Medical Use and Administration Schedule of CCR8 Antibodies

Abstract

The present invention relates to medical uses that involve administering an anti-human CCR8 antibody in a specifically defined dosing regimen, either as monotherapy or in combination with an anti-PD-(L)1 antibody. While the dosing regimen was developed for the anti-human / cynomolgus CCR8 antibody TPP-23411, it can also be used for other antibodies with properties similar to TPP-23411. The medical use or dosing regimen may include a stratification step to select patients with a high probability of successful treatment. Proposed biomarkers are a) tumor proportion score or combined positive score as a measure of PD-(L)1 expression, b) analysis of inflammatory cytokines in blood, plasma, or serum samples, and c) prior cancer treatment with an anti-PD-(L)1 antibody for at least six months. Furthermore, medical uses and treatment methods based on anti-human CCR8 antibodies are provided, including administering Zr-89-labeled anti-CD8 minibodies to determine the abundance and / or distribution of CD8 cells by PET scan for stratification or to monitor treatment success or disease progression. We also provide a method for reliably measuring anti-CCR8 antibodies in cynomolgus monkey or human plasma.Finally, we disclose an anti-mouse CCR8 surrogate antibody that mimics the exceptional half-life of TPP-23411.

Description

[Technical Field]

[0001] Technical Field The present invention relates to medical uses comprising administering anti-human CCR8 antibodies in specifically defined dosing regimens, either as monotherapy or in combination with anti-PD-(L)1 antibodies. The dosing regimen was developed for the anti-human / cynomolgus CCR8 antibody TPP-23411, but it can also be used for other antibodies with properties similar to TPP-23411. In some embodiments, medical uses or treatment methods based on anti-human CCR8 antibodies include a stratification step to select patients with a high probability of successful treatment. The proposed biomarkers are: a. Tumor Proportion Score or Combined Positive Score as a measure of PD-(L)1 expression; b. Analysis of 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 blood, plasma or serum samples; and c. Previous cancer (hereinafter referred to as cancer) treatment with anti-PD-(L)1 antibody for at least 6 months Furthermore, the present invention provides medical uses and treatment methods based on anti-human CCR8 antibodies, including administering Zr-89-labeled anti-CD8 minibodies to determine the abundance and / or distribution of CD8 cells by PET scan for stratification or to monitor treatment success or disease progression. The present invention also provides methods for reliably measuring anti-CCR8 antibodies in cynomolgus monkey or human plasma. Finally, we disclose an anti-mouse CCR8 surrogate antibody that mimics the exceptional half-life of TPP-23411. [Background technology]

[0002] background Targeting regulatory T cells (Tregs) is an attractive approach to enhance antitumor immune responses, either as monotherapy or in combination with immune checkpoint inhibitors (ICIs). This is because Tregs suppress the antitumor immune function of cytotoxic T cells and contribute to the immunosuppressive tumor microenvironment (TME). However, peripheral Tregs are physiologically essential for maintaining immune tolerance. Therefore, systemic depletion of Tregs not only enhances antitumor immune responses but can also lead to potent and undesirable autoimmunity. Essentially, the key to engineering Treg-targeted cancer immunotherapy is ensuring the specific elimination of tumor-infiltrating Tregs without affecting peripheral Tregs.

[0003] Several Treg depletion approaches have demonstrated tumor burden reduction and antitumor immune response enhancement in preclinical models. However, most of these approaches target surface receptors that are not specific to tumor-infiltrating Tregs, such as CD25 or CCR4, and therefore have significant side effects. As a result, there is a strong need for safe and effective medical methods to deplete tumor-infiltrating Tregs while sparing both peripheral Tregs and effector T cells.

[0004] CC motif chemokine receptor 8 (CCR8) has been identified as one of the most differentially and specifically expressed receptors on tumor-infiltrating Tregs compared with peripheral Tregs. CCR8 has four natural ligands, namely, CCL1, CCL8, CCL16, and CCL18, with CCL1 exclusively binding to CCR8. Neither genetic knockout nor functional blockade of CCR8 significantly affected the tumor infiltration, activation, or suppression capabilities of CCR8+ Tregs (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 plays a redundant role with other chemokine receptors in tumor homing of activated Tregs. Therefore, rather than blocking CCR8 function, elimination of tumor-infiltrating CCR8+ Tregs is key to specific immunotherapy with pan-tumor potential (Whiteside, Sarah K. et al., “CCR8 marks highly suppressive Treg cells within tumors but is dispensable for their accumulation and suppressive function.” Immunology 163.4(2021):512-520).

[0005] TPP-23411 is a novel Treg-depleting antibody that specifically depletes tumor-infiltrating Tregs while sparing both peripheral Tregs and effector T cells due to the highly tumor-specific expression profile of its target CCR8. It was first described in U.S. Patent Application No. 17 / 358,841, filed 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. Each of these documents is incorporated herein in its entirety, particularly for the description of specific properties of TPP-23411 and the techniques used to analyze these properties.

[0006] TPP-23411 is a fully human IgG antibody, generated by phage display using a chemically synthesized peptide containing the sulfated N-terminus of human or cynomolgus monkey CCR8 as an epitope. The sequences characterizing TPP-23411 are set forth as SEQ ID NOs: 1 to 18. See also the "Brief Description of the Sequence Listing" section herein.

[0007] TPP-23411 showed highly specific binding to both human and cynomolgus CCR8 expressed in CHO cells, with affinities for each of the same order of magnitude, e.g., in the subnanomolar range. TPP-23411 does not bind to CCR4, the closest paralog of CCR8.

[0008] TPP-23411 is a low / non-internalizing antibody, as demonstrated for human cells expressing endogenous CCR8, a property that may prolong the presentation of TPP-23411 to effector cells and thus improve the efficacy of ADCC- and ADCP-based Treg depletion.

[0009] TPP-23411 is characterized by a relatively high clearance rate in cynomolgus monkeys and humans (see, eg, Examples 12 or 15 herein).

[0010] Antibodies with properties similar to TPP-23411 include: a. an antibody characterized by a KD for binding to CHO cells transfected with human CCR8 that is in the same order of magnitude as the KD of TPP-23411 for binding to CHO cells transfected with human CCR8; b. The antibody induces ADCC and ADCP; Preferably, the antibody binds to human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) that is the same order of magnitude as the KD of TPP-23411 for binding to human Fc gamma receptor IIIA variant V176 (CD16a); and Preferably, the antibody binds to human Fc gamma RIIA (CD32a) with a dissociation constant (KD) that is the same order of magnitude as the KD of TPP-23411 for binding to human Fc gamma RIIA (CD32a); Preferably, the antibody is afucosylated, c. Preferably, the antibody is characterized by a half-life in humans of less than 14 days, preferably less than 10 days, and most preferably less than 7 days.

[0011] TPP-23411, preferably defucosylated, induces both ADCC and ADCP. Consequently, after binding to Tregs, TPP-23411 recruits the respective effector cells (NK cells for ADCC and macrophages for ADCP) via FcR interaction, which then eliminates CCR8-expressing Tregs. Indeed, TPP-23411 induces potent and dose-dependent elimination of human primary CCR8+ Tregs or ectopic human CCR8-expressing HEK293 target cells by engaging either human NK92V cells or human primary M2c macrophages as effector cells.

[0012] TPP-23411 does not block or neutralize CCL1-induced β-arrestin signaling.

[0013] In preclinical studies, the TPP-23411 surrogate antibody was found to demonstrate significant efficacy in syngeneic tumor models, either alone or in combination with a PD-(L)1 inhibitor.

[0014] CCR8 antibodies may be combined with PD-(L)1 inhibitors or other checkpoint inhibitors.

[0015] Pembrolizumab (KEYTRUDA) is a potent humanized IgG4 mAb that binds to the PD-1 receptor with high specificity and inhibits its interaction with PD-L1 and PD-L2. Based on preclinical in vitro data, pembrolizumab exhibits high affinity for PD-1 and potent receptor-blocking activity. 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 concentration of pembrolizumab is a 100 mg / 4 mL (25 mg / mL) solution for injection, provided in a single-dose vial. Pembrolizumab can be administered, for example, at doses of 200 mg every three weeks or 400 mg every six weeks. Therapeutic studies in mouse models have shown that administration of antibodies that block the PD-1 / PD-L1 interaction, either as monotherapy or in combination with other therapeutic modalities, enhances the infiltration of tumor-specific CD8+ T cells, ultimately leading to tumor rejection.

[0016] Nivolumab (OPDIVO) is another PD-1 blocking antibody indicated for the treatment of patients across multiple cancer indications. The dosage form and concentration is a solution for injection provided in a single-dose vial at 10 mg / mL (4 mL, 10 mL). Nivolumab can be administered by intravenous infusion after dilution at doses of, for example, 240 mg every 2 weeks, 360 mg every 3 weeks, or 480 mg every 4 weeks.

[0017] Atezolizumab (TECENTRIQ) is another PD-L1 blocking antibody, also indicated for the treatment of patients across multiple cancer indications. Its dosage and concentration is an injectable solution provided in single-dose vials at 840 mg / 14 mL (60 mg / mL) or 1200 mg / 20 mL (60 mg / mL). After dilution, atezolizumab can be administered by intravenous infusion at doses of, for example, 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every 4 weeks.

[0018] Zimberelimab (Arcus Biosciences) is a monoclonal antibody that binds to PD-1 and restores the anti-tumor activity of T cells. Zimberelimab is currently undergoing clinical trials for various cancer indications, such as for the first-line treatment of metastatic non-small cell lung cancer, in combination with the anti-TIGIT monoclonal antibody domvanalimab and in combination with the dual A2a / A2b adenosine receptor antagonist etormadenant. Zimberelimab can be administered intravenously after dilution, for example, at a dose of 360 mg every three weeks.

[0019] Toripalimab is a recombinant humanized PD-1 monoclonal antibody that binds to PD-1 and prevents its binding to PD-L1 and PD-L2, and is being developed by Shanghai Junshi Bioscience Co., Ltd. for the treatment of various cancers. The approved dose of toripalimab is 3 mg / kg as an intravenous (IV) infusion every 2 weeks.

[0020] Durvalumab (IMFINZI) is a PD-L1 blocking antibody indicated for various cancer types. It is available in single-dose vials at 500 mg / 10 mL or 120 mg / 2.4 mL (50 mg / mL, respectively) as a solution for injection. After dilution, durvalumab can be administered by intravenous infusion at doses of 10 mg / kg every two weeks or 1500 mg every three weeks, for example, as part of a combination regimen.

[0021] Additionally, PD-(L)1 inhibitors and their dosing regimens (approved or in clinical trials) are known in the art and may be useful in the medical uses and methods of treatment provided.

[0022] technical challenges Unless antibodies are properly tested in appropriate model species, it is difficult to propose an appropriate dosing regimen. TPP-23411 cross-reacts with the cynomolgus monkey CCR8 orthologue but not with the mouse CCR8 orthologue. Therefore, established models for finding optimal dosing regimens may not be applicable to finding appropriate solutions for dosing in human patients for monotherapy and combination therapy.

[0023] Furthermore, during characterization of TPP-23411, the inventors observed exceptional PK / PD behavior and increased clearance for this antibody in cynomolgus monkeys. Based on the preliminary translational pharmacokinetic estimates described herein, TPP-23411 is characterized by a half-life of approximately 4 days (a typical antibody has a half-life of 21 days). Therefore, this clearance behavior not only deviates from the expected properties of TPP-23411, but also deviates from the typical half-lives of other antibodies for medical use, making it difficult to identify a safe and efficient dosing regimen for patient treatment.

[0024] To find a suitable solution for administration in human patients, we needed to devise an anti-mouse CCR8 surrogate antibody that could be used to model the short half-life of TPP-23411. This anti-mouse CCR8 surrogate antibody is TPP-29338, provided herein.

[0025] Furthermore, it was necessary to determine and quantify anti-CCR8 antibodies in plasma or serum of cynomolgus monkeys or humans to propose appropriate dosing regimens and perform quality control. The medical use of TPP-23411 provided herein is characterized by a specific dosing regimen that ensures excellent efficacy while meeting necessary safety requirements. With regard to the dosing regimen of the present invention, a successful mode of action is demonstrated in Example 24. Furthermore, the dosing regimen according to the present invention provides ease of handling and administration, thereby reducing dosing errors while improving patient quality of life and compliance.

[0026] Furthermore, administration of Treg depleting agents can be associated with significant side effects and may not be effective in certain patient populations. During testing of the dosing regimens according to the present invention, the inventors devised a specific (pre)dosing scheme that was found to prevent adverse reactions observed upon intravenous administration of anti-CCR8 antibodies (see Example 23). A stratification process is provided herein to identify patients who are likely to benefit from anti-CCR8 antibody treatment and for whom potential side effects are tolerable after benefit-risk assessment.

[0027] Finally, we found that IHC staining to determine the amount of T cells in tumor biopsies as a biomarker for stratification monitoring can lack robustness, for example, when T cell distribution is not normal. Therefore, we present here the application of a specific PET-based method to track T cell recruitment after administration of an anti-human CCR8 antibody.

[0028] background Several companies have initiated or announced plans to initiate clinical trials for administering compounds that target CCR8, each of which provides dosing regimens deviates at least insofar as the CCR8-targeting compounds deviate from TPP-23411, and in various other embodiments, from the dosing schemes of the methods of treatment / medical uses of the present invention described herein.

[0029] Jounce and Gilead are developing the anti-CCR8 antibody JTX-1811 / GS-1811 (see WO2021 / 163064 A1), and Gilead announced the initiation of a "Phase 1 study to evaluate the safety and tolerability of GS-1811, a defucosylated anti-CCR8 monoclonal antibody, as monotherapy and in combination with pembrolizumab in adults with advanced solid tumors" (NCT05007782). During dose escalation, participants will receive escalating dose levels of GS-1811 for up to 12 months to determine the maximum tolerated dose (MTD) and / or recommended Phase 2 dose.

[0030] Shionogi & Co., Ltd. developed the anti-CCR8 antibody S-531011 (see WO2020 / 138489 A1) 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 via intravenous infusion for up to approximately 12 months. For the combination arm, participants will receive escalating doses of S-531011 in combination with pembrolizumab via intravenous infusion for up to approximately 12 months.

[0031] BMS is developing the anti-CCR8 antibody BMS-986340 (see WO2021 / 194942 A1) and initiated a Phase 1 / 2 study of BMS-986340 as monotherapy and in combination with nivolumab in participants with advanced solid tumors in May 2021 (NCT04895709). Completion of the first phase of the study is expected in March 2024. In the dose-escalation phase, subjects will receive 4A19 intravenously at fixed doses of 0.3, 1, 3, 10, 30, 100, 300, and 800 mg once every two weeks (Q2W). In Part IB, subjects will receive 4A19 intravenously at the same fixed dose in combination with nivolumab, administered intravenously at the FDA-approved fixed dose of 480 mg once every four weeks (Q4W).

[0032] International phase application WO2022 / 00443 A1 [entitled "METHODS AND COMPOSITIONS FOR TARGETING TREGS USING CCR8 INHIBITORS"] was filed by Nanjing Immunophage Biotech Co., Ltd. on July 3, 2020, and discloses a small molecule CCR8 inhibitor that blocks the CCR8 / CCL1 axis as demonstrated in a calcium mobilization assay. On November 15, 2021, Nanjing Immunophage initiated a "Phase 1 study to evaluate the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of IPG7236 administered orally as a single agent in patients with advanced solid tumors" (see NCT05142592). The IPG7236 drug product is supplied as an oral tablet dosage form containing two strengths, 25 mg and 100 mg, respectively.

[0033] LM-108 is a humanized monoclonal anti-CCR8 antibody developed by LaNova Medicines, which announced that it will initiate a "Phase I / II, open-label, dose-escalation and dose-expansion clinical trial 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" in August 2022 (NCT05518045).

[0034] Although U.S. Patent Application No. 17 / 280,137 discloses Zr-89 labeled anti-CD8 minibodies for PET scans, it does not disclose the use of these minibodies as part of the medical use of anti-CCR8 antibodies, and this specific method is superior to conventional histopathological approaches for the purpose of reliably tracking T cell recruitment as a biomarker after administration of anti-human CCR8 antibodies.

[0035] Although 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-1 bnAbs." Journal of immunological methods 479 (2020): 112736), the inventors are not aware of a specific method for detecting and quantifying anti-anti-CCR8 antibodies.

[0036] Although various anti-CCR8 murine surrogate antibodies exist, such as those previously described by the inventors in U.S. Patent Application 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, the inventors are not aware that any of these anti-CCR8 murine surrogate antibodies may be suitable for modeling the fast clearance rate of TPP-23411. Summary of the Invention

[0037] Means to solve the problem Based on various experimental data (see Examples 1-16), the present inventors have successfully identified medical uses of anti-CCR8 antibodies, including specific administration schemes, which are described in more detail, inter alia, in Example 17.

[0038] Anti-human CCR8 antibody to patients in need thereof a. Approximately 1, 2.5, 3, 10, 30, 50, 100, 125, or 250 mg once weekly; or b. Approximately 16, 450, 500, 750, 1000, or 1500 mg once every three weeks and administering the antibody to a patient in need thereof intravenously in a total amount of 100 mg of the antibody to a patient in need thereof.

[0039] Optionally, the medical use comprises administering an anti-PD-(L)1 antibody to a patient in need thereof: i. about 200 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or ii. about 400 mg once every 6 weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or iii. about 240 mg once every two weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or iv. about 360 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or v. about 480 mg once every four weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or vi. about 840 mg every two weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or vii. about 1200 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or viii. about 1680 mg every 4 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or ix. about 360 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is zimberelimab), or x. about 3 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is toripalimab), or xi. about 10 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab), or xii. about 1500 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab) This may include administering intravenously in a total amount of

[0040] Also, a. administering an anti-CCR8 antibody intravenously to a patient in need thereof in a total amount of 2.7 mg to 75 mg once weekly; b. Preferably, an anti-PD-(L)1 antibody is administered to the patient: i. about 200 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or ii. about 400 mg once every 6 weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or iii. about 240 mg once every two weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or iv. about 360 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or v. about 480 mg once every four weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or vi. about 840 mg every two weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or vii. about 1200 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or viii. about 1680 mg every 4 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or ix. about 360 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is zimberelimab), or x. about 3 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is toripalimab), or xi. about 10 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab), or xii. about 1500 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab) and administering the antibody to a patient having ADCC activity and / or ADCP activity intravenously in a total amount of 100 mg / mL of the antibody to a patient having CCR8 activity.

[0041] Furthermore, a. administering an anti-CCR8 antibody intravenously to a patient in need thereof in a total dose of 16 mg to 450 mg once every three weeks; b. Preferably, an anti-PD-(L)1 antibody is administered to the patient: i. about 200 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or ii. about 400 mg once every 6 weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or iii. about 240 mg once every two weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or iv. about 360 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or v. about 480 mg once every four weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or vi. about 840 mg every two weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or vii. about 1200 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or viii. about 1680 mg every 4 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or ix. about 360 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is zimberelimab), or x. about 3 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is toripalimab), or xi. about 10 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab), or xii. about 1500 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab) and administering the antibody to a patient having ADCC activity and ADCP activity intravenously to the patient in a total amount of 100 mg of the antibody to a patient having CCR8 activity.

[0042] The particular administration scheme provided herein avoids unacceptable adverse effects while maintaining a sufficient dose of anti-CCR8 antibody in the blood for optimal efficacy.

[0043] In some embodiments, medical uses or treatment methods based on anti-human CCR8 antibodies include a stratification step to select patients with a high probability of successful treatment and / or an improved benefit-risk ratio. a. Tumor Proportion Score or Combined Positive Score as a measure of PD-(L)1 expression; b. Analysis of 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 blood, plasma or serum samples; and c. Prior cancer treatment with an anti-PD-(L)1 antibody for at least 6 months is.

[0044] Furthermore, the present invention provides medical uses and methods of treatment based on anti-human CCR8 antibodies, which include administering Zr-89 labeled anti-CD8 minibodies to determine the abundance and / or distribution of CD8 cells by PET scan for stratification or to monitor the success of treatment or disease progression.

[0045] The present invention also provides a method for reliably measuring anti-CCR8 antibodies in cynomolgus monkey or human plasma using a bridging ELISA method based on anti-CCR8 antibodies. [Brief explanation of the drawings]

[0046] [Figure 1] Overview of the dose escalation portion of the study scheme. [Figure 2] Dose-normalized plasma concentrations of TPP-23411 after intravenous and subcutaneous administration. [Figure 3] Estimated human plasma c / t profile following a single intravenous infusion of 1 mg / kg TPP-23411 over 1 hour. [Figure 4] Simulation of c / t profiles after multiple intravenous infusions of 1 mg / kg TPP-23411 over 1 hour for QW, Q2W, and Q3W dosing. [Figure 5]Correlation between response to anti-CCR8 antibody treatment and anti-PD-L1 antibody (A) or anti-PD-1 (B) treatment in vivo. (A)-(B): The in vivo efficacy of antibody treatment was evaluated in various syngeneic mouse cancer models. The solid and dashed lines indicate the regression line and associated 95% confidence intervals, respectively. [Figure 6] Correlation between PD-L1 mRNA expression (A) and IFN-γ mRNA expression (B) in early-stage, untreated tumors and response to anti-mouse CCR8 antibody treatment. The correlation was assessed between (A) baseline PD-L1 expression and (B) baseline IFN-γ expression and the in vivo efficacy of anti-CCR8 antibody treatment across various syngeneic mouse cancer models. The solid and dashed lines represent the regression line and associated 95% confidence interval, respectively. [Figure 7] The increase in activated, proliferating CD8+ T cells relative to the total number of CD3+ T cells began approximately three days after the first administration of 10 mg of anti-CCR8 antibody to a human patient. A continuous increase was observed, especially from the second week of treatment. This is likely due to the induction of immune activation in the tumor. Treatment ended approximately three weeks after the final infusion. The ratio of these two cell types at the time of screening was set to 1. [Figure 8] The ratio of activated Tregs to the total number of CD3+ T cells decreased approximately 1 day after the first anti-CCR8 antibody administration (10 mg dose). The ratio of these two cell types at the time of screening was set to 1. [Figure 9] Ratio of activated Tregs to the number of activated proliferating CD8+ T cells. The ratio of these two cell types at the time of screening was set to 1. [Figure 10]Reduction of Tregs in patient blood samples upon treatment with 1 mg, 3 mg, 10 mg, or 30 mg of anti-CCR8 antibody. For each patient, the ratio (activated Tregs / total CD3+ T cells) obtained at time points C1D2 to C2D15 was divided by the ratio (activated Tregs / total CD3+ T cells) obtained for the same patient at screening (Scr), i.e., before administration of anti-CCR8 antibody. To obtain data in one box, all patients treated with the indicated doses were grouped together. Even the lowest 1 mg cohort shows a median reduction of activated Tregs to approximately 53% of the screening value. [Figure 11] Box plot of levels of TNF-alpha measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients. [Figure 12] Box plot of levels of IFN-gamma measured in pg / μl in blood or serum samples taken from human patients 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody. [Figure 13] Box plot of IP10 (CXCL10) levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients. [Figure 14] Box plot of IL8 levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients. [Figure 15] Box plot of IL6 levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients. [Figure 16] Box plot of IL-10 levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0047] Brief description of the sequence listing 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 September 3, 2023. [Table 1] TIFF2025530159000002.tif248162TIFF2025530159000003.tif69162

[0048] definition Unless otherwise specified, all scientific and technical terms used in the specification, drawings, and claims have their ordinary meanings as commonly understood by those skilled in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. If two or more documents incorporated by reference contain conflicting and / or inconsistent disclosure, the document having the later effective date will control. When reference is made to a database, the effective date will be the version number applicable as of May 6, 2022, unless otherwise indicated. The materials, methods, and examples are illustrative only and not limiting. Unless otherwise indicated, the following terms used in this document, including the specification and claims, have the definitions set forth below.

[0049] The term "about" as used herein refers to a value within the acceptable error range of a particular value as determined by one skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations, as is customary in the art. The term "about" is also used to indicate that the amount or value in question may be the indicated value or some other value that is approximately the same. This term is intended to convey that similar values ​​will promote results or effects equivalent to those described herein. In this context, "about" can mean a range of up to 10% above or below. Whenever the term "about" is specified with respect to a particular assay or embodiment, that definition will prevail in that particular context.

[0050] Unless otherwise specified, the term "approximately" means + / - 10% of the stated value.

[0051] The words "comprise," "include," "contain," "have," and the like are to be construed as open-ended rather than restrictive. As used herein, the word "comprise" includes "consisting of."

[0052] The singular forms include plural referents unless the context clearly contradicts otherwise. Thus, for example, reference to an "antibody" includes a single monoclonal antibody as well as a plurality of monoclonal antibodies, whether the same or different. Similarly, reference to a "cell" includes a single cell as well as a plurality of cells.

[0053] 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, 1, 2, 3, 4, or 5 or more elements.

[0054] It is further understood that slight variations above and below the stated ranges may be used to achieve substantially the same results as values ​​within the range, and unless otherwise indicated, the disclosure of a range is intended to be a continuous range, including every value between the minimum and maximum values.

[0055] As used herein, the term "amino acid" or "amino acid residue" typically refers to a naturally occurring amino acid. Single-letter codes are used herein to represent each amino acid. As used herein, a "charged amino acid" is a negatively or positively charged amino acid. "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 are 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). The "amphipathic amino acids" are tryptophan (W), tyrosine (Y), and methionine (M). The "aromatic amino acids" are phenylalanine (F), tyrosine (Y), and tryptophan (W). The "hydrophobic amino acids" are glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and cysteine. The "small amino acids" are glycine (G), alanine (A), serine (S), proline (P), threonine (T), aspartic acid (D), and asparagine (N).

[0056] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no maximum limit on the number of amino acids. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are numerous types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0057] Where a gene or protein derived from a particular species, such as mouse, is generally referred to, the human-derived analog is also intended unless otherwise indicated or clearly contradicted, particularly in the case of biomarkers.

[0058] The term "isolated" as applied to a nucleic acid, polypeptide, protein, or antibody means that the nucleic acid, polypeptide, protein, or antibody is substantially free from other cellular components that naturally accompany it. It is preferably in a homogeneous state. It can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. A protein, polypeptide, or antibody that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from adjacent open reading frames encoding proteins other than the gene of interest. However, an isolated polypeptide may also be immobilized, for example, on beads or particles, e.g., via a suitable linker.

[0059] The term "purified" means that a nucleic acid or protein gives rise to substantially one band in an electrophoretic gel. Specifically, 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.

[0060] As used herein, the term "synthetic," e.g., with respect to a synthetic nucleic acid molecule or synthetic gene or synthetic peptide, refers to a nucleic acid molecule or polypeptide molecule produced by recombinant and / or chemical synthesis methods. As used herein, production by recombinant means, using recombinant DNA methods, refers to the use of well-known methods of molecular biology to express proteins encoded by cloned DNA.

[0061] "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 tyrosylprotein sulfotransferases (TPST) 1 and 2, which are Golgi-resident enzymes that transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to context-dependent tyrosines within protein substrates. Synthetic sulfation of tyrosine can be performed by techniques known in the art, for example, as described in 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. A sulfated polypeptide is a polypeptide that contains at least one sulfation. A non-sulfated polypeptide is a polypeptide that does not contain sulfation.

[0062] As used herein, the term "N-terminus" or "N-terminal" of a chemokine receptor refers to the N-terminal amino acids of the chemokine receptor, including at least the TRD. If the polypeptide or protein contains a signal peptide, the N-terminus can also refer to the N-terminal sequence following the natural cleavage site of the polypeptide or protein. According to some preferred embodiments, the N-terminus includes the LID and TRD domains of the chemokine receptor, but does not include the native cysteine ​​between these two domains. Alternatively, the cysteine ​​can be removed or replaced with another amino acid.

[0063] " Sequence identity " or " identity percentage " is the number that indicates how similar a query sequence is to a target sequence, more precisely, how many characters are identical in each sequence after alignment.The most common tool for calculating sequence identity is BLAST (Basic Local Alignment Search Tool, https: / / blast.ncbi.nlm.nih.gov / ), which performs a comparison between sequence pairs to find the area of ​​local similarity.Suitable alignment methods are known in the art, for example, the Needleman-Wunsch algorithm for global-global alignment, using the BLOSUM62 matrix, with a gap opening penalty of 11 and a gap extension penalty of 1.Then, the number of aligned identical residue pairs can be counted, and then divided by the total length of alignment (including gaps, internal and external) to obtain the identity percentage value.

[0064] For "percent similarity" or "sequence similarity" values, the same approach as for percent identity values ​​can be used, except that aligned residue pairs with non-negative (i.e., ≥ 0) BLOSUM62 values ​​are counted rather than pairs of identical residues.

[0065] "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 one of the subfamily of chemokine receptors, a large family of G protein-coupled receptors known 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.

[0066] The term "CCR8" refers to CC chemokine receptor type 8. The CCR8 protein is encoded by the gene CCR8 (NCBI gene ID 1237). Synonyms for CCR8 include, among others, CC-CKR-8, CCR-8, CDw198, CKRL1, CMKBR8, CMKBRL2, GPRCY6, CY6, and TER1. The CCR8 protein includes human, mouse, rat, rhesus monkey, and other mammalian and non-mammalian homologs. The sequence of human CCR8 is accessible via UniProt identifier P51685 (CCR8_HUMAN), including, for example, human isoforms P51685-1 or P51685-2 (UniProt, November 29, 2019). The sequence of mouse CCR8 is accessible via UniProt identifier P56484 (CCR8_MOUSE). The sequence of rhesus monkey CCR8 is accessible via the UniProt identifier O97665 (CCR8_MACMU). Different isoforms and variants may exist depending on the species, all of which are encompassed by the term CCR8. Also included are CCR8 molecules before and after maturation (i.e., independent of cleavage of one or more prodomains). Synthetic variants of the CCR8 protein can be generated and are encompassed by the term CCR8. The CCR8 protein can also undergo various modifications, e.g., synthetic or naturally occurring modifications, e.g., post-translational modifications. Recombinant human CCR8 is commercially available or can be produced as known in the art. CCR8 is a receptor for the chemokine CCL1 / SCYA1 / I-309.Barington et al. reported the importance of conserved disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) of the 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). Furthermore, they found that two distinct aromatic residues in ECL-2, namely, Tyr184 (Cys+1) and Tyr187 (Cys+4), are important for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding.

[0067] "Programmed death-1 (PD-1)" refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes, but is not limited to, human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs that share at least one epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number U64863 (November 29, 2019).

[0068] "Programmed death-ligand 1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding 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 that share at least one epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7 (November 29, 2019).

[0069] The term "PD-(L)1" refers to PD-1 and / or PD-L1.

[0070] The "tumor proportion score" (TPS) is the percentage of viable tumor cells that show partial or complete membrane staining at any intensity. For example, a specimen should be considered to have PD-L1 expression if its TPS is ≥ 1%, and to have high PD-L1 expression if its TPS is ≥ 50%. For example, PD-L1 protein expression in NSCLC is typically determined using the tumor proportion score (TPS).

[0071] A "previous tumor proportion score" is a tumor proportion score obtained during the preparation or monitoring of a previous (cancer) treatment or medical analysis, i.e., without using a sample obtained from a fresh biopsy in preparation for anti-CCR8 antibody treatment.

[0072] The "combined positive score" (CPS) is the number of stained cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells and multiplied by 100. For example, a specimen should be considered to have PD-L1 expression if its CPS is ≥ 1, and to have high PD-L1 expression if its CPS is ≥ 10. The FDA has approved the use of the PD-L1 IHC 22C3 pharmDx assay and the VENTANA PD-L1 (SP263) assay to determine patient eligibility for the therapeutic antibody pembrolizumab.

[0073] A "previous combined positive score" is a combined positive score obtained during the preparation or monitoring of a previous (cancer) treatment or medical analysis, i.e., without using a sample obtained from a fresh biopsy in preparation for anti-CCR8 antibody treatment.

[0074] The "PD-L1 IHC 22C3 pharmDx Assay" is a qualitative immunohistochemistry assay that uses the monoclonal mouse anti-PD-L1 clone 22C3 and is intended for use in detecting PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue using the EnVision FLEX visualization system on the Autostainer Link48 (see, e.g., https: / / www.accessdata.fda.gov / cdrh_docs / pdf15 / p150013s001c.pdf). The "VENTANA PD-L1 (SP263) Assay" is another qualitative immunohistochemistry assay that uses the rabbit monoclonal anti-PD-L1 clone SP142, and may be used on FFPE tissue stained with, for example, the OptiView DAB IHC Detection Kit and OptiView Amplification Kit on a BenchMark ULTRA instrument (see, for example, https: / / www.accessdata.fda.gov / cdrh_docs / pdf16 / p160046c.pdf).

[0075] The term "modulation" refers to any alteration of an existing process or behavior, such as blocking (antagonism) and induction (agonism). For example, modulation of G protein-independent signaling refers to any significant alteration of G protein-independent signaling.

[0076] The term "internalization" of an antibody, fragment, or conjugate refers to the uptake of the antibody, fragment, or conjugate into cells. Preferably, internalization is determined for a cell line with endogenous target expression, for example, for human or mouse CCR8. Preferably, internalization is determined by measuring the total internalization fluorescence intensity per cell and quantified relative to an isotype control. Briefly, the 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 determined and quantified relative to the isotype control.

[0077] A "non-internalizing antibody" is defined as an antibody that exhibits substantially the same internalization as a corresponding isotype control.

[0078] A "low internalizing antibody" is defined as an antibody that exhibits 10-fold or less internalization over the isotype control, preferably less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, less than 1.5-fold, less than 1.4-fold, less than 1.3-fold, less than 1.2-fold or less than 1.1-fold internalization over the isotype control.

[0079] An "isotype control" is an antibody or fragment that does not bind to the target and has the same class and type as a reference antibody or fragment that recognizes the target.

[0080] An antibody or fragment is said to be "cross-reactive" or "cross-reacts" if it binds to antigens from two or more different species with a KD value, for example, of 10-7M or less, more preferably less than 10-8M, and even more preferably in the range of 10-9M to 10-11M.

[0081] The term "specifically binds" as used herein with respect to an antibody contemplates an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. Antibodies characterized by substantially nonspecific binding lack therapeutic applicability, and therefore these embodiments are excluded. However, as is known in the art, specific binding of an antibody or conjugate does not necessarily exclude the antibody or conjugate from binding to other antigens / target molecules. An antibody that specifically binds to an antigen from one species may also bind to antigens of one or more other species. Such cross-species reactivity, in itself, does not alter the classification of an antibody as specific.

[0082] In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species. This means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) in the chemical species. For example, an antibody recognizes and binds to a particular protein structure rather than proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that binds to the antibody.

[0083] In case of doubt, specific binding of an antibody or conjugate is preferably confirmed by a minimum of at least 10% of the antibody, antibody fragment or conjugate to its antigen / target. -7 M (as KD value; i.e., preferably 10 -7M), where the antibody or conjugate has at least a two-fold lower affinity for a non-specific antigen that is not the predetermined antigen / target molecule or a closely related antigen / target molecule.

[0084] The term "affinity" is a term used in the art to describe the strength of binding between a binder, antibody, or antibody fragment and a target. The "affinity" of antibodies and their fragments for a target can be determined using techniques known in the art or described herein, for example, by ELISA, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), flow cytometry, or fluorescence polarization assay. Preferably, affinity is expressed as a dissociation constant, KD.

[0085] The "dissociation constant" (KD) has molar units (M) and corresponds to the concentration of conjugate / antibody at which half of the target protein is occupied at equilibrium. The smaller the dissociation constant, the higher the affinity between the conjugate or antibody and its target.

[0086] According to the present invention, the antibody preferably has a molecular weight of at least 10 -7 M (as KD value), more preferably at least 10 -8 M, even more preferably 10 -9 M~10 -11 The KD values ​​may preferably be determined by surface plasmon resonance spectroscopy, e.g., as described elsewhere herein. If assay conditions are found to affect the determined KD, the assay settings with the smallest standard deviation should be used.

[0087] "Half-maximal effective concentration" (EC50) refers to the concentration of a drug, antibody, fragment, conjugate, or molecule that induces a response halfway between the baseline and maximum after a specified incubation time. Thus, in the case of antibody binding, EC50 represents the antibody concentration required for half-maximal binding. EC50 can be determined when the inflection point can be determined by mathematical modeling (e.g., nonlinear regression) of a dose-response curve that represents the relationship between the concentration of the applied drug, antibody, fragment, conjugate, or molecule and the signal. For example, EC50 can be determined when the dose-response curve follows a sigmoidal curve. When the response is inhibition, EC50 is referred to as the half-maximal inhibitory concentration (IC50). EC80 can be determined mutatis mutandis.

[0088] The (effective) "half-life" of an antibody is the time required for its maximum concentration in plasma (Cmax) to drop to half of that maximum concentration. On average, the serum half-life of IgG subclasses (IgG1, IgG2, and IgG4) is approximately 23 days, while that of IgG3 and other Ig classes is 2-6 days. Various methods for analyzing antibody half-life are known in the art, including, for example, mass spectrometry or ELISA-based approaches.

[0089] The term "antibody" (Ab) refers to an immunoglobulin molecule (e.g., but 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) that specifically binds to or immunologically reacts with a particular antigen. An antibody or antibody fragment contains the complementarity-determining regions (CDRs), also known as hypervariable regions, in both the light and heavy chain variable domains. The more highly conserved portions of variable domains are called framework regions (FRs). As is known in the art, the amino acid positions / boundaries defining the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. As used herein, immunoglobulin amino acid residues are numbered according to the immunoglobulin amino acid residue numbering system of Kabat et al. Each of the naturally occurring heavy and light chain variable domains contains four FR regions. The three CDRs in each chain are closely packed together by their 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." No. 165-462 (1987). As used herein, the term "antibody" also refers to antibody fragments, unless expressly specified otherwise. Depending on the context, the term "antibody" can also refer to any proteinaceous binding molecule with immunoglobulin-like functions.

[0090] The term "CDR" refers to the complementarity-determining region of an antibody. As known in the art, a complementarity-determining region (CDR) is a part of the variable chain of an antibody and a T-cell receptor. A set of multiple CDRs constitutes a paratope. CDRs are crucial for the diversity of antigen specificity. There are three CDRs (CDR1, CDR2, and CDR3) arranged non-contiguously in the amino acid sequence of the variable domain of an antigen receptor. Because an antigen receptor typically consists of two variable domains (on two different polypeptide chains, i.e., heavy and light chains), there are usually six CDRs for each antigen receptor that can come into contact with 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).

[0091] "Constant region" refers to the portion of an antibody molecule that confers effector functions. The heavy chain constant region can be selected from any of five isotypes: alpha (α), delta (δ), epsilon (ε), gamma (g), and mu (μ).

[0092] As used herein, the terms "Fc domain," "Fc region," or "Fc portion" refer to the C-terminal region of an antibody heavy chain containing at least a portion of the constant region. This 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.

[0093] The antibodies or binding fragments according to the invention may be modified to alter at least one constant region-mediated biological effector function. For example, in some embodiments, antibodies may be modified to have at least one constant region-mediated biological effector function that is reduced or enhanced compared to an unmodified antibody, e.g., to have reduced or improved binding to Fc receptors (FcγRs). FcγR binding can be reduced, for example, by mutating the immunoglobulin constant region segment of an antibody in specific regions required 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 can be enhanced, for example, by defucosylation. Reduced FcγR binding may also reduce other effector functions that depend on FcγR interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity (ADCC).

[0094] Furthermore, manipulating the interaction between Fc and FcRn allows for modulation of the half-life of the antibody in vivo. Blocking the interaction, for example by introducing the mutation H435A, results in a significantly shorter half-life because the antibody is no longer protected from lysosomal degradation by FcRn recycling. In some preferred embodiments according to all aspects, the antibodies of the invention comprise the mutation H435A or are otherwise engineered to have a reduced half-life.

[0095] 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 through more efficient recycling from endosomes in both preclinical species and humans (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 of all aspects, antibodies according to the invention include YTE mutations (M252Y / S254T / T256E) and / or equivalent mutations, such as LS (M428L / N434S), or are otherwise engineered for improved half-life. Suitable Fc engineering approaches 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.

[0096] A "defucosylated" antibody is one that has been engineered so that the oligosaccharides in the Fc region of the antibody lack any fucose sugar units. Glycosylation of an antibody can alter its function. For example, complete removal of glycosylation at N297 in the CH2 domain of an IgG results in loss of FcγR binding. However, modulation of the specific carbohydrate composition at N297 can have the opposite effect, enhancing the antibody's ADCC activity. Briefly, the affinity of an antibody for activating FcγRs depends on the composition of the N297 N-linked oligosaccharide. There are 32 different possible combinations of oligosaccharides that can occur at this site. Naturally occurring human IgG, and human IgG produced by hybridomas or other common expression systems, typically consists of N-acetylglucosamine (GlcNAc) and three mannose residues that form the core carbohydrate. This core is linked to two additional GlcNAc groups to form biantennary. Galactose addition at each branch and terminal addition of sialic acid to these galactose molecules can occur. Fucose is often part of the core GlcNAc. This fucose prevents antibody interaction with FcγRIIIA due to steric hindrance. Therefore, removal of this fucose molecule while preserving other forms of glycosylation at this site enhances antibody binding to activating FcγRs and 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). A method for producing fucose-free antibodies involves growth in rat myeloma YB2 / 0 cells (ATCC CRL 1662). YB2 / 0 cells express low levels of FUT8 mRNA, encoding α-1,6-fucosyltransferase, an enzyme required for polypeptide fucosylation. Defucosylated antibodies are preferred in the present invention.

[0097] Antibody-dependent cellular cytotoxicity (ADCC), also referred to as antibody-dependent cell-mediated cytotoxicity, is a cell-mediated immune defense mechanism in which immune cells actively lyse target cells bearing a membrane surface antigen bound by a specific antibody. ADCC is mediated by the interaction of antibodies or fragments with FcγRIIIa. In humans, FcγRIII exists in two distinct forms: FcγRIIIa (CD16a) and FcγRIIIb (CD16b). FcγRIIIa is expressed as a transmembrane receptor on monocytes, neutrophils, mast cells, macrophages, and natural killer cells, whereas FcγRIIIb is expressed only on neutrophils. These receptors bind to the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by human effector cells.

[0098] Various assay systems for measuring 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. investigated various ADCC assay systems, namely, assays based on: (i) natural killer cells (FcγRIIIA + primary NK) derived from human donors, (ii) FcγRIIIA-modified NK-92 cells, and (iii) FcγRIIIA / NFAT-RE / luc2-modified 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-92 and Jurkat T cells," Journal of Immunological Methods 441 (2017): 56-66; the entire contents of which are incorporated herein by reference; in particular, reference is made to the methodology described for these assays). Briefly, all three effector cell lines differentially express FcγRIIIA, resulting in dose-dependent ADCC pathway activity, but only primary NK cells and engineered NK-92 cells can induce ADCC-mediated cell lysis. Therefore, for functional assessment of ADCC activity, primary NK cells or NK-92 (V-158) cells better reflect the physiologically relevant mechanism of action of ADCC. As an engineered (engineered) cell line, NK-92 cells are able to behave more reproducibly than primary NK cells and are therefore the preferred assay system for measuring ADCC responses in human subjects, for example, in cases of doubt.

[0099] An antibody or antigen-binding fragment that induces ADCC is an antibody that can induce substantial cytolysis of target cells in the presence of NK effector cells. Preferably, ADCC induction results in cytolysis 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 the target cells.

[0100] "Antibody-dependent cellular phagocytosis" (ADCP) is a mechanism by which antibody-opsonized target cells activate FcγRs on the surface of macrophages, inducing phagocytosis and resulting in the internalization and degradation of the target cells. In ADCP, binding to macrophages as effector cells typically occurs through the interaction of the Fc portion of the antibody with FcγRIIa (CD32a) expressed by macrophages.

[0101] An ADCP-inducing antibody or antigen-binding fragment is an antibody that can induce phagocytosis of a substantial number of target cells in the presence of 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 the target cells.

[0102] "Complement-dependent cytotoxicity" ("CDC") is an effector function of IgG and IgM antibodies. When they bind to surface antigens on target cells (e.g., bacteria- or virus-infected cells), the binding of the protein C1q to these antibodies initiates the classical complement pathway, resulting in the formation of the membrane attack complex (MAC) and target cell lysis. The complement system is efficiently activated by human IgG1, IgG3, and IgM antibodies, weakly by IgG2 antibodies, and not by IgG4 antibodies. It is one mechanism of action by which therapeutic antibodies (which are also specific embodiments of antibodies according to the invention) can achieve antitumor effects. Several laboratory methods for determining the effectiveness of CDC exist and are known in the art.

[0103] An antibody or antigen-binding fragment that induces CDC is an antibody that can induce membrane attack complex formation and a substantial amount of target cell lysis. Preferably, CDC induction results in cytolysis 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 the target cells.

[0104] Antibodies that include an Fc region may or may not include modifications that promote binding between the first and second subunits of the Fc domain.

[0105] Most preferably, the induction of ADCC and ADCP results in at least 50% Treg depletion.

[0106] As used herein, a "fragment" of an antibody must substantially retain the desired affinity of the full-length antibody. Thus, a suitable fragment of an anti-human CCR8 antibody retains the ability to bind to a target chemokine receptor, for example, the ability to bind to a human CCR8 receptor. An antibody fragment includes a portion of the full-length antibody, generally its antigen-binding region or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single-chain antibody molecules, diabodies (bispecific antibodies), and domain antibodies. See Holt, Lucy J. et al., "Domain antibodies: proteins for therapy." Trends in biotechnology 21.11 (2003):484-490.

[0107] The "Fab fragment" contains the constant domain of the light chain and the first constant domain (CH2) of the heavy chain.

[0108] "Fab' fragments" differ from Fab fragments 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.

[0109] "F(ab') fragments" are generated by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from the animal's circulation, and may exhibit lower 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.

[0110] An "Fv fragment" is the minimum fragment of an antibody which contains a complete target recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs in each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Often, the six CDRs confer antigen-binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) may have the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0111] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody within a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0112] A "single domain antibody" is composed of a single VH or VL domain that exhibits sufficient affinity for a target. In certain embodiments, a single domain antibody is a camelized antibody. See, e.g., Riechmann, Lutz, and Serge Muyldermans. "Single domain antibodies: comparison of camel VH and camelized human VH domains." Journal of immunological methods 231.1-2 (1999):25-38.

[0113] A "minibody" is an antibody form that has a smaller molecular weight than a full-length antibody while maintaining bivalent binding properties to an antigen. For example, a minibody is a bivalent homodimer, with each monomer having a single-chain variable fragment (scFv) linked to a human IgG1 CH3 domain via a modified IgG1 hinge sequence. Due to their small size, minibodies exhibit faster clearance from the system and enhanced penetration when targeting tumor tissue. The ability of potent targeting combined with rapid clearance makes minibodies advantageous for diagnostic imaging and delivery of cytotoxic / radioactive payloads, where long circulation times can result in harmful administration or dosimetry to the patient.

[0114] A "Zr-89-labeled anti-CD8 minibody" is a minibody that specifically binds to CD8 and is further labeled with Zr-89. Preferably, the Zr-89-labeled anti-CD8 minibody binds to human CD8 glycoprotein with an EC50 of less than 1 nM. For example, the minibody can be conjugated via desferrioxamine (Df) and radiolabeled with the positron-emitting radionuclide "zirconium-89" (Zr; Tm 78.4 hours). In a most preferred embodiment, the Zr-89-labeled anti-CD8 minibody is the Zr-89-labeled anti-CD8 minibody described in U.S. Patent Application No. 17 / 280,137.

[0115] A "bispecific antibody" is a monoclonal antibody that has binding specificities for at least two different epitopes on the same or different antigens. In the present disclosure, one of the binding specificities can be for a target chemokine receptor, e.g., CCR8, and the other binding specificity can be for any other antigen, including, but not limited to, a cell surface protein, receptor, receptor subunit, tissue-specific antigen, virus-derived protein, virus-encoded envelope protein, bacterial-derived protein, or bacterial surface protein. Bispecific antibody constructs according to the present invention also include multispecific antibody constructs comprising multiple binding domains / binding sites, e.g., trispecific antibody constructs comprising three binding domains.

[0116] "Derivatized antibodies" are typically modified by glycosylation, acetylation, pegylation, phosphorylation, sulfation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or conjugation to a cellular ligand or other protein. Any of a number 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. Derivatives can also contain one or more unnatural amino acids, for example, using ambrx technology. See, e.g., Wolfson, Wendy. "Amber codon flashing ambrx augments proteins with unnatural amino acids." Chemistry & biology 13.10 (2006):1011-1012. Antibodies of the present invention can be derivatized, for example, by glycosylation or sulfation.

[0117] A "monoclonal antibody" is a substantially homogeneous population of antibodies that bind to a specific antigen. Monoclonal immunoglobulins can be obtained by methods well known to those skilled in the art (see, for example, Kohler, Georges, and Cesar Milstein, "Continuous cultures of fused cells secreting antibody of predefined specificity," Nature 256:5517 (1975):495-497 and U.S. Pat. No. 4,376,110). Immunoglobulins or immunoglobulin fragments with specific binding affinity can be isolated, enriched, or purified from prokaryotes or eukaryotes. Conventional methods known to those skilled in the art enable the production of both immunoglobulins or immunoglobulin fragments and proteinaceous binding molecules with immunoglobulin-like functions in both prokaryotes and eukaryotes. The antibodies of the present invention are preferably monoclonal antibodies.

[0118] A "humanized antibody" contains CDR regions derived from a non-human species, such as mouse, grafted (transplanted) into V regions derived from human sequences, e.g., with any necessary framework backmutations. Thus, in most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. See, e.g., U.S. Patent Nos. 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205 (each of which is incorporated herein by reference). In some cases, framework residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance (e.g., to obtain a desired affinity). In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which 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 those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise 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).

[0119] A fully human antibody (human antibody) comprises CDRs of human origin, i.e., CDRs of human origin. Preferably, a fully human antibody according to the invention is an antibody that shares 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., a sequence extracted from a recommended list and analyzed in an IMGT / domain gap alignment).

[0120] As recognized by conventional nomenclature systems, such as INN species subsystems, which were in effect until 2017, fully human antibodies may have a reduced number of germline deviations compared to the nearest human germline reference, as determined based on the IMGT database (http: / / www.imgt.org; November 29, 2019). For example, a fully human antibody according to the present invention may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, or 15 germline deviations in the CDRs compared to the nearest human germline reference. Fully human antibodies may be obtained from human-derived B cells by cloning techniques combined with cell enrichment or immortalization processes. However, the vast majority of fully human antibodies in clinical use have been isolated from immunized mice transgenic for the human IgG locus or from sophisticated combinatorial libraries using phage display (Bruggemann, Marianne et al., “Human antibody production in transgenic animals.” Archivum immunologiae et therapiae experimentalis 63.2(2015):101-108; Carter, Paul J. “Potent antibody therapeutics by design.” Nature reviews immunology 6.5(2006):343-357; Frenzel, Andre, 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).

[0121] Several techniques are available for generating fully human antibodies or antibodies containing human-derived CDRs (see WO2008112640). Cambridge Antibody Technologies (CAT) and Dyax obtained antibody cDNA sequences from peripheral B cells isolated from immunized humans and devised phage display libraries to identify human variable region sequences with specific specificities. Briefly, antibody variable region sequences are fused to the Gene III or Gene VIII construct of M13 bacteriophage. These antibody variable region sequences are expressed as Fab or single-chain Fv (scFv) structures at the tip of phages containing the respective sequences. Through rounds of panning processes using various levels of antigen-binding conditions (stringency), phages expressing Fab or scFv structures specific to the antigen of interest can be selected and isolated. The antibody variable region cDNA sequences of the selected phages can then be elucidated using standard sequencing methods. These sequences can then be used to reconstruct complete antibodies with the desired isotype using established antibody engineering techniques. Antibodies constructed according to this method are considered fully human antibodies (including CDRs). To improve the immunoreactivity (antigen-binding affinity and specificity) of selected antibodies, an in vitro maturation process can be introduced, including combinatorial binding of different heavy and light chains, deletions / additions / mutations in the CDR3s of the heavy and light chains (to mimic VJ and VDJ recombination), and random mutations (to mimic somatic hypermutation). An example of a "fully human" antibody produced by this method is the anti-tumor necrosis factor alpha antibody Humira (adalimumab).

[0122] "Anti-drug antibodies" (ADAs) are antibodies that bind to therapeutic antibodies, resulting from a subject's or patient's immune response to the therapeutic antibody. ADAs can reduce the effectiveness of treatment and potentially induce side effects.

[0123] The term "quantitative" means to estimate or measure the amount of a molecule, such as an antibody, at least semi-quantitatively.

[0124] The term "polynucleotide" refers to a polymeric deoxyribonucleotide or analogs or modified polynucleotides produced recombinantly or synthetically. The term includes double-stranded and single-stranded DNA or RNA. A polynucleotide can be incorporated, for example, into a minicircle, plasmid, cosmid, minichromosome, or artificial chromosome. A polynucleotide can be isolated or incorporated in another nucleic acid molecule, for example, in an expression vector or the chromosome of a eukaryotic host cell.

[0125] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating a nucleic acid molecule to which it is linked. This term further encompasses plasmid (non-viral) and viral vectors. Certain vectors are capable of directing the expression of a nucleic acid or polynucleotide to which it is operatively linked. Such vectors are referred to herein as "expression vectors." Eukaryotic expression vectors can be constructed by inserting a polynucleotide sequence encoding at least one protein of interest (POI) into an appropriate vector backbone. The vector backbone can contain elements necessary to ensure vector maintenance and, if necessary, amplification within the host. In the case of viral vectors, such as lentiviral or retroviral vectors, additional virus-specific elements, such as structural or other elements, may be required and are well known in the art. These elements can be provided, for example, in cis (on the same plasmid) or trans (on a separate plasmid). Viral vectors may require packaging lines or helper viruses for large-scale transfection. Vectors may contain additional elements such as enhancer elements (e.g., viral, eukaryotic), introns, and viral plasmid replication origins (for replication in mammalian cells). According to the present invention, expression vectors typically have a promoter sequence that directs expression of the POI. Expression of the POI and / or selectable marker protein can be constitutive or regulatable (e.g., inducible by the addition or removal of a small molecule inducer). Preferred regulatory sequences for mammalian host cell expression include regulatory elements, promoters, and / or enhancers derived from viral elements that direct high-level expression of the POI in mammalian cells, such as those derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter Ad LP), or polyoma. 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.

[0126] The term "linker" or "spacer" as used herein refers to any molecule that allows for a direct topological link between two moieties. The moiety can be, inter alia, a polypeptide, a protein, an antibody, an antibody fragment, a cytotoxic moiety, a binding moiety, a detection moiety such as a fluorophore, a moiety for immobilization or retrieval such as a bead or magnetic bead, a reactive moiety, or any other molecule. The two moieties can be of the same type or different. The linker can be part of the conjugate and can even contribute to its function. For example, in the case of a conjugate containing a polypeptide and biotin, the presence of a spacer of approximately 4 angstroms (approximately 5 atoms) between the carboxyl group of biotin and the first bulky amino acid of the peptide allows biotin to reach the (strept)avidin binding pocket. Various linkers are known in the art and can be selected based on the moieties to be linked. Linker lengths typically range from 4 atoms to over 200 atoms. Linkers longer than 60 atoms generally constitute a population of compounds with average lengths.

[0127] The term "linker for polypeptides" refers to a linker that can be attached via an amide bond or any other functional group residue. Linkers for polypeptides can be attached to the N-terminus or C-terminus of the polypeptide, or via a reactive functional group or amino acid side chain. Polypeptides can be coupled to, for example, biotin, proteins such as human serum albumin (HSA), carrier proteins such as keyhole limpet hemocyanin (KLH), ovalbumin (OVA), or bovine serum albumin (BSA), fluorescent dyes, short amino acid sequences such as Flag-tag, HA-tag, Myc-tag, or His-tag, reactive tags such as maleimide, iodoacetamide, alkyl halide, 3-mercaptopropyl, or 4-azidobutyric acid, or a variety of other suitable moieties. Non-limiting examples of suitable linkers, e.g., for conjugating polypeptides, include beta-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric 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 (trioxatridecane-succinamic 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, polyoxyalkylene, or copolymers of polyethylene glycol or polypropylene glycol.

[0128] The term "linker for antibodies" refers to a linker that establishes a covalent bond between different antibody moieties, and includes, but is not limited to, peptide linkers and non-proteinaceous polymers such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol, polypropylene glycol.

[0129] "Treating" a disease in a subject, or "treating" a subject with a disease, means subjecting the subject to pharmaceutical therapy, e.g., administration of a drug, so that at least one symptom of the disease is alleviated or prevented from worsening.

[0130] The terms "prevent," "preventing," "prevention," and the like refer to reducing the likelihood of developing a disease, disorder, or condition in a subject who does not have the disease, disorder, or condition but is at risk or susceptible to developing it.

[0131] The terms "effective amount" and "therapeutically effective amount" are used interchangeably herein and refer to an amount sufficient to achieve a specific biological result or to modulate or ameliorate symptoms or the time to onset of symptoms in a subject, typically by at least about 10%, usually by at least about 20%, preferably by at least about 30%, or more preferably by at least about 50%. The effectiveness of the use of antibodies in cancer therapy can be evaluated based on changes in tumor burden. Both tumor shrinkage (objective response) and time to disease progression are 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. RECIST criteria are typically used in clinical trials where objective response is the primary study endpoint and in trials where stable disease, tumor progression, or time to progression analysis are assessed, because these outcome measures are based on evaluation of anatomical tumor burden and its change over the study period. The effective amount for an individual subject may vary depending on 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. In the case of a combination, the effective amount is in proportion to the combination of the components, and the effect is not limited to each individual component alone.

[0132] Unless otherwise specified, a "complete response" (CR) is defined as the disappearance of all target lesions. Any pathological lymph nodes (target or non-target) must show a shrinkage in their short diameter to less than 10 mm. For a "partial response" (PR), a reduction of at least 30% in the sum of the diameters of the target lesions must be achieved, relative to the baseline summed diameter. For "progressive disease" (PD), a reduction of at least 20% in the sum of the diameters of the target lesions must be achieved, relative to the minimum sum in the study (which includes the baseline sum, if that is the minimum in the study). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. For "stable disease" (SD), neither a shrinkage sufficient to qualify for PR nor an increase sufficient to qualify for PD is observed, relative to the minimum summed diameter in the study.

[0133] Secondary evaluation measures that can be used to assess the therapeutic efficacy of the antibodies of the present invention described herein include the following: "Objective response rate" (ORR) is defined as the proportion of subjects achieving a complete response (CR) or partial response (PR). "Progression-free survival" (PFS) is defined as the period from the date of first administration of the antibody to disease progression or death, whichever occurs first. "Overall survival" (OS) is defined as the period from the date of disease diagnosis or treatment initiation that a patient diagnosed with the disease is still alive. "Duration of overall response" (DOR) is defined as the period from a participant's first CR or PR to disease progression. "Depth of response" (DpR) is defined as the ratio of tumor shrinkage observed at the time of maximum response compared to baseline tumor burden. Clinical endpoints for both ORR and PFS can be determined based on the RECIST 1.1 criteria described above.

[0134] When analyzing non-human subjects, the above parameters for determining therapeutic efficacy and benefit need to be adapted.

[0135] Typical "subjects" according to the present invention include human and non-human subjects. The subject may be a mammal, such as a mouse, rat, cat, dog, primate, and / or human.

[0136] The term "patient" means a human subject having a medical condition.

[0137] A "pharmaceutical composition" (also known as a "therapeutic formulation") of an antibody, fragment, or conjugate can be prepared by mixing an antibody having the desired purity, e.g., in the form of a lyophilized formulation or aqueous solution, with physiologically acceptable carriers, excipients, or stabilizers, as desired, according to, e.g., Remington's Pharmaceutical Sciences (18th ed.; Mack Pub. Co.: Eaton, Pa., 1990). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include the following: buffers, such as phosphate, citrate, and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, 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) polyisoprene; 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, such as 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., Zn-protein complexes); and / or non-ionic surfactants, such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[0138] A "host cell" is a cell used to receive, maintain, replicate, and amplify a vector. Host cells are also used to express a polypeptide, such as an antibody or fragment thereof, encoded by the vector. 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. An even more preferred host cell is rat myeloma YB2 / 0 cell.

[0139] "Cells exhibiting endogenous target expression" are cells that express a target protein at levels comparable to physiological or pathological conditions. Typically, cells engineered to overexpress the target protein express much higher levels.

[0140] As used herein, "lesion" means an area of ​​abnormal tissue. Lesions can be benign or malignant ("cancerous lesion"; also known as "tumor lesion").

[0141] The terms "intratumoral," "intratumor," "tumor-infiltrating," or "tumorous" in the context of a cell, structure, protein, antibody, or marker refer to their localization within tumor tissue.

[0142] A cell that is "positive" or "+" for a particular marker or protein is a cell characterized by substantial expression of that marker or protein. Marker or protein expression can be determined and quantified as known in the art, for example, to define various cell populations. For characterization of (immune) cell populations, marker expression can be determined using FACS or any other technique described herein.

[0143] A "leukocyte" is an immune cell that expresses CD45.

[0144] As used herein, "CD45+ cells" refers to all white blood cells. CD45 can be used as a marker to distinguish between immune and non-immune cells.

[0145] The term "lymphocyte" refers to all white lymphocyte populations in their immature, mature, undifferentiated, and differentiated states, including tissue-specific and specialized variants. It includes, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages, such as pre-B cells, precursor 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, B-1 cells, B-2 cells, and anergic AN1 / T3 cell populations.

[0146] "T cells" are immune cells that express TCRαβ, 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, as well as other T cell populations known in the art. In some embodiments, the presence of a T cell receptor (TCR) on the cell surface distinguishes T cells from other lymphocytes.

[0147] "CD8+ T cells" (also known as "cytotoxic T cells," "TCs," "cytotoxic T lymphocytes," "CTLs," "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 cells), or other damaged cells.

[0148] "CD4+ T cells" (also known as "T helper cells" or "Th cells") are immune cells that express CD3, CD4, and CD45. There are several subsets of T helper cells, including but not limited to Th1, Th2, and Th17. CD4+ T cells help suppress or regulate immune responses. They are essential in B cell antibody class switching, activation and proliferation of cytotoxic T cells, and maximizing the bactericidal activity of phagocytes such as macrophages.

[0149] As used herein, the term "Treg cells" (also known as "Tregs," "regulatory T cells," "T regulatory cells," or "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. Identification of Treg cells can be performed as described elsewhere herein. Treg cells typically also express high levels of CTLA-4, GITR, and LAG-3. In the literature, Tregs have been further classified based on the memory marker CD45RO.

[0150] Under physiological conditions, Treg cells maintain immune tolerance. During an immune response, Treg cells block T cell-mediated immunity and suppress autoreactive T cells that have escaped negative selection in the thymus. Treg cells 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.

[0151] Treg cells also play an important role in immune evasion by suppressing antitumor immunity, thereby providing an environment of immune tolerance. T cells that recognize cancer cells are often present in large numbers within tumors, but their cytotoxic function is suppressed by nearby immunosuppressive cells. Tregs are abundant in many different cancers, are highly abundant within the tumor microenvironment, and are well known for their role in tumor progression.

[0152] "Activated Treg cells" express CD4, CD45, FoxP3, CD69, and CCR8, and also show high expression of CD25 and low expression of CD127. CD69 is a T cell activation marker.

[0153] "CCR8-positive regulatory T cells" or "CCR8+ regulatory T cells" are Tregs that express CCR8.

[0154] "CD4conv cells" are normal CD4+, CD25- T cells.

[0155] The term "gamma delta T cells" refers to T cells that express a distinctive T cell receptor, TCRγδ, on their surface. Gamma delta T cells also express CD3.

[0156] "B cells" are immune cells that express CD19, while mature B cells express CD20 and CD22. B cells activated via CD40 undergo differentiation, where somatic hypermutation and enhanced immunoglobulin class switching occur to generate mature B cells or plasma cells (capable of secreting Abs). B cells are involved in humoral immunity of the adaptive immune system and are antigen-presenting cells.

[0157] The term "macrophage" refers to immune cells that express low CD14 and high CD16, CD11b, CD68, CD163, and CD206. Macrophages engulf and digest cellular debris, foreign bodies, microorganisms, or cancer cells through phagocytosis. In addition to phagocytosis, macrophages also play an important role in innate immunity and help initiate adaptive immunity by recruiting other immune cells. For example, macrophages are important antigen presenters for T cells. Macrophages that promote inflammation are called M1 macrophages, while macrophages that reduce inflammation and promote tissue repair are called M2 macrophages.

[0158] As used herein, "M1 macrophages" are a subset of macrophages that express ACOD1. M1 macrophages have pro-inflammatory, bactericidal, and phagocytic functions.

[0159] As used herein, "M2 macrophages" are a subset of macrophages that express MRC1 (CD206). M2 macrophages secrete anti-inflammatory interleukins, play a role in wound healing, and are required for revascularization and re-epithelialization. Tumor-associated macrophages are predominantly of the M2 phenotype and appear to actively promote tumor growth.

[0160] "Dendritic cells" (DCs) are bone marrow-derived white blood cells and are the most potent type of antigen-presenting cell. DCs are specialized for capturing and processing antigens, converting proteins into peptides that are presented on major histocompatibility complex (MHC) molecules that are recognized by T cells. DCs, as defined herein, are characterized by the expression of CD1c, CD14, CD16, CD141, CD11c, and CD123. Various subpopulations of dendritic cells exist. In humans, DC1 cells are immunogenic, while DC2 cells are tolerogenic. Mature DCs express CD83, while plasmacytoid DCs express CD123.

[0161] "NK cells" (also called natural killer cells) are immune cells that express CD45, CD16, CD56, and NKG2D, but are CD3 negative. NK cells do not require activation to kill cells that lack the MHC class 1 "self" marker. NCR1 (also called CD335 or NKp46) is expressed on NK cells and a subset of NKT cells.

[0162] "Natural killer T (NKT) cells" are a heterogeneous population of T cells that share properties of both T cells and natural killer cells.

[0163] "iNKT cells" (also known as "invariant natural killer T cells") express the invariant αβ TCR (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 influence immune responses by rapidly producing large amounts of cytokines, namely IFNγ.

[0164] As known in the art, "effector cells" are immune cells that actively support immune responses after stimulation. As used herein, effector cells refer to immune cells that express Fcγ receptors and can therefore cause ADCC or ADCP. Non-limiting examples of effector cells include monocytes, neutrophils, mast cells, preferably macrophages and natural killer cells.

[0165] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell surface receptor that is expressed on immune effector cells and engineered to specifically bind to an antigen. CARs can be used as a therapy by adoptive cell transfer. Monocytes are removed from a patient (blood, tumor, or ascites) and modified to express a receptor specific for a particular form of antigen. In some embodiments, a CAR is specifically expressed for a tumor-associated antigen. A CAR can also contain an intracellular activation domain, a transmembrane domain, and an extracellular domain (including a tumor-associated antigen-binding region). In some aspects, a CAR comprises a fusion of a monoclonal antibody derived from a single-chain variable fragment (scFv) fused to the CD3-zeta transmembrane and intracellular domains. The specificity of the CAR design can be derived from the receptor's ligand (e.g., a peptide). In some embodiments, a CAR can target cancer by redirecting monocytes / macrophages expressing a CAR specific for a tumor-associated antigen.

[0166] Dosage schemes are abbreviated as known in the art, e.g., daily (QD), every two days (Q2D), or every three days (Q3D), whereby "QW" means once every 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.

[0167] A "dosing cycle" or "treatment cycle" is a periodic repetition of a treatment period followed by a rest period (no treatment). When this cycle is repeated periodically multiple times, it constitutes a treatment course.

[0168] In pharmacology, the "trough concentration" (abbreviated as "Ctrough") is the concentration reached by a drug just before the next dose is administered.

[0169] "Cytokine release syndrome" (CRS), also known as "cytokine storm" in severe cases, is a supraphysiological reaction that can occur in response to any immunotherapy as a sequela of immune system activation associated with infusion reactions. Binding of mAbs leads to the activation or engagement of endogenous or infused T cells and / or other immune effector cells, which results in the rapid release of proinflammatory cytokines from target immune cells into the circulation. CRS usually occurs within hours to days after monoclonal antibody infusion. While the incidence of CRS is relatively low with monoclonal antibodies, it is high with chimeric antigen receptor (CAR)-T and T-cell engagers, ranging from 17% to 94%.

[0170] An intravenous line or "IV line" is a tube or cannula that can be used for intravenous infusion.

[0171] PET scanning is a diagnostic technique used to observe the function and metabolism of human organs and tissues at the molecular level. For PET scanning, a positron-emitting drug (e.g., 18F-FDG) can be injected into the human body. When FDG is used, fluorodeoxyglucose (FDG) has a metabolic pathway similar to that of glucose, so FDG accumulates in cells that digest glucose. The uptake of radioactive drugs by rapidly growing tumor tissue differs. The positrons released by the decay of 18F undergo an annihilation reaction with electrons in the tissue, generating two gamma photons with the same energy and in opposite directions. In a PET scan, a detector array surrounding the body detects the two photons using coincidence counting techniques to determine the positional information of the positrons. A tomographic image of the positrons in the body can then be constructed by processing the positional information using image reconstruction software. In some situations, immunoPET can be used when a label (e.g., 18F) is attached or bound to an antigen-binding construct. In such an embodiment, the distribution or abundance of the antigen-binding construct can be monitored, which depends on the binding and distribution characteristics of the antigen-binding construct.For example, when using a CD8-directed minibody or Zr-89 labeled anti-CD8 minibody, PET scanning can be used to monitor the distribution and / or abundance of the CD8-directed minibody or Zr-89 labeled anti-CD8 minibody, and thus to monitor the distribution and / or abundance of CD8 molecules throughout the subject's system.PET systems are known in the art, including, 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 their description of PET, PET scanning and its use).

[0172] The term "standardized uptake value" or "standard uptake value" or "SUV" is a term used in the field of nuclear medicine, not only in positron emission tomography (PET) but also in modern calibrated single photon emission computed tomography (SPECT) imaging for semi-quantitative analysis.

[0173] A computed tomography scan or "CT scan" (formerly called a computerized axial tomography scan or CAT scan) is a medical imaging technique used to obtain detailed internal images of the body. CT scanners use a rotating x-ray tube and an array of detectors arranged in a gantry to measure x-ray attenuation by various tissues inside the body. Multiple x-ray measurements taken from various angles are then processed on a computer using tomographic reconstruction algorithms to obtain tomographic (cross-sectional) images (virtual "slices") of the body.

[0174] The term "distribution" in the context of monitoring, detecting, comparing, or observing the distribution of a Zr-89 labeled anti-CD8 minibody administered to a subject means a visual or mathematical image of the biodistribution of the Zr-89 labeled anti-CD8 minibody in relation to a scan of the subject's whole body or partial body, which image can be represented as a planar image (2-dimensional) or a computer-assisted 3-dimensional display (including a hologram), in a format that is useful for an operator or clinician to observe the distribution of the Zr-89 labeled anti-CD8 minibody at the individual tissue level and individual tumor level.

[0175] The term "antihistamines" refers to a class of pharmaceutical compounds designed to alleviate various allergic reactions and symptoms by blocking the actions of histamine in the body. Specific examples include diphenhydramine, loratadine, cetirizine, fexofenadine, desloratadine, and levocetirizine.

[0176] "Diphenhydramine" (DPH) is an antihistamine and sedative well known in the art and is primarily used to treat allergies, insomnia, and cold symptoms. It is also less commonly used to treat tremors and nausea in Parkinson's disease.

[0177] "Paracetamol" (acetaminophen[a] or parahydroxyacetanilide) is a non-opioid analgesic and antipyretic known in the art and is used to treat fever and mild to moderate pain. Common brand names include Tylenol and Panadol.

[0178] "Corticosteroids" are a class of synthetic or natural steroid hormones produced by the adrenal cortex and have potent anti-inflammatory, immunosuppressive, and metabolic effects. Specific examples include prednisone, dexamethasone, or methylprednisolone.

[0179] "Dexamethasone" is a glucocorticoid drug known in the art and is used to treat, among other things, rheumatic diseases, many skin diseases, severe allergies, asthma, chronic obstructive pulmonary disease, croup, or brain swelling.

[0180] Embodiment Aspect 1 Dosing regimen According to a first aspect of the present invention, an anti-human CCR8 antibody is administered to a patient in need thereof by a. 1 to 250 mg once weekly, or b. 16 to 1500 mg once every 3 weeks and administering the antibody to a patient in need thereof intravenously in a total amount of 100 mg of the antibody to a patient in need thereof.

[0181] More specifically, an anti-human CCR8 antibody can be administered to a patient in need thereof, for example, as described in detail in Example 17. a. Approximately 1, 2.5, 3, 10, 30, 50, 100, 125, or 250 mg once weekly; or b. Approximately 16, 450, 500, 750, 1000, or 1500 mg once every three weeks and administering the antibody to a patient in need thereof intravenously in a total amount of 100 mg of the antibody to a patient in need thereof.

[0182] For example, there is provided an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method comprising intravenously administering the anti-CCR8 antibody to a patient in need thereof in a total amount of about 1, 2.5, 3, 10, 30, 50, 100, 125, or 250 mg once weekly, preferably 10, 30, 50, 100, 125, or 250 mg once weekly. In one embodiment, the total amount of the anti-CCR8 antibody is about 1 mg once weekly. In another embodiment, the total amount of the anti-CCR8 antibody is about 2.5 mg once weekly. In another embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once weekly. In a preferred embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once weekly. In a more preferred embodiment, the total amount of the anti-CCR8 antibody is about 50 mg once weekly. In a more preferred embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once weekly. In another preferred embodiment, the total amount of anti-CCR8 antibody is about 125 mg once weekly. In a highly preferred embodiment, the total amount of anti-CCR8 antibody is about 250 mg once weekly.

[0183] For example, an anti-human CCR8 antibody having ADCC activity and ADCP activity is provided for use in a therapeutic method comprising intravenously administering the anti-CCR8 antibody to a patient in need thereof in a total amount of about 500, 750, 1000, or 1500 mg once every three weeks.

[0184] In one 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 one 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.

[0185] The pharmacologically active / effective dose range for the anti-CCR8 antibody TPP-23411, which induces significant ADCC and ADCP but is characterized by a relatively short half-life, is 2.7 mg to 75 mg on a QW schedule and 16 mg to 450 mg on a Q3W schedule for a 70 kg patient (see Example 16). Data demonstrating the success of this mechanism of action are shown, for example, in Example 24. See Figures 7, 8, 9, and 10.

[0186] In accordance with these findings, in a most preferred embodiment, there is provided an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method comprising intravenously administering the anti-CCR8 antibody to a patient in need thereof in a total amount of 2.7 mg to 75 mg once weekly.

[0187] The proposed medical use of a QW dosing schedule is superior because it provides anti-CCR8 antibodies at pharmacologically relevant plasma exposure levels and also because it allows for plasma Ctrough concentrations of anti-CCR8 antibodies that exceed the estimated EC80 value for CCR8+ cell killing that the inventors derived from in vitro studies.

[0188] In another most preferred embodiment, there is provided an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment comprising intravenously administering the anti-CCR8 antibody to a patient in need thereof in a total amount of 16 mg to 450 mg once every three weeks.

[0189] Although medical use of Q3W dosing schedules involves higher doses, it is advantageous because they can be administered less frequently while still achieving the required plasma exposure during the dosing interval to produce the desired pharmacological response (CCR8+ Treg killing). The proposed Q3W dosing schedule also provides convenience of administration and coordination with the infusion of other drugs.

[0190] As will be appreciated by those skilled in the art, the total amounts in the embodiments described herein are designed for a patient of average weight 70 kg and can be adjusted based on the patient's actual weight, i.e., by using the appropriate mg / kg.

[0191] In all cases where an anti-CCR8 antibody is mentioned, the antibody is preferably TPP-23411, most preferably defucosylated TPP-23411.

[0192] In a most preferred example, the anti-CCR8 antibody having ADCC activity and ADCP activity for use in the treatment method according to this embodiment is a. comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 2, 3, 4, 6, 7 and 8; and / or b. comprises a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth 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 set forth in SEQ ID NO:5; and / or c. Comprises a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17 and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.

[0193] However, the anti-CCR8 antibody may also be an alternative anti-human CCR8 antibody, such as an antibody described herein that induces ADCC and ADCP and has a half-life comparable to that of TPP-23411, i.e., an anti-human CCR8 antibody that has a shorter half-life than other antibodies of the same IgG type. For example, the anti-human CCR8 antibodies described herein are characterized by a half-life in humans of less than 14 days, preferably less than 12 days, more preferably less than 10 days, and most preferably less than 7 days, e.g., 0 to 120 hours. Preferably, the anti-human CCR8 antibody is characterized by a half-life of less than 14 days, more preferably less than 10 days, and most preferably less than 7 days in humans.

[0194] Preferably, the anti-CCR8 antibody is a human IgG1 antibody.

[0195] Preferably, the anti-CCR8 antibody is (further) a poorly internalizing antibody or a non-internalizing antibody.

[0196] In a preferred embodiment, the anti-CCR8 antibody is characterized by a dissociation constant (KD) that is the same order of magnitude as the KD of TPP-23411 for binding to CHO cells transfected with human CCR8. As will be appreciated by those skilled in the art, a one order of magnitude difference between the two values ​​is 10-fold. In the most preferred embodiment according to this aspect, the anti-CCR8 antibody is a. characterized by a dissociation constant (KD) for binding to CHO cells transfected with human CCR8 that is the same order of magnitude as the KD of TPP-23411 for binding to CHO cells transfected with human CCR8; b. The antibody induces ADCC and ADCP; Preferably, the antibody binds to human Fc gamma receptor IIIA variant V176 (CD16a) with a dissociation constant (KD) that is the same order of magnitude as the KD of TPP-23411 for binding to human Fc gamma receptor IIIA variant V176 (CD16a); and Preferably, the antibody binds to human Fc gamma RIIA (CD32a) with a dissociation constant (KD) that is the same order of magnitude as the KD of TPP-23411 for binding to human Fc gamma RIIA (CD32a); Preferably, the antibody is afucosylated, c. Preferably, the antibody is characterized by a half-life in humans of less than 14 days, preferably less than 10 days, and most preferably less than 7 days.

[0197] To prepare the intravenous infusion solution, the required amount of anti-CCR8 antibody solution can be removed from the vial and transferred into 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, 1 mg / mL to 10 mg / mL.

[0198] Administration of the anti-CCR8 antibody (e.g., diluted solution) can be intravenously administered over 15 to 120 minutes, preferably over 30 to 60 minutes, and most preferably over 30, 45, 60, or 75 minutes. Administration of the diluted solution can be via an intravenous line containing, for example, a sterile, non-pyrogenic, low protein-binding 0.2 to 5 micron in-line or add-on filter. Because infusion pumps vary by site, a range of -5 to +10 minutes is acceptable (i.e., infusion time is 30 minutes [-5 minutes / +10 minutes]).

[0199] The medical use according to the first aspect further comprises administering an anti-PD-(L)1 antibody to a patient in need thereof: i. about 200 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or ii. about 400 mg once every 6 weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or iii. about 240 mg once every two weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or iv. about 360 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or v. about 480 mg once every four weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or vi. about 840 mg every two weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or vii. about 1200 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or viii. about 1680 mg every 4 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or ix. about 360 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is zimberelimab), or x. about 3 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is toripalimab), or xi. about 10 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab), or xii. about 1500 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab) This may include administering intravenously in a total amount of

[0200] For example, preferably, the medical use according to the first aspect comprises intravenously administering an anti-PD-(L)1 antibody to a patient in need thereof in a total amount of about 200 mg once every three weeks or about 400 mg once every six weeks, wherein the anti-PD-(L)1 antibody is pembrolizumab.

[0201] For example, preferably, the medical use according to the first aspect comprises intravenously administering an anti-PD-(L)1 antibody to a patient in need thereof in 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, wherein the anti-PD-(L)1 antibody is nivolumab.

[0202] For example, preferably, the medical use according to the first aspect comprises intravenously administering an anti-PD-(L)1 antibody to a patient in need thereof in a total amount of about 840 mg every two weeks, about 1200 mg every three weeks, or about 1680 mg every four weeks, wherein the anti-PD-(L)1 antibody is atezolizumab.

[0203] In another example, the medical use according to the first aspect comprises administering an anti-PD-(L)1 antibody intravenously to a patient in need thereof in a total amount of about 360 mg every three weeks, wherein the anti-PD-(L)1 antibody is zimberelimab.

[0204] In another example, the medical use according to the first aspect comprises administering an anti-PD-(L)1 antibody intravenously to a patient in need thereof in a total amount of about 3 mg / kg every two weeks, wherein the anti-PD-(L)1 antibody is toripalimab.

[0205] In another example, the medical use according to the first aspect comprises intravenously administering an anti-PD-(L)1 antibody to a patient in need thereof in a total amount of about 10 mg / kg every two weeks or about 1500 mg every three weeks, wherein the anti-PD-(L)1 antibody is durvalumab.

[0206] In an alternative, the medical use according to the first aspect comprises administering an anti-PD-(L)1 antibody to a patient in need thereof: a. Approximately 200 mg once every 3 weeks, or b. Approximately 480 mg once every 4 weeks, or c. Approximately 480 mg once every 6 weeks This may include administering intravenously in a total amount of

[0207] In this alternative, the anti-PD-(L)1 antibody is pembrolizumab, nivolumab, atezolizumab, avelumab, or durvalumab.

[0208] The use of a fixed dose of the anti-PD-(L)1 antibody reduces the complexity of administration and reduces the possibility of dosing errors. The anti-PD-(L)1 antibody is preferably administered after the anti-CCR8 antibody, although administration in a different order or even simultaneous administration may be possible.

[0209] In the most preferred embodiment of the present invention, a. administering an anti-CCR8 antibody intravenously to a patient in need thereof in a total amount of 2.7 mg to 75 mg once weekly; b. Preferably, an anti-PD-(L)1 antibody is administered to the patient: i. about 200 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or ii. about 400 mg once every 6 weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or iii. about 240 mg once every two weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or iv. about 360 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or v. about 480 mg once every four weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or vi. about 840 mg every two weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or vii. about 1200 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or viii. about 1680 mg every 4 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or ix. about 360 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is zimberelimab), or x. about 3 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is toripalimab), or xi. about 10 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab), or xii. about 1500 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab) and administering the antibody to a patient having ADCC activity and ADCP activity intravenously to the patient in a total amount of 100 mg of the antibody to a patient having CCR8 activity.

[0210] In another most preferred embodiment, a. administering an anti-CCR8 antibody intravenously to a patient in need thereof in a total dose of 16 mg to 450 mg once every three weeks; b. Preferably, an anti-PD-(L)1 antibody is administered to the patient: i. about 200 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or ii. about 400 mg once every 6 weeks (preferably, where the anti-PD-(L)1 antibody is pembrolizumab), or iii. about 240 mg once every two weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or iv. about 360 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or v. about 480 mg once every four weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or vi. about 840 mg every two weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or vii. about 1200 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or viii. about 1680 mg every 4 weeks (preferably, where the anti-PD-(L)1 antibody is atezolizumab), or ix. about 360 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is zimberelimab), or x. about 3 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is toripalimab), or xi. about 10 mg / kg every two weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab), or xii. about 1500 mg every 3 weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab) and administering the antibody to a patient having ADCC activity and ADCP activity intravenously to the patient in a total amount of 100 mg of the antibody to a patient having CCR8 activity.

[0211] To prepare the intravenous infusion solution, the required amount of anti-PD-(L)1 antibody solution can be removed from the vial and transferred into 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, 1 mg / mL to 10 mg / mL.

[0212] For example, intravenous administration of the anti-PD-(L)1 antibody can be performed as a 15- to 60-minute intravenous infusion, preferably as a 30-minute intravenous infusion.

[0213] Anti-PD-(L)1 antibodies may be administered via an intravenous line containing a sterile, non-pyrogenic, low protein-binding 0.2-5 micron in-line or add-on filter. Because infusion pumps vary by site, a range of -5 to +10 minutes is acceptable (i.e., infusion time is 30 minutes [-5 minutes / +10 minutes]).

[0214] 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 configuration is preferred and advantageous because it reduces the complexity of therapeutic administration, which 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., the anti-human PD-(L)1 antibody.

[0215] For example, an anti-human CCR8 antibody with ADCC and ADCP activity for use in a method of treatment according to this embodiment may comprise at least one 21-day administration cycle, and further, the anti-CCR8 antibody and the anti-PD-(L)1 antibody may both be administered on day 1 of a 21-day administration cycle.

[0216] If all previous infusions (e.g., cycles 1, 2, 3, or 4) have been well tolerated by the patient, the PD-(L)1 antibody can be administered without a substantial delay, i.e., without a 15-60 minute pause immediately following administration of the anti-CCR8 antibody. For example, if the medical use involves at least two, and preferably more, administration cycles, the anti-CCR8 antibody and anti-PD-(L)1 antibody can be administered immediately after each other without a substantial delay in the second, third, fourth, fifth, or any subsequent administration cycle. This method is advantageous because it is more time-efficient for patients and medical professionals.

[0217] 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.

[0218] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once every 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.

[0219] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once every 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.

[0220] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once every week, and the anti-PD-(L)1 antibody is pembrolizumab, administered in a total amount of about 200 mg once every three weeks or about 400 mg once every six weeks.

[0221] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once every 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.

[0222] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once every 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.

[0223] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg administered once every week, and the anti-PD-(L)1 antibody is pembrolizumab administered in a total amount of about 200 mg administered once every three weeks or about 400 mg administered once every six weeks.

[0224] In another 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 pembrolizumab and is administered in a total amount of about 200 mg once every three weeks or about 400 mg once every six weeks.

[0225] 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 pembrolizumab and is administered in a total amount of about 200 mg once every three weeks or about 400 mg once every six weeks.

[0226] 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.

[0227] 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, administered in a total amount of about 200 mg once every three weeks or about 400 mg once every six weeks.

[0228] In another preferred embodiment, the anti-PD-(L)1 antibody is nivolumab and is administered at a total amount of about 240 mg every two weeks, about 360 mg every three weeks, about 480 mg every four weeks, or about 480 mg every six weeks.

[0229] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once every 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.

[0230] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once every 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.

[0231] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once every 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.

[0232] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once every 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.

[0233] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once every week, and the anti-PD-(L)1 antibody is nivolumab and is administered at 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.

[0234] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg once every 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.

[0235] In another 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.

[0236] 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.

[0237] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg every three weeks, and the anti-PD-(L)1 antibody is nivolumab and is administered in a total amount of about 240 mg every two weeks, about 360 mg every three weeks, about 480 mg every four weeks, or about 480 mg every six weeks.

[0238] 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 at 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.

[0239] In a preferred embodiment, the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg every two weeks, about 1200 mg every three weeks, or about 1680 mg every four weeks.

[0240] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once weekly, 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.

[0241] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once weekly, 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.

[0242] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once every 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.

[0243] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once every 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.

[0244] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once every 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.

[0245] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 250 mg once every 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.

[0246] In another 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.

[0247] 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.

[0248] In a highly preferred embodiment, the total amount of the anti-CCR8 antibody is about 1000 mg every three weeks, and the anti-PD-(L)1 antibody is atezolizumab and is administered in a total amount of about 840 mg every two weeks, about 1200 mg every three weeks, or about 1680 mg every four weeks.

[0249] 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 at 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.

[0250] In a preferred embodiment, the anti-PD-(L)1 antibody is zimberelimab and is administered in a total amount of about 360 mg once every three weeks.

[0251] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once weekly, and the anti-PD-(L)1 antibody is zimberelimab and is administered in a total amount of about 360 mg once every three weeks.

[0252] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once weekly, and the anti-PD-(L)1 antibody is zimberelimab, administered in a total amount of about 360 mg once every three weeks.

[0253] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg administered once every week, and the anti-PD-(L)1 antibody is zimberelimab administered once every three weeks in a total amount of about 360 mg.

[0254] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once weekly, and the anti-PD-(L)1 antibody is zimberelimab and is administered in a total amount of about 360 mg once every three weeks.

[0255] In another preferred example, the anti-CCR8 antibody is administered in a total amount of about 125 mg once every week, and the anti-PD-(L)1 antibody is zimberelimab, administered in a total amount of about 360 mg once every three weeks.

[0256] In a highly preferred example, the anti-CCR8 antibody is administered in a total amount of about 250 mg once every week, and the anti-PD-(L)1 antibody is zimberelimab, administered in a total amount of about 360 mg once every three weeks.

[0257] In another highly preferred example, the anti-CCR8 antibody is administered in a total amount of about 500 mg once every three weeks, and the anti-PD-(L)1 antibody is zimberelimab in a total amount of about 360 mg once every three weeks.

[0258] In a highly preferred example, the anti-CCR8 antibody is administered in a total amount of about 750 mg once every three weeks, and the anti-PD-(L)1 antibody is zimberelimab, administered in a total amount of about 360 mg once every three weeks.

[0259] In a highly preferred embodiment, the anti-CCR8 antibody is administered in a total amount of about 1000 mg once every three weeks and the anti-PD-(L)1 antibody is zimberelimab in a total amount of about 360 mg once every three weeks.

[0260] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg administered once every three weeks, and the anti-PD-(L)1 antibody is zimberelimab administered once every three weeks in a total amount of about 360 mg.

[0261] 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.

[0262] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once weekly, 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.

[0263] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once weekly, 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.

[0264] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg administered once every week, and the anti-PD-(L)1 antibody is toripalimab administered once every two weeks in a total amount of about 3 mg / kg.

[0265] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once weekly, 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.

[0266] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once weekly, 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.

[0267] In a highly preferred example, the anti-CCR8 antibody is administered in a total amount of about 250 mg once weekly and the anti-PD-(L)1 antibody is toripalimab in a total amount of about 3 mg / kg once every two weeks.

[0268] In another highly preferred example, the total amount of the anti-CCR8 antibody is about 500 mg administered once every three weeks, and the anti-PD-(L)1 antibody is toripalimab administered in a total amount of about 3 mg / kg administered once every two weeks.

[0269] In a highly preferred example, the total amount of the anti-CCR8 antibody is about 750 mg administered once every three weeks, and the anti-PD-(L)1 antibody is toripalimab administered once every two weeks in a total amount of about 3 mg / kg.

[0270] In a highly preferred embodiment, the anti-CCR8 antibody is administered in a total amount of about 1000 mg every three weeks, and the anti-PD-(L)1 antibody is toripalimab and is administered in a total amount of about 3 mg / kg every two weeks.

[0271] In a preferred example, the total amount of the anti-CCR8 antibody is about 1500 mg administered once every three weeks, and the anti-PD-(L)1 antibody is toripalimab administered once every two weeks in a total amount of about 3 mg / kg.

[0272] 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.

[0273] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 10 mg once weekly 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.

[0274] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 30 mg once weekly 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.

[0275] In a preferred example, the total amount of the anti-CCR8 antibody is about 50 mg once weekly, and the anti-PD-(L)1 antibody is durvalumab, administered in a total amount of about 10 mg / kg every two weeks or about 1500 mg every three weeks.

[0276] For example, in one embodiment, the total amount of the anti-CCR8 antibody is about 100 mg once weekly 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.

[0277] In another preferred example, the total amount of the anti-CCR8 antibody is about 125 mg once weekly, 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.

[0278] In a highly preferred example, the anti-CCR8 antibody is administered in a total amount of about 250 mg once weekly and the anti-PD-(L)1 antibody is durvalumab in a total amount of about 10 mg / kg every two weeks or about 1500 mg every three weeks.

[0279] In another 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 at a total amount of about 10 mg / kg every two weeks or about 1500 mg every three weeks.

[0280] 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 durvalumab and is administered at a total amount of about 10 mg / kg every two weeks or about 1500 mg every three weeks.

[0281] 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 at a total amount of about 10 mg / kg every two weeks or about 1500 mg every three weeks.

[0282] 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, administered at a total amount of about 10 mg / kg every two weeks or about 1500 mg every three weeks.

[0283] The medical use according to the first aspect preferably comprises at least one 21-day administration cycle, and in some preferred of these embodiments, the anti-CCR8 antibody and the anti-PD-(L)1 antibody are both administered on day 1 of the 21-day administration cycle.

[0284] Dosage regimens according to the invention are particularly suitable for use with anti-CCR8 antibodies in methods of treating cancer, 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 cancers.

[0285] The administration regimen according to the present invention is particularly suitable for use of anti-CCR8 antibodies in methods of treating patients with tumors or diseases characterized by chemokine receptor-positive cells, preferably CCR8-positive cells, such as CCR8-positive regulatory T cells.

[0286] 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 non-melanoma skin cancer.

[0287] For example, the method of treatment is a method of treating non-small cell lung cancer (NSCLC). For example, the method of treatment is a method of treating triple-negative breast cancer (TNBC). For example, the method of treatment is a method of treating head and neck squamous cell carcinoma (HNSCC). For example, the method of treatment is a method of treating melanoma or non-melanoma skin cancer.

[0288] It has further been found in the present invention that certain adverse reactions to anti-CCR8 antibody administration can be prevented by administering an effective dose of an antihistamine, acetaminophen, a corticosteroid, or a combination thereof. Therefore, the treatment method according to this embodiment of the present invention further comprises administering an effective dose of an antihistamine, acetaminophen, a corticosteroid, or a combination thereof, preferably a. administration of at least 500 mg or at least 650 mg of paracetamol prior to administration of an anti-CCR8 antibody, and / or b. administration of at least 50 mg or at least 100 mg of diphenhydramine prior to administration of an anti-CCR8 antibody, and / or c. Administration of at least 8 mg of dexamethasone prior to administration of anti-CCR8 antibody may include:

[0289] For example, acetaminophen can be administered orally. For example, diphenhydramine can be administered orally.

[0290] Tumor Proportion Score / Combined Positive Score Response rates to immune checkpoint inhibitors (ICIs) vary significantly across cancer types, ranging from less than 15% to more than 60%. The majority of "immune-sensitive" tumor types are resistant to ICI therapy from the start (primary resistance) or eventually acquire resistance after initial clinical benefit (secondary or acquired resistance). Treatment options after failure of frontline standard of care (SoC) ICIs (including in combination with other anticancer drugs) remain limited in most advanced tumor settings. Therefore, because alternative treatment options for these "post-ICI" patients are limited, there is a high medical need in these "post-ICI" patients to define therapies that overcome resistance to immunotherapy (combinations) of malignant diseases.

[0291] The relationship between gene expression and the in vivo efficacy of treatment with CCR8-depleting antibodies was investigated by whole-transcriptome sequencing of untreated syngeneic mouse tumor models. We found that baseline expression of PD-L1 correlated well with the in vivo anti-tumor efficacy of CCR8-surrogate antibodies across 21 tumor models (see Figure 5).

[0292] According to some preferred embodiments of the first aspect, the method of treatment is a method of treating cancer comprising the steps of:

[0293] a. analyzing a tumor proportion score or a combined positive score as a measure of PD-(L)1 expression in the patient's cancer tissue sample; and b. administering an anti-human CCR8 antibody to the patient if the patient has a tumor proportion score of 50% or greater or a combined positive score of 10% or greater or 1% or greater. A method for treating cancer comprising:

[0294] 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 an eligibility criterion for treating patients. For example, in the monotherapy mechanism of action (MoA) expansion arm (Arm 2A) of clinical trials, a historical PD-L1 score of 50% or higher tumor proportion score (TPS) is used as an eligibility criterion. PD-L1 expression appears to have general potential as a predictive biomarker (see also Figure 6).

[0295] According to some preferred embodiments of these embodiments: a. the cancer is non-small cell lung cancer (NSCLC) and a tumor proportion score is analyzed as a measure of PD-(L)1 expression in a patient's cancer tissue sample; or b. the cancer is triple-negative breast cancer and a 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.

[0296] 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 is FDA approved. PD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemistry assay that uses the 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 the Autostainer Link 48.

[0297] Alternatively, the VENTANA PD-L1 (SP142) assay may be used. The VENTANA PD-L1 (SP142) assay is another qualitative immunohistochemistry assay that uses the rabbit monoclonal anti-PD-L1 clone SP142 and may be used on FFPE tissue stained using, for example, the OptiView DAB IHC Detection Kit and OptiView Amplification Kit on a BenchMark ULTRA instrument.

[0298] For assessment of tumor proportion score, please refer to the PD-L1 IHC 22C3 pharmDx Interpretation Manual - NSCLC (Agilent Dako), and for assessment of CPS, please refer to the PD-L1 IHC 22C3 pharmDx Interpretation Manual - Head and Neck Squamous Cell Carcinoma (Agilent Dako).

[0299] For some cancer patients, previous tumor proportion scores and / or previous combined positive scores are available. In the present invention, the previous tumor proportion scores or previous combined positive scores can be used to determine the possibility that a patient will benefit from the administration of an anti-human CCR8 antibody. These previous scores can be used to make a sufficiently reliable, rapid and very convenient stratification decision.

[0300] In particular, there is provided an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment described according to the first aspect, wherein the method of treatment is a method of treating cancer comprising administering the anti-human CCR8 antibody to a patient when the patient has a previous tumor proportion score of 50% or more, or a previous combined positive score of 10% or more, or 1% or more.

[0301] According to some preferred embodiments of these embodiments: 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 has a historical tumor proportion score of 50% or greater; or b. the cancer is triple-negative breast cancer and the method of treatment comprises administering an anti-human CCR8 antibody to the patient if the patient has a prior combined positive score of 10% or greater or 1% or greater; or c. The cancer is head and neck squamous cell carcinoma, and the method of treatment includes administering an anti-human CCR8 antibody to the patient if the patient has a prior combined positive score of 20% or greater or 1% or greater.

[0302] According to some 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 IHC 22C3 pharmDx assay or the VENTANA PD-L1 (SP263) assay.

[0303] Cytokine biomarkers Cytokine release has been identified as an important biomarker in the case of anti-CCR8 antibodies. a. optionally analyzing in the patient's blood, plasma or serum screening sample the level of at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10, pro-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; b. administering to the patient an effective dose of an anti-human CCR8 antibody; c. Analyzing the level of at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above-mentioned pro-inflammatory cytokines in a blood, plasma, or serum sample from the patient, wherein the blood, plasma, or serum sample is taken or collected after administering an effective dose of an anti-human CCR8 antibody according to step b). d. using the cytokine levels obtained according to step c) to identify safety-related events or as surrogate biomarkers for Treg depletion or as biomarkers for treatment success, i. the cytokine level obtained according to step a) or ii. Reference Value Process to compare with The present invention provides an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treating a patient, for example, according to any of the aspects or embodiments described herein, including, but not limited to:

[0304] A screening sample is defined herein as a sample obtained from a patient prior to an actual administration event, preferably a sample obtained from a patient who has not previously been treated with an anti-human CCR8 antibody having ADCC and ADCP activity.

[0305] To use the respective cytokine levels as biomarkers, the respective cytokine levels can be compared, for example, to cytokine levels obtained before the first anti-CCR8 antibody administration to the same patient, or to different baseline values ​​obtained for the same patient or a different subject group.

[0306] Those skilled in the art will appreciate that such reference values ​​can be obtained, for example, by calculating the average or median value from cytokine levels obtained from multiple patients at a defined time point (e.g., but not limited to, before administration of the anti-CCR8 antibody, 1 to 12 hours after administration of the anti-CCR8 antibody, 1 to 3 days after administration of the anti-CCR8 antibody, any time point measured according to Example 24, or any other suitable time point thereafter). Those skilled in the art will also appreciate that when calculating the reference value using a time point after antibody administration, the antibody dose used to treat the subject to identify the reference value should be clearly defined, and may be, for example, any of the doses described herein for administration of the anti-CCR8 antibody. It is not advisable to specify the exact pg / μl values ​​for each cytokine herein, as methods for calibrating each assay for biomarker evaluation are known to those skilled in the art. In either case, a substantial increase in the described biomarker correlates with Treg elimination and / or successful treatment (compare Figure 10 with Figures 11-16).

[0307] For example, a biomarker for treatment success can be a biomarker for monitoring, prediction or stratification, the term stratification referring to the selection of patients for treatment.

[0308] In a preferred embodiment, the at least one, 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 is or comprises IFN-γ.

[0309] In a preferred embodiment, the reference value is a value obtained from a sample taken / collected from the same patient before the start of treatment. For example, a blood, plasma, or serum screening sample for analyzing cytokine levels can be taken / collected 15 to 60 minutes, preferably about 30 minutes, before administration of an effective dose of an anti-human CCR8 antibody.

[0310] Cytokines as safety biomarkers for anti-CCR8 antibodies Briefly, to investigate the potential of anti-CCR8 antibodies alone or in combination with pembrolizumab to activate the secretion of cytokines (analyzed IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and TNFα), cytokine release assays were performed using human whole blood (with soluble antibody added) and human peripheral blood mononuclear cells (PBMCs) (with wet-coated antibody) (see Example 14). Baseline values ​​for comparison of cytokine levels can be obtained for a particular anti-CCR8 antibody at a specific concentration as defined in this example. Alternatively, these baseline values ​​can be obtained based on a surrogate antibody with a known safety profile, as also described in this example.

[0311] Indeed, incubation of whole blood in the presence of soluble anti-human CCR8 antibodies resulted in a dose-dependent release of IFN-γ, IL-1β, IL-6, TNFα, and IL-8. Compared to rituximab, anti-human CCR8 antibodies induced higher IFN-γ, IL-6, TNFα, and IL-8 cytokine levels at a dose of 10 μg / mL, but produced comparable levels of induction at the lower antibody doses tested. IL-1 was not induced by rituximab, but was induced by anti-human CCR8 antibodies. IFN-γ was designated the lead cytokine due to its robust dose response and highest sensitivity.

[0312] Based on these findings, we propose the tested cytokine release panel as a safety biomarker for anti-CCR8 antibody cancer therapy to reduce the potential risk of infusion-associated reactions (including cytokine release syndrome) due to cytokine release and to reduce the risk of immune-related adverse events such as skin toxicity (i.e., rash) observed with other Treg-depleting agents.

[0313] Cytokines as biomarkers for monitoring anti-CCR8 antibodies Fucosylated anti-CCR8 antibody mutants or "silencing" Fc mutants (with reduced binding to FcgR) were tested in two donors and induced no or much lower levels of IFN-γ, IL-1β, IL-6, TNFα, and IL-8 in a whole blood / soluble antibody cytokine release assay format, suggesting that cytokine release is indicative of successful Treg depletion via ADCC and ADCP.

[0314] Because the predicted mechanism of action of defucosylated anti-CCR8 antibodies is dependent on ADCC and ADCP, we concluded that cytokine release is useful as a surrogate biomarker for anti-CCR8 antibody-induced Treg depletion and also as a predictive / stratification biomarker for therapeutic success. Indeed, Figure 6(B) shows the correlation between IFN-γ levels and therapeutic response for multiple mouse models. For human patients, see Figure 10 compared with Figures 11-16.

[0315] In accordance with these findings, in some further embodiments, e. The cytokine levels obtained according to step c) i. is significantly increased compared to the cytokine level obtained according to step a), or ii. If increased compared to baseline, Anti-human CCR8 antibodies having ADCC activity and ADCP activity are described for use in therapeutic methods further comprising administering to a patient at least one additional effective dose of the anti-human CCR8 antibody.

[0316] Cytokine levels were assessed in cynomolgus monkeys over the course of Examples 13.2 and 13.3. Examples 14 and 25 disclose cytokine release assays on human donors and increasing cytokine release with increasing doses of anti-CCR8 antibody in human patients.

[0317] 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 labels conjugated to detection antibodies. These labels emit light when stimulated by electricity in the appropriate chemical environment, which can then be used to measure key proteins and molecules. The detection process begins at electrodes located at the bottom of the platform's microplate; only labels close to the electrode are excited and detected. Electricity is applied to the plate's electrodes, causing light emission by the SULFO-TAG label, an electrochemiluminescent label that allows for ultrasensitive detection. The light intensity is then measured to quantify the analyte in the sample.

[0318] In other words, in principle, each well of the microplate used is pre-coated with a capture antibody (specific for each cytokine to be detected) in a spatially distinct spot. Plasma samples are added to the microplate wells, and bound cytokines from the sample are detected with MSD SULFO-TAG-labeled anti-cytokine detection antibodies. The principle of electrochemiluminescence is used. For reading, a voltage is applied to the plate electrodes, and the intensity of the emitted light allows for quantitative measurement of each cytokine present in the sample.

[0319] In another example, analysis of cytokine levels can be performed using a "single molecule array" (Simoa™). Simoa is based on the isolation of individual immune complexes on paramagnetic beads using standard ELISA reagents. The main difference between Simoa and conventional immunoassays is the ability to capture single molecules in femtoliter-sized wells, which allows for a "digital" readout of each individual bead to determine whether it has bound to the target analyte.

[0320] In some preferred embodiments, the level of at least one, 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 may be compared with baseline levels of the same cytokines obtained from the same patient at an earlier time point or prior to administration of the anti-CCR8 antibody.

[0321] In some preferred embodiments, the level of at least one, 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 compared to a reference value for each of the cytokines. This reference value can be determined by one of skill in the art and may deviate based on the concentration of previously administered anti-CCR8 antibody. See Example 14. The reference value can be a general reference value or a patient-specific reference value obtained from a sample taken before treatment.

[0322] In some embodiments, blood, plasma, or serum samples for analyzing cytokine levels are collected 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. Collecting blood samples on the same day can be convenient, especially when patients can be treated on an outpatient basis.

[0323] In some embodiments, blood, plasma or serum samples for analyzing cytokine levels are collected 1 to 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.

[0324] According to some preferred embodiments, blood, plasma or serum samples for analyzing cytokine levels according to step c) are collected 1 to 24 hours, 24 to 48 hours, 2 to 7 days, 7 to 14 days, 14 to 28 days or more than 28 days after administration of an effective dose of an anti-human CCR8 antibody according to step b).

[0325] According to some preferred embodiments, blood, plasma or serum samples for analyzing cytokine levels according to step c) are collected within the first three days after administration of an effective dose of an anti-human CCR8 antibody. Collecting samples within the first three days after administration of an effective dose of an anti-human CCR8 antibody according to step b) is particularly suitable for monitoring safety-related events.

[0326] According to some preferred embodiments, the blood, plasma or serum sample for analyzing cytokine levels according to step c) is collected within 3 to 21 days after administration of an effective dose of an anti-human CCR8 antibody. Collecting the sample 3 to 21 days after administration of an effective dose of an anti-human CCR8 antibody according to step b) is particularly suitable for monitoring the success of the treatment.

[0327] These embodiments are advantageous because they reduce the risk of infusion reactions and immune-related adverse events and / or improve efficacy / therapeutic success rates by identifying patient populations that will benefit most from anti-CCR8 antibody treatment.

[0328] Preferably, the fold change in cytokine levels can be measured by an immune-based assay during treatment compared to a baseline serum sample as a reference value.

[0329] Responsiveness Tregs are known to promote tumor growth by suppressing the function of cytotoxic T cells and contribute to the immunosuppressive tumor microenvironment (TME) through various mechanisms. In line with this, Tregs have also been identified as one of the major resistance mechanisms to ICIs across multiple tumor types. Furthermore, established PD-1 / PD-L1 inhibitors can not only induce the recovery of PD-1-positive dysfunctional cytotoxic T cells but also enhance the proliferation and suppressive activity of PD-1-positive Tregs. Consequently, PD-1+ Tregs, which exhibit upregulated PD-1 expression, were more frequently detected in patients with tumors unresponsive to ICIs. These observations support targeting Tregs as an attractive approach to enhance antitumor immune responses, both as monotherapy and in combination with ICIs.

[0330] This preclinical rationale also suggests that treatment with TPP-23411 may provide an innovative and effective therapy to counter immunosuppressive pathways in tumors by overcoming resistance to PD-(L)1 inhibitors and improving the efficacy of established PD-(L)1 inhibitors when combined with anti-PD-(L)1 inhibitors in patients after ICI failure.

[0331] A non-responder is a patient who has not received treatment with an anti-PD-(L)1 antibody for at least six months. This is due to the fact that the effect of anti-PD-(L)1 antibody treatment is monitored and treatment is discontinued if no response is observed. Consequently, a responder is a patient who has received treatment with an anti-PD-(L)1 antibody for at least six months.

[0332] According to another 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, wherein the method of treatment comprises: a. stratifying patients based on prior cancer treatment with an anti-PD-(L)1 antibody for at least 6 months; and b. Administer anti-human CCR8 antibodies only if the patient has received prior treatment with an anti-PD-(L)1 antibody for at least 6 months. A method for treating cancer, comprising:

[0333] In some embodiments, the anti-human CCR8 antibody is an anti-human CCR8 antibody for use in the method of treatment according to the first aspect.

[0334] Aspect 2 Mini Body We have shown that anti-mouse CCR8 surrogate antibodies eliminate tumor-infiltrating CCR8+ Tregs via ADCC and ADCP mechanisms and induce potent in vivo single-agent tumor growth inhibition in immunogenic mouse tumor models. Furthermore, ex vivo analysis demonstrated that these antitumor effects are associated not only with efficient elimination of CCR8+ Tregs but also with a substantial expansion of CD8+ T cells within the tumor microenvironment.

[0335] Thus, in U.S. Patent Application No. 17 / 358,841, filed 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 presented molecules that bind to T cell markers for use in a method of diagnosing / stratifying a subject as having a tumor that is sensitive to treatment with an anti-CCR8 antibody, the method comprising: a. Determining the level of T cell marker expression in the tumor (sample); comparing the level of bT cell marker expression to a reference sample or value; and If the level of the cT cell marker is higher than or equal to the reference sample or value, the subject is diagnosed / stratified as having a tumor that is susceptible to treatment with an anti-CCR8 antibody. Includes.

[0336] Since there is a strong interest in determining the levels of appropriate T cell markers in a highly reliable manner to analyze the amount of T cells in tumor biopsy samples, the present inventors have developed a method in the present invention that uses PET technology instead of biopsy tissue for evaluation, thereby achieving a more comprehensive, reliable, and less error-prone situation for assessing treatment success or determining patient eligibility for treatment with (additional administration of) anti-CCR8 antibodies.

[0337] According to an aspect of the present invention, a. Administering a Zr-89 labeled anti-CD8 minibody to a subject; b. performing at least one PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 minibody in the subject and obtain a first image of the subject; c. determining the abundance and / or distribution of Zr-89 labeled anti-CD8 minibody in one or more of the subject's cancer lesions based on the first subject image; and d. administering to the subject an effective dose of an anti-human CCR8 antibody if the first subject image shows the amount and / or distribution of the Zr-89 labeled anti-CD8 minibody in any one or more cancer lesions and indicates that the subject is likely to benefit from administration of the anti-human CCR8 antibody. The present invention provides an anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer, comprising:

[0338] Minibodies can be bivalent homodimers containing each monomer with a single-chain variable fragment (scFv) linked to a human IgG1 CH3 domain via a modified IgG1 hinge sequence. Minibodies preferably lack Fc receptor interacting domains and have a small size compared to full monoclonal antibodies.

[0339] Preferably, the Zr-89 labeled anti-CD8 minibody binds to human CD8 glycoprotein with an EC50 of less than 1 nM. For example, the minibody can be conjugated via desferrioxamine (Df) and radiolabeled with the positron-emitting radionuclide zirconium-89 (Zr; Tm 78.4 hours). According to a most preferred embodiment, the Zr-89 labeled anti-CD8 minibody is the Zr-89 labeled anti-CD8 minibody described in U.S. Patent Application No. 17 / 280,137.

[0340] The uptake of Zr-Df-clefmirlimab in tumor lesions correlates with CD8 expression in these lesions and can be used in accordance with the present invention to monitor the influx of T cells into tumor lesions after the first administration of an anti-human CCR8 antibody. This influx demonstrates successful activation of the immune system by the anti-human CCR8 antibody and can be used, for example, as a surrogate biomarker for treatment success, a monitoring biomarker, or a predictive / stratification biomarker. More specifically, an increase in the abundance and / or altered distribution of Zr-Df-clefmirlimab, indicating a substantial influx or substantially greater increase in T cells into at least one tumor lesion or tumor microenvironment, is indicative of a substantial likelihood that the subject will benefit from administration of an anti-human CCR8 antibody.

[0341] A Zr-89 labeled anti-CD8 minibody or pharmaceutical formulation thereof can be produced by conjugating a minibody to desferrioxamine to form a Df-minibody, radiolabeling the Df-minibody with 89Zr to form a radiolabeled minibody, purifying the radiolabeled minibody, and mixing the radiolabeled minibody with a non-radioactive (cold) minibody to form a diagnostic composition, wherein the minibody and the non-radioactive minibody bind to the same epitope on CD8. A detailed description of this process can be found in U.S. Patent Application Serial No. 17 / 280,137.

[0342] Preferably, the abundance and / or distribution of CD8 minibodies in a subject is analyzed within 6-36 hours of administering the CD8 minibody to the patient, for example, within 24 hours of administering the CD8 minibody to the patient.

[0343] In some embodiments, a method of positron emission tomography ("PET scan") comprises: a. Administering a Zr-89 labeled anti-CD8 minibody to a subject; b. Preparing a scintillator; c. Using a scintillator to detect photon pairs generated by the Zr-89 labeled anti-CD8 minibody; and d. utilizing detection of photon pairs to locate a Zr-89 labeled anti-CD8 minibody source by a list of coincidence events processed via a processor configured to obtain output from the scintillator and convert it into a list of coincidence events; e. where 1 mm of tissue or cancerous lesion within the subject 3 Approximately 300 to 500 CD8-positive cells can be detected per sample. Includes.

[0344] In some embodiments, an MRI and / or CT scan may be performed to determine the location of the tumor and obtain more precise spatial information.

[0345] In some embodiments, the method comprises: a. Obtaining an image of the distribution of Zr-89 labeled anti-CD8 minibody; b. obtaining an image of the distribution of the FDG marker via a second PET image of the subject; c. creating a third PET image comprising an overlay of the first PET image onto the second PET image. Includes.

[0346] For example, a method for determining the standard uptake value is a. applying a Zr-89 labeled anti-CD8 minibody to a subject; determining br [where r is the radioactivity concentration (kBq / ml) measured by a PET scanner within the region of interest of radiation from the Zr-89 labeled anti-CD8 minibody]; c. Determining a', where a' is the decay-corrected amount (kBq) of injected radiolabeled tracer; d. Determining the subject's body weight, w; and The SUV is determined as the calculation result of er(a' / W).

[0347] For example, if the standard uptake value (SUV) is greater than 1, more preferably greater than 2, greater than 3, or greater than 4, and most preferably greater than 5, greater than 6, greater than 7, or greater than 8, the amount of Zr-89 labeled anti-CD8 minibody can be considered to indicate a substantial likelihood that the patient will benefit from administration of the anti-human CCR8 antibody.

[0348] In some embodiments, the method of analyzing an image comprises: a. Obtaining an image; b. defining a first region of interest (ROI) on the image by marking the image; c. determining signal intensities for data points within the first ROI; d. Determining the maximum signal intensity within the first ROI; e. determining a mean value of signal intensity within the first ROI; f. summing each signal intensity within the first ROI to obtain a first total signal level for the first ROI. where the first ROI represents data regarding the amount of Zr-89 labeled anti-CD8 minibody administered to the subject.

[0349] In some preferred embodiments, the amount and / or distribution of Zr-89 labeled anti-CD8 minibody in any one or more cancer lesions is determined by: i. Assessing the abundance and / or distribution of Zr-89 labeled anti-CD8 minibodies in healthy tissues of the patient, or ii. Assessing abundance and / or distribution of Zr-89 labeled anti-CD8 minibody relative to one or more benchmarks.

[0350] According to some embodiments, a. administering a first dose of a Zr-89 labeled anti-CD8 minibody to a subject; b. performing a first PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 minibody in the subject to obtain a first subject image; c. determining a first abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancer lesions in the subject based on the first subject image; d. administering an effective dose of an anti-human CCR8 antibody to the subject; e. administering a second dose of Zr-89 labeled anti-CD8 minibody to the subject; f. performing a second PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 minibody in the subject to obtain a second image of the subject; g. determining a second abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancerous lesions in the subject based on the second subject image; h. Comparing the second subject image to the first subject image to assess whether the abundance of the Zr-89 labeled anti-CD8 minibody has increased substantially or the distribution of the Zr-89 labeled anti-CD8 minibody has changed substantially in one or more cancer lesions to monitor disease progression or the success of anti-human CCR8 antibody treatment. The present invention provides an anti-human CCR8 antibody having ADCC activity and / or ADCP activity for use in a method for treating cancer, comprising:

[0351] According to some of these embodiments, an anti-human CCR8 antibody having ADCC activity and ADCP activity is provided for use in a method for treating cancer, comprising the further step of administering at least one additional effective dose of the anti-human CCR8 antibody to the patient if the abundance of the Zr-89-labeled anti-CD8 minibody is substantially increased in one or more cancer lesions or if the distribution of the Zr-89-labeled anti-CD8 minibody is substantially changed.

[0352] For example, a Zr-89 labeled anti-CD8 minibody can yield approximately 0.5-3.6 mCi of radioactivity.

[0353] For example, a PET scan can be performed approximately 6 hours to 36 hours after administration of each dose of Zr-89 labeled anti-CD8 minibody.

[0354] In some highly preferred embodiments, the Zr-89 labeled anti-CD8 minibody is 89Zr-Df-clefmirlimab.

[0355] 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.

[0356] In some highly preferred embodiments, the anti-human CCR8 antibody for use in the methods of treating cancer is any of TPP-23411, TPP-29338, TPP-27454, TPP-31741, TPP-31742, TPP-31743, or TPP-31744.

[0357] The anti-CCR8 antibody according to this embodiment is preferably an isolated anti-CCR8 antibody or antigen-binding fragment thereof comprising six CDR sequences, wherein each CDR sequence is 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, 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, 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, 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; 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; 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 and having at least 98% or 100% sequence identity to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the present invention.

[0358] Preferably, the antibody further comprises a. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth 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 set forth in SEQ ID NO:5; b. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41; c. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53; d. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 65; e. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 73; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 77; or f. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 85; and / or A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 89. may include:

[0359] Preferably, the antibody further comprises a. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18; b. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48; c. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60; d. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72; e. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 83; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84; f. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 95; and / or A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. may include:

[0360] Aspect 3 plasma method Described herein is a method for determining anti-CCR8 antibodies in cynomolgus monkey plasma, the method comprising: a. diluting the plasma or serum sample with a buffer; b. capturing anti-CCR8 antibodies in the plasma using immobilized biotinylated anti-human IgG-Fc antibodies as capture molecules; c. Detecting the amount of captured molecules present using a fluorescently labeled anti-human IgG antibody as a detection reagent. Includes.

[0361] This method was used in preclinical pharmacokinetic studies and pivotal nonclinical safety studies in monkeys, and it was found to be suitable for reliable measurement of anti-CCR8 antibodies in plasma.

[0362] Anti-CCR8 antibodies were found to be measurable in plasma with an adequate detection range for cynomolgus monkey subjects using the Gyrolab Generic PK Kit assay. Briefly, samples (calibration standards, quality control, qualification samples, or unknown samples), antibodies, and buffers are placed on a microplate. Biotinylated antibodies are then used for capture, and fluorescently tagged antibodies are used for detection. The microplate can then be loaded onto a Gyrolab instrument and a Gyrolab Bioaffy CD with sufficient buffer (Bioaffy Pump Liquid, Bioaffy Wash Buffers 1 and 2). The resulting fluorescent signal was found to be directly proportional to the concentration of anti-CCR8 antibodies present in the sample. The lower limit of quantitation was 1.25 ng / mL. The measurement range is 1.25–250 ng / mL in MRD (12.5–2500 ng / mL in 100% plasma). The fluorophore can be any fluorophore known in the art, and is preferably an Alexa Dye, such as AF647.

[0363] The immobilized biotinylated anti-human IgG-Fc antibody can be CaptureSelect™ Human IgG-Fc PK Biotin Conjugate (Thermo Scientific, Catalog No. 7103322100). CaptureSelect™ Human IgG-Fc PK Biotin Conjugate consists of a 13 kDa recombinant single-domain antibody fragment (VHH affinity ligand) that specifically binds to the Fc portion of all four human IgG subclasses without cross-linking mouse, rat, rhesus, and cynomolgus IgG. The affinity ligand is chemically conjugated to biotin via an appropriate spacer that preserves the binding responsiveness of the ligand when used in combination with a streptavidin-based conjugate or a surface pre-coated with streptavidin.

[0364] Aspect 4 ADA law Biologic agents, including therapeutic antibodies, are known to be immunogenic, and their administration to patients can induce an immune response, resulting in the formation of anti-drug antibodies (ADAs). Such ADAs can reduce the efficacy of anti-CCR8 antibodies. For example, they can bind to and / or neutralize anti-CCR8 antibodies, altering their pharmacokinetics or pharmacodynamics, thereby modifying their efficacy. ADAs can cause serious side effects, including allergic reactions, cross-reactions with endogenous proteins by neutralizing antibodies (NAbs), and complement activation. Therefore, there was a need to develop a reliable assay for monitoring the formation of anti-CCR8 antibodies.

[0365] The present inventors have developed a method for reliably determining and (semi-)quantifying the formation of anti-anti-CCR8 antibodies in the plasma or serum of cynomolgus monkeys or humans in the present invention. This method has been found to be superior to other tested approaches. More specifically, a method for determining and quantifying the formation of anti-anti-CCR8 antibodies in the plasma or serum of cynomolgus monkeys or humans is provided, which method comprises a bridging ELISA method based on anti-CCR8 antibodies.

[0366] Briefly, we found that anti-drug antibodies (ADAs) against anti-CCR8 antibodies can be reliably detected using a bridging 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 prediluted (e.g., 1:8) in dilution buffer, mixed with dilution buffer, and preincubated in a polypropylene plate, for example, on an orbital shaker (e.g., RT, 600 rpm) for 1 hour. A master mix containing biotinylated anti-CCR8 antibodies (e.g., 1 μg / mL) and SULFO-tagged anti-CCR8 antibodies (e.g., 1 μg / mL) can be added to the sample mixture and incubated for 2 hours (RT, 600 rpm). From the incubated samples, 25 μL can be transferred in duplicate into wells of a blocked MSD Streptavidin Gold plate (150 μL of blocking buffer, at least 30 minutes, 600 rpm) to which biotinylated anti-CCR8 antibodies can bind. If functional anti-drug antibodies are present, they will bridge the biotinylated and SULFO-tagged anti-CCR8 antibodies. When voltage is applied, the SULFO-tagged anti-CCR8 antibodies generate an electrochemiluminescence (ECL) signal that correlates with the amount of ADA in the wells. The plate can be read (e.g., using a MesoQuickPlex SQ 120) and the data can be analyzed (e.g., with MSD® Workbench™ software).

[0367] According to this embodiment, a signal is generated when the anti-anti-CCR8 antibody crosslinks a) the biotinylated anti-CCR8 antibody and b) the SULFO-tagged anti-CCR8 antibody. Methods for obtaining biotinylated antibodies are known in the art, and methods for obtaining SULFO-tagged antibodies are also available to those skilled in the art (e.g., based on NHS ester chemistry).

[0368] The antibody used to obtain the SULFO-tagged anti-CCR8 antibody and the biotinylated anti-CCR8 antibody can be any of TPP-23411, TPP-27454, TPP-31741, TPP-31742, TPP-31743 or TPP-31744.

[0369] Thus, the anti-CCR8 antibody according to this embodiment is an isolated anti-CCR8 antibody or antigen-binding fragment thereof comprising six CDR sequences, wherein each CDR sequence is 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, 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, 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, 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; 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; 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 and having at least 98% or 100% sequence identity to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of the present invention.

[0370] Preferably, the antibody further comprises a. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth 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 set forth in SEQ ID NO:5; b. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 41; c. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 49; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 53; d. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 61; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 65; e. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 73; and / or a variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 77; or f. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 85; and / or A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 89. may include:

[0371] Preferably, the antibody further comprises a. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18; b. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48; c. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 59; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 60; d. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 71; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 72; e. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 83; and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 84; f. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 95; and / or A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 96. may include:

[0372] Aspect 5 TPP-29338 When the present inventors characterized the properties of the clinical candidate TPP-23411, they surprisingly found that this antibody exhibited unexpected clearance behavior in cynomolgus monkeys. Although anti-mouse CCR8 surrogate antibodies, such as TPP-15285, have already been generated to model the behavior of TPP-23411, these surrogate antibodies have not yet captured TPP-23411's unexpected clearance behavior. Therefore, in order to derive a safe and effective administration scheme for the clinical candidate TPP-23411, the present inventors first needed to identify a mouse surrogate antibody that exhibits PK / PD behavior very similar to TPP-23411. Indeed, using TPP-29338, they were finally able to obtain such a surrogate antibody. They generated a mutant TPP-29338 with a short half-life, which proved suitable for modeling the high clearance of TPP-23411. TPP-29338 is an anti-mouse CCR8 antibody in which the human VH / VL chains are chimerized to mIgG2a with H310Q / H330N mutations. It induces both ADCC and ADCP and has a short half-life, i.e., less than 10 days.

[0373] Thus, according to another aspect, there is provided 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.

[0374] For example, the isolated anti-CCR8 antibody or antigen-binding fragment thereof may further comprise: a. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, and / or b. A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 23 Includes.

[0375] For example, the isolated anti-CCR8 antibody or antigen-binding fragment thereof may further comprise: a. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35, and / or b. A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 36 Includes.

[0376] In some highly preferred embodiments, the antibody according to the above another aspect is defucosylated.

[0377] Further 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 polynucleotide.

[0378] Example

[0379] To demonstrate a suitable administration scheme for (defucosylated) TPP-23411, the following example was performed. 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.

[0380] Because TPP-23411 is cross-reactive only to cynomolgus monkeys and not to the mouse CCR8 orthologue, a mouse surrogate antibody was used to further evaluate its mechanism of action and efficacy in depleting CCR8+ Tregs in mice in vitro and in vivo. Table 1.1 summarizes the antibodies used in the in vitro and in vivo studies. [Table 2]

[0381] Example 1: Generation of a murine surrogate antibody to model the high clearance of TPP-23411 When the present inventors characterized the properties of the clinical candidate TPP-23411, they surprisingly found that this antibody exhibited unexpected clearance behavior in cynomolgus monkeys. Although anti-mouse CCR8 surrogate antibodies, such as TPP-15285, have already been generated to model the behavior of TPP-23411, these surrogate antibodies have not yet captured TPP-23411's unexpected clearance behavior. Therefore, in order to derive a safe and effective administration scheme for the clinical candidate TPP-23411, the present inventors first needed to identify a mouse surrogate antibody that exhibits PK / PD behavior very similar to TPP-23411. Indeed, using TPP-29338, they were finally able to obtain such a surrogate antibody. They generated a mutant TPP-29338 with a short half-life, which proved suitable for modeling the high clearance of TPP-23411. TPP-29338 is an anti-mouse CCR8 antibody in which human VH / VL chains are chimerized to mIgG2a with H310Q / H330N mutations.

[0382] Example 2: Pharmacological evaluation of TPP-23411 A series of primary and secondary pharmacodynamic, safety pharmacology, and pharmacodynamic drug interaction studies were conducted 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 described in further detail below.

[0383] Table 2.1: Selected non-clinical pharmacology in vitro studies with TPP-23411. For further details of 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. [Table 3] TIFF2025530159000006.tif250150

[0384] Table 2.2: Nonclinical pharmacology in vivo studies with TPP-23411. Q3 / 4D, twice weekly every 3 or 4 days; SD = single dose (single administration); T / C = treatment / control ratio (calculated from mean tumor volume compared to isotype control). [Table 4] TIFF2025530159000008.tif252149TIFF2025530159000009.tif251148

[0385] Example 3: Characterization of Murine Surrogate Antibodies: Binding Affinity The binding of murine surrogate antibodies to murine Fc receptor mutants (mFcγR and mFcRn) was analyzed by SPR. The binding properties of TPP-15285 (mIgG2a surrogate antibody with a normal half-life of 141 hours), TPP-29338 (mIgG2a surrogate antibody with H310Q / H330N mutations that prevent interaction with murine FcRn, resulting in a short antibody half-life of 27 hours), and the mIgG2a isotype control TPP-10748 were evaluated by SPR analysis. In this study, TPP-15285, TPP-29338, and TPP-10748 all showed similar binding properties to the mFcγR mutants (Table 3.1), but only TPP-15285 and TPP-10748 showed binding to mFcRn (Table 3.2). [Table 5]

[0386] Example 4: Characterization of Murine Surrogate Antibodies: Cell Binding We evaluated the binding properties of murine surrogate antibodies using flow cytometry in HEK293 cells ectopically expressing murine CCR8. We compared the binding properties of the surrogate antibodies TPP-15285 (chimerized to mIgG2a), TPP-29338 (chimerized to murine IgG2a with H310Q / H330N mutations that prevent interaction with murine FcRn), TPP-14099 (human IgG1), and TPP-18208 (a deglycosylated hIgG1 form of TPP-14099; in which the N297A mutation completely removes the N-glycosylation site of the antibody) with those of non-mIgG2a and hIgG1 isotype controls (TPP-10748 and TPP-9809, respectively).

[0387] The TPP-15285, TPP-29338, TPP-14099, and TPP-18208 surrogate antibodies demonstrated specific binding to mouse CCR8. Surprisingly, TPP-29338 exhibited 10-fold higher affinity compared to TPP-15825 (Table 4.1). In summary, these antibodies are suitable mouse surrogate antibodies for the anti-human CCR8 antibody candidate TPP-23411, which will be used for in vivo efficacy and mechanism of action studies in mice. [Table 6]

[0388] Example 5: In vitro mechanism of action study: ADCC evaluation of anti-human CCR8 antibody TPP-23411 The ADCC mechanism of action of the defucosylated TPP-23411 antibody was evaluated in a CytoTox-Glo assay using CCR8-expressing target cells and NK92V (NK-like cell line) effector cells at an effector-to-target cell ratio (E:T) of 4:1. Also analyzed were the wild-type fucosylation variant of TPP-23411, hIgG1, and hIgG1 isotype control antibodies (both defucosylated and wild-type fucosylated forms).

[0389] The higher the CCR8 expression level on target cells, the higher the maximal ADCC response. The maximal ADCC response to TPP-23411 was 52.7% when activated human Tregs with 85.0% CCR8 expression were used, whereas it was 19.5% when Tregs with 31.4% CCR8 expression were used. Defucosylation of TPP-23411 enhanced the antibody's affinity for FcγRIII, thereby significantly enhancing the ADCC potential of the therapeutic antibody. In summary, TPP-23411 induced potent and dose-dependent depletion of both CCR8-expressing human Tregs and human CCR8-HEK293 cells with EC50 values ​​ranging from 55.9 pM to 12.8 pM. [Table 7]

[0390] Example 6: In vitro mechanism of action study: ADCP evaluation of anti-human CCR8 antibody TPP-23411 The ADCP mechanism of action of defucosylated TPP-23411 was evaluated by a flow cytometry-based assay using activated human Tregs endogenously expressing CCR8 or HEK293 cells ectopically expressing human CCR8 (as target cells) and human M2c macrophages (as effector cells) at an effector-to-target cell ratio (E:T) of 4:1. A wild-type fucosylation mutant of TPP-23411 and an hIgG1 isotype control antibody with defucosylated and wild-type fucosylated forms were also analyzed.

[0391] Neither antibody defucosylation nor the CCR8 expression level on target cells affected ADCP activity. Both wild-type and defucosylated TPP-23411 antibodies induced comparable dose-dependent ADCP responses against human Tregs in the presence of M2c macrophages. The maximal response to TPP-23411 was 49.9% when human Tregs with 63% CCR8 expression were used, whereas it was 49.3% when Tregs with 40% CCR8 expression were used. The corresponding EC50 values ​​were 143 pM and 440 pM, respectively. In summary, TPP-23411 induced potent and dose-dependent phagocytosis of both CCR8-expressing human Tregs and human CCR8-HEK293 cells, with maximal ADCP responses ranging from 49.9% to 26.6% (Table 6.1). [Table 8]

[0392] Example 7: In vitro mechanism of action study: ADCC evaluation of murine surrogate antibodies of TPP-23411 The ADCC mechanism of action of the mouse surrogate antibody TPP-23411 was evaluated using mouse CCR8-expressing HEK293 cells (as target cells) and mouse NK cells (as effector cells) at an effector-to-target ratio (E:T) of 10:1. Mouse CCR8-HEK293 target cells were double-labeled with Cytolight Red Rapid Dye to label live cells and Caspase 3 / 7 Green Dye to label apoptotic cells, and the cells were imaged for 24 hours in an Incucyte system. The normal half-life anti-mouse CCR8 antibody TPP-15285, an artificially modified short-half-life variant of TPP-15285 (termed TPP-29338) (both mIgG2a), and an mIgG2a derivative isotype control (TPP-10748) were evaluated in a dose-response assay. Anti-mouse CCR8 antibodies with human IgG1 or deglycosylated human IgG1 forms (TPP-14099 and TPP-18208, respectively) were also evaluated.

[0393] The TPP-15285, TPP-29338, and TPP-14099 surrogate antibodies induced clear dose-dependent ADCC responses compared to isotype controls. TPP-15285 had an EC of 227.38 pM. 50 and 24.64% maximum ADCC response, while the short half-life variant TPP-29338, which has a higher cellular affinity for mouse CCR8, was consequently more potent, with an EC of 9.28 pM. 50 and a maximum ADCC response of 31.92%. TPP-14099 had an EC 50 and showed a maximum ADCC response of 16.43%, while its N297A deglycosylated mutant TPP-18208 showed no ADCC activity (Table 7.1).

[0394] In summary, efficient ADCC typically requires a fully functional Fc portion, as in the anti-mouse CCR8 surrogate antibodies TPP-15285, TPP-29338, and TPP-14099. These antibodies are suitable mouse surrogates of the anti-human CCR8 antibody TPP-23411, used in in vivo efficacy and mechanism-of-action studies in mice. [Table 9]

[0395] Example 8: In vitro mechanism of action study: ADCP evaluation of murine surrogate antibodies to TPP-23411 We evaluated the ADCP mechanism of action of the mouse surrogate antibody TPP-23411 using mouse CCR8-expressing HEK293 cells (as target cells) and mouse M2 macrophages (as effector cells) at an effector-to-target cell ratio (E:T) of 4:1. Mouse CCR8-HEK293 target cells were dual-labeled with Cytolight Rapid Green Dye to label live cells and pHrodo Red Cell Labeling Dye to label phagocytes, and the cells were imaged in an Incucyte system for up to 12 hours. The normal half-life anti-mouse CCR8 antibody TPP-15285, an artificially modified short-life variant of TPP-15285 (termed TPP-29338) (both mIgG2a), and the mIgG2a derivative isotype control TPP-10748 were evaluated. Anti-mouse CCR8 antibodies with human IgG1 or deglycosylated human IgG1 forms (TPP-14099 and TPP-18208, respectively) and a non-binding hIgG1 isotype control (TPP-9809) were also evaluated for ADCP.

[0396] The TPP-15285, TPP-29338, and TPP-14099 surrogate antibodies induced comparable dose-dependent ADCC responses. After 4 hours of co-culture, the surrogate antibodies induced 67%-75% of the maximum ADCP response, and after 12 hours of co-culture, the maximum ADCP response was 92%-94%. TPP-15285 exhibited EC50 values ​​of 379.8 pM and 158.1 pM, while its short-half-life mutant, TPP-29338, exhibited EC50 values ​​of 518.3 pM and 197.3 pM at 4 and 12 hours, respectively. TPP-14099 exhibited an EC50 of 584 pM and a maximum ADCC response of 16.43%, while its N297A deglycosylated mutant, TPP-18208, exhibited no ADCC activity (Table 8.1).

[0397] In summary, efficient ADCPs typically require fully functional Fc portions of the anti-mouse CCR8 surrogate antibodies TPP-15285, TPP-29338, and TPP-14099. These antibodies are suitable mouse surrogates of the anti-human CCR8 antibody TPP-23411, which are used in in vivo efficacy and mechanism-of-action studies in mice. [Table 10]

[0398] Example 9: Primary Pharmacodynamics In Vivo Because TPP-23411 is cross-reactive only with cynomolgus monkeys and not with the mouse CCR8 orthologue, a murine surrogate antibody was used to evaluate the in vivo mechanism of action and efficacy of CCR8+ Treg depletion in mice. In vivo studies were performed in monotherapy or in combination with ICIs, such as anti-PD-1 and anti-PD-L1 antibodies.

[0399] Example 9.1: In vivo efficacy of normal and short half-life murine surrogate antibodies in a syngeneic EMT6 mouse cancer model The pharmacokinetics of two anti-mouse CCR8 surrogate antibodies, namely, TPP-15285 (an mIgG2a surrogate of TPP-23411) and TPP-29338 (an mIgG2a surrogate with H310Q / H330N mutations that prevent interaction with mouse FcRn), were investigated in mice after a single intravenous administration of a 5 mg / kg dose. For TPP-15285, the plasma clearance was 0.0012 L / (h * The plasma clearance of TPP-29338 was 0.0054 L / (h * The dose was 0.14 L / kg, the volume of distribution (Vss) was 0.14 L / kg, and the plasma elimination half-life was 27 hours.

[0400] The in vivo antitumor efficacy of both the anti-mouse CCR8 surrogate antibodies TPP-15285 (a mIgG2a surrogate antibody with a normal half-life) and TPP-29338 (a mIgG2a surrogate antibody with a short half-life) was analyzed in monotherapy using a syngeneic EMT6 mouse breast cancer model. The changes in intratumoral CCR8+ Tregs under study were also determined. TPP-29338, a short-half-life variant of TPP-15285, was used to mimic the predicted short human half-life of the anti-human antibody TPP-23411 in mice.

[0401] Treatment with the short-half-life surrogate antibody TPP-29338 at 0.3, 1, or 3 mg / kg resulted in T / C values ​​of 0.90, 0.72, and 0.22, respectively, demonstrating dose-dependent antitumor efficacy. Treatment with the normal-half-life surrogate antibody TPP-15285 at 1 mg / kg resulted in a T / C value of 0.26. Both 3 mg / kg TPP-29338 and 1 mg / kg TPP-15285 depleted intratumoral CCR8+ Tregs. All treatments were well tolerated, as indicated by increases in mean body weight in all treatment groups.

[0402] In conclusion, both murine surrogate antibodies of TPP-23411 exhibited potent in vivo activity, with the short half-life surrogate anti-CCR8 antibody TPP-29338 requiring a higher dose than the normal half-life surrogate anti-CCR8 antibody TPP-15285 to achieve comparable exposure (Table 9.1.1) and to induce comparable intratumoral CCR8+ Treg depletion and in vivo anti-tumor efficacy. [Table 11]

[0403] Example 9.2: PK / PD Relationship of Murine Surrogate Antibodies TPP-15285 and TPP-29338 in a Syngeneic EMT6 Mouse Cancer Model To assess the time course of Treg depletion and repopulation and to better understand the PK / PD relationship of the TPP-15285 and TPP-29338 antibodies (both mIgG2a; surrogates of the anti-human CCR8 antibody TPP-23411), we conducted further studies using a syngeneic EMT6 mouse breast cancer model. TPP-29338, a short-half-life variant of TPP-15285, was used to mimic the expected short human half-life of TPP-23411 in mice. Female BALB / cAnN mice from Charles River were randomized to receive intraperitoneal (ip) treatment with TPP-15285 (single doses of 0.25, 1, and 4 mg / kg), TPP-29338 (single doses of 0.25, 1, and 4 mg / kg), or the mIgG2a isotype control TPP-10748, beginning 8 days after subcutaneous (sc) inoculation of EMT6 mouse breast cancer cells. 24, 48, 120, 192, and 336 hours after a single dose of compound or when tumors reached 225 mm 2 Mice (n=5 / group / time point) were sacrificed when tumors reached a predetermined size. Tumors and blood were collected for pharmacokinetic analysis and flow cytometric quantification of T cell populations.

[0404] Treatment with TPP-15285 and TPP-29338 resulted in intratumoral depletion of CCR8+ Treg cells compared with isotype control, as assessed by flow cytometry. CCR8+ Tregs repopulated the TME more rapidly when mice were treated with the short-half-life mutant TPP-29338 compared with the normal-half-life mutant TPP-15285.

[0405] In conclusion, for both murine surrogate antibodies TPP-15285 and TPP-29338, a direct correlation was observed between antibody plasma concentrations and efficacy of intratumoral CCR8+ Treg depletion (Table 9.2.1, Table 9.2.2), suggesting that persistent coverage of threshold antibody concentrations may be required to ensure Treg depletion and downstream effects. [Table 12]

[0406] Example 9.3: IFN-γ concentrations measured in tumors or blood of EMT6 tumor-bearing mice At the end of the study, on day 19, ELISA analysis of IFN-γ concentrations was performed in the 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. [Table 13]

[0407] Example 10: Evaluation of Pharmacokinetic / Pharmacodynamic (PK / PD) Relationships and Effective Exposure To establish the PK / PD relationship, the concentration producing the maximum effect (EC80 for prediction of effective dose) and the concentration producing the minimum effect (EC20 for prediction of the minimum predicted effect level) were derived from human in vitro ADCC and ADCP assays (see Examples elsewhere herein).

[0408] These concentrations were corrected by an additional in vitro to in vivo correlation (IVIVC) factor obtained from mice by in vitro ADCC and ADCP assays and observed in vivo Treg depletion (see Examples elsewhere herein).

[0409] Furthermore, an empirical correlation between Treg depletion and tumor regression in mice in vivo was considered, with a T / C ratio of less than 0.5 indicated when Treg depletion was greater than 50% from baseline.

[0410] Because limited information was available regarding Treg dynamics in humans, it was assumed that persistent Treg depletion was necessary for antitumor efficacy. Because a direct correlation between further exposure and Treg depletion was observed in dedicated in vivo exposure-response studies in mice, it was assumed that persistent coverage of the threshold antibody concentration would ensure sustained Treg depletion necessary for antitumor efficacy. Therefore, the steady-state trough concentration (Ctrough) was assumed to be the driving force behind Treg depletion and further effects on antitumor immunity in humans.

[0411] In summary, for the prediction of effective doses in humans, the estimated EC80 associated with an expected Treg depletion of approximately 50% should be targeted as Ctrough. For the prediction of minimally effective dose levels associated with approximately 10% Treg depletion, the estimated EC20 should be targeted as Ctrough. Table 10.1 shows the estimated range of effective concentrations in humans, in addition to the estimated range of minimally effective concentrations. [Table 14]

[0412] Example 11: Safety Pharmacology In accordance with ICH guidelines S6 and S9, the effects of TPP-23411 on vital organ function (central nervous system [CNS], cardiovascular system including ECG, and respiratory system) were investigated, i.e., each endpoint was included in a pivotal 4-week toxicity study in cynomolgus monkeys.

[0413] Safety pharmacology endpoints included detailed clinical observations, physical / neurological examinations [abdominal palpation, temperature, cardiopulmonary auscultation, cerebral (pupillary, orbicularis oculi) and spinal (patellar, anal) reflexes, and general sensation including foot grasp reflex], respiratory rate, arterial blood pressure (high-resolution oscillometry), and ECG (30-60 second recordings). These investigations were performed once during the pre-dose phase, once during weeks 1 and 4 of the dosing phase (before dosing and 1-2 hours after dosing), and once during week 2 of the recovery phase. Systemic exposure to TPP-23411 was also measured.

[0414] TPP-23411 was administered by intravenous (iv) slow bolus injection once or twice weekly to three to five male / female cynomolgus monkeys per group at doses of 2 × 0 (vehicle), 15, 50, and 2 × 40 mg / kg. None of these parameters were significantly affected by treatment, with mean plasma concentrations up to 1550 mg / L (50 mg / kg). These plasma concentrations are more than 500-fold higher than those currently expected for human therapeutic efficacy (dose range 38-1100 μg / kg, Cmax range 0.7-20 mg / L).

[0415] Example 12: Nonclinical Pharmacokinetics and Drug Metabolism The pharmacokinetics of TPP-23411 was studied in vivo in male cynomolgus monkeys after single intravenous and subcutaneous administration of TPP-23411. TPP-23411 was measured in monkey plasma using an anti-human IgG universal assay (IgG-ELISA). Anti-TPP-23411 antibody formation was monitored using a validated bridging ELISA method based on TPP-23411 (described elsewhere herein). [Table 15]

[0416] Table 12.2 shows the pharmacokinetics of TPP-23411 after intravenous and subcutaneous administration. Figure 2 shows the dose-normalized plasma concentrations of TPP-23411 after intravenous and subcutaneous administration.

[0417] Following single intravenous bolus administration of 1 mg / kg and 10 mg / kg TPP-23411 to male cynomolgus monkeys, exposure, as expressed by AUCnorm, increased slightly more than dose-proportionally, from 391 kg·h / L to 617 kg·h / L, without evidence of target-mediated drug disposition. Plasma elimination was biphasic, with mean plasma clearance of 2.55 mL / (h·kg) at the 1 mg / kg dose and 1.62 mL / (h·kg) at the 10 mg / kg dose. The mean volumes of distribution reached 0.154 L / kg and 0.110 L / kg at the 1 mg / kg and 10 mg / kg doses, respectively. The effective half-lives were short, 41.9 and 46.8 hours, reflecting the relatively rapid elimination of the antibody, while the less pharmacologically relevant terminal elimination half-lives were 108 and 148 hours.

[0418] Following single subcutaneous administration of 3 mg / kg and 10 mg / kg TPP-23411, a dose-proportional increase in exposure was observed, with mean AUCnorm increasing from 331 kg·h / L to 403 kg·h / L and mean Cmax increasing from 2.22 kg / L to 2.95 kg / L. Cmax was reached 8 to 30 hours post-dose (mean). TPP-23411 was eliminated from plasma with terminal half-lives of 81 and 109 hours (mean) at the 3 mg / kg and 10 mg / kg dose levels, respectively, and plasma concentrations paralleled the intravenous profile. Bioavailability was moderate to high at both dose levels, ranging from 54% to 103%. [Table 16]

[0419] Example 13: Toxicology The general objectives of the toxicology program were to identify target organs of toxicity, assess reversibility, determine late-onset findings and exposure-response relationships.

[0420] TPP-23411 is a human monoclonal antibody with no cross-reactivity in animal species other than non-human primates. Therefore, the in vivo toxicology program included comprehensive evaluation in cynomolgus monkeys only. The amino acid sequence of CCR8 is 94% identical between humans and cynomolgus monkeys, and TPP-23411 binds to CCR8 in humans and cynomolgus monkeys with similar affinity. Furthermore, monkeys are considered to be the species most similar to humans in terms of immune function.

[0421] The toxicology program in monkeys consisted of a high-dose toxicokinetic study, a 4-week pilot study with repeated doses, and a central 4-week repeated-dose toxicity study under immune-stimulating conditions (KLH immunization) with a washout / recovery phase. The in vivo toxicity study included several routine immunological parameters, such as immunophenotyping, cytokine levels, and inflammatory markers, and mRNA sequencing assessment of CCR8 mRNA expression in selected tissues.

[0422] Additionally, comprehensive tissue cross-reactivity studies and in vitro cytokine release assays in human whole blood and human PBMCs were performed.

[0423] Pivotal studies were performed in accordance with OECD GLP principles at the Charles River Laboratories (Evreux, France) testing facilities and at Labcorp Early Development Services GmbH (formerly Covance Preclinical Services GmbH) (Munster, Germany) testing facilities. A detailed summary of the toxicology studies performed with TPP-23411 is provided below in Table 13.1. [Table 17]

[0424] Local tolerability was evaluated in a repeat-dose toxicity study in monkeys. No signs of local intolerance reactions were observed by histopathological evaluation of the injection site after administration of the TPP-23411 formulation.

[0425] Example 13.1: Repeated-Dose Toxicity Study in Monkeys: Toxicokinetic Study in Cynomolgus Monkeys with Subcutaneous Administration Once Weekly and Intravenous Administration Twice Weekly A preliminary high-dose toxicokinetic study was conducted to evaluate the toxicokinetic profile of TPP-23411 in plasma after one subcutaneous dose and two intravenous doses in cynomolgus monkeys. The animals received a single subcutaneous dose of 50 mg / kg or twice-weekly doses of 17 mg / kg or 50 mg / kg of TPP-23411 on days 1 and 4.

[0426] TPP-23411 was well tolerated systemically and locally up to the highest dose. Decreased food intake was observed in all animals in all dose groups during the morning feeding. The second feeding (evening) was normal. The decrease in food intake, accompanied by slight weight loss (1%-5%), was not due to the test substance but was attributed to treatment with repeated blood sampling. Necropsy and histopathology were not performed in this study. The mean plasma concentrations and pharmacokinetic parameters of TPP-23411 are summarized below in Tables 13.1.1 and 13.1.2. [Table 18]

[0427] Example 13.2: Repeat-Dose Toxicity Study in Monkeys: A 4-Week Repeat-Dose Toxicity Study in Cynomolgus Monkeys by Intravenous Administration Once Weekly [Table 19]

[0428] Four groups of two female cynomolgus monkeys were administered TPP-23411 intravenously at 0, 6, 17, or 50 mg / kg / injection in a fixed dose volume of 2 mL / kg / dose on days 1, 8, 15, and 22. Toxicity assessments were based on survival, clinical signs, body weight, food intake, electrocardiography, blood pressure recording, ophthalmology, temperature recording, functional observational board (FOB), hematology, coagulation and blood chemistry analysis, urinalysis, blood immunophenotyping (IPT), C-reactive protein (CRP) and cytokine / chemokine levels, organ weights, and macroscopic and microscopic examinations. Blood samples were collected for toxicokinetic and immunogenicity assessments. Blood and selected tissue samples were also collected for deep RNA sequencing.

[0429] Clinical observations No unscheduled deaths occurred during the study. No test article-related clinical signs were observed. Body weight, food intake, and quantitative and qualitative parameters of electrocardiogram were not affected at any dose level during the study. No ophthalmological findings were observed at the end of the treatment period.

[0430] clinical pathology There were no test article-related changes in any hematology, coagulation, blood chemistry, and urine parameters or CRP levels throughout the study. There was no effect of the test article on the levels of any of the cytokines measured during the study (IFNγ, TNFα, IL-1β, IL-2, IL-4, IL-10, IL-6, and IL-8).

[0431] immunotoxicity Immunophenotypic (IPT) analysis of T lymphocytes, cytotoxic T lymphocytes (conventional, activated, and regulatory), helper T lymphocytes (conventional, activated, and regulatory), CD4- / CD8- T lymphocytes (conventional, activated, and regulatory), natural killer T lymphocytes, B lymphocytes, and natural killer cells showed no evidence of immunotoxicity. Except for a slight trend (not statistically significant) for a decrease in natural killer cells after administration of TPP-23411, the cell populations examined were within normal ranges.

[0432] Cross-sectional pathology, histopathology TPP-23411 at doses of 6 mg / kg / injection or higher induced a non-toxic, minimal to slight decrease in lymphoid cellularity in the iliac and axillary lymph nodes, particularly in germinal centers, i.e., secondary lymphoid follicles, in treated females; no dose-related effect was observed. In the thymus, one of two females in each of the 6 mg / kg / injection or higher groups showed a minimal decrease in lymphoid cellularity, which correlated with a decrease in thymus weight. It remains unclear whether this finding is related to TPP-23411 or due to physiological thymic involution. A minimal increase in tingible body macrophages was also observed in one of two females in the 17 mg / kg / injection group and in both females in the 50 mg / kg / injection group. No TPP-23411-related changes were observed at the injection site, indicating that the test article was well tolerated locally.

[0433] Deep RNA sequencing analysis To investigate whether anti-CCR8 treatment significantly eliminated Tregs or altered the expression of immune cell type-specific markers, we performed RNA sequencing on various tissue samples from cynomolgus monkeys. Samples were collected from the following tissues: intestine (ileum, jejunum, cecum, and colon), lymph nodes (mandibular, mesenteric, axillary, and iliac), liver, spleen, lung, skin, thymus, and tonsils. As expected, CCR8 mRNA was preferentially detected in thymus tissue and various lymph node samples. No associated reduction in CCR8 mRNA levels was detected in any of the tissues, except for two thymus samples from the highest-dose treatment group. After correction for multiple testing, none of the changes were significant or consistently dose-dependent.

[0434] Assessment of the expression of the pro-inflammatory markers CXCL9 / 10 and IFNγ, the cytotoxic T cell marker CD8A / B, the NK cell marker NCR1 / SH2D1B, the B cell markers CD19 / CD20 / CD22, the M2 macrophage marker CSF1R / MRC1 / CD163 and the FCg receptors FCGR2A / 2B showed no significant changes in any of these markers in any of the tissues evaluated. [Table 20]

[0435] Toxicokinetic evaluation The results of the measurements are summarized in Table 13.2.2. The low and mid dose groups show clear dose linearity at steady state. Based on AUC(0-tlast)norm, the highest dose group showed over-proportional exposure on day 22. Cmax,norm increased in a dose-proportional manner on day 22. Based on Cmax,norm, no associated accumulation was observed in any dose group. Based on AUC(0-tlast)norm, a slight increase was observed in all dose groups. The coefficient of AUC(0-tlast)norm was most significant in the highest dose group (factor 1.5). At the end of the 168-hour post-dose observation period, no associated concentrations remained. [Table 21]

[0436] Example 13.3: A 4-week repeat-dose toxicity study in cynomolgus monkeys with intravenous administration once or twice weekly, KLH immunization, and a 2-week treatment-free period A pivotal GLP repeat-dose study with a 4-week treatment period was conducted in male and female cynomolgus monkeys. TPP-23411 was administered intravenously at doses of 15 mg / kg and 50 mg / kg once weekly, and at doses of 0 mg / kg (vehicle control) and 40 mg / kg twice weekly. Dose levels were selected based on pilot toxicokinetic and repeat-dose toxicity studies supplemented by simulation modeling to compensate for the short half-life of TPP-23411 in cynomolgus monkeys. [Table 22]

[0437] The objectives of this pivotal study were to determine the potential toxicity of TPP-23411, formulated in 5% dextrose for the treatment of cancer, when administered intravenously to cynomolgus monkeys once or twice weekly for 4 weeks, including the highest dose not causing significant toxicity, and to evaluate the potential reversibility of any findings during a 2-week recovery period. Toxicity assessments were based on survival, clinical signs, body weight, food intake, electrocardiogram (ECG), blood pressure recording, ophthalmological examination, temperature recording, FOB, respiratory rate, hematology, coagulation and blood chemistry analysis, urinalysis, organ weights, and gross and microscopic examinations. A broad range of immunotoxicity endpoints, including 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) in blood, and KLH immunization, were also included. Blood samples were collected for toxicokinetic and immunogenicity (anti-drug antibodies [ADA]) assessment, and blood and selected tissue samples (mesenteric lymph nodes, thymus, and tonsils) were collected for deep RNA sequencing. [Table 23]

[0438] Clinical observations Mortality was not affected by TPP-23411 administration. No overt signs of toxicity or effects on food intake or body weight were observed during the study.

[0439] clinical pathology No relevant effects on hematology, coagulation, clinical chemistry, electrocardiograms, or fundus examinations were found.

[0440] Immunotoxicological parameters No adverse effects of the test article were observed on any of the cytokine levels measured during the study. IL-2, IL-4, IL-5, IL-6, and TNFα levels were generally low throughout the study and considered within normal physiological variation. A small increase in IFN-γ at the high dose observed in a few animals cannot be ruled out as potentially related to TPP-23411. Increased levels of IL-1β were observed in one high-dose animal and IL-10 in two high-dose animals, but these were above the individual pretreatment or control groups.

[0441] Evaluation of the humoral immune response to antigen challenge with KLH showed no signs of alteration due to administration of the test article.

[0442] Treatment with TPP-23411 at 15-50 mg / kg once weekly or 40 mg / kg twice weekly did not adversely affect any leukocyte subpopulations measured by flow cytometry during this study. Throughout the treatment phase, decreases in absolute CD16+ NK cell counts were observed at 15 mg / kg and above on days 15 and 29 in females and on days 8 and 15 in males. Mean NK cell counts were reduced by 74% and 90% in males and females, respectively, at the 40 mg / kg twice weekly dose on day 29 compared with the mean of the second pre-treatment group. This effect was not reversible by the end of the 2-week recovery period.

[0443] Cross-sectional pathology, histopathology Moderate increases in spleen weight were observed during necropsy in males at all dose levels and in females at 40 mg / kg twice weekly or 50 mg / kg once weekly, corresponding to an increased incidence of gross enlargement of the organ. In the absence of microscopic correlation, this finding was considered incidental.

[0444] All histopathological changes were consistent with the range of spontaneous pathologies commonly seen in cynomolgus monkeys of this age and origin.

[0445] TPP-23411 was well tolerated at the injection site. Treatment-related changes, such as fibrosis, edema, hemorrhage, or inflammatory cell infiltration, were observed with similar incidence and severity in control and TPP-23411-treated animals.

[0446] Minimal ADA formation was observed in all treatment groups, including the control group. Only a moderate ADA response was observed in one animal from control group 1, indicating the presence of pre-existing antibodies. In all other animals showing ADA formation, 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 the plasma concentration of TPP-23411 in affected animals could be detected. Therefore, the sporadic occurrence of ADA in all study groups was considered incidental without any biological relevance.

[0447] Regarding toxicokinetics, no relevant gender differences in systemic exposure to TPP-23411 were observed. On day 1, C max However, on the 22nd day, the C level was reached after about an 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, the AUC 0-168 and C maxBoth AUC and TC increased approximately dose-proportionally. 0-144 was calculated for the 15.0 mg / kg group and the 50.0 mg / kg group, and the AUC 0-72 was estimated for Group 4 by combining day 4 or day 22 with day 25.

[0448] On day 22, the AUC of the 40 mg / kg dose group 0-144 was significantly increased compared to the 15.0 mg / kg dose group and moderately increased compared to the 50 mg / kg dose group (due to the difference between twice-weekly and once-weekly administration).

[0449] On the 22nd and 25th days, C max increased almost dose-proportionally in the 40.0 mg / kg dose group compared with the 15.0 mg / kg and 50.0 mg / kg dose groups on day 22.

[0450] Repeated dosing was associated with a significant increase in AUC 0-168 and C max However, when TPP-23411 was administered twice weekly, with slight accumulation at each administration, total AUC levels were slightly elevated (compared to Day 1 / Day 4 levels). max Levels were slightly elevated compared to day 1. [Table 24]

[0451] Deep mRNA analysis To assess whether TPP-23411 treatment has a general effect on gene expression in specific tissues of cynomolgus monkeys, such as thymus, tonsils, and mesenteric lymph nodes, RNA sequencing (RNA-sequencing) studies were performed.The gene CCR8 showed elevated baseline expression in the thymus compared with tonsils and mesenteric lymph nodes, but did not show significant changes in gene expression upon treatment with TPP-23411.Other genes of interest only showed small, statistically insignificant changes in expression upon treatment across all three tissues, but showed considerable inter-animal variability in expression values. [Table 25]

[0452] Example 14: Cytokine Release Assay (CRA) To investigate the potential for activation of cytokine secretion (e.g., IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and TNFα analyzed) by TPP-23411 alone or in combination with pembrolizumab, cytokine release assays were performed using human whole blood (with soluble antibody added) and human PBMCs (containing wet-coated antibody).

[0453] Example 14.1: Cytokine Release Assay - Study 1 Heparinized human peripheral blood samples from 10 healthy donors were incubated for 24 hours 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 TPP9809. All antibodies were tested at three different concentrations (50, 10, and 2 μg / mL). TPP-23411 and the isotype control antibody TPP9809 were also tested in combination with pembrolizumab at 2 μg / mL. Cytokine concentrations in the supernatants were measured by multiplex electrochemiluminescence.

[0454] Incubation of whole blood in the presence of soluble TPP-23411 antibody resulted in a clear dose-dependent release of IFN-γ to median concentration levels (across all donors) higher than those observed with the control antibody MabThera and lower than those observed with the control antibody ANC28.1. IL-1β, IL-6, and TNFα were induced to levels similar to or slightly higher than those observed with the control antibody MabThera and significantly lower than those observed with the control antibody ANC28.1. Incubation of PBMCs in the presence of wet-coated TPP-23411 antibody induced a clear release of the 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 the control antibody MabThera and lower than or similar to those observed with the control antibody OKT3.

[0455] The combination of pembrolizumab and TPP-23411 in both assay formats resulted in cytokine patterns, released cytokine concentration levels, and response frequencies comparable to assays with TPP-23411 alone. [Table 26] TIFF2025530159000032.tif144162TIFF2025530159000033.tif218162TIFF2025530159000034.tif150164

[0456] Example 14.2: In vitro cytokine release assay - Study 2 As a follow-up study, cytokine release assays were performed using lower concentrations of TPP-23411 ranging from 10 μg / mL to 0.128 ng / mL under the same assay conditions.

[0457] Material from eight healthy donors was incubated for 24 hours 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.

[0458] TPP-23411, its isotype control, and Poteligeo were tested at eight 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 four or five different concentrations (10 / 2 / 0.4 / 0.08 / 0.016 μg / mL).

[0459] Cytokine concentrations were measured in the supernatants, and flow cytometry measurements of cell numbers (monocytes, T cells, NK cells, granulocytes, and lymphocytes) in the cell fractions of this cytokine release assay setup were performed after 24 hours of incubation from two of the donors (stimulated with only TPP-23411, isotype control antibody, Lemtrada, and Poteligeo).

[0460] 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. Cytokines were induced to lower median concentration levels (across all donors) than those observed with alemtuzumab, ANC28.1, and mogamulizumab (although at 10 μg / mL, IL-8 concentrations were higher than with mogamulizumab, and IL-1β levels were comparable to mogamulizumab). Compared to rituximab, TPP-23411 induced higher IFN-γ, IL-6, TNFα, and IL-8 cytokine levels at the 10 μg / mL dose, but comparable levels at lower antibody doses tested. IL-1β was induced by TPP-23411, but not by rituximab. IFN-γ was established as the lead cytokine due to its strong dose response and highest sensitivity, with little effect observed at concentrations starting from 0.4 μg / mL.

[0461] Analysis of cell pellets by flow cytometry from two donors in this cytokine release assay setting (using only the test article TPP-23411, isotype control antibody, alemtuzumab, and mogamulizumab) showed a dose-dependent reduction in monocytes, T cells, NK cells, and lymphocytes with Lemtrada, as expected. None of the other test articles resulted in a reduction in any of these cell types. Only one of the two donors tested showed less than 50% monocyte reduction when treated with TPP-23411, at the highest concentration of 10 μg / mL.

[0462] Incubation of PBMCs in the presence of wet-coated TPP-23411 antibody induced a clear dose-dependent release of IFN-γ, IL-1β, IL-6, IL-8, IL-10, and TNFα, with very little IL-2 and no IL-4. The comparator antibodies mogamulizumab and alemtuzumab showed cytokine induction but did not demonstrate a clear dose-response relationship. Compared with OKT3, a more dose-dependent inducer, TPP-23411 produced much lower median cytokine levels (across all donors) of IFN-γ, IL-1β, IL-6, IL-10, TNFα, and IL-2. For IL-6, the median cytokine levels obtained with 10 μg / mL antibody were comparable between OKT3 and TPP-23411. At all lower doses tested (2 μg / mL to 0.128 ng / mL), IL-6 levels were lower than those induced by OKT3.

[0463] Intracellular staining and subsequent flow cytometry analysis after 5 h of incubation of whole blood in the presence of TPP-23411 or its isotype control antibody showed IFN-γ production (and degranulation detected by the CD107a marker) by CD3− CD16+ CD56+ NK cells (but not CD3+ T cells) at this early stage.

[0464] Precautions will be taken during patient administration to reduce the potential risk of infusion-associated reactions due to cytokine release (including cytokine release syndrome) and to reduce the risk of immune-related adverse events such as skin toxicity (i.e., rash) observed with other Treg-depleting agents.

[0465] Fucosylated anti-CCR8 antibody variants or "silencing" Fc variants (with reduced binding to FcgR) were tested in two donors, and the variants induced no or much lower levels of IFN-γ, IL-1β, IL-6, TNFα, and IL-8 in a whole blood / soluble antibody cytokine release assay format. Data in the PBMC / wet coating format were less clear and showed high donor dependence. Data for the commercially available anti-CCR8 antibodies 433H or L263G8 could not be interpreted due to high background from the respective isotype control antibodies. [Table 27] TIFF2025530159000036.tif149163TIFF2025530159000037.tif91164TIFF202 5530159000038.tif245159TIFF2025530159000039.tif42162TIFF20255301590 00040.tif242162TIFF2025530159000041.tif135165TIFF2025530159000042. tif84163TIFF2025530159000043.tif245160TIFF2025530159000044.tif41160

[0466] conclusion Based on the results of the CRA, precautions were recommended in clinical trials to reduce the potential risk of infusion reactions (see also Example 23).

[0467] Example 15: Estimation of human pharmacokinetic parameters The evaluation of human PK parameters was based on data obtained from in vivo experiments following 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 two-compartment model using Phoenix 8.0. The fitted single parameters for total plasma clearance (CL), distribution clearance (CLD), central compartment volume (Vc), and tissue compartment volume (Vt) were allometrically scaled to humans. The scaling was performed by assuming an average body weight of 70 kg and using a fixed-exponential approach, using an exponent of 1 for volume scaling and an exponent of 0.8 for clearance scaling. The resulting human PK parameters are summarized in Table 15.1. The parameters were used to model the c / t profile in humans following a single intravenous infusion of 1 mg / kg over 1 hour (see Figure 3).

[0468] In humans, the resulting CL of TPP-23411 was moderately high at 1.1 mL / h / kg, and the resulting volume of distribution at steady state (Vss) was low at 133 mL / kg, corresponding to a fairly short effective half-life (t 1 / 2 , effective), while the resulting terminal half-life (t 1 / 2 , terminal) is long at 284 hours (see Table 15.1). [Table 28]

[0469] Using the obtained human PK parameters, we further obtained the corresponding concentration-time profiles after single and multiple intravenous infusions of 1 mg / kg TPP-23411 over 1 hour for QW, Q2W, and Q3W dosing. The results are summarized in Figure 4. After three to four consecutive infusions, steady state was well approached for all evaluated dosing regimens (see Figure 4), with less accumulation observed with longer dosing intervals. For QW dosing, for example, a slight increase in AUC (120% accumulation rate) and a two-fold increase in trough concentration (224% accumulation rate) are predicted (see Table 15.2). For Q3W dosing, both accumulation rates are reduced, with no significant accumulation predicted for AUC (107% accumulation rate) and only slight accumulation predicted for Ctrough (131% accumulation rate).

[0470] Table 15.2 lists the estimated relevant human PK parameters. [Table 29]

[0471] Example 16: Dose and exposure estimation in humans: Pharmacologically active dose and effective dose in humans Taking into account human PK predictions based on interpretation of monkey data, as described elsewhere herein, human effective dose ranges were estimated for various dosing schedules, as shown in Table 16.1. The predicted EC for Treg depletion in vivo for TPP-23411 in humans is shown in Table 10.1. 80 concentration exceeds steady state C trough The predicted dose was estimated to maintain

[0472] Considering human PK predictions based on monkey data, human doses are suggested to range from 38 μg / kg (2.7 mg / 70 kg) to 1100 μg / kg (75 mg / 70 kg) for once-weekly (QW) administration and 230 (16 mg / 70 kg) to 6400 μg / kg (450 mg / 70 kg) for an every-3-weekly schedule (Q3W). [Table 30]

[0473] The minimum effective dose was estimated using the predicted EC20 values ​​for TPP-23411 in vivo Treg reduction in humans. 20 Concentration (1 / 4×EC 50 ) were considered (see Table 10.1). We also considered a receptor occupancy (RO)-based approach. EC 20 The ROI in mice (3.8%) was estimated and the corresponding EC in humans was determined using in vitro measured binding affinity of TPP-23411 in mouse and human cells. 20 This is in line with the estimated EC2R based on RO of 0.019 μg / mL in vivo in humans. 20 Concentrations were given.

[0474] C at steady state trough Estimated EC 20 A range of concentrations was considered for estimating the minimum effective dose and is shown in Table 16.2 for the QW schedule. [Table 31]

[0475] Example 17: A First-in-Human Dose Escalation and Expansion Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of the Anti-CCR8 Antibody TPP-23411, as Monotherapy and in Combination With Pembrolizumab, in Participants with Selected Advanced Solid Tumors This study is an open-label, multicenter, randomized, Phase 1, first-in-human (FiH) study to determine the maximum tolerated dose (MTD) / maximum administered dose (MAD), recommended expanded dose (RDE), and recommended phase 2 dose (RP2D) of TPP-23411 in combination with the PD-(L)1-targeting monoclonal antibody (mAb) pembrolizumab.

[0476] The maximum tolerated dose (MTD) is defined as the highest dose at which 30% or less of participants are expected to experience dose-limiting toxicity (DLT) during the DLT observation period. The DLT observation period is 21 days (Cycle 1) after the first dose of TPP-23411. If the MTD is not reached, the maximum dose will be the MAD. The maximum dose (MAD) is the highest dose administered.

[0477] Research purpose This study will evaluate TPP-23411 as a novel immunotherapeutic agent in the setting of advanced solid tumors 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.

[0478] The main objectives of this study are, among others: a. Determine the safety and tolerability of TPP-23411 in participants with advanced solid tumors when administered as monotherapy and in combination with pembrolizumab; b. Determination of the MTD / MAD or RDE of TPP-23411 when administered as monotherapy and in combination with pembrolizumab, and c. Single- and multiple-dose PK characterization of TPP-23411 when administered as monotherapy and in combination with pembrolizumab is.

[0479] The secondary objectives of this study are, among others: d. Preliminary estimates of the antitumor activity of TPP-23411 when administered as monotherapy and in combination with pembrolizumab; e. Evaluation of target engagement and pharmacodynamic effects of TPP-23411 by routine blood and tumor biomarkers using a backfill cohort; and f. Determination of the RP2D of TPP-23411 in combination with pembrolizumab is.

[0480] Research Plan This study includes two parts: a dose escalation and an expansion part. Dose escalation will be performed for TPP-23411 both as monotherapy and in combination with pembrolizumab. To characterize the safety and preliminary antitumor activity of TPP-23411 as a single agent, a monotherapy dose escalation will be performed and a dedicated TPP-23411 monotherapy-MoA expansion will be recruited. Additionally, to potentially provide participants with a greater therapeutic benefit, TPP-23411 will be administered in combination with a fixed dose and schedule of pembrolizumab (e.g., 200 mg Q3W) in the dose escalation and disease-specific combination expansion. For an overview of the dose escalation part of the study scheme, please refer to Figure 1 or Table 17.1.

[0481] The recommended starting dose was selected using predicted minimum effective doses based on the median EC20 of pharmacological ADCC / ADCP and receptor occupancy, supplemented by results from in vitro cytokine release assays in human whole blood and human PBMCs. The minimum effective dose, based on consideration of the median EC20 of TPP-23411, is approximately 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 was selected to balance safety considerations with minimizing patient exposure to subtherapeutic doses.

[0482] The dose-escalation portion of the study will begin in TPP-23411 monotherapy escalation arm 1A. For TPP-23411 QW administration, combination with pembrolizumab (arm 1B) will be initiated at dose levels of 100 mg or 125 mg QW after the 250 mg dose level has been shown to be safe in arm 1A, or at one dose level below the MTD after the MTD is established as less than 250 mg in arm 1A. For TPP-23411 Q3W administration, combination with pembrolizumab (arm 1B) will be initiated at dose level of 500 mg Q3W after the 1000 mg dose level has been shown to be safe in arm 1A.

[0483] The study will enroll participants with advanced solid tumors, preferably ICI-sensitive tumor types, in the dose-escalation portion and with ICI-relapsed / refractory tumor indications, including NSCLC, HNSCC, TNBC, and melanoma, in the expansion portion. During the FiH study, TPP-23411 will be administered as a 1-hour intravenous (IV) infusion on a QW or Q3W schedule during a 21-day treatment cycle.

[0484] The dose escalation part will include two arms: in both arms, dose escalation will begin with a QW dosing schedule and then switch to a Q3W dosing schedule.

[0485] Arm 1A: Dose escalation of TPP-23411 as monotherapy to characterize the safety, tolerability, PK and PD profile, and to determine the maximum tolerated dose (MTD) / maximum administered dose (MAD) and recommended dose for expansion (RDE) of TPP-23411.

[0486] Arm 1B: Dose escalation of TPP-23411 in combination with pembrolizumab to characterize the safety, tolerability, PK and PD profile, and to determine the MTD / MAD or RDE of TPP-23411 in combination with pembrolizumab at a fixed, approved dose and schedule (e.g., 200 mg Q3W). [Table 32]

[0487] In Arm 1B, the IV line will be flushed with saline immediately after the completion of the TPP-23411 infusion to prepare the IV line for the pembrolizumab infusion. Sixty minutes later, pembrolizumab can then be infused using the same IV line at the planned dose of 200 mg administered once every three weeks.

[0488] Preferably, on days when both the anti-CCR8 antibody and the PD-(L)1 inhibitor are administered (e.g., day 1 of each cycle for both the QW and Q3W administration schedules), the dose of the PD-(L)1 inhibitor is administered after a 60-minute pause following completion of the anti-CCR8 antibody infusion to allow for monitoring of safety events (infusion-related reactions). After completion of the IV infusion of the anti-CCR8 antibody, the PD-(L)1 inhibitor may be administered via the same IV line. It is strongly recommended to flush the IV line with saline before injecting the PD-(L)1 inhibitor.

[0489] If a dose lower than 250 mg QW is indicated as the MTD for the QW schedule, this dose level will be considered for the Q3W schedule if it is within the predicted effective dose range for the Q3W schedule (17 mg to 450 mg TPP-23411). However, if the QW MTD is below the lowest predicted effective dose for the Q3W schedule (i.e., less than 17 mg TPP-23411), the Q3W schedule may not be considered.

[0490] If 500 mg TPP-23411 Q3W as monotherapy is not tolerated, and 250 mg QW is clearly safe and well tolerated as monotherapy, the next lower dose level (250 mg) may be considered in combination with pembrolizumab on a Q3W dosing schedule.

[0491] Separate dose escalation for TPP-23411 monotherapy and TPP-23411 in combination with pembrolizumab will help understand and recognize differences between the safety, tolerability, and pharmacodynamic effects of TPP-23411 monotherapy and the effects of the combination with pembrolizumab, and will help identify the MTD / MAD and RDE (dose levels selected after review of all available pharmacokinetic, pharmacodynamic, and safety data) of the separate monotherapy and combination therapies.

[0492] After the 250 mg dose level (or alternative MTD) of TPP-23411 monotherapy escalation (Arm 1A) on a QW dosing schedule has been shown to be safe in at least three evaluable participants, dose escalation of TPP-23411 in combination with pembrolizumab (fixed dose of 200 mg Q3W) will begin at the next lower TPP-23411 dose level (i.e., 100 mg or 125 mg TPP-23411 QW). If the MTD of TPP-23411 monotherapy (as determined in Arm 1A) is lower than 250 mg QW, TPP-23411 dose escalation in Arm 1B will begin at one dose level below the MTD.

[0493] Similarly, once the 1000 mg dose of TPP-23411 monotherapy (Arm 1A) on a Q3W dosing schedule has been shown to be safe in at least three evaluable participants, dose escalation of TPP-23411 Q3W in combination with pembrolizumab (200 mg Q3W) will begin at the next lower TPP-23411 dose level (i.e., 500 mg TPP-23411 Q3W).

[0494] The extension part includes two arms.

[0495] Arm 2A: TPP-23411 monotherapy mechanism of action (Monotherapy-MoA) expansion in NSCLC participants with first-line (ICI-refractory) or second-line (ICI-relapsed) resistance to prior ICI therapy (PD-L1 Tumor Proportion Score [TPS] ≥ 50%) to obtain a preliminary estimate of TPP-23411 antitumor activity and to evaluate TPP-23411 target engagement and pharmacodynamic effects by predefined blood and tumor biomarkers.

[0496] Arm 2B: Disease-specific combination expansion in separate cohorts in four ICI-recurrent tumor types (NSCLC, TNBC, HNSCC, and melanoma) to obtain preliminary efficacy assessments, as well as to obtain further safety and pharmacokinetic / pharmacodynamic (PK / PD) data and to confirm the RP2D of TPP-23411 in combination with pembrolizumab at a fixed, approved dose and schedule (e.g., 200 mg Q3W).

[0497] Based on the results obtained during the dose escalation part, one of these two fixed-dose schedules of TPP-23411 (QW or Q3W) will be selected for the TPP-23411 monotherapy-MoA expansion and disease-specific combination expansion (in combination with pembrolizumab 200 mg Q3W).

[0498] Treatment duration The treatment period or start of the first treatment cycle will be defined by the first administration of study treatment (i.e., intravenous [IV] infusion of TPP-23411 as monotherapy or in combination with a fixed, approved IV dose of pembrolizumab [e.g., 200 mg Q3W]).

[0499] The cycle length is 21 days, with study treatment administration on days 1, 8, and 15 in the dose escalation part (dose level on a QW schedule) or on day 1 (Q3W schedule).

[0500] Participants will receive study treatment until disease progression or unacceptable toxicity occurs, or another specified discontinuation criterion is met.

[0501] The active follow-up (FU) period begins after completion of the end-of-treatment (EOT) visit and includes a safety FU visit / contact and, if applicable, an efficacy FU visit. The safety FU visit / contact occurs 90 days (± 7 days) after the last dose of study treatment. Efficacy FU visits occur every 12 weeks (± 14 days) after the last dose for participants who permanently discontinue study treatment for reasons other than disease progression and who do not initiate a new anticancer therapy (ACT). After completion of the active FU period, participants enter the long-term FU period, during which all participants are contacted every 6 months (± 14 days) to determine vital status and subsequent systemic ACT for up to 24 months after the last participant's EOT or until the end of the study (whichever occurs first).

[0502] Monitors and Biomarkers The TPP-23411 monotherapy-MoA extension will include evaluation of paired biopsies for TME analysis. Furthermore, to characterize the baseline CD8 immune cell status of tumor lesions and changes induced by TPP-23411 treatment, 89-Zr-anti-CD8 minibody PET / CT scans using 89Zr-Df-clefmirlimab will be used as a biomarker for quantitative imaging of CD8 T cells in the TPP-23411 monotherapy-MoA extension (Arm 2A). The Zr89 anti-CD8 PET / CT scans will subject participants to moderate additional radiation exposure. Baseline quantification and distribution of tracer uptake in tumor lesions will be performed to classify lesions as radioactive (hot), immune-excluded, or non-radioactive (cold) by central review. Changes in tracer uptake / distribution between baseline and the end of cycle 2 will be examined by central review, and these changes will be compared for individual participants in terms of BOR and DOR based on RECIST 1.1 criteria.

[0503] To closely monitor study participants for the potential occurrence of CRS, blood samples will be taken for assessment of inflammatory cytokines (e.g., IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL-13, and TNF-α) at ​​least pre-dose, 4 hours, and 24 hours after the start of each TPP-23411 infusion in Cycle 1 at the first three dose levels.

[0504] T cells, B cells, NK cells and T cell subpopulations are quantified and the activation status of T cell populations is determined by flow cytometry in peripheral blood.

[0505] Fold change in peripheral IFN-γ will be measured by immune-based assay during treatment compared to baseline serum samples as reference. Fold change in intratumoral CD8+ T cell / Treg ratio will be measured by IHC during treatment compared to baseline biopsy.

[0506] Biomarker samples will be collected from participants, including: Archived tumor tissue and / or fresh pre-treatment biopsies, Paired tumor tissue biopsies under study, Blood samples before and during study treatment for: cytokines and chemokines, flow cytometry analysis, RNA and ctDNA preparation.

[0507] Pharmacodynamic biomarkers will be assessed in samples collected before and during study treatment to determine the effect of TPP-23411 on these biomarkers.

[0508] Baseline candidate biomarkers will be assessed for correlation with response to study treatment (to further validate the predictive biomarkers). Samples from tumor biopsies and blood will be analyzed retrospectively. Candidate predictive biomarkers include (but are not limited to): Tumor immune infiltrate (e.g., immune gene expression profile; presence, phenotype, and activation state of tumor-infiltrating leukocytes with special emphasis on CCR8-positive Treg cells, CD8 T cells, NK cells, and macrophages), PD-L1 expression level as a surrogate for immune susceptibility Tumor mutation status (microsatellite instability [MSI], tumor mutations, tumor mutation burden [TMB]), Imaging-based biomarkers for tumor-infiltrating CD8 T cells.

[0509] PD-L1 expression for patient selection Because CD8 T cells are essential for IO-associated antitumor efficacy, the functional relevance of Treg depletion is likely to be highest in tumors with already high T cell infiltration. Furthermore, macrophages and NK cells are essential for anti-CCR8-mediated Treg depletion via ADCC or ADCP. Based on mRNA expression, PD-L1 positivity correlates with the presence of immune cells, including Tregs, macrophages, and CD8 Teffs, in the TME across all indications from The Cancer Genome Atlas (TCGA) project. The relevance of PD-L1 expression is further supported by the observation that PD-L1 mRNA expression is a significant positive predictive biomarker for anti-CCR8 efficacy across mouse tumor models. Therefore, patients with PD-L1-positive tumors from immune-sensitive indications are expected to respond best to TPP-23411 as monotherapy.

[0510] As a result, the monotherapy-MoA expansion arm (arm 2A) enrolling patients with NSCLC will use PD-L1 expression level as an inclusion criterion: patients must therefore have a historical PD-L1 score of TPS ≥ 50% (previously determined by a locally approved assay).

[0511] Alternatively, the arm 2A may be NSCLC participants (PD-L1 TPS ≥ 50%) or TNBC (PD-L1 CPS ≥ 10%) or HNSCC (PD-L1 CPS ≥ 1%) or HNSCC (PD-L1 CPS ≥ 20%) or Melanoma (no PD-L1 cutoff) This includes the MoA expansion of anti-CCR8 antibody monotherapy in

[0512] PET / CT imaging of CD8 cells In the monotherapy-MoA expansion (arm 2A), zirconium-89 (89Zr) anti-CD8 minibody PET / CT was performed, utilizing 89Zr-Df-clefmirlimab, an 89Zr-labeled minibody that targets CD8 cells. CD8 tracer uptake has been shown to correlate with CD8 expression by IHC and positively correlate with tumor response assessment by RECIST 1.1.

[0513] The exploratory results of this study are as follows:

[0514] At baseline / screening, tumor uptake and biodistribution of 89Zr CD8 tracer using whole-body PET / CT allows classification of whole-body tumor lesions as radioactive, immunoexcluded, or non-radioactive with respect to CD8 cell infiltration.

[0515] Evaluate changes in CD8 cell quantity and distribution pattern in tumor lesions / TME induced by TPP-23411 using 89Zr anti-CD8 minibody PET / CT early in treatment (end of cycle 2). Correlate CD8 patterns with best overall response (BOR) and progression-free time data obtained from tumor response imaging for each participant assessed using RECIST 1.1 for each participant in monotherapy-MoA expansion (arm 2A).

[0516] At each time point, a whole-body PET / CT scan (from skull vertex to mid-thigh) will be performed approximately 24 hours ± 3 hours after infusion of 89Zr-Df-clefmirlimab.

[0517] Radiomics analysis Radiomics is a field of imaging biomarker research defined as the use of automated or semi-automated analytical methods to extract quantitative measures (often referred to as radiomics features) from medical images (Mayerhoefer et al., 2020). These measures capture tissue and lesion characteristics (e.g., 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 objective and will 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 1.0 mm reconstruction slice thickness. Elimination of CCR8-expressing Tregs by TPP-23411 may lead to an increase in tumor-infiltrating CD8 lymphocytes. Changes in CD8 radiomics measures from baseline may be used as an exploratory endpoint to evaluate the pharmacodynamic effects of TPP-23411. CD8 radiomics measures will also be correlated with paired tumor biopsy results (e.g., CD8 IHC) when possible. Similarly, radiomics measures derived from research images (e.g., tumor assessments) will be compared with other research biomarkers and endpoints.

[0518] Example 18: Biomarkers: Association between response to murine surrogate antibodies TPP-14099 and TPP-15285 and gene expression in syngeneic mouse cancer models The relationship between gene expression and the efficacy of treatment with a CCR8-depleting antibody was investigated by whole-transcriptome sequencing of early-stage, untreated tumors from 21 syngeneic mouse cancer models. The relationship between anti-CCR8 treatment and treatment that inhibits the immune checkpoints PD-1 and PD-L1 was also evaluated.

[0519] The anti-mouse CCR8 antibodies TPP-14099 (hIgG1) and TPP-15285 (mIgG2a) studied were effective in immune checkpoint-sensitive models. Anti-mouse CCR8 antibodies almost always demonstrated superior efficacy compared with anti-PD-1 or anti-PD-L1 antibody treatment. The efficacy of anti-mouse CCR8 antibodies was independent of mouse strain and did not correlate with the mutational burden of each cancer cell line.

[0520] Figure 5 shows that response to anti-mouse CCR8 antibody treatment correlates with response to anti-PD-L1 antibody and anti-PD1 antibody treatment.

[0521] Figure 6 shows that baseline mRNA expression of PD-L1 and inflammatory markers such as IFN-γ correlate with response to anti-mouse CCR8 antibody treatment in early-stage, untreated tumors.

[0522] Example 19: Measurement of unchanged compound in plasma 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).

[0523] In this protocol, TPP-23411 was measured in plasma after dilution with buffer by the Gyrolab method with fluorescence reading using immobilized biotinylated anti-human IgG-Fc antibody as the capture molecule and Alexa fluorescein-labeled anti-human IgG antibody as the detection reagent. Method-specific parameters are listed in Table 19.1. [Table 33]

[0524] TPP-23411 has proven stable as shown under the above conditions associated with sample handling (see Table 19.2). [Table 34]

[0525] Example 20: Measurement of anti-CCR8 antibodies in plasma Antibody binding to TPP-23411 (anti-drug antibodies) was measured in monkey plasma using a Meso Scale Discovery (MSD)-based bridging assay, which uses biotinylated TPP-23411 as the antigen. Anti-TPP-23411 antibodies present in positive control or study-specific plasma samples bound to biotinylated TPP-23411 and were captured on a streptavidin plate, while other plasma components were washed away. Bound antibodies were then detected using SULFO-tagged TPP-23411 and an ECL readout (see U.S. Patent Nos. 7,855,287 and 7,803,573 for ECL reagents).

[0526] More specifically, positive and negative control samples (monkey serum in the presence and absence of the positive control, respectively) and unknown samples were prediluted (1:8) with dilution buffer, mixed with the dilution buffer, and preincubated in a polypropylene plate on an orbital shaker (room temperature, 600 rpm) 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 (room temperature, 600 rpm). Then, 25 μL of each incubated sample was transferred in duplicate to wells of a blocked MSD Streptavidin Gold plate (150 μL of blocking buffer, at least 30 minutes, 600 rpm) to which biotinylated TPP-23411 could bind. Functional anti-drug antibodies crosslink the biotinylated and SULFO-tagged TPP-23411. The sulfonated TPP-23411 generates an electrochemiluminescence (ECL) signal when voltage is applied, which correlates with the amount of ADA in the well. Plates were read using a Meso QuickPlex SQ 120 and data were analyzed using MSD® Workbench™ software. All samples were measured in duplicate.

[0527] The positive control antibody was an affinity pure goat anti-human IgG antibody, and the sensitivity of this positive control in the assay was 4.79 μg / L (assay cutoff concentration).

[0528] Method validation and analysis of study samples were performed in accordance with in-house SOPs and the relevant guidelines of "Assay Development for Immunogenicity Testing of Therapeutic Protein Products" (FDA, 2019) and "Guideline on Immunogenicity Assessment of Therapeutic Proteins" (EMA, 2017). Bioanalytical methods applied in pivotal nonclinical safety studies were fully validated, and samples from these studies were analyzed according to Good Laboratory Practice (GLP). [Table 35]

[0529] The stability of a positive control antibody mimicking the study sample in plasma was examined under various storage conditions, including various steps in sample processing during the assay and storage intervals appropriate for actual study samples. TPP-23411 (the positive control antibody) was stable under all conditions relevant to sample handling. [Table 36]

[0530] Example 21: Tissue cross-reactivity A preliminary tissue cross-reactivity (TCR) study was performed to validate the immunohistochemistry (ICH) method for detecting TPP-23411-FITC bound to CCR8. This was a non-GLP study, but was conducted in accordance with current scientific and regulatory standards.

[0531] The purpose of this preliminary TCR study was to evaluate the potential cross-reactivity of a fluorescein isothiocyanate (FITC)-conjugated form of TPP-23411 using immunohistochemistry (IHC) techniques on a panel of frozen tissue and blood smear specimens from three human and three cynomolgus donors (per tissue). 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.

[0532] TPP-23411-FITC at 0.1-9 μg / mL resulted in positive membrane / cytoplasmic staining of monkey CCR8-positive cells but did not induce any staining in CCR8-negative cells. No staining was observed in any of the positive or negative cells incubated in the presence of IgG1-FITC at any concentration.

[0533] The FDA's full tissue list was examined in human and cynomolgus species with the following results at 3 μg / mL and 9 μg / mL:

[0534] human tissue TPP-23411-FITC yielded the following: Membranous, variably cytoplasmic positive staining of mononuclear cells within some germinal centers of the ileal GALT at 3 μg / mL and 9 μg / mL (suggestive of dendritic cells and / or macrophages; other cell types cannot be excluded). Membranous, variably cytoplasmic staining in subepithelial cells of the small intestinal villi. These cells were thought to represent myoepithelial cells or pericytes (other cell types cannot be excluded). Staining in the peritubular interstitial tissue of the kidney (i.e., lamina propria of tubules / vessels and / or fibroblasts). Minimal to moderate staining in histological glandular compartments with secretory function, such as the mammary gland (breast), prostate, parotid gland, gallbladder, and stomach. TPP-23411-FITC also caused minimal staining in umbrella cells in the ureter.

[0535] Cynomolgus monkeys TPP-23411-FITC caused staining in: Pancreatic interstitial spindle cells (suggestive of endothelial cells), Scattered large cells in the parathyroid glands Umbrella cells (lumen side) of the urothelium (bladder), ·Adrenal medulla (plasma protein).

[0536] Example 22: Tissue cross-reactivity (GLP study) The optimized IHC assay was validated in a preliminary non-GLP study using Ventana's DAB Map kit as the detection system and automated technology. Two concentrations of the test article TPP-23411-FITC were examined in the GLP study: 3 μg / mL and 10 μg / mL. The tissues examined are listed in Table 22.1. [Table 37]

[0537] TPP-23411-FITC at 3 μg / mL and 10 μg / mL caused membranous and variably cytoplasmic staining of human and cynomolgus monkey CCR8-positive cells, but no staining in negative cells. The negative control antibody TPP-9809-FITC at 10 μg / mL caused no staining at all in human or cynomolgus monkey CCR8-positive or CCR8-negative cells.

[0538] Tissue integrity was shown to be adequate: all tissues were deemed acceptable for microscopic evaluation in terms of morphology and tissue element inclusion.

[0539] In human tissues, TPP-23411-FITC tested at 3 μg / mL and / or 10 μg / mL resulted in the following: Membranous staining in platelets (rare to frequent, 3 / 3 donors), Membranous / cytoplasmic staining of interstitial cells in the liver (2 / 3 donors) (suggestive of Kupffer cells), Staining of mixed inflammatory cells (macrophages and granulocytes / neutrophils) in the fallopian tube lumen (2 / 3 donors), Occasional glial cell staining in the spinal cord (1 / 3 donors), Membranous, variably cytoplasmic staining of mononuclear cells, suggestive of macrophages, in the ureteral serosa and adjacent tissue (1 / 3 donors). Interstitial spindle cell staining in the small intestine (villi, 2 / 3 donors) (suggesting smooth cells and / or fibroblasts), Diffuse staining in fibrous and / or fibrovascular tissue (mainly extracellular matrix) in several organs: adrenal glands (1 / 3 donors), heart (1 / 3 donors), kidneys (3 / 3 donors), ovaries (3 / 3 donors), stomach (1 / 3 donors) and tonsils (1 / 3 donors).

[0540] In cynomolgus monkeys, TPP-23411-FITC tested at 3 μg / mL and / or 10 μg / mL resulted in: Diffuse staining in fibrous and / or fibrovascular tissue (mainly extracellular matrix) in several organs [breast (2 / 3 donors), corneal stroma (2 / 3 donors), kidney (3 / 3 donors), ovary (3 / 3 donors), parotid gland (3 / 3 donors) (more prominent 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 gland (3 / 3 donors), bladder (3 / 3 donors), cervix and endometrium (3 / 3 donors)], Occasional scattered cell staining in the parathyroid glands (2 / 3 donors), Staining of cells within the red pulp in the spleen (3 / 3 donors; most cells; mixture of mononuclear cells, granulocytes, and mesenchymal cells).

[0541] Based on immunohistochemistry, TPP-23411-FITC tested at 3 μg / mL and / or 10 μg / mL induced staining in fibrovascular tissue (primarily extracellular matrix) in both human and monkey tissues. Some staining was also observed in inflammatory cells (granulocytes and / or monocytes, suggestive of macrophages) in both species (more prominent in humans and confined to the spleen in monkeys). Staining seen in humans but not in monkeys was associated with platelets and spinal cord (nerve tissue).

[0542] Comprehensive histopathological examination of monkey toxicity studies revealed no morphological changes in tissues with positive staining in the TCR assay. Cytoplasmic staining (e.g., in spleen, spinal cord) was deemed to pose little or no safety concern because monoclonal antibody access to the cytoplasmic compartment is thought to be limited in vivo.

[0543] Example 23: Administration of antihistamines, acetaminophen, and corticosteroids for side effect management If an infusion-related reaction occurred during administration of the anti-CCR8 antibody, participants received additional medications, and if necessary, the study treatment infusion time for subsequent doses was extended. Additional medications included, for example, antihistamines, acetaminophen, or corticosteroids.

[0544] After an infusion reaction occurred during administration of anti-CCR8 antibody, 650 mg to 1000 mg of paracetamol was administered before the next infusion. This premedication prevented infusion reactions from occurring during subsequent administrations of anti-CCR8 antibody.

[0545] Based on these data, it is understood that paracetamol at a dose of 500 mg to 1000 mg may be administered prior to the first administration of anti-CCR8 antibody or prior to subsequent administrations of anti-CCR8 antibody to prevent or reduce side effects.

[0546] After an infusion reaction occurred with the first dose of anti-CCR8 antibody, 650 mg of paracetamol and 100 mg of diphenhydramine were administered before subsequent infusions. This premedication prevented the occurrence of infusion reactions with subsequent doses of anti-CCR8 antibody.

[0547] Alternatively, dexamethasone can be administered before a dose of anti-CCR8 antibody. A suitable dose would be, for example, two doses of 8 mg dexamethasone, 3 hours before and 4 hours after administration of anti-CCR8 antibody.

[0548] Based on the severity of the adverse reaction, pembrolizumab may be discontinued and corticosteroids may be administered. Once the adverse reaction improves to Grade 1 or less, a corticosteroid taper may be initiated and continued for at least 1 month.

[0549] Example 24: Demonstration of Treg Depletion and Mechanism of Action of Recommended Dosing Regimen Expression of CD45, CD45RA, CD3, CD4, CD8, CD25 (IL-2RA), CD127 (IL7RA), CD69, CCR7, GZMB, Ki67, PDL1, OX40, and CD137 (4-1BB = activation marker) was analyzed by flow cytometry to monitor, among other things, the percentage of activated proliferating CD8+ T cells and the percentage of Treg cells. Based on the assumed mechanism of action, an increase in the percentage of activated proliferating CD8+ T cells and a decrease in the percentage of remaining Treg cells are prerequisites for successful treatment and can be demonstrated with the proposed dosing regimen.

[0550] Activated proliferating CD8+ T cells were defined by expression of the proliferation marker Ki67, the activation markers 4-1BB (CD137), CD8, and CD3.

[0551] Activated Tregs were defined by expression of CD137 (4-1BB), CD25, CD127low, CD4, and CD3. To allow assessment of Treg levels and depletion, 4-1BB was used instead of CCR8 because binding of the therapeutic antibody may compete with binding of the flow cytometry marker antibody to CCR8.

[0552] Figure 7 shows the increase in activated, proliferating CD8+ T cells relative to the total number of CD3+ T cells, beginning approximately 3 days after the first administration of 10 mg of anti-CCR8 antibody. A continuous increase was observed, especially from the second week of treatment, likely due to the induction of immune activation in the tumor. Treatment ended approximately 3 weeks after the final infusion. The ratio of these two cell types at the time of screening was set to 1.

[0553] The abbreviations listed on the x-axis of Figures 7, 8 and 9 are as follows: Scr: Screening value (during patient screening, i.e., before the first anti-CCR8 antibody administration) C1D1: Cycle 1, Day 1, Time 0 (just before starting anti-CCR8 antibody administration) C1D2: Cycle 1, Day 2, Time 0 (approximately 1 day after the first anti-CCR8 antibody administration) C1D3: Cycle 1, Day 3, Time 0 (approximately 2 days after the first anti-CCR8 antibody administration) C1D8: Cycle 1, Day 8, Time 0 (approximately 7 days after the first anti-CCR8 antibody administration, immediately before the second anti-CCR8 antibody administration) C1D15: Cycle 1, Day 15, Time 0 (approximately 7 days after the second dose of anti-CCR8 antibody, immediately before the third dose of anti-CCR8 antibody) C2D1: Cycle 2, Day 1, Time 0 (approximately 7 days after the third dose of anti-CCR8 antibody, immediately before the fourth dose of anti-CCR8 antibody) C2D3: Cycle 2, Day 3, Time 0 (approximately 1 day after the fourth dose of anti-CCR8 antibody) C2D8: Cycle 2, Day 8, Time 0 (approximately 7 days after the fourth dose of anti-CCR8 antibody, immediately before the fifth dose of anti-CCR8 antibody) C2D15: Cycle 2, Day 15, Time 0 (approximately 7 days after the fifth dose of anti-CCR8 antibody, immediately before the sixth dose of anti-CCR8 antibody) EOT: End of treatment (see protocol; e.g., 30 days after last infusion).

[0554] Figure 8 shows a substantial decrease in the ratio of activated Tregs to total CD3+ T cells beginning approximately one day after the first anti-CCR8 antibody administration (10 mg dose). The ratio of the two cell types at the time of screening was set to 1.

[0555] Figure 9 shows the ratio of activated Tregs to the number of activated proliferating CD8+ T cells. The ratio of the two cell types at the time of screening was set to 1.

[0556] Other doses tested showed similar trends.

[0557] Figure 10 shows the reduction in Treg counts in patient blood samples upon treatment with 1 mg, 3 mg, 10 mg, or 30 mg of anti-CCR8 antibody. For each patient, the ratio (activated Treg / total CD3+ T cells) obtained at time points C1D2–C2D15 was divided by the ratio (activated Treg / total CD3+ T cells) obtained for the same patient during screening (Scr), i.e., before administration of anti-CCR8 antibody. To obtain data in one box, all patients treated with the indicated doses were grouped together. Even the lowest 1 mg cohort shows a median reduction in activated Treg counts to approximately 53% of the screening value.

[0558] Example 25: Biomarkers for detecting immune cell activation in blood Human blood / serum samples were collected from patients as known in the art and as described elsewhere herein. TNF-α and cytokine levels were then measured using commercially available V-plex assays (V-PLEX Proinflammatory Panel 1 Human Kit, MSD; https: / / www.mesoscale.com / en / products / v-plex-proinflammatory-panel-1-human-kit-k15049d / (see references).

[0559] Figure 11 shows a box plot of TNF-alpha levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg, or 30 mg of anti-CCR8 antibody to human patients. Biomarker levels increased with increasing doses of anti-CCR8 antibody.

[0560] Figure 12 shows a box plot of IFN-gamma levels measured in pg / μl in blood or serum samples taken from human patients 4 hours after administration of 1 mg, 3 mg, 10 mg, or 30 mg of anti-CCR8 antibody. Biomarker levels increased with increasing doses of anti-CCR8 antibody.

[0561] FIG. 13 shows a box plot of IP10 levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0562] FIG. 14 shows a box plot of IL8 levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0563] FIG. 15 shows a box plot of IL6 levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients.

[0564] FIG. 16 shows a box plot of IL10 levels measured in pg / μl in blood or serum samples taken 4 hours after administration of 1 mg, 3 mg, 10 mg or 30 mg of anti-CCR8 antibody to human patients. [Table 38] TIFF2025530159000056.tif246162TIFF2025530159000057.tif246162TIFF2025530159000058.tif244163TIFF2025530159000059.tif245162TIFF2025530159000060.tif192163

Claims

1. anti-CCR8 antibody to a patient in need thereof, a. about 1-250 mg once weekly, preferably 3, 10, 30, 50, 100, 125, or 250 mg once weekly, or b. About 16-1500 mg once every three weeks, preferably 16, 450, 500, 750, 1000, or 1500 mg once every three weeks 10. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method comprising intravenously administering to a subject a total amount of:

2. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment comprising intravenously administering the anti-CCR8 antibody to a patient in need thereof in a total amount of 2.7 mg to 75 mg once weekly.

3. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment comprising intravenously administering the anti-CCR8 antibody to a patient in need thereof in a total amount of 16 mg to 450 mg once every three weeks.

4. anti-PD-(L)1 antibody to a patient in need thereof, 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 6 weeks (preferably, wherein the anti-PD-(L)1 antibody is pembrolizumab), or c. about 240 mg once every two weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or d. about 360 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is nivolumab), or e. about 480 mg once every four weeks (preferably, where 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 atezolizumab), or g. about 1200 mg once every three weeks (preferably, wherein the anti-PD-(L)1 antibody is atezolizumab), or h. about 1680 mg once every four weeks (preferably, wherein the anti-PD-(L)1 antibody is atezolizumab), or i. about 360 mg once every three weeks (preferably, wherein the anti-PD-(L)1 antibody is zimberelimab), 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 l. about 1500 mg once every three weeks (preferably, where the anti-PD-(L)1 antibody is durvalumab) 4. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the therapeutic method according to claim 1, further comprising intravenously administering the antibody in a total amount of:

5. 10. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment according to any one of the preceding claims, wherein the anti-human CCR8 antibody is characterized by a half-life in humans of less than 14 days, preferably less than 10 days, most preferably less than 7 days.

6. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the 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 activity and ADCP activity for use in the method of treatment according to any one of the preceding claims, wherein the intravenous administration of the anti-human CCR8 antibody is carried out as an intravenous infusion over a period of 15 to 120 minutes, preferably 30 to 60 minutes.

8. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment according to any one of claims 4 to 7, wherein the anti-PD-(L)1 antibody is administered intravenously as an intravenous infusion over a period of 15 to 60 minutes, preferably 30 minutes.

9. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment according to any one of claims 4 to 8, wherein the intravenous administration of the anti-PD-(L)1 antibody is carried out 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 activity and ADCP activity for use in the method of treatment according to claim 9, wherein the IV line is flushed with saline before intravenous administration of the anti-human PD-(L)1 antibody.

11. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment according to any one of the preceding claims, wherein the medical use comprises at least one 21-day administration cycle, and preferably both the anti-CCR8 antibody and the anti-PD-(L)1 antibody are administered on day 1 of the 21-day administration cycle.

12. 12. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the 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 in the second, third, fourth, fifth or any subsequent administration cycle, the anti-human CCR8 antibody and the anti-PD-(L)1 antibody are administered immediately after each other without a substantial delay.

13. The anti-CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment according to any one of the preceding claims, wherein the anti-CCR8 antibody is a human IgG1 antibody.

14. The anti-CCR8 antibody is a low-internalizing antibody or a non-internalizing antibody. An anti-CCR8 antibody having ADCC activity and ADCP activity for use in a therapeutic method according to any one of the preceding claims.

15. 10. The anti-CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment according to any one of 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 dissociation constant (KD) of TPP-23411 for binding to CHO cells transfected with human CCR8.

16. The anti-CCR8 antibody having ADCC activity and ADCP activity for use in the therapeutic method according to any one of claims 4 to 15, wherein the anti-PD-(L)1 antibody is pembrolizumab, nivolumab, atezolizumab, avelumab, zimberelimab, toripalimab, or durvalumab.

17. The anti-CCR8 antibody a. comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NOs: 2, 3, 4, 6, 7 and 8; and / or b. comprises a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth 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 set forth in SEQ ID NO: 5; and / or c. An anti-CCR8 antibody having ADCC activity and ADCP activity for use in the therapeutic method of any one of claims 4 to 16, comprising a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17 and / or a light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

18.

18. 10. The anti-CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment according to any one of 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 non-melanoma skin cancer.

19. The method of treatment may further comprise administering an effective dose of an antihistamine, acetaminophen, a corticosteroid, or a combination thereof, preferably a. administration of at least 500 mg or at least 650 mg of paracetamol prior to administration of an anti-CCR8 antibody, and / or b. administration of at least 50 mg or at least 100 mg of diphenhydramine prior to administration of an anti-CCR8 antibody, and / or c. Administration of at least 8 mg of dexamethasone prior to administration of an anti-CCR8 antibody 10. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the therapeutic method according to claim 1 , comprising:

20. The treatment method is a. Analyzing a tumor proportion score or a combined positive score as a measure of PD-(L)1 expression in a patient's cancer tissue sample; and b. administering an anti-human CCR8 antibody to the patient if the patient has a tumor proportion score of 50% or greater or a combined positive score of 10% or greater or 1% or greater.

10. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a cancer treatment method according to any one of the preceding claims, the method comprising:

21. a. the cancer is non-small cell lung cancer and analyzing the tumor proportion score as a measure of PD-(L)1 expression in the patient's cancer tissue sample; or b. the cancer is triple-negative breast cancer and a combined positive score is analyzed as a measure of PD-(L)1 expression in the patient's cancer tissue sample; or c. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the treatment method according to claim 20, wherein the cancer is head and neck squamous cell carcinoma and a combined positive score is analyzed as a measure of PD-(L)1 expression in the patient's cancer tissue sample.

22. The treatment method is a. A prior tumor proportion score of 50% or greater; or b. A previous combined positive score of 10% or more or 1% or more 20. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment of cancer according to any one of claims 1 to 19, comprising administering the anti-human CCR8 antibody to a patient when the patient has:

23. a. the cancer is non-small cell lung cancer and the method of treatment comprises administering an anti-human CCR8 antibody to the patient if the patient has a historical tumor proportion score of 50% or greater; or b. the cancer is triple-negative breast cancer and the method of treatment comprises administering an anti-human CCR8 antibody to the patient if the patient has a prior combined positive score of 10% or greater or 1% or greater; or c) An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment according to claim 22, wherein the cancer is head and neck squamous cell carcinoma, and the method of treatment comprises administering the anti-human CCR8 antibody to a patient when the patient has a past combined positive score of 20% or more or 1% or more.

24. 24. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment 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. The treatment method is a. optionally analyzing in the patient's blood, plasma or serum screening sample the level of at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10, pro-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; b. administering to the patient an effective dose of an anti-human CCR8 antibody; c. analyzing the level of at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of said pro-inflammatory cytokines in a blood, plasma or serum sample from the patient, wherein the blood, plasma or serum sample is taken after administering an effective dose of an anti-human CCR8 antibody according to step b); d. Using the cytokine levels obtained according to step c) to identify safety-related events or as surrogate biomarkers for Treg depletion or as biomarkers for treatment success, i. the cytokine levels obtained according to step a) or ii. Reference Value Process to compare with 10. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating a patient, preferably according to any one of the preceding claims, which is a method for treating cancer comprising:

26. Furthermore e. The cytokine levels obtained according to step c) are: i. if the cytokine level is increased compared to the level obtained according to step a), or ii. If increased compared to baseline, administering to the patient at least one additional effective dose of an anti-human CCR8 antibody.

26. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the therapeutic method of claim 25, comprising:

27. 27. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treatment according to claim 25 or 26, wherein the blood, plasma or serum sample for analyzing cytokine levels according to step c) is taken / collected 1 to 24 hours, 24 to 48 hours, 2 to 7 days, 7 to 14 days, 14 to 28 days or more than 28 days after administration of an effective dose of the anti-human CCR8 antibody according to step b).

28. 28. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method of treatment according to any one of claims 25 to 27, wherein a blood, plasma or serum sample for analyzing cytokine levels according to step a) is taken / collected 15 to 60 minutes, preferably about 30 minutes, before administering an effective dose of the anti-human CCR8 antibody according to step b).

29. The treatment method is a. stratifying patients based on prior cancer treatment with an anti-PD-(L)1 antibody for at least 6 months; and b. Administer anti-human CCR8 antibodies only if the patient has received prior treatment with an anti-PD-(L)1 antibody for at least 6 months.

1. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a cancer treatment method, the method comprising:

30. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method for treating cancer according to claim 29, wherein the anti-human CCR8 antibody is an anti-human CCR8 antibody for use in the method for treating cancer according to any one of claims 1 to 28.

31. a. administering a Zr-89 labeled anti-CD8 minibody to a subject; b. performing at least one PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 minibody in the subject and obtain a first image of the subject; c. determining the abundance and / or distribution of Zr-89 labeled anti-CD8 minibody in one or more of the subject's cancer lesions based on the first subject image; and d. administering to the subject an effective dose of an anti-human CCR8 antibody if the first subject image shows the amount and / or distribution of the Zr-89 labeled anti-CD8 minibody in any of the one or more cancer lesions and indicates that the subject is likely to benefit from administration of the anti-human CCR8 antibody.

1. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer, comprising:

32. determining the amount and / or distribution of Zr-89 labeled anti-CD8 minibody in any one or more cancer lesions; i. Assess the abundance and / or distribution of Zr-89 labeled anti-CD8 minibody in healthy tissues of the patient, or ii. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer according to claim 31, which is assessed in comparison with one or more reference values ​​for the abundance and / or distribution of a Zr-89 labeled anti-CD8 minibody.

33. a. administering a first dose of a Zr-89 labeled anti-CD8 minibody to a subject; b. Performing a first PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 minibody in the subject to obtain a first image of the subject. c. determining a first abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancer lesions in the subject based on the first subject image; d. administering an effective dose of an anti-human CCR8 antibody to the subject; e. administering a second dose of Zr-89 labeled anti-CD8 minibody to the subject; f. performing a second PET scan and optionally a CT scan to detect the Zr-89 labeled anti-CD8 minibody in the subject to obtain a second image of the subject; g. determining a second abundance and / or distribution of the Zr-89 labeled anti-CD8 minibody in one or more cancerous lesions in the subject based on the second subject image; h. Comparing the second subject image to the first subject image to assess whether the abundance of the Zr-89 labeled anti-CD8 minibody has increased substantially or the distribution of the Zr-89 labeled anti-CD8 minibody has changed substantially in one or more cancer lesions to monitor disease progression or the success of anti-human CCR8 antibody treatment.

1. An anti-human CCR8 antibody having ADCC activity and / or ADCP activity for use in a method for treating cancer, comprising:

34. An anti-human CCR8 antibody having ADCC activity and ADCP activity for use in a method for treating cancer according to claim 33, comprising the further step of administering at least one additional effective dose of the anti-human CCR8 antibody to the patient when the abundance of the Zr-89-labeled anti-CD8 minibody in one or more cancer lesions is substantially increased or the distribution of the Zr-89-labeled anti-CD8 minibody is substantially altered.

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. 36. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the method of treating cancer according to any one of claims 31 to 35, wherein a PET scan is performed about 6 to 36 hours after administration of each 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 minibody is 89Zr-Df-clefmirlimab.

38. The anti-human CCR8 antibody having ADCC activity and ADCP activity for use in the cancer treatment method according to any one of claims 1 to 30, wherein the anti-human CCR8 antibody is an anti-human CCR8 antibody for use in the cancer treatment method according to any one of claims 31 to 37.

39. A method for determining and quantifying anti-CCR8 antibody formation in cynomolgus monkey or human plasma, including a bridging ELISA method.

40. 40. The method of claim 39, wherein a signal is generated when the anti-anti-CCR8 antibody crosslinks 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. a. a variable heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, and / or b. A variable light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

23.

42. The isolated anti-CCR8 antibody or antigen-binding fragment thereof of claim 41, further comprising:

43. a. a heavy chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35, and / or b. A light chain sequence having at least 98% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:

36.

43. The isolated anti-CCR8 antibody or antigen-binding fragment thereof of claim 41 or 42, further comprising:

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