibodies in plasma,

[0594] c. Use fluorophore-labeled anti-human IgG antibody as detection reagent to detect the abundance of captured molecules.

[0595] This method is used for preclinical pharmacokinetic studies and key nonclinical safety studies in monkeys. It was found that it is suitable for reliable measurement of anti-CCR8 antibodies in plasma. It was found that the Gyrolab Generic PK kit method assay can be used to measure anti-CCR8 antibodies in plasma with a suitable detection range for cynomolgus monkey subjects. In brief, samples (calibration standards, quality control controls, qualified samples or unknown samples), antibodies and buffers are placed on microplates. Then, biotinylated antibodies are used for capture and antibodies with fluorophore tags are used for detection. The microplate can be loaded into the Gyrolab device, as well as the Gyrolab Bioaffy CD, and sufficient buffer (Bioaffy Pump Liquid, BioaffyWash buffer 1 and 2). It was found that the fluorescent signal generated is directly proportional to the concentration of anti-CCR8 antibodies present in the sample. The lower limit of quantification is 1.25ng / mL. The working range of MRD is 1.25 to 250ng / mL (12.5 to 2500ng / mL in 100% plasma). The fluorophore may be any fluorophore known in the art, and is preferably Alexa Dye, such as AF647.

[0596] Immobilized biotinylated anti-human IgG-Fc antibody can be CaptureSelect TM Human IgG-Fc PKBiotin Conjugate (Thermo Scientific, catalog number 7103322100). CaptureSelect TMHumanIgG-Fc PK Biotin Conjugate consists of a 13kDa recombinant single domain antibody fragment (VHH affinity ligand) that specifically binds to the Fc portion of all four human IgG subclasses without cross-binding to mouse, rat, rhesus and cynomolgus monkey IgG. The affinity ligand is chemically conjugated to biotin via an appropriate spacer, retaining the binding reactivity of the ligand when used in combination with a streptavidin-based conjugate or a streptavidin-precoated surface.

[0597] Aspect 4

[0598] ADA Method

[0599] It is known that biological agents (including therapeutic antibodies) have immunogenic potential, and administration to patients can induce an immune response, resulting in the formation of anti-drug antibodies ("ADA"). Such ADA can reduce the effectiveness of anti-CCR8 antibodies. For example, they can bind to and / or neutralize anti-CCR8 antibodies, causing changes in the pharmacokinetics or pharmacodynamics of the drug, thereby changing the efficacy of the drug. ADA may cause serious side effects, including allergic reactions, cross-reactivity against endogenous proteins through neutralizing antibodies (Nabs), and complement activation. Therefore, it is necessary to develop a reliable assay to monitor the formation of anti-anti-CCR8 antibodies.

[0600] The present inventors have now developed a method for reliably determining and (semi-)quantifying anti-anti-CCR8 antibody formation in cynomolgus monkey or human plasma or serum. The method was found to be superior compared to other tested methods. In more detail, provided is a method for determining and quantifying anti-anti-CCR8 antibody formation in cynomolgus monkey or human plasma or serum, the method comprising a bridging ELISA method based on anti-CCR8 antibodies.

[0601] In brief, it was found that anti-drug antibodies (ADA) against anti-CCR8 antibodies were reliably detectable using a bridged ligand binding assay on the Meso Scale Discovery platform. Affinity Pure goat anti-human IgG can be used as a positive control in cynomolgus monkey plasma or serum. Positive and negative control samples and unknown samples can be pre-diluted (e.g., 1:8) with dilution buffer, mixed with dilution buffer, and pre-incubated in a polypropylene plate, for example, on an orbital shaker (e.g., RT, 600rpm) for 1 hour. To the sample mixture, a master mix containing biotinylated anti-CCR8 antibody (e.g., 1 μg / mL) and SULFO-tagged anti-CCR8 antibody (e.g., 1 μg / mL) can be added and incubated for 2 hours (RT, 600rpm). From the incubated samples, 25 μL can be transferred in duplicate to the wells of a blocked MSD Streptavidin Gold plate (150 μL blocking buffer for at least 30 minutes, 600 rpm), to which the biotinylated anti-CCR8 antibody can bind. If a functional anti-drug antibody is present, it will bridge the biotinylated and SULFO-tagged anti-CCR8 antibody. When a voltage is applied, the SULFO-tagged anti-CCR8 antibody produces an electrochemiluminescent (ECL) signal that correlates with the number of ADA in the well. The plate can be read (e.g., using a MesoQuickPlex SQ 120), and the data analyzed (e.g., using a WorkbenchTM software).

[0602] According to this aspect, a signal is generated if the anti-anti-CCR8 antibody bridges a) a biotinylated anti-CCR8 antibody, and b) a SULFO-tagged anti-CCR8 antibody. Methods for producing biotinylated antibodies are known in the art, and methods for producing SULFO-tagged antibodies are likewise available to those skilled in the art (e.g., based on NHS ester chemistry).

[0603] The antibody used to generate the SULFO-tagged anti-CCR8 antibody and the biotinylated anti-CCR8 antibody may be any one of TPP-23411, TPP-27454, TPP-31741, TPP-31742, TPP-31743, or TPP-31744.

[0604] Thus, the anti-CCR8 antibody according to this aspect may be an isolated anti-CCR8 antibody or antigen-binding fragment thereof comprising six CDR sequences, wherein each CDR sequence has at least 98% or 100% sequence identity to the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences:

[0605] a) SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8,

[0606] b) SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44,

[0607] c) SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56,

[0608] d) SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67 and SEQ ID NO: 68,

[0609] e) SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80,

[0610] f) SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92.

[0611] Preferably, the antibody may further comprise

[0612] a. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and / or

[0613] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 5,

[0614] b. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 37, and / or

[0615] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 41,

[0616] c. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 49, and / or

[0617] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 53,

[0618] d. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 61, and / or

[0619] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 65,

[0620] e. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 73, and / or

[0621] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 77, or

[0622] f. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 85, and / or

[0623] A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:89.

[0624] Preferably, the antibody may further comprise

[0625] a. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 17, and / or

[0626] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18,

[0627] b. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 47, and / or

[0628] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 48,

[0629] c. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 59, and / or

[0630] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 60,

[0631] d. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 71, and / or

[0632] A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:72.

[0633] e. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 83, and / or

[0634] A light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 84,

[0635] f. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, and / or

[0636] A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:96.

[0637] Aspect 5

[0638] TPP-29338

[0639] When the inventors characterized the properties of the clinical candidate TPP-23411, it was surprisingly found that the antibody had an unexpected clearance behavior in cynomolgus monkeys. Although anti-mouse CCR8 surrogate antibodies such as TPP-15285 have been created before simulating the behavior of TPP-23411, these surrogate antibodies have not achieved the unexpected clearance behavior of TPP-23411. Therefore, in order to obtain a safe and effective dosage regimen for the clinical candidate TPP-23411, the first step is that the inventors must identify a murine surrogate antibody with a very similar PK / PD behavior to TPP-23411. In fact, with TPP-29338, such a surrogate antibody can eventually be obtained. A short half-life variant TPP-29338 has been produced and found to be suitable for simulating the high clearance rate of TPP-23411. TPP-29338 is an anti-mouse CCR8 antibody in which the human VH / VL chain has been chimerized to the mIgG2a with H310Q / H330N mutations. It induces both ADCC and ADCP and has a short half-life, ie, <10 days.

[0640] According to a further aspect, therefore provided is an isolated anti-CCR8 antibody or antigen-binding fragment thereof comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID Nos.: 20, 21, 22, 24, 25, 26.

[0641] For example, the isolated anti-CCR8 antibody or antigen-binding fragment thereof further comprises

[0642] a. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 19, and / or

[0643] b. A variable light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 23.

[0644] For example, the isolated anti-CCR8 antibody or antigen-binding fragment thereof further comprises

[0645] a. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 35, and / or

[0646] b. a light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:36.

[0647] In some highly preferred embodiments, the antibody according to the further aspect is non-fucosylated. In addition, provided is a polynucleotide encoding the antibody or antigen-binding fragment according to the second aspect. Also provided is a vector comprising the polynucleotide. Finally, provided is an isolated host cell comprising the aforementioned polynucleotide. Example

[0648] The following examples were performed to suggest appropriate dosing regimens for (non-fucosylated) TPP-23411. In particular, a series of in vitro and in vivo pharmacodynamic (PD) and in vivo pharmacokinetic (PK) studies were performed using TPP-23411 and its murine surrogate antibody.

[0649] Since TPP-2341 only cross-reacts with cynomolgus monkeys and not with mouse CCR8 orthologs, murine surrogate antibodies were used for further in vitro and in vivo mode of action in mice and efficacy evaluation of CCR8+ Treg depletion. Table 1.1 summarizes the antibodies used in the in vitro and in vivo studies.

[0650] Table 1.1: Antibodies used in in vitro and in vivo studies

[0651]

[0652] Example 1: Generation of murine surrogate antibodies to mimic the high clearance rate of TPP-23411

[0653] When the inventors characterized the properties of the clinical candidate TPP-23411, it was surprisingly found that the antibody had an unexpected clearance behavior in cynomolgus monkeys. Although anti-mouse CCR8 surrogate antibodies such as TPP-15285 have been created before simulating the behavior of TPP-23411, these surrogate antibodies have not achieved the unexpected clearance behavior of TPP-23411. Therefore, in order to obtain a safe and effective dosage regimen for the clinical candidate TPP-23411, the first step is that the inventors must identify a murine surrogate antibody with a very similar PK / PD behavior to TPP-23411. In fact, with TPP-29338, such a surrogate antibody can eventually be obtained. A short half-life variant TPP-29338 has been produced and found to be suitable for simulating the high clearance rate of TPP-23411. TPP-29338 is an anti-mouse CCR8 antibody in which the human VH / VL chain has been chimerized to the mIgG2a with H310Q / H330N mutations.

[0654] Example 2: Pharmacological evaluation of TPP-23411

[0655] A series of primary and secondary pharmacodynamic, safety pharmacology, and pharmacodynamic drug interaction studies were performed to characterize and evaluate the efficacy, specificity, and safety of TPP-23411. Notable findings are presented in Table 2.1 (in vitro studies) and Table 2.2 (in vivo studies), and are further described below.

[0656] Table 2.1: Selected non-clinical pharmacology in vitro studies with TPP-23411. For more details on the individual experiments, see PCT / EP2021 / 067504, PCT / EP2021 / 067578, PCT / EP2021 / 067574, PCT / EP2021 / 067579 and PCT / EP2021 / 067580, which are incorporated herein by reference. SPR: surface plasmon resonance; N / A: not applicable.

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663] Example 3: Characterization of murine surrogate antibodies: binding affinity

[0664] The binding of mouse surrogate antibodies to mouse Fc receptor variants (mFcγR and mFcRn) was analyzed by SPR. TPP-15285 (mIgG2a surrogate antibody with a normal half-life of 141 hours), TPP-29338 (mIgG2a surrogate antibody with H310Q / H330N mutations, interfering with the interaction with murine FcRn, resulting in a short antibody half-life of 27 hours) and mIgG2a isotype control TPP-10748 were evaluated by SPR analysis. In this study, TPP-15285, TPP-29338 and TPP-10748 all showed similar binding characteristics to mFcγR variants (Table 3.1), but only TPP-15285 and TPP-10748 showed binding to mFcRn (Table 3.2).

[0665] Table 3.1: Binding of murine surrogate antibodies TPP-15285 and TPP-29338 to mouse FcγR variants as determined by SPR. a TPP-15285, anti-mouse CCR8 antibody, a normal half-life mouse surrogate antibody of the anti-human CCR8 antibody TPP-23411; TPP-29338, anti-mouse CCR8 antibody, a short half-life mouse surrogate antibody of the anti-human CCR8 antibody TPP-23411; TPP-10748, mIgG2a isotype control. b Use K D As an approximation when saturation is not reached. c K D >25,000nM: The fitted value exceeds the saturation curve. .

[0666]

[0667] Table 3.2: Binding of murine surrogate antibodies TPP-15285 and TPP-29338 to mouse FcRn as determined by SPR

[0668]

[0669] Example 4: Characterization of murine surrogate antibodies: cell binding

[0670] The binding properties of murine surrogate antibodies were evaluated using flow cytometry on HEK293 cells ectopically expressing murine CCR8. The binding properties of surrogate antibodies TPP-15285 (chimerized with mIgG2a), TPP-29338 (chimerized with mouse IgG2a with H310Q / H330N mutations that interfere with interaction with mouse FcRn), TPP-14099 (human IgG1), and TPP-18208 (an aglycosylated hIgG1 version of TPP-14099 in which the N297A mutation completely removes the N-glycosylation site of the antibody) were compared to mIgG2a and hIgG1 non-binding isotype controls (TPP-10748 and TPP-9809, respectively).

[0671] TPP-15285, TPP-29338, TPP-14099 and TPP-18208 surrogate antibodies showed specific binding to murine CCR8. TPP-29338 had an unexpectedly 10-fold higher affinity than TPP-15825 (Table 4.1). In summary, these antibodies are suitable murine surrogates for the anti-human CCR8 antibody candidate TPP-23411 and can be used for in vivo efficacy and mode of action studies in mice.

[0672] Table 4.1: Binding affinities of the tested antibodies to murine CCR8 transfected HEK293 cells. 50 : Concentration required for 50% effect.

[0673]

[0674] Example 5: In vitro mode of action study: ADCC evaluation of anti-human CCR8 antibody TPP-23411

[0675] The ADCC mode of action of the non-fucosylated TPP-23411 antibody was evaluated in the CytoTox-Glo assay using CCR8-expressing target cells and NK92V (NK-like cell) effector cells at an effector cell to target cell ratio (E:T) of 4: 1. In addition, wild-type fucosylated variants of TPP-23411 (all hIgG1) and hIgG1 isotype control antibodies with non-fucosylated and wild-type fucosylated forms were analyzed.

[0676] The higher the level of CCR8 expression on the target cells, the higher the maximum ADCC response. Using activated human Tregs with 85.0% CCR8 expression, the maximum ADCC response to TPP-23411 was 52.7%, while using Tregs with 31.4% CCR8 expression it was 19.5%. Non-fucosylation of TPP-23411 enhances the affinity of the antibody to FcγRIII, thereby significantly enhancing the ADCC potential of therapeutic antibodies. In summary, TPP-23411 triggers potent and dose-dependent depletion of human Tregs expressing CCR8 and human-CCR8-HEK293 cells, with EC50 values ​​ranging from 55.9pM to 12.8pM.

[0677] Table 5.1: Summary of human ADCC parameters for the antibodies tested. Non-fucosylated and wild-type TPP-23411, anti-CCR8 antibody; TPP-9809, isotype control; N / A: not applicable; EC 50 : Concentration required for 50% effect; a 85.0% CCR8 expression; b 31.4% CCR8 expression.

[0678]

[0679] Example 6: In vitro mode of action study: ADCP evaluation of anti-human CCR8 antibody TPP-23411

[0680] The ADCP mode of action of non-fucosylated TPP-23411 was evaluated by flow cytometry-based assay using activated human Tregs endogenously expressing CCR8 or HEK293 cells ectopically expressing CCR8 as target cells and M2c macrophages as effector cells at an effector cell to target cell ratio (E:T) of 4: 1. In addition, the wild-type fucosylated variant of TPP-23411 and the hIgG1 isotype control antibody in both non-fucosylated and wild-type fucosylated forms were also analyzed.

[0681] Neither antibody non-fucosylation nor CCR8 expression levels on target cells affect ADCP activity. In the presence of M2c macrophages, both antibodies (wild-type and non-fucosylated TPP-23411) induce comparable dose-dependent ADCP responses against human Tregs. The maximum response to TPP-23411 using human Tregs with 63% CCR8 expression was 49.9%, while that using Tregs with 40% CCR8 expression was 49.3%. The corresponding EC50 values ​​were 143pM and 440pM, respectively. In summary, TPP-23411 triggers a strong and dose-dependent phagocytosis of both, human Tregs expressing CCR8 and human-CCR8-HEK293 cells, with a maximum ADCP response range of 49.9% to 26.6% (Table 6.1).

[0682] Table 6.1: Summary of human ADCP parameters for the antibodies tested. TPP-9809, isotype control; N / A: not applicable; EC 50 : Concentration required for 50% effect; a 63% CCR8 expression; b 40% CCR8 expression.

[0683]

[0684] Example 7: In vitro mode of action study: ADCC evaluation of murine surrogate antibodies to TPP-23411

[0685] HEK293 cells expressing mouse CCR8 were used as target cells and mouse NK cells as effector cells, and the ADCC action mode of TPP-23411 was evaluated by an effector cell to target cell ratio (E:T) of 10:1. Mouse-CCR8-HEK293 target cells were double-labeled with Cytolight Red Rapid Dye to mark live cells and Caspase 3 / 7Green Dye to mark apoptotic cells, and cells were imaged in Incucyte for 24 hours. Normal half-life anti-mouse CCR8 antibody TPP-15285, an artificially modified short half-life variant of TPP-15285 named TPP-29338 (all mIgG2a), and mIgG2a derived isotype control (TPP-10748) were evaluated in dose response. In addition, anti-mouse CCR8 antibodies (TPP-14099 and TPP-18208, respectively) with human IgG1 or non-glycosylated human IgG1 forms were also evaluated.

[0686] Compared to isotype controls, TPP-15285, TPP-29338, and TPP-14099 surrogate antibodies induced clear dose-dependent ADCC responses. TPP-15285 demonstrated EC 50 The maximum ADCC response was 227.38 pM, while the short half-life variant TPP-29338, which has a higher cell affinity for mouse CCR8, is more potent and has an EC 50 The maximum ADCC response of TPP-14099 was 9.28 pM and 31.92%. 50 The p-value of TPP-18208 was 584 pM, and the maximum ADCC response was 16.43%, while its N297A non-glycosylated variant TPP-18208 showed no ADCC activity (Table 7.1).

[0687] In summary, efficient ADCC generally requires the fully functional Fc portion of the anti-mouse CCR8 surrogate antibodies TPP-15285, TPP-29338, and TPP-14099. These antibodies are suitable murine surrogates for the anti-human CCR8 antibody TPP-23411 and can be used for in vivo efficacy and mode of action studies in mice.

[0688] Table 7.1: Summary of ADCC parameters for surrogate antibodies. ADCC measured at 20 hours. a Calculated relative to no antibody control. b Normal 1 / 2 Mouse surrogate antibody. c Short T 1 / 2 Mouse surrogate antibody. d Isotype controls. e Conventionally glycosylated mouse surrogate antibodies. f Non-glycosylated mouse surrogate antibody. N / A, not applicable.

[0689] Parameter a <![CDATA[TPP-15285 b ]]> <![CDATA[TPP-29338 c ]]> <![CDATA[TPP-10748 d ]]> <![CDATA[TPP-14099 e ]]> <![CDATA[TPP-18208 f ]]> <![CDATA[EC 50 (ng / mL)]]> 32.60 1.33 N / A 87.64 N / A <![CDATA[EC 50 (pM)]]> 227.38 9.28 N / A 584 N / A Maximum response (%) 24.64 31.92 N / A 16.43 N / A

[0690] Example 8: In vitro mode of action study: ADCP evaluation of murine surrogate antibodies to TPP-23411

[0691] Using HEK293 cells expressing mouse CCR8 as target cells and mouse M2 macrophages as effector cells, the ADCP mode of action of the murine surrogate antibody of TPP-23411 was evaluated at an effector cell to target cell ratio (E:T) of 4:1. Mouse-CCR8-HEK293 target cells were double labeled with Cytolight Red Rapid Dye to label live cells and pHrodo Red cell Labeling Dye to label phagocytes, and cells were imaged for up to 12 hours in Incucyte. The ADCP potential of the normal half-life anti-mouse CCR8 antibody TPP-15285, an artificially modified short half-life variant of TPP-15285 named TPP-29338 (both mIgG2a), and the mIgG2a-derived isotype control TPP-10748 was evaluated. In addition, ADCP of anti-mouse CCR8 antibodies in human IgG1 or aglycosylated human IgG1 format (TPP-14099 and TPP-18208, respectively), and a non-binding hIgG1 isotype control (TPP-9809) were also evaluated.

[0692] TPP-15285, TPP-29338 and TPP-14099 surrogate antibodies induce comparable dose-dependent ADCP responses. After 4 hours of co-culture, the surrogate antibodies produce a maximum ADCP response between 67% and 75%, and after 12 hours of co-culture, the maximum ADCP response is between 92% and 94%. At 4 hours and 12 hours, it was confirmed that the EC50 of TPP-15285 was 379.8pM and 158.1pM, respectively, while the EC50 of its short half-life variant TPP-29338 was 518.3pM and 197.3pM, respectively. It was confirmed that the EC50 of TPP-14099 was 584pM and the maximum ADCC response was 16.43%, while its N297A non-glycosylated variant TPP-18208 showed no ADCC activity (Table 8.1).

[0693] In summary, efficient ADCC generally requires the fully functional Fc portion of the anti-mouse CCR8 surrogate antibodies TPP-15285, TPP-29338, and TPP-14099. These antibodies are suitable murine surrogates for the anti-human CCR8 antibody TPP-23411 and can be used for in vivo efficacy and mode of action studies in mice.

[0694] Table 8.1: Summary of cumulative ADCP evaluations for TPP-15285, TPP-29338, TPP-14099, and TPP-18208. a Calculated relative to the lowest concentration of isotype control. b Normal half-life mouse surrogate antibody with mIgG2a format.c A short half-life mouse surrogate antibody with mIgG2a format. d Mouse surrogate antibody with hIgG1 format. e Aglycosylated mouse surrogate antibody in hIgG1 format. N / A: Not applicable.

[0695]

[0696] Example 9: In vivo main pharmacodynamics

[0697] Since TPP-23411 cross-reacts only with cynomolgus monkeys and not with mouse CCR8 orthologs, the in vivo mode of action and efficacy evaluation of CCR8+ Treg depletion was performed in mice using a murine surrogate antibody. In vivo studies were performed as monotherapy or in combination with ICIs (e.g., anti-PD-1 and anti-PD-L1 antibodies).

[0698] Example 9.1: In vivo efficacy of normal half-life and short half-life murine surrogate antibodies in a syngeneic EMT6 murine carcinoma model

[0699] After a single iv administration at a dose of 5 mg / kg, the pharmacokinetics of two anti-mouse CCR8 replacement antibodies, TPP-15285 (mIgG2a replacement antibody for TPP-23411) and TPP-29338 (mIgG2a replacement antibody with H3 10Q / H330N mutations, interfering with the interaction with murine FcRn) were studied in mice. For TPP-15825, the plasma clearance was 0.0012 L / (h*kg), the distribution volume (Vss) was 0.16 L / kg, and the plasma elimination half-life was 141 hours. For TPP-29338, the plasma clearance was 0.0054 L / (h*kg), the distribution volume (Vss) was 0.14 L / kg, and the plasma elimination half-life was 27 hours.

[0700] The in vivo anti-tumor efficacy of two anti-mouse CCR8 replacement antibodies TPP15285 (mIgG2a replacement antibody with normal half-life) and TPP-29338 (mIgG2a replacement antibody with short half-life) was analyzed using a syngeneic EMT6 murine breast cancer model in a monotherapy setting. In addition, changes in CCR8+Tregs within the tumor during the study were also determined. TPP-29338 (a short half-life variant of TPP15285) was used to mimic the short half-life predicted by the anti-human antibody TPP-23411 in mice.

[0701] Treatment with TPP-29338 (a short half-life surrogate antibody) at 0.3, 1, or 3 mg / kg produced T / C values ​​of 0.90, 0.72, and 0.22, respectively, showing dose-dependent antitumor efficacy. TPP-15285 (a normal half-life surrogate antibody) produced a T / C value of 0.26 at 1 mg / kg. Both TPP-29338 at 3 mg / kg and TPP-15285 at 1 mg / kg depleted CCR8+Tregs in the tumor. All treatments were well tolerated, as shown by the average weight gain in all treatment groups.

[0702] In summary, the murine surrogate antibodies of TPP-23411 all showed potent in vivo activity, and the short half-life surrogate anti-CCR8 antibody TPP-29338 required a higher dose than the normal half-life surrogate anti-CCR8 antibody TPP-15285 to achieve comparable exposure (Table 9.1.1) and induced comparable intratumoral CCR8+ Treg depletion, as well as in vivo antitumor efficacy.

[0703] Table 9.1.1: Plasma concentrations of TPP-15285 and TPP-29338 in EMT6 tumor-bearing mice treated with TPP-15285 or TPP-29338. N = 3 / time point, except a n=1. GM: geometric mean; GSD: geometric standard deviation; LLQ: lower limit of quantitation; TPP-15285, a murine surrogate antibody of the anti-human CCR8 hIgG1 antibody TPP-23411 with a normal half-life (141 hours), TPP-29338, a murine surrogate antibody of the anti-human CCR8 IgG1 antibody TPP-23411 with a short half-life (27 hours). Blood samples were collected 24, 48 and 72 hours after the first treatment for pharmacokinetic analysis by ELISA.

[0704]

[0705]

[0706] Example 9.2: PK / PD relationship of murine surrogate antibodies TPP-15285 and TPP-29338 in a syngeneic EMT6 mouse cancer model

[0707] In order to assess the time course of Treg depletion and repopulation, and better understand the PK / PD relationship of TPP-15285 and TPP-29338 antibodies (all mIgG2a, a substitute for anti-human CCR8 antibody TPP-23411), isogenic EMT6 mouse breast cancer model is used for further study. TPP-29338 (a short half-life variant of TPP-15285) is used to simulate the short half-life of the prediction of TPP-23411 in mice. Female BALB / cAnN mice from Charles River are randomly divided into groups, and TPP-15285 (with a single dose of 0.25, 1 and 4 mg / kg), TPP-29338 (with a single dose of 0.25, 1 and 4 mg / kg) or mIgG2a isotype control TPP-10748 are used for ip treatment after sc inoculation of EMT6 murine breast cancer cells for 8 days. Mice were sacrificed at 24, 48, 120, 192, and 336 hours after a single dose of compound (n=5 / group / time point), or when tumors reached a predetermined size of 225 mm 2 Mice were sacrificed at 4 ℃ and 1 ℃. Tumors and blood were collected for pharmacokinetic analysis and flow cytometry quantification of T cell populations.

[0708] Treatment with TPP-15285 and TPP-29338 resulted in intratumoral depletion of CCR8+ Treg cells when compared to isotype controls as determined by flow cytometry. CCR8+ Tregs repopulated more rapidly in the TME when mice were treated with the short half-life variant TPP-29338 compared to the normal half-life variant TPP-15285.

[0709] In summary, a direct correlation was observed between antibody plasma concentrations and the efficacy of intratumoral CCR8-positive Treg depletion for two murine surrogate antibodies, TPP-15285 and TPP-29338 (Table 9.2.1, Table 9.2.2), suggesting that a threshold antibody concentration may need to be permanently covered to ensure Treg depletion and downstream effects.

[0710] Table 9.2.1: TPP-15285 and TPP-29338 plasma concentrations in EMT6 tumor-bearing mice. GM: geometric mean; GSD: geometric standard deviation; LLQ: lower limit of quantitation. a The normal half-life (t 1 / 2 = 141 hours) murine replacement antibody variant. b The short half-life (t 1 / 2 = 27 hours) murine surrogate antibody variant. c n=4.d n=2. e n=3. f n = 1. Note: n = 5 / time point unless otherwise stated.

[0711]

[0712] Table 9.2.2: Percentage of CCR8-positive cells in tumors of EMT6 tumor-bearing mice at various time points and after treatment with varying amounts of TPP-15285 and TPP-29338.

[0713]

[0714] Example 9.3: IFN-γ concentration measured in tumor or blood of EMT6 tumor-bearing mice

[0715] At the end of the study, day 19, IFN-γ concentrations in tumors (Table 9.3.1) and blood (Table 9.3.2) of EMT6 tumor-bearing mice tested with TPP-10748 isotype control or TPP-15285 were measured by ELISA analysis.

[0716] Table 9.3.1: Intratumoral IFN-γ concentrations (pg / L) in EMT6 tumor-bearing mice treated with TPP-10748 or anti-mouse CCR8 antibody TPP-13285.

[0717]

[0718] Table 9.3.2: Blood IFN-γ concentrations (pg / L) in EMT6 tumor-bearing mice treated with TPP-10748 or anti-mouse CCR8 antibody TPP-13285.

[0719]

[0720] Example 10: Evaluation of Pharmacokinetic / Pharmacodynamic (PK / PD) Relationship and Effective Exposure

[0721] To establish PK / PD relationships, concentrations producing maximal efficacy (EC80 predicted for effective dose) and minimal efficacy (EC20 predicted for minimum expected efficacy level) were derived from human in vitro ADCC and ADCP assays (see Examples provided elsewhere herein).

[0722] These concentrations were corrected by additional in vitro versus in vivo correlation (IVIVC) factors reported from mice, based on Treg depletion observed by in vitro ADCC and ADCP assays as well as in vivo (see Examples provided elsewhere herein).

[0723] Furthermore, given the empirical correlation of Treg depletion with tumor shrinkage in mice, it was demonstrated that when Treg depletion was greater than 50% relative to baseline, the T / C ratio was < 0.5.

[0724] Since the information about Treg dynamics in humans is too limited, persistent Treg depletion is assumed to be required for anti-tumor efficacy. Because a direct correlation between more exposure and Treg depletion was observed in indicative in vivo exposure-response studies in mice, it is assumed that persistence covers the threshold antibody concentration to ensure sustained Treg depletion required for anti-tumor efficacy. Therefore, in humans, the trough concentration at steady state (Ctrough) is assumed to be the driving factor for Treg depletion and any further effects on anti-tumor immunity.

[0725] In summary, for human effective dose predictions, the estimated EC80 associated with an expected -50% Treg depletion should target the C trough. For the minimum effective dose level predicted to be associated with -10% Treg depletion, the estimated EC20 should target the C trough. In addition to the estimated range of minimum effective concentrations, Table 10.1 provides the estimated range of effective concentrations in humans.

[0726] Table 10.1: Extrapolated results of effective and minimal biologically effective concentrations for Treg depletion of TPP-23411 in humans.

[0727]

[0728] Example 11: Safety Pharmacology

[0729] The effects of TPP-23411 on vital organ functions (central nervous system [CNS], cardiovascular system including ECG, respiratory system) were investigated according to ICH guidelines S6 and S9, i.e., the corresponding endpoints were included in the pivotal 4-week toxicity study in cynomolgus monkeys.

[0730] Safety pharmacology endpoints included detailed clinical observations, physical / neurological examinations (abdominal palpation, temperature, cardiopulmonary auscultation, general sensory aspects including cerebral [pupil, orbicularis oculi] and spinal reflexes [knee, anus], and foot grasp reflex), respiratory rate, arterial blood pressure (high-resolution oscillometric), and ECG (30 to 60 second recordings). These studies were conducted once during the pre-dose phase, during weeks 1 and 4 of the dosing phase (pre-dose and 1 to 2 hours post-dose), and during week 2 of the recovery phase. Systemic exposure to TPP-23411 was also determined.

[0731] TPP-23411 was administered to 3 to 5 male / female cynomolgus monkeys per group at doses of 2×0 (vehicle), 15, 50 and 2×40 mg / kg by iv slow push once / twice weekly. None of the above parameters were significantly affected by treatment at mean plasma concentrations up to 1550 mg / L (50 mg / kg). These plasma concentrations are >500-fold higher than those currently expected for human therapeutic efficacy (C values ​​in the dose range 38-1100 mg / kg). max The range is 0.7-20mg / L).

[0732] Example 12: Nonclinical Pharmacokinetics and Drug Metabolism

[0733] The pharmacokinetics of TPP-23411 were studied in vivo in male cynomolgus monkeys after single iv and sc administration of TPP-23411. TPP-23411 was measured using an anti-human IgG universal assay (IgG-ELISA) in monkey plasma. Anti-TPP24311 antibody formation was monitored using a validated TPP-23411-based bridging ELISA method (described elsewhere herein).

[0734] Table 12.1: Summary of single-dose pharmacokinetic data, in vivo studies. iv: intravenous; sc: subcutaneous; PK: pharmacokinetic; m: male. The observation interval was 336 hours for the low dose and 504 hours for the high dose.

[0735] Type of study Single-dose pharmacokinetics, in vivo studies Test system Monkey, 3 males / dose group, strain: Cynomolgus monkey dose TPP-23411: 1 and 10 mg / kg iv and 3 and 10 mg / kg sc Analysis, Matrix IgG ELISA, plasma samples

[0736] Table 12.2 shows the pharmacokinetics of TPP-23411 after intravenous and subcutaneous administration. Figure 2 Shown are dose-normalized plasma concentrations of TPP-23411 following intravenous and subcutaneous administration.

[0737] Following single intravenous bolus administration of 1 and 10 mg / kg of TPP-23411 to male cynomolgus monkeys, exposure of AUCnorm increased slightly beyond dose proportion from 391 kg·h / L to 617 kg· / L, with no suggestion of target-mediated drug disposition. Plasma elimination was biphasic, and plasma clearance was 2.55 mL / (h·kg) for the 1 mg / kg dose and 1.62 mL / (h·kg) for the 10 mg / kg dose (mean). The volume of distribution for the 1 and 10 mg / kg doses reached 0.154 and 0.110 L / kg (mean), respectively. The effective half-life was short, 41.9 hours and 46.8 hours, reflecting the relatively rapid elimination of the antibody, and the pharmacologically less relevant terminal elimination half-life was 108 and 148 hours.

[0738] After a single subcutaneous administration of 3 and 10 mg / kg of TPP-23411, the exposure according to AUCnorm increased from 331 kg·h / L to 403 kg·h / L in a dose-proportional manner, and Cmax,norm was observed to increase from 2.22 kg / L to 2.95 kg / L (mean value). Cmax (mean value) was reached 8 to 30 hours after administration. TPP-23411 was eliminated from plasma with a terminal half-life of 81 and 109h (mean value) at 3 and 10 mg / kg dose levels, respectively, and the plasma concentration run was synchronized with the intravenous spectrum. The bioavailability of both dose groups was moderate to high and ranged from 54% to 103%.

[0739] Table 12.2: Pharmacokinetics of TPP-23411 after a single dose (non-rodents). a : Bioavailability, calculated at an IV AUCnorm value of 1 mg / kg bw at 3 mg / kg sc and at an IV AUCnorm value of 10 mg / kg bw at 10 mg / kg sc; b : Not calculated.

[0740]

[0741] No significant anti-drug antibody formation was detected.

[0742] Example 13: Toxicology

[0743] The overall goals of the toxicity program are to identify target organs of toxicity, assess reversibility, delayed findings, and determine exposure-response relationships.

[0744] TPP-23411 is a human monoclonal antibody that is not cross-reactive in animal species other than non-human primates. Therefore, the in vivo toxicology program included a comprehensive evaluation only in cynomolgus monkeys. The amino acid sequence of CCR8 is 94% identical between humans and cynomolgus monkeys, and TPP-23411 binds human and cynomolgus monkey CCR8 with similar affinity. In addition, monkeys are considered the species most similar to humans in terms of immune function.

[0745] The toxicology program in monkeys consisted of a high-dose toxicokinetic study, a 4-week exploratory study with repeated administration, and a pivotal 4-week repeated-dose toxicity study under immunostimulatory conditions (KLH immunization) including a washout / recovery phase. In vivo toxicity studies included some non-routine immunological parameters such as immunophenotyping, cytokine levels, and inflammatory markers, and mRNA sequence evaluation on CCR8 mRNA expression was evaluated for selected tissues.

[0746] In addition, comprehensive tissue cross-reactivity studies and in vitro cytokine release assays were performed using human whole blood and human PBMCs.

[0747] Pivotal studies were performed in accordance with OECD principles for GLP at the testing facilities of Charles River Laboratories Evreux, France, and Labcorp Early Development Services GmbH (formerly Covance Preclinical Services GmbH) Münster, Germany. A detailed overview of the toxicology studies performed with TPP-23411 is provided in Table 13.1 below.

[0748] Table 13.1: Toxicology procedures. GLP: good laboratory practice; TK: toxicokinetics; KLH: keyhole limpet hemocyanin; PBMC: peripheral blood mononuclear cell; TCR: tissue cross-reactivity; CRA: cytokine release assay; iv: intravenous; sc: subcutaneous. a 17 or 50 mg / kg were given iv twice weekly and 50 mg / kg was given sc as a single dose.

[0749]

[0750] Local tolerance was evaluated in a repeat-dose toxicity study in monkeys. No signs of local intolerance reactions were observed following administration of the TPP-23411 formulation by histopathological evaluation of the injection site.

[0751] Example 13.1: Repeated Dose Toxicity Study in Monkeys: Toxicokinetic Study in Cynomolgus Monkeys with Once-Weekly sc Administration and Twice-Weekly iv Administration

[0752] A preliminary high-dose toxicokinetic study was conducted to evaluate the toxicokinetic profile of TPP-23411 in plasma following one sc and two iv administrations in cynomolgus monkeys. Animals received a single sc administration of 50 mg / kg on days 1 and 4 or twice weekly doses of 17 mg / kg or 50 mg / kg of TPP-23411.

[0753] TPP-23411 was well tolerated both systemically and locally up to the highest dose. During the morning feeding, reduced food consumption was observed in all animals in all dose groups. The second feeding (overnight) was normal. The reduced food intake, accompanied by a slight loss of body weight (1% to 5%) was not due to the test material but to the handling of repeated blood draws. Necropsy and histopathology were not performed in this study. The mean plasma concentrations and pharmacokinetic parameters of TPP-23411 are compiled in Tables 13.1.1 and 13.1.2 below.

[0754] Table 13.1.1: TPP-23411 Exposure on Day 1 in the High-Dose Pharmacokinetic Study in Monkeys

[0755]

[0756] Table 13.1.2: TPP-23411 exposure in a high-dose pharmacokinetic study in monkeys on Day 4. RA-AUC = cumulative ratio (AUC(0-last) 第4天 / AUC(0-last) 第1天 );RA-C max = Cumulative ratio (C max，第4天 / C max，第1天 )

[0757]

[0758] Example 13.2: Repeated Dose Toxicity Study in Monkeys: Four-week Repeated Dose Toxicity Study in Cynomolgus Monkeys with Weekly IV Administration

[0759] Table 13.2.1: Key data from the exploratory repeated-dose (4-week) toxicity study in monkeys

[0760]

[0761] Two female cynomolgus monkeys in four groups were administered by iv at 0, 6, 17 or 50 mg / kg / injection at a constant dose volume of 2 mL / kg / administered on the 1st, 8th, 15th and 22nd days to receive TPP-23411. The assessment of toxicity is based on survival, clinical signs, body weight, food consumption, electrocardiogram, blood pressure recording, ophthalmological examination, temperature recording, functional observation group system (functionalobservation battery, FOB), hematology, coagulation and blood biochemistry analysis, urine analysis, immunophenotyping (IPT) of blood, C-reactive protein (CRP) and cytokine / chemokine level determination, organ weight and macroscopic and microscopic examination. Blood samples were collected for toxicokinetics and immunogenicity evaluation. In addition, samples of blood and selected tissues were also collected for deep RNA sequencing.

[0762] Clinical observation

[0763] There were no unplanned deaths during the study. No clinical signs related to the test item were observed. Body weight, food consumption and ECG quantitative and qualitative parameters were not affected at any dose level during the study. No ophthalmological findings were observed at the end of the treatment period.

[0764] Clinical Pathology

[0765] There were no test item-related changes in any hematology, coagulation, blood chemistry, and urine parameters or CRP levels throughout the study. The test items had no effect on any cytokine levels measured during the study (IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-10, IL-6, and IL-8). Immunotoxicity

[0766] T lymphocytes, cytotoxic T lymphocytes (conventional, activated, and regulatory profiles), helper T lymphocytes (conventional, activated, and regulatory profiles), CD4- / CD8-T lymphocytes (conventional, activated, and regulatory profiles), natural killer T lymphocytes, B lymphocytes, and immunophenotyping (IPT) of natural killer cells did not reveal evidence of immunotoxicity. All populations studied were within normal ranges, with the exception of a trend toward a slight decrease in natural killer cells after administration of TPP-23411 (not statistically significant).

[0767] Cross-Pathology, Histopathology

[0768] In all treated females, administration of TPP-23411 at ≥6 mg / kg / injection induced non-adverse minimal or slightly reduced lymphoid cellularity in the iliac and axillary lymph nodes, particularly in the germinal centers (i.e., in the secondary lymphoid follicles), however without any dose relationship. In the thymus, at ≥6 mg / kg / injection, lymphoid cellularity was minimally reduced in 1 of 2 females in each group, associated with lower thymus weight. It is still unclear whether this finding is related to TPP-23411 or due to physiological thymic involution. In addition, a slight increase in stainable macrophages in vivo was noted in 1 of 2 females injected at 17 mg / kg / , and a slight increase was seen in both females injected at a dose of 50 mg / kg. No changes associated with TPP-23411 were observed at the injection site, indicating that the local tolerance of the test item was good.

[0769] Deep RNA sequencing analysis

[0770] Whether anti-CCR8 treatment can lead to significant exhaustion of Treg or changes in immune cell type-specific marker expression, RNA sequencing of different tissue samples from cynomolgus monkeys was performed. Samples collected from the following tissues: intestine (ileum, jejunum, cecum and colon), lymph nodes (mandible, mesentery, axilla and iliac), liver, spleen, lung, skin, thymus and tonsil. In line with expectations, CCR8mRNA is preferably detected in thymus tissue and different lymph node samples. Except for 2 thymus samples from the highest dose treatment group, CCR8mRNA levels were not detected in any tissue and there was a corresponding decrease. After multiple test corrections, these changes were not significant and did not show consistent dose dependence.

[0771] Evaluation of the expression of the pro-inflammatory markers CXCL9 / 10 and IFN-γ, the cytotoxic T-cell marker CD8A / B, the NK-cell marker NCR1 / SH2D1B, the B-cell marker CD19 / CD20 / CD22, the M2 macrophage marker CSF1R / MRC1 / CD163, and the FCg receptor FCGR2A / 2B did not indicate significant changes in any of these markers in any of the tissues evaluated.

[0772] Table 13.2.3: Notable findings from an exploratory repeated dose (4 week) toxicity study in male (M) and female (F) cynomolgus monkeys.

[0773]

[0774]

[0775] Toxicokinetic evaluation

[0776] The results of the assays are summarized in Table 13.2.2. The low and mid dose groups showed clear dose linearity at steady state. Based on AUC(0-tlast)norm, the highest dose group showed out-of-proportional exposure on Day 22. On Day 22, Cmax,norm increased in a dose-proportional manner. No relevant accumulation was seen in all dose groups based on Cmax,norm. Slight increases were seen in all dose groups based on AUC(0-tlast)norm. The factor for AUC(0-tfinal)norm was most significant in the highest dose group (factor 1.5 times). At the end of the observation interval of 168 hours after dosing, no relevant concentrations remained.

[0777] Table 13.2.2: TPP-23411 exposure in an exploratory 4-week toxicology study in monkeys on Day 22. RA-AUC = cumulative ratio (AUC(0-last) Day 22 / AUC(0-last) Day 1); RA-Cmax = cumulative ratio (Cmax, Day 22 / Cmax, Day 1).

[0778]

[0779] Example 13.3: Four-week Repeated-dose Toxicology Study in Cynomolgus Monkeys Using Once-weekly or Twice-weekly IV Immunization with KLH and a 2-week Treatment-free Period

[0780] A pivotal GLP repeated dose study was conducted in male and female cynomolgus monkeys with a treatment period of 4 weeks. TPP-23411 was administered intravenously once a week at doses of 15 mg / kg and 50 mg / kg and twice a week at doses of 0 mg / kg (vehicle control) and 40 mg / kg. The dose level was selected based on exploratory toxicokinetics and repeated dose toxicology studies, supplemented with simulation models to compensate for the short half-life of TPP-23411 in cynomolgus monkeys.

[0781] Table 13.3.1: Treatment Groups. 2qw = twice a week. a : Group 1 was administered vehicle only control; b : Animals were dosed at a volume of 2 mL / kg; c : Group 1 and Group 4: twice weekly (2qw) on days 1, 4, 8, 11, 15, 18, 22, 25, and 29. Group 2 and Group 3: once weekly on days 1, 8, 15, 22, and 29; d : Three animals / sex / group were designated as terminal animals, and 2 animals / sex / Group 1 and Group 4 were designated as recovery animals (based on survival); e : Total 80 mg / kg / week.

[0782]

[0783] The goal of this pivotal study is to determine potential toxicity, including the highest non-severe toxic dose of TPP-23411 formulated in 5% glucose solution when administered intravenously once or twice a week to cynomolgus monkeys for 4 weeks to treat cancer, and to evaluate the potential reversibility of any findings during the 2-week recovery period. Evaluation of toxicity is based on survival, clinical signs, body weight, food consumption, electrocardiogram, blood pressure recording, ophthalmology, temperature recording, FOB, respiratory rate, hematology, coagulation and blood biochemistry analysis, urine analysis, organ weight, and macroscopic and microscopic examination. In addition, a wide range of immunotoxicological endpoints such as blood IPT, cytokine / chemokine levels (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, MCP-1, IFN-γ and TNF-α) and KLH immunization are included. Blood samples are collected for toxicokinetics and immunogenicity (anti-drug antibodies [ADA]) evaluation. Additionally, samples of blood and selected tissues (mesenteric lymph nodes, thymus, and tonsils) were collected for deep RNA sequencing.

[0784] Table 13.3.2: Key Data from the Pivotal Repeated-Dose (4-Week) Toxicity Study in Monkeys

[0785]

[0786] Clinical observation

[0787] Deaths were not affected by TPP-23411 administration. No overt signs of toxicity were observed during the study, as well as no effects on food consumption or body weight.

[0788] Clinical Pathology

[0789] No relevant effects were found on hematology, coagulation, clinical chemistry, ECG, or ophthalmology.

[0790] Immunotoxicology parameters

[0791] The test items had no adverse effects on the levels of any of the cytokines measured during the study. Levels of IL-2, IL-4, IL-5, IL-6, and TNFα were generally low throughout the study and considered to be within normal physiological variation. Slight elevations in IFN-γ were noted in a few animals at high doses, and a potential relationship to TPP-23411 cannot be ruled out. Elevated levels of IL-1β were noted in 1 high-dose animal and IL-10 in 2 high-dose animals, above those before treatment alone or in the control group.

[0792] Evaluation of humoral immune responses following antigenic stimulation with KLH showed no evidence of alterations based on test item administration.

[0793] Treatment with TPP-23411 between 15 and 50 mg / kg once a week or 40 mg / kg twice a week had no adverse effects on any leukocyte subsets as measured by flow cytometry during this study. Throughout the dosing phase, absolute CD16+NK cell counts were noted to decrease at ≥15 mg / kg in females on days 15 and 29 and in males on days 8 and 15. Compared to the group mean before the second dose, the average NK cell counts in males and females were reduced by 74% and 90% at a dose of 40 mg / kg twice a week on day 29, respectively. This effect was not reversible until the end of the 2-week recovery period.

[0794] Cross-Pathology, Histopathology A modest increase in spleen weights was observed at necropsy in males at all dose levels and in females at 40 mg / kg twice weekly or 50 mg / kg once weekly. This was consistent with a higher incidence of gross organomegaly. In the absence of microscopic correlation, this finding is considered incidental.

[0795] All histopathological changes matched the spectrum of spontaneous pathology commonly seen in cynomolgus monkeys of this age and origin.

[0796] TPP-23411 was well tolerated at the injection site. Surgery-related changes such as fibrosis, edema, hemorrhage, or inflammatory cell infiltration were observed with similar incidence and severity in control and TPP-23411-administered animals.

[0797] Minimal ADA formation was observed in all dose groups including the control group. A moderate ADA response was observed only in one animal in control group 1, suggesting the presence of pre-existing antibodies. In all other animals with ADA formation, only low responses were detected at 0 hours on day 1, 0 hours and 168 hours on day 22, and 336 hours on day 29. No effect on TPP-23411 plasma concentrations was detected in the affected animals. Therefore, the sporadic occurrence of ADA in all study groups is considered to be accidental and without any biological relevance.

[0798] With regard to toxicokinetics, no relevant sex differences in systemic exposure to TPP-23411 were observed. For all dosing levels, C was reached after 0.25 h on Day 1. max However, on day 22, C was reached mainly after 1 hour. max On day 22, when the dose of TPP-23411 was increased from 15.0 mg / kg to 50.0 mg / kg in male and female monkeys, AUC 0-168 and C maxThe AUC values ​​of the 15.0 mg / kg group and the 50.0 mg / kg group were calculated by comparing all groups. 0-144 and by combining the AUC on days 1 and 4 or days 22 and 25 0-72 Estimate Group 4. On Day 22, the AUC of the 40 mg / kg dose group 0-144 The significant increase compared to the 15.0 mg / kg dose group and the modest increase compared to the 50 mg / kg dose group were due to twice weekly versus once weekly dosing.

[0799] On days 22 and 25, C max The increase was approximately dose proportional compared to Day 22 for the 15.0 and 50.0 mg / kg dose groups.

[0800] If animals are dosed once weekly, repeated dosing will not affect the AUC 0-168 and C max However, if TPP-23411 was administered twice weekly, the combined AUC levels were slightly upregulated (compared to the levels on day 1 / 4) with slightly cumulative doses per week. max The level was slightly increased compared to the first day.

[0801] Table 13.3.3: Summary of exposures at Day 22 from the repeat dose (4 week) toxicity study in male (M) and female (F) monkeys. a : extrapolated value. AUC: area under the plasma concentration versus time curve; AUC (0-144) : AUC from 0 to 144 hours; AUC (0-144)norm : AUC normalized to dose and body weight (0-144) ; C max : Maximum drug concentration in plasma; C max,norm : C normalized to dose and body weight max ;t max : time to reach maximum plasma drug concentration; C(tint): concentration at 168 hours; RA AUC : Cumulative ratio calculated from AUCτ after multiple administrations and AUCτ after single administration; RA cmax : Relative maximum drug concentration in plasma; RA AUC(0-168) : RA from 0 to 168 hours AUC . .

[0802] dose [mg / kg] 15 50 2x40 gender M+F M+F M+F <![CDATA[C max ]]> [μg / mL] 418 1490 1400 <![CDATA[t max ]]> [h] 1.00 0.625 1.00 <![CDATA[C max,norm ]]> [kg / L] 27.9 29.8 17.5 <![CDATA[C (tint) -C max ]]> [％] 2.65 4.99 18.4 <![CDATA[AUC (0-144) ]]> [μg·h / mL] 11400 41300 <![CDATA[78800 a ]]> <![CDATA[AUC (0-144)norm ]]> [kg·h / L] 761 825 <![CDATA[985 a ]]> <![CDATA[AUC all ]]> [μg·h / L] 11700 43300 78800 <![CDATA[AUC all,norm ]]> [kg·h / L] 781 865 985 <![CDATA[RA-AUC all ]]> [％] 101 87.0 144 <![CDATA[RA-C max ]]> [％] 89.6 86.8 128

[0803] Deep mRNA analysis

[0804] RNA sequencing studies were performed to evaluate whether the treatment with TPP-23411 had any general effects on gene expression in specific tissues of cynomolgus monkeys, such as the thymus, tonsils, and mesenteric lymph nodes. The gene CCR8 showed elevated baseline expression in the thymus when compared to the tonsils and mesenteric lymph nodes, but there was no significant change in gene expression after treatment with TPP-23411. Other target genes showed only minor, statistically insignificant expression changes after treatment in all three tissues, but the inter-animal variability in expression values ​​was considerable.

[0805] Table 13.3.4: Notable Findings from the Pivotal Repeated-Dose (4-Week) Toxicity Study in Cynomolgus Monkeys

[0806]

[0807]

[0808] Example 14: Cytokine Release Assay (CRA)

[0809] Cytokine release assays were performed using human whole blood (with added soluble antibody) and human PBMCs (with wet-coated antibody) to investigate the potential of TPP-23411 alone or in combination with pembrolizumab to activate the secretion of cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and TNFα were analyzed).

[0810] Example 14.1: Cytokine Release Assay - Study 1

[0811] Materials from 10 healthy donors (heparinized human peripheral blood) were incubated with PBS (negative control, NC), LPS / PHA (positive control, PC), commercially available control antibodies (anti-CD3 antibody OKT3 or anti-CD28 antibody ANC28.1, anti-CD20 antibody rituximab (MabThera), anti-EGFR antibody cetuximab (Erbitux)), TPP-23411 or the corresponding isotype control antibody TPP-9809 for 24 hours. All antibodies were tested at 3 different concentrations (50, 10 and 2 μg / mL). In addition, TPP-23411 and isotype control antibody TPP-9809 were tested in combination with 2 μg / mL of pembrolizumab. Cytokine concentrations in the supernatant were determined by multiple electrochemiluminescence methods.

[0812] Incubation of whole blood with soluble TPP-23411 antibody resulted in a significant dose-dependent release of IFN-γ to median concentration levels (among all donors) that were higher than those observed with control antibody MabThera, but lower than those observed with control antibody ANC28.1. IL-1β, IL-6, and TNFα were induced to levels comparable to or slightly higher than those observed with control antibody MabThera, but significantly lower than those observed with control antibody ANC28.1. Incubation of PBMCs with wet-coated TPP-23411 antibody induced a significant release of all analyzed cytokines (IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and TNFα) to levels higher than those obtained with control antibody MabThera, but lower than or comparable to those observed with control antibody OKT3.

[0813] In both assay formats, the combination of pembrolizumab and TPP-23411 produced comparable cytokine patterns, released cytokine concentration levels, and responder frequencies to the assay with TPP-23411 alone.

[0814] Table 14.1.1: CRA using PBMC / wet-coated antibodies. Median cytokine concentrations (pg / ml) after stimulation with the indicated antibodies and concentrations (50 μg / mL, 10 μg / mL, 2 μg / mL, or 50 μg / mL, 10 μg / mL, 2 μg / mL of TPP-23411 in combination with 2 μg / ml pembrolizumab (Pembro)).

[0815]

[0816]

[0817] Table 14.1.2: 95th percentile of mean cytokine concentrations (pg / ml) after stimulation of PBMCs with wet-coated Erbitux antibody (concentrations 50 μg / ml, 10 μg / ml, 2 μg / ml); N=8.

[0818] IFN-γ IL-10 IL-1β IL-2 IL-4 IL-6 IL-8 TNF-α Erbitux–50 14.16 0.49 6.44 1.76 0.02 35.76 5364.55 58.78 Erbitux–10 25.50 0.50 5.68 2.13 0.09 32.41 5058.72 41.62 Erbitux–2 16.98 0.34 5.93 2.63 0.07 29.22 4751.53 53.78

[0819] Table 14.1.3: CRA using PBMC / wet coated antibodies. Frequency of positive responses after stimulation with indicated antibodies and concentrations (50 μg / mL, 10 μg / mL, 2 μg / mL, or 50 μg / mL, 10 μg / mL, 2 μg / mL of TPP-23411 in combination with 2 μg / ml of pembrolizumab (Pembro)); N=8.

[0820]

[0821]

[0822] Table 14.1.4: CRA using whole blood / soluble antibodies. Median cytokine concentrations (pg / ml) after stimulation with the indicated antibodies and concentrations (50 μg / mL, 10 μg / mL, 2 μg / mL, or 50 μg / mL, 10 μg / mL, 2 μg / mL of TPP-23411 in combination with 2 μg / ml of pembrolizumab (Pembro)); N=10. .

[0823]

[0824]

[0825] Table 14.1.5: 95th percentile of mean cytokine concentrations (pg / ml) after stimulation of whole blood with soluble Erbitux antibody (concentrations 50 μg / ml, 10 μg / ml, 2 μg / ml); N=10. .

[0826] Antibody-μg / mL IFN-γ IL-10 IL-1β IL-2 IL-4 IL-6 IL-8 TNF-α Erbitux–50 17.10 0.50 0.84 0.53 0.11 1.68 388.40 1.57 Erbitux–10 14.19 0.57 0.82 0.66 0.02 1.15 330.38 1.89 Erbitux–2 18.12 0.08 2.18 1.32 0.03 3.77 300.83 1.95

[0827] Table 14.1.6: Frequency of positive responses after stimulation with the indicated antibodies and concentrations (50 μg / mL, 10 μg / mL, 2 μg / mL, or 50 μg / mL, 10 μg / mL, 2 μg / mL of TPP-23411 in combination with 2 μg / ml of pembrolizumab (Pembro)); N=10.

[0828] Antibody-μg / mL IFN-γ IL-10 IL-1β IL-2 IL-4 IL-6 IL-8 TNF-α MabThera–50 40 0 30 20 10 70 10 40 MabThera–10 40 0 90 0 70 100 50 40 MabThera–2 50 90 30 10 40 50 40 80 TPP-23411–50 100 40 90 20 0 100 30 100 TPP-23411–10 100 20 100 0 20 100 10 80 TPP-23411–2 80 70 0 0 10 0 10 30 IgG1 Iso Ctrl–50 20 30 50 20 10 40 20 30 IgG1 Iso Ctrl–10 20 10 20 20 30 40 20 30 IgG1 Iso Ctrl–2 10 80 0 20 40 10 10 10 Pembrolizumab-2 30 0 30 100 70 60 10 20 TPP-23411-Pembro–50 / 2 100 50 90 60 20 100 20 100 TPP-23411-Pembro–10 / 2 100 40 70 30 10 100 10 90 TPP-23411-Pembro–2 / 2 90 80 0 10 20 10 0 40 IgG1 Iso Ctrl / Pembro–50 / 2 20 30 60 10 0 30 30 20 IgG1 Iso Ctrl / Pembro–10 / 2 30 10 50 20 20 30 40 20 IgG1 Iso Ctrl / Pembro–2 / 2 10 80 10 0 30 0 20 10 Erbitux–50 0 0 0 0 0 0 0 0 Erbitux–10 0 0 0 0 0 0 0 0 Erbitux–2 0 0 0 0 0 0 0 0 ANC28.1–50 100 100 100 100 100 100 100 100 ANC28.1–10 90 90 90 90 90 90 80 90 ANC28.1–2 100 100 30 90 80 80 50 80

[0829] Example 14.2: In vitro cytokine release assay - Study 2

[0830] As a follow-up study, cytokine release assays were performed under the same assay conditions using lower concentrations of TPP-23411 ranging from 10 μg / mL to 0.128 ng / mL.

[0831] Materials from eight healthy donors were incubated for 24 h with PBS (negative control, NC), LPS / PHA (positive control, PC), or commercially available control antibodies (anti-CD3 antibody OKT3 or anti-CD28 antibody ANC28.1, anti-CD20 antibody rituximab (MabThera), anti-EGFR antibody cetuximab (Erbitux), anti-CD52 antibody alemtuzumab (Lemtrada), and anti-CCR4 antibody mogamulizumab (Poteligeo)), TPP-23411, or the corresponding isotype control antibody TPP-9809.

[0832] TPP-23411, its isotype control and Poteligeo were tested at 8 different concentrations (10 / 2 / 0.4 / 0.08 / 0.016 / 0.0032 / 0.00064 / 0.000128 μg / mL). All other commercially available antibodies were tested at 4 or 5 different concentrations (10 / 2 / 0.4 / 0.08 / 0.016 μg / mL).

[0833] The concentration of cytokines in the supernatant was determined. In addition, flow cytometry determination of the number of cells (monocytes, T cells, NK cells, granulocytes and lymphocytes) in the cell fractions set up for the cytokine release assay was performed after 24 hours of culture in two donors (stimulated with TPP-23411 alone, isotype control antibody, Lemtrada and Poteligeo).

[0834] Incubation of whole blood with soluble TPP-23411 antibody resulted in a dose-dependent release of IFN-γ, IL-1β, IL-6, TNFα, and IL-8. The median concentration levels (among all donors) at which cytokines were induced were lower than those observed with alemtuzumab, ANC28.1, and moglizumab (except that IL-1β levels were comparable to moglizumab, when compared to moglizumab, IL-8 concentrations at 10 μg / mL were higher). When compared to rituximab, TPP-23411 induced higher IFN-γ, IL-6, TNFα, and IL-8 cytokine levels at a dose of 10 μg / mL, but at comparable levels at the lower antibody doses tested. IL-1β was not induced by rituximab, but was induced by TPP-23411. IFN-γ is defined as the leading cytokine because its dose response is robust and has the highest sensitivity. Slight effects were observed at concentrations starting at 0.4 μg / mL.

[0835] Analysis of cell pellets from this cytokine release assay setup (using only the test item TPP-23411, isotype control antibody, alemtuzumab and moglizumab) by flow cytometry from 2 donors confirmed the expected dose-dependent reduction of monocytes, T cells, NK cells and lymphocytes due to Lemtrad. None of the other test items resulted in a reduction in any of those cell types. Only one of the 2 donors tested showed a reduction of less than 50% in monocytes at the highest concentration of 10 μg / mL when treated with TPP-23411.

[0836] Incubation of PBMCs with wet-coated TPP-23411 antibody induced a clear dose-dependent release of IFN-γ, IL-1β, IL-6, IL-8, IL-10, and TNFα, very little IL-2, and no induction of IL-4. Comparative antibodies moglizumab and alemtuzumab did show cytokine induction, but without a clear dose-response relationship. Median cytokine levels (across all donors) of IFN-γ, IL-1β, IL-6, IL-10, TNFα, and IL-2 obtained with TPP-23411 were much lower than those induced by OKT3, a more potent dose-dependent inducer. For IL-6, median cytokine levels obtained with 10 μg / mL of the antibody were comparable between OKT3 and TPP-23411. For all lower doses tested (from 2 μ / mL to 0.128 ng / mL), IL-6 levels were below those induced by OKT3.

[0837] Intracellular staining and subsequent flow cytometric analysis after 5 h incubation of whole blood with TPP-23411 or its isotype control antibody demonstrated that during this initial phase, CD3-CD16+CD56+ NK cells, but not CD3+ T cells, produced IFN-γ (and degranulation as detected by the CD107a marker).

[0838] During administration to patients, precautions will be taken to reduce the potential risk of infusion-related reactions due to cytokine release, including cytokine release syndrome, and to mitigate the risk of immune-related adverse events, such as skin toxicity (i.e., rash) observed with other Treg-depleting agents.

[0839] When fucosylated anti-CCR8 antibody variants or "silent" Fc variants (reduced binding to FcgR) were tested on two donors, those variants did not - or to a lesser extent - induce IFN-γ, IL-1β, IL-6, TNFα, and IL-8 in the whole blood / soluble antibody cytokine release assay format. The data for the PBMC / wet-coat format were less clear and showed a high degree of donor dependence. The data for the commercially available anti-CCR8 antibodies 433H or L263G8 may not be interpreted because of the high background of the respective isotype control antibodies.

[0840] Table 14.2.1: CRA using PBMC / wet coated antibodies. Median cytokine concentrations (pg / ml) after stimulation with indicated antibodies and concentrations (maximum range for TPP-23411 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL, 0.016 μg / mL, 0.0032 μg / mL, 0.00064 μg / mL, 0.000128 μg / mL); N=8.

[0841]

[0842]

[0843] Table 14.2.2: 95th percentile of mean cytokine concentrations (pg / ml) of PBMCs stimulated with wet-coated Erbitux antibody (concentrations 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL); N=8.

[0844] Antibody-μg / mL IFN-γ IL-10 IL-1β IL-2 IL-4 IL-6 IL-8 TNF-α Erbitux 10 16.73 1.00 17.72 5.53 0.00 208.67 4441.76 20.41 Erbitux 2 6.37 1.79 24.86 2.78 7.86 350.50 4902.00 26.78 Erbitux 0.4 10.14 5.12 60.31 3.20 0.28 1042.14 6126.36 47.63 Erbitux 0.08 18.61 9.38 93.52 6.47 0.87 1358.85 7146.00 68.82

[0845] Table 14.2.3: 95th percentile of mean cytokine concentrations (pg / ml) of PBMCs stimulated with wet-coated IgG1 Iso Ctrl Afuco antibody (concentrations 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL, 0.016 μg / mL, 0.0032 μg / mL, 0.00064 μg / mL, 0.000128 μg / mL); N=8.

[0846] Antibody-μg / mL IFN-γ IL-10 IL-1β IL-2 IL-4 IL-6 IL-8 TNF-α IgG1 Iso Ctrl, afuco–10 93.17 0.64 0.77 6.14 0.00 8.43 1369.71 26.08 IgG1 Iso Ctrl, afuco–2 13.44 0.31 1.06 2.93 0.00 2.63 265.54 4.03 IgG1 Iso Ctrl, afuco–0.4 5.12 0.40 0.09 1.08 0.00 0.32 159.18 1.55 IgG1 Iso Ctrl, afuco–0.08 5.23 0.31 0.08 1.85 0.01 1.17 176.70 1.20 IgG1 Iso Ctrl, afuco–0.016 3.61 0.22 0.44 1.17 0.02 0.55 116.88 1.19 IgG1 Iso Ctrl, afuco–0.0032 7.52 0.29 0.22 2.31 0.02 0.56 107.82 1.25 IgG1 Iso Ctrl, afuco–0.00064 7.86 0.24 0.10 1.89 0.02 1.10 147.03 1.33 IgG1 Iso Ctrl,afuco–0.000128 6.05 1.74 14.86 2.76 0.05 2.66 190.41 6.70

[0847] Table 14.2.4: Frequency of positive responses after stimulation with indicated antibodies and concentrations* (maximum range for TPP-23411 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL, 0.016 μg / mL, 0.0032 μg / mL, 0.00064 μg / mL, 0.000128 μg / mL); N=8. .

[0848]

[0849]

[0850] Table 14.2.5: CRA using whole blood / soluble antibodies. Median cytokine concentrations (pg / ml) after stimulation with indicated antibodies and concentrations (maximum range for TPP-23411 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL, 0.016 μg / mL, 0.0032 μg / mL, 0.00064 μg / mL, 0.000128 μg / mL); N=8.

[0851]

[0852]

[0853] Table 14.2.6: 95th percentile of mean cytokine concentrations (pg / ml) after stimulation of whole blood with soluble Erbitux antibody (concentrations 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL); N=8.

[0854] IFN-γ IL-10 IL-1β IL-2 IL-4 IL-6 IL-8 TNF-α Erbitux–10 20.18 1.36 0.39 0.81 0.17 1.80 109.48 1.26 Erbitux–2 21.85 4.72 0.96 2.00 0.11 3.10 178.83 2.31 Erbitux–0.4 19.50 1.19 0.42 0.54 0.17 1.66 110.68 0.93 Erbitux – 0.08 18.62 2.96 0.81 1.70 0.14 2.30 106.90 0.96

[0855] Table 14.2.7: 95th percentile of mean cytokine concentrations (pg / ml) of whole blood stimulated with soluble IgG1 Iso Ctrl Afuco antibody (concentrations 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL); N=8.

[0856] Antibody-μg / mL IFN-γ IL-10 IL-1β IL-2 IL-4 IL-6 IL-8 TNF-α IgG1 Iso Ctrl, afuco–10 87.69 1.17 1.02 0.56 0.04 2.54 547.36 1.93 IgG1 Iso Ctrl, afuco–2 32.75 2.11 5.52 3.40 0.22 42.78 1702.04 8.39 IgG1 Iso Ctrl, afuco–0.4 25.54 0.96 0.68 0.51 0.11 3.55 124.62 0.90 IgG1 Iso Ctrl, afuco–0.08 21.77 0.99 0.60 0.57 0.09 1.96 112.84 0.55 IgG1 Iso Ctrl, afuco–0.016 23.00 0.90 0.53 0.41 0.14 1.87 103.49 0.52 IgG1 Iso Ctrl, afuco–0.032 20.91 0.96 0.42 0.58 0.14 2.06 105.06 0.83 IgG1 Iso Ctrl, afuco-0.0064 25.57 0.88 0.47 0.35 0.15 1.97 105.71 0.55 IgG1 Iso Ctrl,afuco–0.000128 23.10 1.01 0.61 0.46 0.24 2.56 106.43 0.56

[0857] Table 14.2.8: Frequency of positive responses after stimulation with the indicated antibodies and concentrations (maximum range for TPP-23411 10 μg / mL, 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL, 0.016 μg / mL, 0.0032 μg / mL, 0.00064 μg / mL, 0.000128 μg / mL); N=8.

[0858]

[0859]

[0860] in conclusion

[0861] Based on the results of the CRA, it is recommended that precautions be taken in clinical trials to reduce the potential risk of infusion reactions, see also Example 23.

[0862] Example 15: Estimation of human pharmacokinetic parameters

[0863] The assessment of human PK parameters was based on data obtained from in vivo experiments after administration of TPP-23411 to cynomolgus monkeys. To simulate human pharmacokinetic parameters and concentration-time (c / t) profiles, plasma concentrations after intravenous administration were fitted to a 2-compartment model using Phoenix 8.0. The fitted single parameters of total plasma clearance (CL), distribution clearance (CLD), central compartment volume (Vc), and tissue compartment volume (Vt) were allometrically scaled to humans by assuming an average body weight of 70 kg and using a fixed exponential method (exponent of 1 for scaling conversion and 0.8 for scaling clearance). The obtained human PK parameters are summarized in Table 15.1 and used to simulate the c / t profile in humans after a single intravenous infusion of 1 mg / kg within one hour, see Figure 3 In humans, the CL achieved with TPP-23411 was moderately high (1.1 mL / h / kg), while the volume of distribution achieved at steady state (V SS ) is low (133 mL / kg). This results in an effective half-life (t 1 / 2, effective) is quite short at 84h, while the terminal half-life (t1 / 2, terminal) obtained is as long as 284h, see Table 15.1.

[0864] Table 15.1: Estimated key human pharmacokinetic parameters of TPP-23411. CL: total plasma clearance; CLD: clearance by distribution; Vc: volume of central compartment; Vt: volume of tissue compartment; Vss: volume of distribution; t 1 / 2,有效 : effective half-life; t 1 / 2,终末 : The half-life of the terminal phase. .

[0865] parameter unit Predicted value CL [mL / h / kg] 1.1 CLD [mL / h / kg] 0.27 Vc [mL / kg] 53 Vt [mL / kg] 81 <![CDATA[V ss ]]> [mL / kg] 133 <![CDATA[t 1 / 2,有效 ]]> [hours] 84 <![CDATA[t 1 / 2,终末 ]]> [hours] 284

[0866] The obtained human PK parameters were further used to obtain the corresponding concentration time profiles after single and multiple iv infusions of 1 mg / kg TPP-23411 over 1 hour for QW, Q2W and Q3W administration. The results are summarized in Figure 4 After three to four consecutive infusions, steady state was very close in all dosing regimens evaluated (see Figure 4 ), less accumulation is observed with longer dosing intervals. For QW administration, for example, a slight increase in AUC (accumulation ratio of 120%) and a 2-fold increase in trough concentration (accumulation ratio of 224%) are expected (see Table 15.2). For Q3W administration, the accumulation ratios are all reduced, no significant accumulation is expected in AUC (accumulation ratio of 107%), and only a slight accumulation is expected in the case of C trough (accumulation ratio of 131%).

[0867] Table 15.2 lists the estimated relevant human PK parameters.

[0868] Table 15.2: Estimated human PK parameters following single and multiple iv infusions of 1 mg / kg TPP-23411 over 1 hour for QW, Q2W, and Q3W administration. §: AUCτ refers to the AUC between the first and second doses at a dosing interval τ, and AUCτ,ss refers to the AUC at steady state between 2 consecutive doses at a dosing interval τ, which corresponds to 168 hours for QW administration and 504 hours for Q3W administration, respectively.

[0869] parameter unit QW Q2W Q3W <![CDATA[AUC τ § ]]> [μg*h / mL] 758 817 850 <![CDATA[AUC τ,ss § ]]> [μg*h / mL] 913 913 913 <![CDATA[C max ]]> [μg / mL] 19 19 19 <![CDATA[C max,ss ]]> [μg / mL] 20 19 19 <![CDATA[C 谷 ]]> [μg / mL] 0.58 0.24 0.16 <![CDATA[C 谷,ss ]]> [μg / mL] 1.3 0.41 0.21

[0870] Example 16: Estimation of human dose and exposure:

[0871] Pharmacologically active dose and human effective dose

[0872] Considering human PK predictions based on conversion of monkey data as described elsewhere herein, human efficacious dose ranges were estimated for different dosing schedules, as provided in Table 16.1. The predicted doses were estimated to maintain a steady-state C 谷 Higher than the predicted EC for Treg depletion by TPP-23411 in humans 80 Concentrations, as provided in Table 10.1.

[0873] Considering human PK predictions based on monkey data, human doses are recommended to be from 38 μg / kg (2.7 mg / 70 kg) to 1100 μg / kg (75 mg / 70 kg) for once-weekly (QW) administration, and from 230 (16 mg / 70 kg) to 6400 μg / kg (450 mg / 70 kg) for a three-week schedule (Q3W).

[0874] Table 16.1: Estimated effective dose ranges

[0875]

[0876]

[0877] To estimate the minimum effective dose, the predicted EC for Treg depletion by TPP-23411 in humans was considered. 80 Concentration (1 / 4×EC 50 ), see Table 10.1. In addition, a method based on receptor occupancy (RO) was also considered: estimating Treg depletion in EC in mouse in vivo experiments 20 The RO of 1.5 (3.8%) was reprojected to the corresponding EC in humans using the TPP-23411 binding affinity measured in vitro in mouse and human cells. 20 This yields an EC based on the RO estimate 20The concentration in the human body is 0.019 μg / mL.

[0878] Covering the steady state C 谷 Estimated EC 20 The concentrations were considered for estimation of minimum effective concentrations and are presented in Table 16.2 for use in QW planning.

[0879] Table 16.2: Estimated minimum effective dose range. ADCC: antibody-dependent cellular / cell-mediated cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; RO: receptor occupancy

[0880] Example 17: First-in-Human Dose Escalation and Expansion Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of Anti-CCR8 Antibody TPP-23411 as Monotherapy and in Combination with Pembrolizumab in Participants with Selected Advanced Solid Tumors

[0881] The study is an open-label, multicenter, non-randomized Phase 1 first-in-human (FiH) study to determine the maximum tolerated dose (MTD) / maximum administered dose (MAD), recommended expansion dose (RDE), and recommended phase 2 dose (RP2D) of TPP-23411 in combination with pembrolizumab, a monoclonal antibody (mAb) targeting PD-(L)1.

[0882] The maximum tolerated dose (MTD) is defined as the maximum dose at which ≤30% of participants are expected to experience DLT during the dose-limiting toxicity (DLT) observation period. The DLT observation period will be 21 days after the first administration of TPP-23411 (Cycle 1). If the MTD is not reached, the MAD is the highest administered dose. The maximum administered dose (MAD) is the highest dose administered.

[0883] Research objectives

[0884] This study evaluates TPP-23411 as a novel immunotherapeutic agent in advanced solid tumor settings with high medical need by assessing the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of TPP-23411 as monotherapy and in combination with pembrolizumab.

[0885] The main objectives of this study are, in particular,

[0886] a. Determine the safety and tolerability of TPP-23411 when administered as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors

[0887] b. determine the MTD / MAD or RDE of TPP-23411 when administered as monotherapy and in combination with pembrolizumab, and

[0888] c. Characterization of single-dose and multiple-dose PK of TPP-23411 when administered as monotherapy and in combination with pembrolizumab.

[0889] The secondary objectives of this study were specifically

[0890] d. Preliminary estimates of the anti-tumor activity of TPP-23411 when administered as monotherapy and in combination with pembrolizumab, and

[0891] e. evaluating target engagement and pharmacodynamic effects of TPP-23411 by predefined blood and tumor biomarkers using backfill cohorts, and

[0892] f. Determine the RP2D for the combination of TPP-23411 and pembrolizumab.

[0893] Study plan

[0894] The study consists of 2 parts: a dose escalation and an expansion part. For TPP-23411, dose escalation is performed both as monotherapy and in combination with pembrolizumab. To characterize the safety and preliminary anti-tumor activity of TPP-23411 as a single agent, monotherapy dose escalation is performed and a dedicated TPP-23411 monotherapy-MoA expansion is recruited. To provide participants with potentially greater treatment benefit, TPP-23411 is also administered in combination with a fixed dose and planned pembrolizumab (e.g., 200 mg Q3W) in a combination dose escalation and disease-specific combination expansion manner. For an overview of the dose escalation portion of the study protocol, see Figure 1 Or Table 17.1.

[0895] Based on the median EC20 and receptor occupancy readings of pharmacological ADCC / ADCP, supplemented with the results of in vitro cytokine release assays in human whole blood and human PBMC, the recommended starting dose is selected using the predicted minimum effective dose. The minimum effective dose considered based on the median EC20 of TPP-23411 is about 0.14 mg to 0.66 mg for ADCC, 1.98 mg to 4.02 mg for ADCP, 0.84 mg for receptor occupancy (RO), and 1.2 mg for CRA. Therefore, the starting dose is selected to balance safety aspects and minimize patient exposure to subtherapeutic doses.

[0896] The dose escalation portion of the study began with TPP-23411 monotherapy escalation in Cohort 1A. For TPP-23411 QW dosing, after the dose level 250 mg had been declared safe in Cohort 1A, the combination with pembrolizumab (Cohort 1B) was initiated at dose levels of 100 mg or 125 mg QW, or after the MTD was defined as <250 mg in Cohort 1A, at 1 dose level below the MTD. For TPP-23411 Q3W dosing, after the dose level 1000 mg had been declared safe in Cohort 1A, the combination with pembrolizumab (Cohort 1B) was initiated at dose level 500 mg Q3W.

[0897] The study enrolled participants with advanced solid tumors (preferably ICI-sensitive tumor types) in the dose-escalation portion and participants in ICI-relapsed / refractory tumor indications (including NSCLC, HNSCC, TNBC, and melanoma) in the expansion portion. During the FiH study, TPP-23411 was administered as a 1-hour intravenous (IV) infusion on a QW or Q3W schedule during a 21-day treatment cycle.

[0898] The dose-escalation part consisted of 2 groups. In both groups, dose escalation started with a QW dosing schedule and then switched to a Q3W dosing schedule.

[0899] Cohort 1A: Dose escalation of TPP-23411 as monotherapy to characterize the safety, tolerability, PK, and PD profiles and to determine the maximum tolerated dose (MTD) / maximum administered dose (MAD) and recommended dose expansion (RDE) of TPP-23411.

[0900] - Cohort 1B: Dose escalation of TPP-23411 in combination with pembrolizumab to characterize the safety, tolerability, PK, and PD profiles and determine the MTD / MAD or RDE of TPP-23411 in combination with a fixed, approved dose and planned pembrolizumab (e.g., 200 mg Q3W).

[0901] Table 17.1: Dose levels of the monotherapy dose escalation regimen in Group 1A. QW = once a week; Q3W = once every 3 weeks.

[0902]

[0903] Table 17.2: Dose levels of the dose escalation schedule for combination therapy in Group 1 B. QW = once a week; Q3W = once every 3 weeks.

[0904] Dosage Levels TPP-23411 Dosage and Administration Schedule Pembrolizumab Dosage and Administration Schedule 1 125 mg qW or 100 mg qW 200 mg every 3 weeks 2 250mg QW 200 mg every 3 weeks 3 500 mg every 3 weeks 200 mg every 3 weeks 4 1000 mg every 3 weeks 200 mg every 3 weeks 5 1500 mg every 3 weeks 200 mg every 3 weeks

[0905] For Group 1B, the IV line was flushed with saline immediately after the infusion was completed for TPP-23411 to prepare the IV line for pembrolizumab infusion. After 60 minutes, the same IV line could then be used to infuse pembrolizumab at the planned dose of 200 mg administered once every 3 weeks.

[0906] Preferably, on the day that both the anti-CCR8 antibody and the PD-(L)1 inhibitor are administered (e.g., day 1 of each cycle for both QW and Q3W dosing schedules), the dose of the PD-(L)1 inhibitor is administered after a 60-minute pause after the completion of the anti-CCR8 antibody infusion to monitor for safety events (infusion-related reactions). After the IV infusion of the anti-CCR8 antibody is completed, the PD-(L)1 inhibitor can be administered via the same IV line. It is strongly recommended to flush the IV line with saline before infusing the PD-(L)1 inhibitor.

[0907] If a dose lower than 250 mg QW is declared as the MTD for the QW dosing schedule, this dose level is explored in the Q3W dosing schedule provided that this dose level falls within the predicted effective dose range for the Q3W dosing schedule (17 mg to 450 mg of TPP-23411). However, if the QW MTD is lower than the predicted minimum effective dose for the Q3W dosing schedule (i.e., <17 mg of TPP-23411), the Q3W dosing schedule may not be studied.

[0908] If 500 mg of TPP-23411 Q3W as monotherapy is not tolerated, the next lowest dose level (250 mg) could still be explored in combination with pembrolizumab on a Q3W dosing schedule, provided that 250 mg QW is demonstrated to be safe and well tolerated as monotherapy.

[0909] Separate dose escalation of TPP-23411 monotherapy and TPP-23411 in combination with pembrolizumab will help understand and distinguish the safety, tolerability, and pharmacodynamic effects of TPP-23411 monotherapy and the effects in combination with pembrolizumab, and will help determine separate monotherapy and combination treatment MTD / MAD and RDE (dose levels selected following review of all available pharmacokinetic, pharmacodynamic, and safety data).

[0910] After the 250 mg dose level (or alternative MTD) of TPP-23411 monotherapy escalation (Group 1A) in the QW dosing schedule has been declared safe in at least 3 evaluable participants, dose escalation of TPP-23411 in combination with pembrolizumab (fixed dose of 200 mg Q3W) will be initiated at 1 dose level lower (i.e., 100 mg or 125 mg of TPP-23411 QW). If the MTD of TPP-23411 monotherapy (as defined in Group 1A) is less than 250 mg QW, then dose escalation of TPP-23411 in Group 1B will be initiated at 1 dose level lower than the MTD.

[0911] Likewise, once the 1000 mg dose of TPP-23411 monotherapy (Group 1A) on a Q3W dosing schedule had been declared safe in at least 3 evaluable participants, dose escalation of TPP-23411 Q3W in combination with pembrolizumab (200 mg Q3W) was initiated at a TPP-23411 dose level that was one dose level lower.

[0912] The extension contains 2 groups.

[0913] Cohort 2A: TPP-23411 monotherapy mode of action (monotherapy-MoA) expansion in participants with primary (ICI-refractory) or secondary (ICI-recurrent) NSCLC resistant to prior ICI therapy (PD-L1 tumor proportion score [TPS] ≥ 50%) to provide a preliminary estimate of TPP-23411 antitumor activity and assess target engagement and pharmacodynamic effects of TPP-23411 by predefined blood and tumor biomarkers.

[0914] Cohort 2B: Disease-specific combination expansion in separate cohorts for 4 ICI-relapsed tumor types (NSCLC, TNBC, HNSCC, and melanoma) to obtain preliminary efficacy assessments, provide further safety and pharmacokinetic / pharmacodynamic (PK / PD) data, and confirm the RP2D of TPP-23411 in combination with a fixed, approved dose and planned dose of pembrolizumab (e.g., 200 mg Q3W).

[0915] Based on the results obtained during the dose escalation part, 1 of the 2 fixed-dose schedules of TPP-23411 (QW or Q3W) was selected for TPP-23411 monotherapy-MoA expansion and disease-specific combination expansion (combination with pembrolizumab 200 mg Q3W).

[0916] Treatment cycle

[0917] The start of the treatment period or first treatment cycle was defined as the first administration of study treatment (ie, intravenous [IV] infusion of TPP-23411 as monotherapy or in combination with a fixed, approved IV dose of pembrolizumab [eg, 200 mg Q3W]).

[0918] Cycle length was 21 days, with study treatment administered on Days 1, 8, and 15 (dose levels for the QW schedule) or Day 1 (Q3W schedule) during the dose escalation portion.

[0919] Participants received study treatment until disease progression or unacceptable toxicity or until another specified withdrawal criterion was met.

[0920] The Active Follow-up (FU) Period began after completion of the End-of-Treatment (EOT) visit and included safety FU visits / contacts and, if applicable, efficacy FU visits. Safety FU visits / contacts occurred 90 days (±7 days) after the last administration of study treatment; for participants who permanently discontinued study treatment for reasons other than disease progression and did not start a new anticancer therapy (ACT), efficacy FU visits were conducted every 12 weeks (±14 days) from the last dose. After completion of the Active FU Period, participants entered the Long-term FU Period, during which all participants were contacted every 6 months (±14 days) to determine survival status and subsequent systemic ACTs, continuing up to 24 months after the last participant's EOT, or until the end of the study, whichever occurred first. Monitoring & Biomarkers

[0921] TPP-23411 monotherapy-MoA extension includes the evaluation of paired biopsies for TME analysis. In addition, in order to characterize the CD8 immune cell status of tumor lesions at baseline and the changes induced by TPP-23411 89-ZR-anti-CD8 mini-antibody treatment, PET / CT using 89Zr-Df-crefmirlimab as a biomarker for TPP-23411 monotherapy-MoA extension is used for quantitative imaging of CD8 T cells (Group 2A). Zr89 anti-CD8PET / CT scanning will expose participants to moderate additional radiation exposure. Baseline quantification and distribution of tracer uptake in tumor lesions reviewed centrally are performed to classify lesions as hot lesions, immune rejection lesions or cold lesions. For individual participants, based on RECIST 1.1 standards, changes in tracer uptake / distribution between baseline and the end of cycle 2 and comparison of these changes with BOR and DOR are performed by centralized review.

[0922] To closely monitor study participants for the possibility of CRS, blood will be collected at least prior to dosing and 4 and 24 hours after the start of each TPP-23411 infusion for the first 3 dose levels in Cycle 1 for the assessment of inflammatory cytokines (e.g., IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL 12p70, IL-13, and TNF-α).

[0923] T cells, B cells, NK cells, and T cell subsets will be quantified, and the activation status of T cell populations will be determined by flow cytometry in peripheral blood.

[0924] The fold change of peripheral IFN-γ was measured on-treatment by an immunoassay compared to baseline serum samples (as reference). The fold change of intratumoral CD8+ T cell / Treg ratio was measured on-treatment by IHC compared to baseline biopsies.

[0925] Biomarker samples will be collected from participants and will include:

[0926] Archival tumor tissue and / or fresh pretreatment biopsies

[0927] Tumor biopsies in paired studies

[0928] For the following pre- and during-study treatment blood samples:

[0929] ○ Cytokines and chemokines

[0930] ○ Flow cytometry analysis

[0931] ○ RNA and ctDNA preparations

[0932] Pharmacodynamic biomarkers will be evaluated in samples collected before and during study treatment to investigate the effects of TPP-23411 on these biomarkers.

[0933] Baseline candidate biomarkers (further validation of predictive biomarkers) will be evaluated for correlation with response to study treatment. Samples from tumor biopsies and blood will be analyzed retrospectively. Candidate predictive biomarkers include (but are not limited to):

[0934] Tumor immune infiltration (e.g., immune gene expression profile; presence, phenotype, and activation status of tumor-infiltrating leukocytes, with a particular focus on CCR8-positive Treg cells, CD8 T cells, NK cells, and macrophages)

[0935] PD-L1 expression level as a surrogate for immune sensitivity

[0936] Tumor mutation status (microsatellite instability [MSI], tumor mutations, tumor mutation burden [TMB])

[0937] Imaging-based biomarkers for tumor-infiltrating CD8 T cells

[0938] PD-L1 expression for patient selection

[0939] Since CD8 T cells are essential for IO-related anti-tumor effects, it is assumed that the functional relevance of Treg depletion is the highest in tumors where T cell infiltration is already high. In addition, macrophages and NK cells are essential for anti-CCR8-mediated Treg depletion via ADCC or ADCP. Based on mRNA expression, PD-L1 positivity is associated with the presence of immune cells including Treg, macrophages, and CD8 Teff in the TME between all indications planned from the Cancer Genome Atlas (TCGA). The correlation of PD-L1 expression is further supported by the following observations: PD-L1 mRNA expression is a significant positive predictive biomarker for anti-CCR8 efficacy between mouse tumor models. Therefore, it is expected that patients with PD-L1-positive tumors with immune-sensitive indications will respond best to TPP-23411 as a monotherapy.

[0940] Thus, in the monotherapy-MoA expansion group (Group 2A) recruiting NSCLC patients, PD-L1 expression level will be used as an inclusion criterion. Therefore, patients must have a historical PD-L1 score of TPS ≥ 50% (as previously determined according to a locally approved assay).

[0941] Alternatively, Group 2A includes anti-CCR8 antibody monotherapy-MoA extensions in the following

[0942] Participants with NSCLC (with PD-L1 TPS ≥ 50%), or

[0943] TNBC (with PD-L1 CPS ≥10%), or

[0944] HNSCC (with PD-L1 CPS ≥1%), or

[0945] HNSCC (with PD-L1 CPS ≥ 20%), or

[0946] Melanoma (no PD-L1 cutoff)

[0947] PET / CT imaging of CD8 cells

[0948] Zirconium-89 (89Zr) anti-CD8 miniantibody PET / CT was performed in the monotherapy-MoA extension (cohort 2A) with 89Zr-Df-crefmirlimab (a miniantibody labeled with 89Zr that targets CD8 cells). CD8 tracer uptake has been shown to correlate with CD8 expression by IHC and was positively correlated with tumor response assessment per RECIST 1.1.

[0949] In this study, the exploratory outputs are as follows:

[0950] Tumor uptake and biodistribution of the 89Zr CD8 tracer using whole-body PET / CT at baseline / screening allows classification of whole-body tumor lesions as hot, immune-rejected, or cold lesions based on CD8 cell infiltration.

[0951] · Use 89Zr anti-CD8 microantibody PET / CT early during treatment (end of cycle 2) to assess TPP-23411-induced changes in CD8 cell quantification and distribution patterns in tumor lesions / TME. For Monotherapy - MoA Extension (Arm 2A)

[0952] For each participant in the study, CD8 patterns were correlated with best overall response (BOR) and time to progression data using tumor response imaging assessed using RECIST 1.1 for each patient.

[0953] At various time points, infusion of 89Zr-Df-crefmirlimab was followed by a whole-body PET / CT scan (vertex of the skull to mid-thigh) approximately 24 hours ± 3 hours later.

[0954] Radiomics analysis

[0955] Radiomics is a field of imaging biomarker research defined as the use of automated or semi-automated analysis methods to derive quantitative measures (often referred to as radiomic features) from medical images (Mayerhoefer et al. 2020). These measures capture tissue and lesion characteristics, such as shape and heterogeneity, and can be used alone or in combination with histological, genetic, or proteomic data to help better understand the clinical activity of experimental drugs. In this study, radiomics analysis is an exploratory goal and can be performed on all image data, including images collected for tumor response assessment. CT images from tumor response assessment will be centrally collected in DICOM format with a reconstructed slice thickness of 1.0 mm. Depletion of CCR8-expressing Tregs by TPP-23411 may result in an increase in tumor-infiltrating CD8 lymphocytes. The pharmacodynamic effects of TPP-23411 can be evaluated using changes in CD8 radiomics measures relative to baseline as an exploratory endpoint. When possible, CD8 radiomics measures will also be correlated with paired tumor biopsy results (e.g., CD8 IHC). Likewise, radiomics measures from study images (eg, tumor assessments) will be compared with other study biomarkers and endpoints.

[0956] Example 18: Biomarkers: Association between gene expression and response to the murine surrogate antibodies TPP-14099 and TPP-15285 in a syngeneic murine cancer model

[0957] We investigated the association between gene expression and the efficacy of treatment with a CCR8-depleting antibody by whole transcriptome sequencing of early-stage, untreated tumors from 21 syngeneic murine carcinoma models. We also assessed the association between anti-CCR8 treatment and treatment that inhibits the immune checkpoints PD-1 and PD-L1.

[0958] The investigated anti-mouse CCR8 antibodies TPP-14099 (hIgG1) and TPP-15285 (mIgG2a) were effective in immune checkpoint sensitivity models. Anti-mouse CCR8 antibodies almost always showed superior efficacy compared to anti-PD-1 antibody or anti-PD-L1 antibody treatment. The efficacy of anti-mouse CCR8 antibodies did not depend on the mouse strain or correlate with the mutation load of the respective cancer cell lines.

[0959] FIG. 5 shows that the response to anti-mouse CCR8 antibody treatment correlates with the response to anti-PD-L1 antibody and anti-PD-1 antibody treatment.

[0960] FIG. 6 shows that baseline mRNA expression of PD-L1 and inflammatory markers such as IFN-γ in early-stage untreated tumors correlates with response to anti-mouse CCR8 antibody treatment.

[0961] Example 19: Determination of unchanged compounds in plasma

[0962] In preclinical pharmacokinetic studies and pivotal nonclinical safety studies in monkeys, TPP-23411 was measured in plasma using an anti-human IgG universal assay format (IgG-ELISA).

[0963] In this protocol, TPP-23411 in plasma was measured by the Gyrolab method using fluorescence reading after dilution with buffer using immobilized biotinylated anti-human IgG-Fc antibody as capture molecule and Alexa fluorophore labeled anti-human IgG antibody as detection reagent. The method specific parameters are listed in Table 19.1.

[0964] Table 19.1: Method specifications for monkey plasma. Accuracy and precision were calculated from validation quality control samples (including LLOQ and ULOQ results).

[0965]

[0966] TPP-23411 was demonstrated to be stable as indicated under the conditions listed below, which were correlated with sample processing, see Table 19.2.

[0967] Table 19.2: Stability data.

[0968]

[0969]

[0970] Example 20: Determination of anti-CCR8 antibodies in plasma

[0971] Using biotinylated TPP-23411 as an antigen, the binding antibodies (anti-drug antibodies) to TPP-23411 were determined in monkey plasma using a bridging assay based on Meso Scale Discovery (MSD). Anti-TPP-23411 antibodies present in positive controls or in study-specific plasma samples bind to biotinylated TPP-23411 and are captured on a streptavidin plate, while other plasma components are washed away. Then, SULFO-tagged TPP-23411 and ECL readings are used to detect bound antibodies (for ECL reagents, see U.S. Pat. No. 7,855,287 and U.S. Pat. No. 7,803,573).

[0972] In more detail, positive and negative control samples (monkey serum with and without positive control, respectively) and unknown samples were pre-diluted (1:8) with dilution buffer, mixed with dilution buffer, and pre-incubated on an orbital shaker (RT, 600rpm) in a polypropylene plate for 1 hour. A master mix containing biotinylated TPP-23411 (1 μg / mL) and SULFO-tagged TPP-23411 (1 μg / mL) was added to the sample mixture and incubated for 2 hours (RT, 600rpm). Then, 25μ was transferred in duplicate from the incubated sample to the wells of the blocked MSD streptavidin gold plate (biotinylated TPP-23411 can bind to it) (150 μL of blocking buffer, at least 30 minutes, 600rpm). Functional anti-drug antibodies bridge biotinylated and SULFO-tagged TPP-23411. When a voltage is applied, the sulfonated TPP-23411 produces an electrochemiluminescent (ECL) signal that correlates with the amount of ADA in the well. The plates were read using a Meso QuickPlex SQ 120 and the Workbench TM Data were analyzed using the ANALYZER software. All samples were assayed in duplicate.

[0973] The positive control antibody was an affinity-purified goat anti-human IgG antibody. In the assay, the sensitivity of this positive control was 4.79 μg / L (assay cut-off concentration).

[0974] Method validation and analysis of study samples were performed in accordance with internal SOPs and relevant guidelines on "Assay Development for Immunogenicity Testing of Therapeutic Protein Products" (FDA, 019) and "Guideline on Immunogenicity Assessment of Therapeutic Proteins" (EMA, 2017). The bioanalytical methods used in key nonclinical safety studies were fully validated, and samples from these studies were analyzed in accordance with good laboratory practices (GLP).

[0975] Table 20.1: Method specifications for the anti-TPP-23411 antibody method in monkey plasma. Precision was calculated based on low-, mid-, and high-mass control samples.

[0976]

[0977] The stability of the positive control antibody simulating the study samples in plasma was studied under different storage conditions, covering different steps of sample handling during the assay execution, and storage intervals relevant to the actual study samples. TPP-23411 (positive control antibody) was stable under all conditions relevant to sample handling.

[0978] Table 20.2: Selected plasma stability data for polyclonal goat anti-human IgG (positive control antibody)

[0979] Stability Type Species temperature period Stable short term Cynomolgus monkey RT 24 hours yes Freeze-thaw Cynomolgus monkey -75℃ / -20℃ to RT 5 cycles yes

[0980] Table 20.3: Selected plasma stability data for polyclonal goat anti-human IgG (positive control antibody)

[0981] Stability Type Species temperature period Stable short term Cynomolgus monkey RT 24 hours yes Freeze-thaw Cynomolgus monkey -75℃ / -20℃ to RT 5 cycles yes

[0982] Example 21: Tissue Cross-Reactivity

[0983] A preliminary tissue cross-reactivity (TCR) study was conducted to validate the immunohistochemistry (ICH) method for detecting TPP-23411-FITC bound to CCR8. This was a non-GLP study but was conducted according to current scientific and regulatory standards. The goal of this preliminary TCR study was to evaluate the potential cross-reactivity of TPP-23411 in a fluorescein isothiocyanate (FITC)-conjugated form using an immunohistochemistry (IHC) technique using a panel of frozen tissues and blood smears (per tissue) from three human and three cynomolgus macaque donors. The IHC method for detecting TPP-23411-FITC bound to CCR8 was successfully validated in terms of specificity, sensitivity, range, linearity, precision (repeatability), and reproducibility.

[0984] TPP-23411-FITC produced positive membrane / cytoplasmic staining of monkey CCR8-positive cells at 0.1 to 9 μg / mL and did not cause any staining in negative cells. No staining was observed in any positive or negative cells incubated with IgG1-FITC at any concentration.

[0985] The full FDA tissue listing was studied in human and cynomolgus monkey species and the following results were obtained at 3 and 9 μg / mL:

[0986] People Organization

[0987] TPP-23411-FITC generation:

[0988] At 3 and 9 μg / mL, there was positive membrane, variable cytoplasmic staining of mononuclear cells within some germinal centers of the GALT in the ileum (suggestive of dendritic cells and / or macrophages; other cell types could not be excluded).

[0989] Membranous, variable cytoplasmic staining of subepithelial cells in the jejunal villi. These cells are thought to represent myoepithelial cells or pericytes (other cell types cannot be excluded).

[0990] • Staining in the peritubular interstitial tissue of the kidney (ie, fibroblasts and / or the lamina propria of the tubes / vessels).

[0991] - Minimal to moderate staining in histological glandular compartments with secretory function, such as mammary gland (breast), prostate, parotid gland, gall bladder and stomach. TPP-23411-FITC also produces minimal staining in umbrella cells of the ureter.

[0992] Cynomolgus monkey

[0993] TPP-23411-FITC induced staining in:

[0994] In interstitial spindle cells (suggestive of endothelial cells) of the pancreas

[0995] In the scattered large cells of the parathyroid glands

[0996] In the umbrella cells (luminal side) of the urothelium (bladder)

[0997] In the adrenal medulla (plasmin).

[0998] Example 22: Tissue Cross-Reactivity (GLP Study)

[0999] The optimized IHC assay was validated in a preliminary non-GLP study using automated technology and the DAB Map kit from Ventana as the detection system. In the GLP-compliant study, two concentrations of the test item TPP-23411-FITC were studied: 3 μg / mL and 10 μg / mL. The tissues studied are shown in Table 22.1.

[1000] Table 22.1: Tissue cross-reactivity - GLP studies. a Evaluation was performed from peripheral blood smears. b Evaluate from all tissues in which it exists. c Includes the esophagus, large intestine / colon, small intestine (duodenum or jejunum), and stomach (including the underlying smooth muscle).

[1001]

[1002] TPP-23411-FITC induced membrane and variable cytoplasmic staining in human and cynomolgus CCR8-positive cells at 3 μg / mL and 10 μg / mL, but no staining in negative cells. Negative control antibody TPP-9809-FITC did not induce any staining in human and cynomolgus CCR8-positive cells or CCR8-negative cells at 10 μg / mL.

[1003] Tissue integrity appeared adequate. All tissues were considered acceptable by microscopic evaluation in terms of morphology and inclusion of tissue components.

[1004] In human tissues, TPP-23411-FITC studied at 3 μg / mL and / or 10 μg / mL produced:

[1005] Membrane staining in platelets (rare to frequent, 3 / 3 donors)

[1006] Staining of interstitial cells and membrane / cytoplasm (2 / 3 donors) in the liver, suggestive of Kupffer cells

[1007] Staining of mixed inflammatory cells (macrophages and granulocytes / neutrophils) in the fallopian tube lumen (2 / 3 donors)

[1008] · Occasional staining of glial cells in the spinal cord (1 / 3 donors)

[1009] Membranous and variable cytoplasmic staining of mononuclear cells in the serosa of the ureters and adjacent tissues, suggestive of macrophages (1 / 3 donors)

[1010] Staining of stromal spindle cells in the small intestine (villi, 2 / 3 donors), suggestive of smooth cells and / or fibroblasts

[1011] Diffuse staining in fibrous and / or fibrovascular tissue (primarily extracellular matrix) of multiple organs (adrenal glands (1 / 3 donors), heart (1 / 3 donors), kidneys (3 / 3 donors), ovaries (3 / 3), stomach (1 / 3 donors), and tonsils (1 / 3 donors))

[1012] In cynomolgus monkeys, TPP-23411-FITC studied at 3 μg / mL and / or 10 μg / mL produced:

[1013] Diffuse staining in fibrous and / or fibrovascular tissue (primarily extracellular matrix) of multiple organs (breast (2 / 3 donors), corneal stroma (2 / 3 donors), kidney (3 / 3 donors), ovary (3 / 3 donors), parotid gland (3 / 3 donors, more prominently around some ducts, possibly including some myoepithelial cells), pituitary gland (3 / 3 donors), prostate (3 / 3 donors), stomach (3 / 3 donors), testis (3 / 3 donors), thyroid (3 / 3 donors), bladder (3 / 3 donors), cervix and endometrium (3 / 3 donors))

[1014] · Staining in occasional scattered cells in the parathyroid glands (2 / 3 donors)

[1015] Staining of cells in the red pulp of the spleen (3 / 3 donors, majority of cells, mixture of monocytes, granulocytes, and mesenchymal cells)

[1016] Based on immunohistochemistry, TPP-23411-FITC tested at 3 μg / mL and / or 10 μg / mL produced staining in fibro-vascular tissue (primarily extracellular matrix) in both human and monkey tissues. In addition, some staining was also observed in inflammatory cells (granulocytes and / or monocytes, suggesting macrophages) of both species (more obvious in humans and limited to the spleen in monkeys). The staining seen in humans but not in monkeys involved platelets and spinal cord (nervous tissue).

[1017] Comprehensive histopathology studies in monkey toxicology studies did not reveal any morphological changes in tissues with positive staining in the TCR assay. Cytoplasmic staining (e.g., in spleen, spinal cord) is considered to be of little to no safety concern, as in vivo, entry of monoclonal antibodies into the cytoplasmic compartment is thought to be limited.

[1018] Example 23: Administration of Antihistamines, Acetaminophen, Corticosteroids for Side Effect Management

[1019] If an infusion-related reaction occurred after administration of the anti-CCR8 antibody, participants received additional medications, optionally extending the study treatment infusion time for subsequent administrations. Additional medications included, for example, antihistamines, acetaminophen, or corticosteroids.

[1020] Following an infusion reaction that occurred after administration of anti-CCR8 antibody, between 650 mg and 1000 mg of paracetamol was administered prior to subsequent infusions. With this premedication, no infusion reactions were observed with subsequent administration of anti-CCR8 antibody.

[1021] Based on these data, a dose of 500 mg to 1000 mg of paracetamol may be administered either before the first dose of anti-CCR8 antibody or before subsequent doses of anti-CCR8 antibody to prevent or reduce adverse reactions.

[1022] After an infusion reaction occurred with the first administration of anti-CCR8 antibody, 650 mg of paracetamol and 100 mg of diphenhydramine were administered prior to subsequent infusions. With this premedication, no infusion reactions were observed with subsequent administrations of anti-CCR8 antibody.

[1023] In an alternative approach, dexamethasone may be administered prior to administration of one dose of anti-CCR8 antibody. A suitable dose is, for example, 2 doses of 8 mg dexamethasone, for example 3 h before and 4 h after administration of the anti-CCR8 antibody.

[1024] Based on the severity of the adverse reaction, pembrolizumab may be withheld and corticosteroids may be administered. At improvement to grade ≤ 1, corticosteroids may be tapered and continued for at least 1 month.

[1025] Example 24: Demonstration of Treg depletion & mode of action of the proposed dosing regimen

[1026] The expression of CD45, CD45RA, CD3, CD4, CD8, CD25 (IL2RA), CD127 (IL7RA), CD69, CCR7, GZMB, Ki67, PDL1, OX40 and CD137 (4-1BB = activation marker) was analyzed by flow cytometry to monitor in particular the percentage of activated proliferative CD8+ T cells and the percentage of Treg cells. Based on the assumed mode of action, an increase in the percentage of activated proliferative CD8+ T cells and a decrease in the percentage of residual Treg cells are prerequisites for therapeutic success and can be confirmed for the proposed dosing regimen:

[1027] Activated proliferative CD8+ T cells were defined as those expressing the proliferation marker Ki67, the activation marker 4-1BB (CD137), CD8, and CD3.

[1028] Activated Tregs were defined as expressing CD137 (4-1BB), CD25, CD127low, CD4, and CD3. 4-1BB was used instead of CCR8 to evaluate Treg levels and Treg depletion because binding of therapeutic antibodies may compete with the binding of flow cytometry marker antibodies directed to CCR8.

[1029] Figure 7The increase in activated proliferative CD8+ T cells relative to the total number of CD3+ T cells is shown starting ~3 days after the first administration of 10 mg of anti-CCR8 antibody. In particular, a sustained increase was observed starting from the second week of treatment, presumably due to the induction of immune activation in the tumor. The end of treatment was ~3 weeks after the last infusion. The ratio of the two cell types was set to 1 at the screening time point.

[1030] Figure 7 , Figure 8 and Fig. 9 The abbreviations listed on the x-axis are:

[1031] Scr: screening value (during patient screening, i.e. before the first anti-CCR8 antibody administration);

[1032] C1D1: Cycle 1, Day 1, 0 h (immediately before the start of anti-CCR8 antibody administration);

[1033] C1D2: Cycle 1, Day 2, 0 h (~1 day after first anti-CCR8 antibody administration);

[1034] C1D3: Cycle 1, Day 3, 0 h (~2 days after first anti-CCR8 antibody administration);

[1035] C1D8: Cycle 1, Day 8, 0 h (~7 days after the first anti-CCR8 antibody administration, immediately before the second anti-CCR8 antibody administration)

[1036] C1D15: Cycle 1, Day 15, 0 h (~7 days after the first anti-CCR8 antibody administration, immediately before the third anti-CCR8 antibody administration)

[1037] C2D1: Cycle 2, Day 1, 0 h (~7 days after the third anti-CCR8 antibody administration, immediately before the fourth anti-CCR8 antibody administration)

[1038] C2D3: Cycle 2, day 3, 0 h (~1 day after the fourth anti-CCR8 antibody administration);

[1039] C2D8: Cycle 2, Day 8, 0 h (~7 days after the fourth anti-CCR8 antibody administration, immediately before the fifth anti-CCR8 antibody administration)

[1040] C2D15: Cycle 2, Day 15, 0 h (~7 days after the fifth anti-CCR8 antibody administration, immediately before the sixth anti-CCR15 antibody administration)

[1041] • EOT: End of treatment (see protocol, eg 30 days after last infusion).

[1042] Figure 8 A dramatic decrease in the ratio of activated Tregs relative to the total number of CD3+ T cells is shown starting ~1 day after the first anti-CCR8 antibody administration (10 mg dose). The ratio of the two cell types was set to 1 at the screening time point.

[1043] Fig. 9 The ratio of activated Tregs relative to the total number of activated proliferating CD8+ T cells is shown. The ratio of the two cell types was set to 1 at the screening time point.

[1044] Other doses tested showed similar trends.

[1045] Fig.10 The Tregs in the patient's blood samples were reduced when treated with 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibodies. For each patient, the ratio of C1D2 to C2D15 (activated Tregs / total CD3+ T cells) obtained at the time point was divided by the ratio (activated Tregs / total CD3+ T cells) obtained for the same patient at screening (Scr) (ie, before the anti-CCR8 antibodies were administered). In order to obtain data for one box, all patients treated with the specified dose were grouped together. Even the lowest 1 mg group showed a reduction in the median activated Tregs to ~53% of the screening value. [1046...

Claims

1. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method, comprising intravenously administering to a patient in need thereof an anti-CCR8 antibody in a total amount of: a. about 1 to 250 mg once a week, preferably 3, 10, 30, 50, 100, 125 or 250 mg once a week, or b. About 16 to 1500 mg once every three weeks, preferably 16, 450, 500, 750, 1000 or 1500 mg once every three weeks.

2. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method, comprising intravenously administering a total amount of 2.7 mg to 75 mg of the anti-CCR8 antibody once a week to a patient in need thereof.

3. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method, comprising intravenously administering to a patient in need thereof a total amount of 16 mg to 450 mg of the anti-CCR8 antibody once every three weeks.

4. An anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to the preceding claims, further comprising intravenously administering to a patient in need thereof an anti-PD-(L)1 antibody in a total amount of: a. about 200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or b. about 400 mg once every six weeks, preferably wherein the anti-PD-(L)1 antibody is pembrolizumab, or c. about 240 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or d. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or e. about 480 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is nivolumab, or f. about 840 mg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or g. about 1200 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or h. about 1680 mg once every four weeks, preferably wherein the anti-PD-(L)1 antibody is atelizumab, or i. about 360 mg once every three weeks, preferably wherein the anti-PD-(L)1 antibody is sepalizumab, or j. about 3 mg / kg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is toripalimab, or k. about 10 mg / kg once every two weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab, or 1. About 1500 mg once every 3 weeks, preferably wherein the anti-PD-(L)1 antibody is durvalumab.

5. An anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to the preceding claims, wherein the anti-human CCR8 antibody is characterized by a half-life in humans of <14 days, preferably <10 days, most preferably <7 days. 6 . The anti-human CCR8 antibody having ADCC and ADCP activities for use in a therapeutic method according to any one of claims 4 to 5 , wherein the anti-PD-(L)1 antibody is administered after the anti-CCR8 antibody.

7. An anti-human CCR8 antibody having ADCC and ADCP activity for use in a therapeutic method according to the preceding claims, wherein the intravenous administration of the anti-human CCR8 antibody is performed as a 15 to 120 minute and preferably 30 to 60 minute intravenous infusion.

8. An anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to any one of claims 4 to 7, wherein the intravenous administration of the anti-PD-(L)1 antibody is performed as a 15 to 60 minute and preferably 30 minute intravenous infusion.

9. The anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to any one of claims 4 to 8, wherein the intravenous administration of the anti-PD-(L)1 antibody is performed using the same IV line previously used for the intravenous administration of the anti-human CCR8 antibody.

10. The anti-human CCR8 antibody having ADCC and ADCP activity for use in a therapeutic method according to claim 9, wherein the IV line is flushed with saline prior to intravenous administration of the anti-human PD-(L)1 antibody.

11. An anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to the preceding claims, wherein the medical use comprises at least one 21-day dosing cycle, and preferably wherein both the anti-CCR8 antibody and the anti-PD-(L)1 antibody are administered on day 1 of the 21-day dosing cycle.

12. An anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to any one of claims 4 to 11, wherein the medical use comprises at least two and preferably more administration cycles, and wherein for the second, third, fourth, fifth or any subsequent administration cycle, the anti-human CCR8 antibody and the anti-PD-(L)1 antibody are administered directly after each other without substantial delay.

13. An anti-CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to the preceding claims, wherein the anti-CCR8 antibody is a human IgG1 antibody.

14. An anti-CCR8 antibody having ADCC and ADCP activity for use in a therapeutic method according to the preceding claims, wherein the anti-CCR8 antibody is a low internalizing or non-internalizing antibody.

15. An anti-CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to the preceding claims, wherein the anti-CCR8 antibody is characterized by a dissociation constant (KD) for binding to CHO cells transfected with human CCR8 that is of the same order of magnitude as the KD for binding of TPP-23411 to CHO cells transfected with human CCR8.

16. The anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to any one of claims 4 to 15, wherein the anti-PD-(L)1 antibody is pembrolizumab, nivolumab, atezolizumab, avelumab, sepalizumab, toripalimab or durvalumab.

17. An anti-CCR8 antibody having ADCC and ADCP activity for use in a therapeutic method according to any one of claims 4 to 16, wherein the anti-CCR8 antibody a. comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID Nos.: 2, 3, 4, 6, 7 and 8, and / or b. comprising a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 5, and / or c. comprising a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 17, and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:

18.

18. An anti-CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to the preceding claims, wherein the method of treatment is a method of treating cancer, preferably wherein the cancer is non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), head and neck squamous cell carcinoma (HNSCC), melanoma or skin cancer other than melanoma.

19. An anti-human CCR8 antibody having ADCC and ADCP activity for use in a method of treatment according to the preceding claims, wherein the method of treatment further comprises administering an effective dose of an antihistamine, acetaminophen, a corticosteroid or a combination thereof, preferably a. at least 500 mg or at least 650 mg of paracetamol prior to administration of the anti-CCR8 antibody, and / or b. at least 50 mg or at least 100 mg of diphenhydramine prior to administration of the anti-CCR8 antibody, and / or c. At least 8 mg of dexamethasone prior to administration of the anti-CCR8 antibody.

20. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to the preceding claims, wherein the therapeutic method is a method for treating cancer, comprising the following steps: a. analyzing the tumor proportion score or combined positive score as a measure of PD-(L)1 expression in a patient's cancer tissue sample, and b. If the patient's tumor proportion score is ≥50% or the combined positive score is ≥10% or ≥1%, administer an anti-human CCR8 antibody to the patient.

21. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to claim 20, wherein a. the cancer is non-small cell lung cancer, and the tumor proportion score is analyzed as a measure of PD-(L)1 expression in a cancer tissue sample of the patient, or b. the cancer is triple negative breast cancer and the combined positive score is analyzed as a measure of PD-(L)1 expression in the patient's cancer tissue sample, or c. The cancer is head and neck squamous cell carcinoma, and the combined positive score is analyzed as a measure of PD-(L)1 expression in the patient's cancer tissue sample.

22. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to any one of claims 1 to 19, wherein the therapeutic method is a method for treating cancer, comprising administering the anti-human CCR8 antibody to a patient if the patient has: a. Historical tumor proportion score ≥ 50%, or b. Historical combined positive score ≥10% or ≥1%.

23. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to claim 22, a. wherein the cancer is non-small cell lung cancer and the method of treatment comprises administering to the patient an anti-human CCR8 antibody if the patient's historical tumor proportion score is ≥ 50%, or b. wherein the cancer is triple negative breast cancer and the method of treatment comprises administering to the patient an anti-human CCR8 antibody if the patient's historical combined positive score is ≥ 10% or ≥ 1%, c. Wherein the cancer is head and neck squamous cell carcinoma and the method of treatment comprises administering an anti-human CCR8 antibody to the patient if the patient's historical combined positive score is ≥20% or ≥1%.

24. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treating a patient according to any one of claims 20 to 23, wherein the tumor proportion score is analyzed or obtained using an FDA-approved PD-L1 assay, such as the PD-L1 IHC 22C3 pharmDx assay or the VENTANA PD-L1 (SP263) assay.

25. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treating a patient, preferably according to the preceding claims, wherein the method of treatment is a method of treating cancer, comprising the steps of: a. Optionally, analyzing the patient's blood, plasma or serum screening sample for the level of at least one and preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inflammatory cytokines selected from the group consisting of IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL 12p70, IL-13 and TNF-α, b. administering to the patient an effective dose of an anti-human CCR8 antibody, c. analyzing a patient's blood, plasma or serum sample for at least one and preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 inflammatory cytokines, wherein the blood, plasma or serum sample is drawn after administering an effective dose of anti-human CCR8 antibody according to step b), d. comparing the cytokine levels obtained according to step c), i. with the cytokine levels obtained according to step a), or ii. With reference value, To identify safety-related events, or as a surrogate biomarker of Treg exhaustion, or as a biomarker of treatment success.

26. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to claim 25, further comprising e. If the cytokine level obtained according to step c): i. the cytokine level is increased relative to that obtained according to step a), or ii. is increased relative to the reference value, At least one further effective dose of an anti-human CCR8 antibody is then administered to the patient.

27. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to any one of claims 25 or 26, wherein the blood, plasma or serum sample for analyzing cytokine levels according to step c) is drawn / collected 1-24 hours, 24-48 hours, 2-7 days, 7-14 days, 14-28 days or more than 28 days after administration of an effective dose of the anti-human CCR8 antibody according to step b).

28. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to any one of claims 25 to 27, wherein the blood, plasma or serum sample for analyzing cytokine levels according to step a) is drawn / collected 60-15 minutes and preferably ~30 minutes before administering an effective dose of the anti-human CCR8 antibody according to step b).

29. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method, wherein the therapeutic method is a method for treating cancer, comprising: a. stratify patients based on prior treatment of cancer with anti-PD-(L)1 antibodies for at least 6 months, and b. Only if the patient has been previously treated with anti-PD-(L)1 antibody for at least 6 months, An anti-human CCR8 antibody is then administered to the patient. .

30. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer according to claim 29, wherein the anti-human CCR8 antibody is the anti-human CCR8 antibody for use in a therapeutic method according to any one of claims 1 to 28.

31. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer, comprising the following steps: a. administering to a subject a Zr-89 labeled anti-CD8 minibody, b. performing at least one PET scan and optionally a CT scan to detect Zr-89 labeled anti-CD8 miniantibodies in the subject, thereby generating a first image of the subject, c. determining the abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancer lesions of the subject based on the first subject image, and d. If the first subject image indicates that the amount and / or distribution of Zr-89 labeled anti-CD8 minibody in any of the one or more cancer lesions indicates a substantial likelihood that the subject will benefit from administration of an anti-human CCR8 antibody, administering an effective dose of an anti-human CCR8 antibody to the subject.

32. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer according to claim 31, wherein the amount and / or distribution of the Zr-89 labeled anti-CD8 minibody in any one or more cancer lesions is assessed relative to: i. the abundance and / or distribution of Zr-89 labeled anti-CD8 minibody in healthy tissues of patients, or ii. One or more reference values ​​for the abundance and / or distribution of Zr-89 labeled anti-CD8 minibody.

33. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer, comprising the following steps: a. administering to the subject a first dose of Zr-89 labeled anti-CD8 minibody, b. performing at least one PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 miniantibody in the subject, thereby generating a first image of the subject, c. determining the abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancer lesions of the subject based on the first subject image, d. administering to the subject an effective dose of an anti-human CCR8 antibody, e. administering to the subject a second dose of Zr-89 labeled anti-CD8 minibody, f. performing a second PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 miniantibody in the subject, thereby generating a second image of the subject, g. determining a second abundance and / or distribution of the Zr-89 labeled anti-CD8 miniantibody in one or more cancer lesions of the subject based on the second subject image, h. Comparing the second subject image with the first subject image to evaluate whether the abundance of Zr-89 labeled anti-CD8 miniantibodies is substantially increased in one or more cancer lesions, or if the distribution of Zr-89 labeled anti-CD8 miniantibodies is substantially changed, for monitoring disease progression or the success of anti-human CCR8 antibody treatment.

34. An anti-human CCR8 antibody with ADCC activity and ADCP activity for use in a method for treating cancer according to claim 33, comprising the further step of administering to the patient at least one further effective dose of the anti-human CCR8 antibody if the abundance of the Zr-89-labeled anti-CD8 miniantibody is substantially increased in one or more cancer lesions, or if the distribution of the Zr-89-labeled anti-CD8 miniantibody is substantially changed.

35. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer according to any one of claims 31 to 34, wherein the Zr-89 labeled anti-CD8 minibody provides a radioactivity of about 0.5 to 3.6 mCi.

36. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer according to any one of claims 31 to 35, wherein a PET scan is performed about 6 hours to 36 hours after administration of a corresponding dose of the Zr-89 labeled anti-CD8 minibody.

37. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer according to any one of claims 31 to 36, wherein the Zr-89 labeled anti-CD8 miniantibody is 89Zr-Df-crefmirlimab.

38. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment according to any one of claims 1 to 30, wherein the anti-human CCR8 antibody is an anti-human CCR8 antibody for use in a method of treating cancer according to any one of claims 31 to 37.

39. A method for measuring and quantifying anti-anti-CCR8 antibody formation in cynomolgus monkey or human plasma, the method comprising a bridging ELISA method.

40. The method according to claim 39, wherein a signal is generated if the anti-anti-CCR8 antibody bridges a) the biotinylated anti-CCR8 antibody and b) the SULFO-tagged anti-CCR8 antibody.

41. An isolated anti-CCR8 antibody or antigen-binding fragment thereof comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID Nos. 20, 21, 22, 24, 25 and 26.

42. The isolated anti-CCR8 antibody or antigen-binding fragment thereof according to claim 41, further comprising a. a variable heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 19, and / or b. A variable light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

23.

43. The isolated anti-CCR8 antibody or antigen-binding fragment thereof according to claim 41 or 42, further comprising a. a heavy chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 35, and / or b. a light chain sequence having at least 98% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 36.

Images