IL15 / IL15R alpha heterodimer FC fusion protein for cancer treatment

Abstract

We provide cancer treatment methods. [Solution] A method for treating cancer is provided by administering a heterodimer protein comprising a first monomer containing an IL-15 protein-Fc domain fusion and a second monomer containing an IL-15Rα tancinus-Fc domain fusion.

Description

Technical Field

[0001]

[0001] This disclosure relates to the field of cancer treatment using an IL15-IL15R heterodimeric Fc fusion protein.

[0002] Cross-reference of related applications

[0002] This application claims priority benefit of U.S. Provisional Application No. 62 / 966,976, filed on January 28, 2020, the entire content of which is incorporated herein by reference.

[0003] Sequence List

[0003] This application includes a sequence listing submitted electronically in ASCII format, the entire content of which is incorporated herein by reference. The ASCII copy created on January 28, 2021, is named 000218-0006-WO1_SL.txt and has a size of 110,469 bytes.

Background Art

[0004]

[0004] Cancer is the most common cause of death worldwide, with an estimated 14 million new cases and 8 million deaths worldwide in 2012 (Torre et al. Cancer Epidemiol Biomarkers Prev. 2016;25(1):16-27). By 2018, this trend had increased, with new cases exceeding 18 million and deaths exceeding 9 million (New global cancer data: GLOBOCAN 2018. https: / / www.uicc.org / news / new-global-cancer-data-globocan-2018). Such a trend suggests an increasing crisis and the need for effective therapies for cancer treatment. Cancer immunotherapy (CIT) has developed in recent years as a promising approach in oncology, broadly including checkpoint inhibitors, adoptive cell transfer, targeted antibodies (T / NK cell engagers), cancer vaccines, and cytokines.

[0005]

[0005] Cytokines can boost immune cells by regulating the proliferation, differentiation, and survival of leukocytes (Berraondo et al. Br J Cancer 2019;120(1):6-15). Despite the known biology of cytokines and their role in the immune system and cancer biology, a limited number of cytokines have been approved for the treatment of cancers with limited symptoms, including IFNα (e.g., particularly pilocytic cell leukemia and chronic myeloid leukemia) and IL-2 (e.g., progressive melanoma and metastatic RCC). This is partly due to the low tolerability, narrow therapeutic index, and poor PK behavior of these cytokines (Berraondo et al. 2019, cited above).

[0006]

[0006] For example, recombinant IL-2, also known as aldesleukin (Proleukin®), has been used clinically as a CIT agent for over 20 years. Despite its proven clinical utility as an antitumor agent, Proleukin® can induce major toxicities such as capillary leak syndrome (CLS), and patients receiving Proleukin require extensive monitoring in a hospital setting. IL-2 is differentiation-4 positive (CD4 + IL-2 is a secreted cytokine that acts on cells such as regulatory T cells (Tregs), endothelial cells, and clusters of activated T cells. These cells express IL-2Rα (CD25) in a high-affinity trimer receptor complex along with CD122 and CD132. IL-2 is also known to induce activation-induced cell death (AICD). Increased Treg function and induction of AICD are two ways in which antitumor immunity is thought to decrease over time.

[0007]

[0007] Interleukin (IL)-15, like other common gamma chain (CD132) cytokines, such as IL-2, IL-4, IL-7, IL-9, and IL-21, plays a crucial role in regulating the immune response. In addition to the common gamma chain, IL-15 and IL-2 also share a β subunit (CD122) in their heterotrimeric receptor complex and have overlapping biological effects. However, IL-15 and IL-2 have their own α receptor subunits for downstream signaling. IL-15 and IL-2 are known to play important roles in cancer immunity, particularly with natural killer (NK) cells and differentiation-8 positive (CD8) cells. + It has been shown that this boosts the immune system by inducing proliferation and activation of T cell clusters.

[0008]

[0008] IL-15, in association with IL-15Rα (CD215), is expressed by monocytes and dendritic cells, as well as by other cells that primarily express CD122 and CD132, such as NK cells and memory CD8 + It is presented in trans to T cells (moderate affinity heterodimer receptor complex). Therefore, when IL-15 / IL-15Rα binds to CD122 and CD132 on NK and T cells, it is transmitted to CD8 + T cell proliferation and memory CD8 + It enhances the sustained T cell response by maintaining T cells and inducing increased proliferation and cytotoxicity of NK cells. Importantly, the biological effects of IL-15 / IL-15Rα are minimal in CD25-expressing Tregs, suggesting that IL-15 / IL-15Rα reduces vascular leakage induced by IL-2, and is not known to induce AICD.

[0009]

[0009] Therefore, IL-15 has potential advantages over IL-2 as a CIT agent. Over the past decade, several IL-2 and IL-15-based therapies, recombinant human IL-15 (rhIL-15), and modified IL-15 / IL-15Rα-Fc superagonists (ALT-803), among others, have been tested in various clinical trials aimed at achieving improved clinical utility and reduced toxicity. However, there have been limitations to the clinical impact of pharmacokinetic (PK) exposure, pharmacodynamic (PD) response, or acute toxicity. For example, IV bolus administration of rhIL-15 or rhIL-15 / rhIL-15Rα complexes resulted in low PK exposure due to high target-mediated pharmacokinetics (TMDD) and rapid renal clearance (CL) (due to small molecular size of approximately 60 kDa); frequent administration was required. Furthermore, IV bolus administration was limited by acute toxicity, including CLS and hypotension. Due to the pharmacokinetic (PK) and safety limitations associated with IV bolus administration, alternative administration routes such as subcutaneous (SC) injection or continuous IV infusion have been explored to improve tolerability and PD efficacy. Some of these methods have been shown to improve PD response (i.e., NK and CD8). + While T cell enlargement and tolerability were improved, SC administration of rhIL-15 and ALT-803 was associated with frequent injection site reactions, requiring frequent administration (SC) or continuous infusions over multiple days in each treatment cycle. Available clinical data on IL-15 pathway agonists provided rationale for developing IL-15 therapies with optimized PK profiles and improved therapeutic indices.

[0010]

[0010] Therefore, there is still a demand for CIT agents, particularly for IL-15 pathway agonists. [Overview of the project]

[0011]

[0011] In a first embodiment, the present disclosure provides a method for treating a solid tumor in a subject requiring treatment of a solid tumor, the method comprising administering to the subject a therapeutically effective amount of a heterodimer protein, the heterodimer protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364 K / E357Q;S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q36 2E:D401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K3 It contains a set of amino acid substitutions selected from the group consisting of 70S;L368D / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

[0012]

[0012] In a second aspect, the present disclosure relates to CD8 in the subject. +A method is provided for inducing the proliferation of effector memory T cells, the method comprising administering an effective amount of heterodimer protein to a subject, the heterodimer protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L 368D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L36 It contains a set of amino acid substitutions selected from the group consisting of 8D / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

[0013]

[0013] In a third aspect, the present disclosure provides a method for inducing the proliferation of NK cells in a subject, the method comprising administering an effective amount of a heterodimer protein to the subject, the heterodimer protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357 Q;S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D 401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S This includes a set of amino acid substitutions selected from the group consisting of ;L368D / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

[0014]

[0014] In a fourth aspect, the present disclosure relates to CD8 in the subject. +A method is provided for inducing the proliferation of effector memory T cells and NK cells, the method comprising administering an effective amount of heterodimer protein to a subject, the heterodimer protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S36 4K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K ;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L3 It contains a set of amino acid substitutions selected from the group consisting of 68D / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

[0015]

[0015] In a fifth aspect, the present disclosure provides a method for inducing IFNγ production in a subject, the method comprising administering an effective amount of heterodimeric protein to the subject, the heterodimeric protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357 Q;S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D 401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S This includes a set of amino acid substitutions selected from the group consisting of ;L368D / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

[0016]

[0016] In some embodiments, each of the first and / or second Fc domains independently further comprises amino acid substitutions Q295E, N384D, Q418E and N421D (according to EU numbering).

[0017]

[0017] In some embodiments, each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / L234V / L235A / G236del / S239K;E233P / L234V / L235A / G236del / S267K;E233P / L234V / L235A / G236del / S239K / A327G;E233P / L234V / L235A / G236del / S267K / A327G; and E233P / L234V / L235A / G236del (as assigned by EU numbering), wherein the Fc domain is derived from the Fc domain of IgG1 or IgG3. In some embodiments, each of the first Fc domain and / or the second Fc domain independently further comprises an amino acid substitution selected from the group consisting of L328R;S239K; and S267K (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG2. In some embodiments, each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K; and E233P / F234V / L235A / G236del (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG4.

[0018]

[0018] In some embodiments, the IL-15 protein comprises one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E.

[0019]

[0019] In some embodiments, the IL-15 protein and the IL-15Rα protein include a set of amino acid substitutions or additions selected from E87C:65DPC;E87C:65DCA;V49C:S40C;L52C:S40C;E89C:K34C;Q48C:G38C;E53C:L42C;C42S:A37C and L45C:A37C, respectively.

[0020]

[0020] In some embodiments, the IL-15 protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 2 (full-length human IL-15) and SEQ ID NO: 1 (cleaved human IL-15). In some embodiments, the IL-15Rα protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 (full-length human IL-15Rα) and SEQ ID NO: 4 (sucrose domain of human IL-15Rα).

[0021]

[0021] In some embodiments, the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain comprises amino acid substitutions S364K and E357Q; each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4.

[0022]

[0022] In some embodiments, the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions L368D and K370S; each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4.

[0023]

[0023] In some embodiments, the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain comprises amino acid substitutions K246T, S364K and E357Q; each of the first and second Fc domains comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4.

[0024]

[0024] In some embodiments, the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions K246T, L368D and K370S; each of the first and second Fc domains comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4.

[0025]

[0025] In some embodiments, the IL-15 protein is covalently bound to the N-terminus of the first Fc domain via a first linker. In some embodiments, the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain via a second linker. In some embodiments, the IL-15 protein is covalently bound to the N-terminus of the first Fc domain via a first linker, and the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain via a second linker.

[0026]

[0026] In some embodiments, the first linker and / or the second linker are independently variable-length Gly-Ser linkers. In some embodiments, the first linker and / or the second linker independently include a linker selected from the group consisting of (Gly-Gly-Gly-Gly-Ser)n (Sequence ID 39), (Ser-Ser-Ser-Ser-Gly)n (Sequence ID 40), (Gly-Ser-Ser-Gly-Gly)n (Sequence ID 41), and (Gly-Gly-Ser-Gly-Gly)n (Sequence ID 42), where n is an integer between 1 and 5.

[0027]

[0027] In some embodiments, the heterodimer protein is selected from the group consisting of XENP22822, XENP23504, XENP24045, XENP24306, XENP22821, XENP23343, XENP23557, XENP24113, XENP24051, XENP24341, XENP24052, XENP24301, and XENP32803 proteins. In some embodiments, the heterodimer protein is XENP24306. In some embodiments, the heterodimer protein is XENP32803. In some embodiments, the heterodimer protein is a combination of XENP24306 and XENP32803.

[0028]

[0028] In a sixth aspect, the present disclosure provides a method of treating a solid tumor in a subject that requires treatment of the solid tumor, the method comprising administering to the subject a therapeutically effective amount of a heterodimeric protein, the heterodimeric protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising the sushi domain of an IL-15Rα protein and a second Fc domain, wherein the sushi domain of the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises an N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

[0029]

[0029] In a seventh aspect, the present disclosure + provides a method for inducing the proliferation of CD8 effector memory T cells in a subject, the method comprising administering to the subject an effective amount of a heterodimeric protein, the heterodimeric protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising the sushi domain of an IL-15Rα protein and a second Fc domain, wherein the sushi domain of the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises an N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

[0030]

[0030] In an eighth aspect, the present disclosure provides a method for inducing the proliferation of NK cells in a subject, the method comprising administering an effective amount of a heterodimer protein to the subject, the heterodimer protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain The IL-15 protein comprises a monomer, wherein the sucoid domain of the IL-15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first and second Fc domains comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

[0031]

[0031] In the ninth aspect, the present disclosure relates to CD8 + A method is provided for inducing the proliferation of effector memory T cells and NK cells, the method comprising administering an effective amount of heterodimeric protein to a subject, the heterodimeric protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain. The IL-15 protein comprises a second monomer in which the sucrose domain of the IL-15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

[0032]

[0032] In a tenth aspect, the present disclosure provides a method for inducing the production of IFNγ in a subject, the method comprising administering to the subject an effective amount of a heterodimer protein, the heterodimer protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain The IL-15 protein comprises a monomer, wherein the sucoid domain of the IL-15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first and second Fc domains comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

[0033]

[0033] In some embodiments, the first Fc domain further comprises amino acid substitutions L368D and K370S, and the second Fc domain further comprises amino acid substitutions S364K and E357Q (according to EU numbering).

[0034]

[0034] In some embodiments, the first Fc domain further comprises amino acid substitutions S364K and E357Q, and the second Fc domain further comprises amino acid substitutions L368D and K370S (according to EU numbering).

[0035]

[0035] In some embodiments, the first Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E and N421D (according to EU numbering).

[0036]

[0036] In some embodiments, the second Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E and N421D (according to EU numbering).

[0037]

[0037] In some embodiments, the second Fc domain further comprises the amino acid substitution K246T (according to EU numbering).

[0038]

[0038] In some embodiments, the IL-15 protein includes amino acid substitutions D30N, E64Q and N65D.

[0039]

[0039] In some embodiments, the IL-15 protein comprises the amino acid sequence shown in SEQ ID NO: 5.

[0040]

[0040] In some embodiments, the sucrose domain of the IL-15Rα protein contains the amino acid sequence shown in SEQ ID NO: 4.

[0041]

[0041] In some embodiments, the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 10.

[0042]

[0042] In some embodiments, the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 16.

[0043]

[0043] In some embodiments, the IL-15 protein is covalently bound to the N-terminus of the first Fc domain via a first linker.

[0044]

[0044] In some embodiments, the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain via a second linker. In some embodiments, the IL-15 protein is covalently bound to the N-terminus of the first Fc domain via a first linker, and the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain via a second linker.

[0045]

[0045] In some embodiments, the first linker and / or the second linker are independently variable-length Gly-Ser linkers. In some embodiments, the first linker and / or the second linker independently include a linker selected from the group consisting of (Gly-Gly-Gly-Gly-Ser)n (Sequence ID 39), (Ser-Ser-Ser-Ser-Gly)n (Sequence ID 40), (Gly-Ser-Ser-Gly-Gly)n (Sequence ID 41), and (Gly-Gly-Ser-Gly-Gly)n (Sequence ID 42), where n is an integer between 1 and 5.

[0046]

[0046] In some embodiments of the methods disclosed herein, the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments of any of the methods disclosed herein, the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 16. In some embodiments of any of the methods disclosed herein, the heterodimer protein is XENP24306. In some embodiments of any of the methods disclosed herein, the heterodimer protein is XENP32803. In some embodiments of any of the methods disclosed herein, a combination of XENP24306 and XENP32803 is used.

[0047]

[0047] In some embodiments of any of the methods disclosed herein, the XENP24306 protein accounts for about 50–about 100%, about 70–about 95%, about 80–about 90%, or about 80–about 85% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP32803 protein accounts for about 1–about 50%, about 5–about 30%, about 10–about 20%, or about 15–about 20% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP24306 protein accounts for about 85% of the combined heterodimer protein, and the XENP32803 protein accounts for about 15% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP24306 protein accounts for approximately 84% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 16% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP24306 protein accounts for approximately 83% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 17% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP24306 protein accounts for approximately 82% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 18% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP24306 protein accounts for approximately 81% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 19% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP24306 protein accounts for approximately 80% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 20% of the combined heterodimer protein.

[0048]

[0048] In some embodiments of any of the methods disclosed herein, a combination of two or more heterodimer proteins is administered to a subject. In some embodiments, a combination of a first heterodimer protein and a second heterodimer protein is administered to a subject.

[0049]

[0049] In some embodiments, the first heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 10; the second heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 16.

[0050]

[0050] In some embodiments, the first heterodimer protein and the second heterodimer protein are administered simultaneously. In some embodiments, the first heterodimer protein and the second heterodimer protein are administered sequentially. In some embodiments, the first heterodimer protein and the second heterodimer protein are administered in the same composition. In some embodiments, the first heterodimer protein and the second heterodimer protein are administered in separate compositions.

[0051]

[0051] In some embodiments, the solid tumors treated by any of the methods disclosed herein are locally progressive, recurrent, or metastatic. In some embodiments, the solid tumors are selected from the group consisting of squamous cell carcinoma, cutaneous squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liposarcoma, soft tissue sarcoma, urothelial carcinoma, ureter and renal pelvis, multiple myeloma, osteosarcoma, hepatocellular carcinoma, melanoma, stomach cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cell carcinoma, liver cancer, esophageal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, Merkel cell carcinoma, germ cell carcinoma, high-frequency microsatellite instability cancer, and head and neck squamous cell carcinoma. In some embodiments, the solid tumor is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and triple-negative breast cancer.

[0052]

[0052] In some embodiments, the subject has not previously been administered any medication for the treatment of the condition. In some embodiments, the subject is currently being administered a checkpoint inhibitor. In some embodiments, the subject has previously been administered a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor targets PD-1. In some embodiments, the checkpoint inhibitor targets PD-L1. In some embodiments, the checkpoint inhibitor targets CTLA-4.

[0053]

[0053] In some embodiments, the heterodimer protein is administered in doses selected from the group consisting of approximately 0.0025 mg / kg, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.015 mg / kg, approximately 0.02 mg / kg, approximately 0.025 mg / kg, approximately 0.03 mg / kg, approximately 0.04 mg / kg, approximately 0.05 mg / kg, approximately 0.06 mg / kg, approximately 0.08 mg / kg, approximately 0.10 mg / kg, approximately 0.12 mg / kg, approximately 0.16 mg / kg, approximately 0.20 mg / kg, approximately 0.24 mg / kg, and approximately 0.32 mg / kg, based on body weight. In some embodiments, the heterodimer protein is administered at a dose selected from the group consisting of approximately 0.01 mg / kg, approximately 0.02 mg / kg, approximately 0.04 mg / kg, approximately 0.06 mg / kg, approximately 0.09 mg / kg, approximately 0.135 mg / kg, and approximately 0.2025 mg / kg by body weight. In some embodiments, the heterodimer protein is administered at a frequency selected from the group consisting of Q1W, Q2W, Q3W, Q4W, Q5W, and QW6. In some embodiments, the heterodimer protein is administered in doses selected from the group consisting of 0.0025 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.08 mg / kg, 0.10 mg / kg, 0.16 mg / kg, 0.20 mg / kg, 0.24 mg / kg, and 0.32 mg / kg, based on body weight. In some embodiments, the heterodimer protein is administered in doses selected from the group consisting of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.06 mg / kg, 0.09 mg / kg, 0.135 mg / kg, and 0.2025 mg / kg, based on body weight. In some embodiments, the heterodimeric protein is administered at a frequency selected from the group consisting of Q1W, Q2W, Q3W, Q4W, Q5W, and Q6W.

[0054]

[0054] In some embodiments, the heterodimer protein combination (e.g., XENP24306 + XENP32803) is administered in doses selected from the group consisting of approximately 0.0025 mg / kg, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.015 mg / kg, approximately 0.02 mg / kg, approximately 0.025 mg / kg, approximately 0.03 mg / kg, approximately 0.04 mg / kg, approximately 0.05 mg / kg, approximately 0.06 mg / kg, approximately 0.08 mg / kg, approximately 0.10 mg / kg, approximately 0.12 mg / kg, approximately 0.16 mg / kg, approximately 0.20 mg / kg, approximately 0.24 mg / kg, and approximately 0.32 mg / kg, based on body weight. In some embodiments, the heterodimer protein combination (e.g., XENP24306+XENP32803) is administered at doses selected from the group consisting of approximately 0.01 mg / kg, approximately 0.02 mg / kg, approximately 0.04 mg / kg, approximately 0.06 mg / kg, approximately 0.09 mg / kg, approximately 0.135 mg / kg, and approximately 0.2025 mg / kg by body weight. In some embodiments, the heterodimer protein combination is administered at frequencies selected from the group consisting of Q1W, Q2W, Q3W, Q4W, Q5W, and Q6W. In some embodiments, the heterodimer protein combination (e.g., XENP24306 + XENP32803) is administered in doses selected from the group consisting of 0.0025 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.08 mg / kg, 0.10 mg / kg, 0.16 mg / kg, 0.20 mg / kg, 0.24 mg / kg, and 0.32 mg / kg by body weight. In some embodiments, the heterodimer protein combination (e.g., XENP24306+XENP32803) is administered at doses selected from the group consisting of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, 0.06 mg / kg, 0.09 mg / kg, 0.135 mg / kg, and 0.2025 mg / kg by body weight. In some embodiments, the heterodimer protein combination is administered at frequencies selected from the group consisting of Q1W, Q2W, Q3W, Q4W, Q5W, and Q6W.

[0055]

[0055] In some embodiments, the methods disclosed herein further include administering a PD-L1 / PD-1 axis-targeting agent to a subject. In some embodiments, the PD-L1 / PD-1 axis-targeting agent is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, pidilizumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, MDX-1106, AMP-514, and AMP-224. In some embodiments, the PD-L1 / PD-1 axis-targeting agent is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from avelumab, durvalumab, atezolizumab, BMS-936559, BMS-39886, KN035, CK-301, and MSB0010718C.

[0056]

[0056] These and other embodiments will be obvious to those skilled in the art in light of the entirety of this disclosure. [Brief explanation of the drawing]

[0057] [Figure 1]

[0057] The combination of XENP24306 (~82%) and XENP32803 (~18%) promotes dose-dependent proliferation of human NK cells (A) and CD8+ T cells (B) in human PBMCs. PBMCs from 22 individual human donors were treated for 4 days with the total concentrations of XENP24306 (~82%) and XENP32803 (~18%) shown, and the frequency of Ki67+ (a marker of cell proliferation) was determined by flow cytometry for CD3-CD56+ NK cells (A) or CD3+CD8+CD16- T cells (B). Each point represents the mean value for the 22 donors, and the error bars represent SEM. Curve fitting was generated using the least squares method. EC50 values ​​were determined by nonlinear regression analysis using agonist-pair responses with a variable gradient (4-parameter) equation. [CD = differentiated cluster; NK = natural killer; PBMC = peripheral blood mononuclear cell]. [Figure 2]

[0058] This study compares CD8+ terminal effector T cell proliferation induced in human PBMCs by combinations of XENP24306 (~82%) and XENP32803 (~18%), recombinant wild-type IL-15 (rIL15), and wild-type IL-15 / wild-type IL-15Rα heterodimer Fc fusion (XENP22853). [EC50 = half-effect concentration]. [Figure 3A-B]

[0059] This graph shows the absolute number of CD8β+ T cells (A (male) and B (female)) in the whole blood of cynomolgus monkeys treated with repeated doses of XENP24306 (~82%) and XENP32803 (~18%), as well as different doses (0; 0.03 mg / kg; 0.2 mg / kg and 0.6 mg / kg). [Figure 3C-D] This graph shows the absolute number of NK cells (C (male) and D (female)) in the whole blood of cynomolgus monkeys treated with repeated doses of XENP24306 (~82%) and XENP32803 (~18%), as well as different doses (0; 0.03 mg / kg; 0.2 mg / kg and 0.6 mg / kg). Whole blood from cynomolgus monkeys was stained with antibodies, and CD8+ T cells were identified as CD45+ CD3+ CD8β+ CD4- CD16-, and NK cells as CD45+ CD3- CD16+. Each data point represents the mean value from 3 to 5 cynomolgus monkeys per group; error bars indicate standard deviation (SD). [Figure 4]

[0060] This graph shows the mean (±SD) serum concentration (ng / mL) versus time (days) profile of the heterodimer protein combination (XENP24306 (~82%) and XENP32803 (~18%)) in cynomolgus monkeys (male and female combinations) after a total of three intravenous administrations of heterodimer proteins (0.03 mg / kg; 0.2 mg / kg and 0.6 mg / kg) during Q2W. [Figure 5]

[0061] This graph shows weight loss in non-obese diabetic / severely immunodeficient gamma (NSG) mice transplanted with human PBMCs. A combination of XENP24306 (~82%) and XENP32803 (~18%) was administered at various concentrations, with or without the anti-PD1 bivalent antibody, XENP16432, at a dose of 3 mg / kg. Samples: (A) PBS; (B) 3.0 mg / kg of XENP16432; (C) 0.3 mg / kg of a combination of XENP24306 (~82%) and XENP32803 (~18%); (D) 0.1 mg / kg of a combination of XENP24306 (~82%) and XENP32803 (~18%); (E) 0.03 mg / kg of a combination of XENP24306 (~82%) and XENP32803 (~18%); (F) 0.01 mg / kg of a combination of XENP24306 (~82%) and XENP32803 (~18%); (G) 0.3 mg / kg of XENP24306 (~ (H) XENP16432 at 3.0 mg / kg in combination with XENP32803 (~18%) (82%); (I) XENP24306 (~82%) at 0.03 mg / kg in combination with XENP32803 (~18%) (0.03 mg / kg in combination with XENP24306 (~82%) and XENP32803 (~18%) (0.03 mg / kg in combination with XENP16432 at 3.0 mg / kg in combination with XENP24306 (~82%) and XENP32803 (~18%) (0.01 mg / kg in combination with XENP16432 at 3.0 mg / kg in combination with XENP32803 (~18%). [Figure 6]

[0062] This graph shows the group median change in tumor volume in non-obese diabetic / severely immunodeficient gamma (NSG) mice transplanted with human tumor cells (pp65-MCF7) and huPBMCs as a human leukocyte source. A combination of XENP24306 (~82%) and XENP32803 (~18%) was administered at various concentrations, either in the presence or absence of 3 mg / kg of XENP16432. Samples: (A) PBS; (B) 3.0 mg / kg of XENP16432; (C) 1.0 mg / kg of a combination of XENP24306 (~82%) and XENP32803 (~18%); (D) 0.3 mg / kg of a combination of XENP24306 (~82%) and XENP32803 (~18%); (E) 0.1 mg / kg of a combination of XENP24306 (~82%) and XENP32803 (~18%); (F) 1.0 mg / kg of X A combination of ENP24306 (~82%) and XENP32803 (~18%) + 3.0 mg / kg of XENP16432; (G) A combination of 0.3 mg / kg of XENP24306 (~82%) and XENP32803 (~18%) + 3.0 mg / kg of XENP16432; and (H) A combination of 0.1 mg / kg of XENP24306 (~82%) and XENP32803 (~18%) + 3.0 mg / kg of XENP16432. [Figure 7]

[0063] This is a study schema for monotherapy with IL15 / IL15Rα heterodimer protein (e.g., XENP24306, XENP32803, or a combination of XENP24306 (~82%) and XENP32803 (~18%)), showing patients enrolled in two phases: dose escalation and dose expansion, and details for these two phases. DL = dose level; DLT = dose-limiting toxicity; MTD = maximum tolerated dose; PD = pharmacodynamics; Q2W = every 2 weeks; Q3W = every 3 weeks; Q4W = every 4 weeks; RCC = renal cell carcinoma; RED = recommended dose expansion. a PD effect is evaluated by enumeration of peripheral blood NK cells and CD8+ T cells and Ki67 staining. b The safety threshold for changing from 1 / dose level to 3+3+3 design is defined in Example 6. The safety threshold for changing from ≤100% dose expansion to ≤50% dose expansion is defined in Example 6. d. If cumulative toxicity results in unacceptable tolerability (e.g., frequent dose delays of IL15 / IL15Rα heterodimer protein), the frequency of administration of IL15 / IL15Rα heterodimer protein may be reduced. [Figure 8]

[0064] This is a combination therapy study schema for IL15 / IL15Rα heterodimer proteins (e.g., XENP24306, XENP32803, or a combination of XENP24306 (~82%) and XENP32803 (~18%) in combination with atezolizumab (anti-PD-L1 antibody)), and it shows patients enrolled in two phases: the dose escalation phase and the dose expansion phase, along with details about these two phases. Bx=biopsy; CIT=cancer immunotherapy; cSCC=cutaneous squamous cell carcinoma; DL=dose level; DLT=dose-limiting toxicity; GC=gastric cancer; HNSCC=head and neck squamous cell carcinoma; MCC=Merkel cell carcinoma; MSI-H=high frequency microsatellite instability; MTD=maximal tolerated dose; NSCLC=non-small cell lung cancer; PD=pharmacodynamics; Q2W=every 2 weeks; Q3W=every 3 weeks; Q4W=every 4 weeks; RCC=renal cell carcinoma; RED=recommended expanded dose; SCLC=small cell lung cancer; TBD=undetermined; TNBC=triple-negative breast cancer; UCC=urothelial carcinoma. The safety threshold for switching from a≦00% dose increase to ≦50% is defined in Example 6. b If a dose level of 0.01 mg / kg of IL15 / IL15Rα heterodimer protein in initial monotherapy shows PD activity, the starting dose of IL15 / IL15Rα heterodimer protein in the atezolizumab combination cohort in initial combination therapy should not exceed 0.005 mg / kg. c If cumulative toxicity results in unacceptable tolerability (e.g., frequent dose delays of IL15 / IL15Rα heterodimer protein), the frequency of administration of IL15 / IL15Rα heterodimer protein / atezolizumab may be reduced. d The PD effect indicating the initial dose level of IL15 / IL15Rα heterodimer protein is defined in Example 6. e Patients must have previously received an anti-PD-L1 / PD-1 inhibitor as monotherapy or in combination and have obtained clinical benefit from the previous treatment. f. Symptoms include melanoma, NSCLC, HNSCC, TNBC, UCC, RCC, SCLC, GC, MCC, cSCC, and MSI-H cancer. g. Patients with melanoma, RCC, UCC, NSCLC, HNSCC, and TNBC will be enrolled. The hPD-L1 threshold may vary depending on the symptoms and will be determined thereafter. [Figure 9]

[0065] The amino acid sequences of XENP24306 monomer 1 (SEQ ID NO: 9), XENP24306 monomer 2 (SEQ ID NO: 10), XENP32803 monomer 1 (SEQ ID NO: 9), and XENP32803 monomer 2 (SEQ ID NO: 16) are provided. In the sequence of monomer 1, the IL15 portion is underlined, the linker is offset by a slash, and it is shown in bold and underlined. The Fc portion is after the second slash and does not contain any formatting. In the sequence of monomer 2, the IL15Rα portion is underlined, the linker is offset by a slash, and it is shown in bold and underlined. The Fc portion is after the second slash and does not contain any formatting. [Figure 10A]

[0066] This document provides the amino acid sequences of the human IL-15 precursor protein (full-length human IL-15) (SEQ ID NO: 2), mature or cleaved human IL-15 protein (SEQ ID NO: 1), full-length human IL-15Rα protein (SEQ ID NO: 3), and the extracellular domain of the human IL-15Rα protein (SEQ ID NO: 54). [Figure 10B] This document provides the amino acid sequences of the sucrose domain (SEQ ID NO: 4) of the human IL-15Rα protein, the full-length human IL-15Rβ protein (SEQ ID NO: 55), and the extracellular domain (SEQ ID NO: 56) of the human IL-15Rβ protein. [Figure 11A]

[0067] This provides the amino acid sequence of the first monomer (SEQ ID NO: 11) of XENP2853 wild-type IL-15-Fc. [Figure 11B] The amino acid sequences of the XENP2822 protein (SEQ ID NO: 19 and SEQ ID NO: 20) and the XENP23504 protein (SEQ ID NO: 29 and SEQ ID NO: 30) are provided. [Figure 11C] The amino acid sequences of the XENP24045 protein (SEQ ID NOs. 23 and 24) and the XENP22821 protein (SEQ ID NOs. 17 and 18) are provided. [Figure 11D]The amino acid sequences of the XENP23343 protein (SEQ ID NOs. 31 and 32) and the XENP23557 protein (SEQ ID NOs. 21 and 22) are provided. [Figure 11E] The amino acid sequences of the XENP24113 protein (SEQ ID NOs. 33 and 34) and the XENP24051 protein (SEQ ID NOs. 25 and 26) are provided. [Figure 11F] The amino acid sequences of the XENP24341 protein (SEQ ID NOs. 35 and 36) and the XENP24052 protein (SEQ ID NOs. 27 and 28) are provided. [Figure 11G] The amino acid sequences for the XENP24301 protein (SEQ ID NOs. 37 and 38) are provided. [Modes for carrying out the invention]

[0058] general

[0068] The methods disclosed herein, as well as the preparation and use of the compositions, will employ, unless otherwise specified, general techniques within the scope of the art in molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA, and related fields. These techniques are well described in the literature. For example, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, “Chromatin See "Protocols" (PB Becker, ed.), Humana Press, Totowa, 1999.

[0059]

[0069] The term "this specification" refers to the entire specification.

[0060]

[0070] Any embodiment described herein can be combined with one or more other embodiments disclosed herein, including those described in different aspects of this disclosure and different parts of the specification (including embodiments described only in the Examples), unless expressly stated otherwise or deemed inappropriate. The combinations of embodiments are not limited to any particular combination claimed by any of the dependent claims.

[0061]

[0071] Any publications, patents, and published patent applications referenced herein are incorporated herein by reference. In case of any conflict, this specification, including its specific definitions, shall prevail.

[0062]

[0072] Throughout this specification, the word “comprise” or its grammatical variations implicitly includes the integer (or component) or group of integers (or components) mentioned, and does not exclude any other integer (or component) or group of integers (or components).

[0063]

[0073] Throughout this specification, when a composition is described as having, containing, or comprising (or a variation thereof) a particular component, it should be considered that the composition may be essentially composed of or comprise such a component. Similarly, when a method or process is described as having, containing, or comprising a particular step, the process may be essentially composed of or comprise such a step. Furthermore, it should be understood that the order of steps or the order in which particular actions are performed is not important, as long as the compositions and methods described herein are operable. Moreover, two or more steps or actions can be performed simultaneously.

[0064]

[0074] The term "~includes" is used to mean "includes ~ without limitation." "~includes" and "includes ~ without limitation" are interchangeable.

[0065]

[0075] The one or more examples following the term “e.g.” or “for example” do not imply that they are exclusive or limiting.

[0066]

[0076] The articles ("a" and "an") are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, "element" means one or more elements.

[0067]

[0077] When used herein, the term “about” modifying quantities of components, parameters, calculations, or measurements of compositions used in the methods of the disclosure means variations in quantities that may occur without substantially affecting the chemical or physical properties of the compositions or methods of the disclosure, for example, due to common measurement and liquid handling procedures used to prepare isolated polypeptides or pharmaceutical compositions in the real world; due to careless errors in these procedures; or due to differences in the manufacture, source, or purity of components used to prepare the compositions or perform the methods. Such variations may be within a range of generally no more than 10%, and more generally no more than 5%, of a given value or range. The term “about” also includes quantities that differ due to different equilibrium conditions of a composition resulting from a particular initial mixture. Whether modified by the term “about” or not, a paragraph includes the equivalent quantity. References to values ​​or parameters following “about” herein include (and describe) embodiments that apply to that value or parameter itself. For example, a statement referring to “about X” includes a statement of “X.” A numerical range includes the numerical value that defines that range.

[0068]

[0078] As used herein, the term "or" should be understood to mean "and / or" unless the context makes it clear otherwise.

[0069]

[0079] Although the numerical ranges and parameters representing the broad scope of this disclosure are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors that inevitably arise from the standard deviation observed in each test measurement. Furthermore, all ranges disclosed herein should be understood to encompass all subranges contained therein. For example, a range described as "1 to 10" should be considered to include all subranges between the minimum value of 1 and the maximum value of 10 (including 1 and 10); that is, all subranges beginning with a minimum value of 1 or greater, e.g., 1 to 6.1, and all subranges ending with a maximum value of 10 or less, e.g., 5.5 to 10. Disclosure of a range should also be considered as disclosure of the endpoints of that range.

[0070]

[0080] Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this application. The materials, methods, and examples are illustrative and not intended to be limiting.

[0071] definition

[0081] The following terms should be understood to have the following meanings unless otherwise indicated:

[0072]

[0082] As used herein, the term "ablation" refers to a reduction or removal of activity. Therefore, for example, "ablation of FcγR binding" means that the Fc region amino acid variant has less than 50% initiation binding compared to an Fc region without the specific variant, and preferably less than 70%, 80%, 90%, 95%, or 98% loss of activity, and generally, the activity is below the level of binding detectable in the BIACORE® assay (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). Unless otherwise specified, the Fc domains described herein retain binding to the FcRn receptor.

[0073]

[0083] "Administering" a substance, compound, or drug to a subject, or "administering" a substance, compound, or drug to a subject, means contact of the substance, compound, or drug with the subject or with the subject's cells, tissues, organs, or bodily fluids. Such administration can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or drug may be administered sublingually or intranasally by inhalation into the lungs or rectum. Administration may also be carried out, for example, once, multiple times, and / or over one or more extended periods. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug or to have another person administer the drug, and / or a physician who provides a patient with a prescription for a drug, is administering the drug to the patient.

[0074]

[0084] As used herein, the term "affinity" of a molecule refers to the strength of the interaction between the molecule and its binding partner, such as a receptor, ligand, or antigen. The affinity of a molecule for its binding partner is typically expressed as the binding affinity equilibrium dissociation constant (KD) for a particular interaction, with lower KDs indicating higher affinity. The KD binding affinity constant can be measured by surface plasmon resonance, for example, using the BIACORE® system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). See also Jonsson et al., Ann. Biol. Clin. 51:19 26 (1993); Jonsson et al., Biotechniques 11:620 627 (1991); Jonsson et al., J. Mol. Recognit. 8:125 131 (1995); Johnsson et al., Anal. Biochem. 198:268 277 (1991); and Hearty S et al., Methods Mol Biol. 907:411-42 (2012), each incorporated herein by reference. KD may also be measured using the KinExA® system (Sapidyne Instruments, Hanover, Germany and Boise, ID). In some embodiments, the IL-15 variants of the heterodimer protein described herein exhibit reduced binding affinity to the IL-2 / IL-15βγ receptor compared to wild-type IL-15. In some embodiments, the first and / or second Fc variants of the heterodimer protein described herein exhibit reduced affinity to human, cynomolgus monkey, and mouse Fcγ receptors. In some embodiments, the first and / or second Fc variants of the heterodimer protein described herein do not bind to human, cynomolgus monkey, and mouse Fcγ receptors.

[0075]

[0085] As used herein, the terms "amino acid" and "amino acid identity" refer to one of the 20 naturally occurring amino acids encoded by DNA and RNA.

[0076]

[0086] As used herein, the terms “amino acid substitution” or “substitution” refer to the replacement of an amino acid with a different amino acid at a specific position in the parent polypeptide sequence. In particular, in some embodiments, the substitution is for an amino acid that does not naturally exist at a particular position and is not naturally present in any organism or any organism. For example, substitution E272Y refers to a variant polypeptide, in this case the Fc variant, in which glutamic acid at position 272 is replaced with tyrosine. For clarity, a protein that has been manipulated to change the nucleic acid coding sequence but not the starting amino acid (for example, replacing CGG (coding arginine) with CGA (still encoding arginine) to increase the expression level in a host organism) is not an “amino acid substitution.” That is, if a new gene encoding the same protein is generated, but the protein has the same amino acid at a particular position that is the starting position, it is not considered an amino acid substitution.

[0077]

[0087] As used herein, the terms “amino acid insertion,” “amino acid addition,” “addition,” and “insertion” refer to the addition of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, -233E or 233E indicates the insertion of glutamic acid after position 233 and before position 234. Furthermore, -233ADE or 233ADE indicates the insertion of AlaAspGlu after position 233 and before position 234.

[0078]

[0088] As used herein, the terms "amino acid deletion" or "deletion" refer to the removal of an amino acid sequence at a specific position in the parent polypeptide sequence. For example, E233- or E233#, E233() or E233del refer to the deletion of glutamic acid at position 233. Furthermore, EDA233- or EDA233# indicates the deletion of the sequence GluAspAla beginning at position 233.

[0079]

[0089] As used herein, the term “antibody” or “Ab” refers to an immunoglobulin molecule (e.g., a complete antibody, antibody fragment, or modified antibody) that can recognize a specific target or antigen located in the variable region of the immunoglobulin molecule, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., and bind to it through at least one antigen recognition site. As used herein, the term “antibody” can include all types of antibodies, including, but are not limited to, monoclonal antibodies, polyclonal antibodies, human antibodies, and engineered antibodies (including humanized antibodies, fully human antibodies, chimeric antibodies, single-chain antibodies, artificially selected antibodies, CDR-granting antibodies, etc.) that specifically bind to a given antigen. In some embodiments, “antibody” and / or “immunoglobulin” (Ig) refers to a polypeptide comprising at least two heavy (H) chains (about 50–70 kDa) and two light (L) chains (about 25 kDa) optionally interconnected by disulfide bonds. There are two types of light chains: λ and κ. In humans, the λ and κ light chains are similar, but only one type exists in each antibody. The heavy chain is classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. For an overview, see Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)) (the entire text is incorporated by reference).

[0080]

[0090] As used herein, the term "checkpoint inhibitor" refers to a compound that targets and blocks a checkpoint protein. Checkpoint inhibitors interfere with the interaction between a checkpoint protein and its partner protein. Examples of checkpoint inhibitors include, but are not limited to, drugs that target the PD-1 / PD-L1 axis and drugs that target CTLA-4.

[0081]

[0091] As used herein, the term “effector function” refers to a biochemical event resulting from the interaction between the Fc region of an antibody and an Fc receptor or another effector molecule (e.g., an Fc receptor-like (FcRL) molecule, complement component C1q, and trifidative motif-containing protein 21 (TRIM21)). Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). As used herein, “ADCC” or “antibody-dependent cell-mediated cytotoxicity” refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize a bound antibody on a target cell, subsequently causing lysis of the target cell. ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa results in increased ADCC activity. As discussed herein, many embodiments of this disclosure completely ablate ADCC activity. As used herein, the term “ADCP” or “antibody-dependent cell-mediated phagocytosis” refers to a cell-mediated response in which nonspecific cytotoxic cells expressing FcγR recognize bound antibodies on target cells, subsequently causing phagocytosis of the target cells. As used herein, the term “CDC” or “complement-dependent cell-mediated cytotoxicity” refers to an effector function resulting in the activation of the classical complement pathway, which is triggered by the binding of antibodies to antigens on target cells and activates a cascade of complement-related proteins in the bloodstream.

[0082]

[0092] As used herein, the terms “Fc,” “Fc region,” or “Fc domain” are interchangeable and, in some cases, refer to a polypeptide containing the constant region of an antibody excluding the immunoglobulin domain of the first constant region (e.g., CH1) or a portion thereof, and in some cases refer to a portion of the hinge. Thus, Fc can refer to the immunoglobulin domains of the last two constant regions of IgA, IgD, and IgG (e.g., CH2 and CH3), and to the immunoglobulin domains of the last three constant regions of IgE and IgM, and the flexible hinge N-terminus to these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, the Fc domain includes the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, Fc refers to the cleaved CH1 domain of the immunoglobulin and CH2 and CH3. While the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is typically defined as containing residues E216, C226, or P230 at its carboxyl terminus, and its numbering follows EU numbering. In some embodiments, as described further in this specification, amino acid modifications are made to the Fc region to alter binding, for example, to one or more FcγR receptors or FcRn receptors. In some embodiments, the Fc domain is derived from the Fc domain of a human IgG monohelic acid heavy chain. In some embodiments, the Fc domain is derived from the Fc domain of a human IgG dihelic acid heavy chain. "EU format as defined by Edelman," "EU numbering," or "EU index" refers to the numbering of residues in the human Fc domain as described by Edelman GM et al. (Proc. Natl. Acad. USA (1969), 63, 78-85, which is incorporated herein by reference in its entirety).

[0083]

[0093] As used herein, the terms “Fc fusion protein” and “immunoadhesin” are interchangeable and generally refer to a protein containing an Fc region that is bound to a different protein described herein, for example, IL-15 and / or IL-15R (optionally via a linker portion described herein). In some examples, two Fc fusion proteins can form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, the latter being preferred.

[0084]

[0094] As used herein, the terms “Fc variant” or “variant Fc” refer to a protein containing amino acid modifications in its Fc domain. The Fc variants of this invention are defined according to the amino acid modifications that constitute them. For example, N434S or 434S is an Fc variant having a substituted serine at position 434 compared to the parent Fc polypeptide, and the numbering follows the EU index. Similarly, M428L / N434S defines an Fc variant having the substitutions M428L and N434S compared to the parent Fc polypeptide. The identification of the WT amino acids may be unspecified, in which case the variant is referred to as 428L / 434S. Note that the order in which substitutions are provided is arbitrary, i.e., for example, 428L / 434S is the same Fc variant as M428L / N434S. For all positions discussed in this invention relating to antibodies, unless otherwise stated, the numbering of amino acid positions follows the EU index. Modifications may be additions, deletions, or substitutions. Substitutions may include naturally occurring amino acids and, in some cases, synthetic amino acids. Examples include, but are not limited to, U.S. Patent No. 6,586,207; International Publication No. 98 / 48032; International Publication No. 03 / 073238; U.S. Patent Application Publication No. 2004-0214988; International Publication No. 05 / 35727A2; International Publication No. 05 / 74524A2; JW Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027; JW Chin, & PG Schultz, (2002), ChemBioChem 11:1135-1137; JW Chin, et al., (2002), PICAS United States of America 99:11020-11024; and L. Wang, & PG Schultz, (2002), Chem. 1-10, all of which are incorporated in their entirety by reference.

[0085]

[0095] As used herein, the terms “Fc gamma receptor,” “FcγR,” and “Fc gamma R” refer to any member of a family of proteins that bind to the Fc region of an IgG antibody and are used interchangeably, and are encoded by the FcγR gene. FcγR may be of any biological origin. In some embodiments, FcγR is human FcγR. In humans, this family includes, but is not limited to, FcγRI(CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII(CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; FcγRIII(CD16) including isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65 (the whole is incorporated by reference)); and any undiscovered human FcγR or FcγR isoform or allotype.

[0086]

[0096] As used herein, the terms “FcRn” or “neonatal Fc receptor” refer to a protein that binds to the Fc region of an IgG antibody and is at least partially encoded by the FcRn gene. FcRn may originate from any organism. In some embodiments, FcRn is human FcRn. As is known in the art, functional FcRn proteins often consist of two polypeptides called a heavy chain and a light chain. The light chain is beta-2 microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein refers to the complex of the FcRn heavy chain and beta-2 microglobulin. Various FcRn variants can be used to increase binding to the FcRn receptor and, in some cases, increase the serum half-life. In general, unless otherwise specified herein, the Fc monomers disclosed herein retain binding to the FcRn receptor (and may include amino acid variants that increase binding to the FcRn receptor, as described below).

[0087]

[0097] As used herein, the term “modification” refers to amino acid substitutions, insertions, and / or deletions in a polypeptide sequence, or alterations to a portion chemically bound to a protein. For example, a modification may be an altered carbohydrate or PEG structure bound to a protein. In this specification, “amino acid modification” means the substitution, insertion, and / or deletion of an amino acid in a polypeptide sequence. For clarity, unless otherwise specified, amino acid modification always refers to the amino acids encoded by DNA, e.g., the 20 amino acids that have codons in DNA and RNA.

[0088]

[0098] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used interchangeably and refer to deoxyribonucleotide or ribonucleotide polymers in linear or circular conformation and in single-stranded or double-stranded form. For the purposes of this disclosure, these terms are not construed as limitations relating to the length of the polymer. These terms may encompass natural nucleotides, as well as known analogs of nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate skeletons). Generally, analogs of a particular nucleotide have the same base-pairing specificity; i.e., an analog of A would be base-paired with T.

[0089]

[0099] As used herein, the term "modification not found in nature" refers to non-isotype amino acid modifications. For example, since none of the IgG molecules contain serine at position 434, the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or their hybrids) is considered a modification not found in nature.

[0090]

[0100] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to a human or non-human animal in need of treatment. These terms include mammals, e.g., humans, and primates (e.g., monkeys). In some embodiments, the subject is human. In some embodiments, the subject is in need of treatment for cancer. As used herein, the terms “to treat / administer” and “treatment / administer” refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or their causes, preventing the onset and / or causes of symptoms, and improving or repairing damage.

[0091]

[0101] As used herein, “amino acid sequence identity percentage (%)” for a protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular (parent) sequence, after the sequence has been aligned to obtain the maximum percentage of sequence identity, gaps have been introduced as necessary, and no conservative substitutions have been considered as part of the sequence identity. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. One particular program is the ALIGN-2 program outlined in paragraphs

[0279] -

[0280] of U.S. Patent Application Publication No. 20160244525, which is incorporated herein by reference.

[0092]

[0102] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to polymers of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally occurring amino acids. Expression of fusion proteins in cells can result from the delivery of the fusion protein to the cell or from the delivery of polynucleotides encoding the fusion protein to the cell, where the polynucleotides are transcribed, and the transcripts are translated to produce the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation may also be involved in protein expression in cells. Methods for the delivery of polynucleotides and polypeptides to cells are known in the prior art.

[0093]

[0103] As used herein, the term “position” refers to a location within a protein sequence. Positions may be numbered sequentially or according to an established format, such as the EU index for antibody numbering. Positions may be defined relative to a reference sequence. In such cases, the reference sequence is provided for comparative purposes, and the heterodimer protein (or portion thereof) of this disclosure may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the reference sequence. In some embodiments, the heterodimer protein (or portion thereof) of this disclosure does not include any additional amino acid modifications relative to the reference sequence.

[0094]

[0104] As used herein, the term "residue" refers to a position in a protein and the associated amino acid identity. For example, asparagine 297 (also known as Asn297 or N297) is a residue at position 297 in a particular protein.

[0095]

[0105] As used herein, the term “therapeutic effective dose” refers to the amount of a therapeutic agent administered alone or in combination with one or more additional agents that will alleviate, to some extent, one or more of the symptoms of the condition being treated. In some embodiments, the therapeutic effective dose is an amount sufficient to produce an effective or desired clinical outcome. In relation to the treatment of cancer, the therapeutic effective dose refers to an amount having at least one of the following effects: mitigating, improving, stabilizing, reversing, preventing, delaying, or slowing the progression of cancer (and / or associated symptoms). The effective doses that may be used herein vary depending on the method of administration, the age, weight, and overall health of the subject. The appropriate dose and dosage regimen can be determined using skills commonly used in the art.

[0096]

[0106] As used herein, the term “effective dose” refers to the amount of an agent administered alone or in combination with one or more additional agents that would be fully or partially effective or sufficient to produce the desired result. The effective dose as used herein may vary depending on the method of administration, the age, weight, and overall health of the subject. The appropriate dose and dosage regimen can be determined using skills commonly used in the art.

[0097]

[0107] The terms “wild-type” or “WT” are used interchangeably herein and refer to naturally occurring amino acid or nucleotide sequences, including allelic variants. WT proteins have an unmodified amino acid sequence or are encoded by an unmodified nucleotide sequence.

[0098] general

[0108] This disclosure relates to a method for treating solid tumors in subjects requiring treatment of solid tumors, the method comprising administering to the subject a therapeutically effective amount of a heterodimeric Fc fusion protein (or a combination of heterodimeric Fc fusion proteins) containing IL-15 and IL-15 receptor alpha (IL-15Rα) protein domains. This disclosure relates to CD8 + The present invention relates to a method for subjectively inducing the proliferation of effector memory T cells and / or NK cells, or for subjectively inducing IFNγ production, wherein the method comprises administering to a subject an effective amount of a heterodimeric Fc fusion protein (or a combination of heterodimeric Fc fusion proteins) containing IL-15 and IL-15 receptor alpha (IL-15Rα) protein domains. The Fc domain can be obtained from an IgG Fc domain, for example, the Fc domain of IgG1, IgG2, IgG3, or IgG4.

[0099] IL15-IL15Rα heterodimer Fc fusion protein

[0109] Any or a combination thereof of the IL15-IL15Rα heterodimeric Fc fusion proteins disclosed in U.S. Patent Application Publication No. 2018 / 0118805, whose entire disclosure is incorporated herein by reference, may be used in the methods disclosed herein. These include, in particular, Fc variants such as stereovariants (e.g., “knob-hole,” “skew,” “electrostatic steering,” and “electrostatic pair” variants), pI variants, isotype variants, FcγR variants, and ablation variants (e.g., “FcγR ablation variant” or “Fc knockout (FcKO or KO)” variants), as well as various IL-15 and IL15Rα proteins disclosed herein.

[0100]

[0110] Accordingly, in some embodiments, the heterodimer protein useful in the methods disclosed herein includes (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first Fc domain and the second Fc domain are respectively S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357Q:L368 D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K37 0S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D / K3 It contains a set of amino acid substitutions selected from the group consisting of 70S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

[0101]

[0111] In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution set S267K / L368D / K370S:S267K / S364K / E357Q (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution set S364K / E357Q:L368D / K370S (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution set L368D / K370S:S364K (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution set L368E / K370S:S364K (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution T411E / K360E / Q362E:D401K (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution L368D / K370S:S364K / E357L (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution K370S:S364K / E357Q (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution S267K / S364K / E357Q:S267K / L368D / K370S (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution pair L368D / K370S:S364K / E357Q (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution pair S364K:L368D / K370S (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution pair S364K:L368E / K370S (according to EU numbering).In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution set D401K:T411E / K360E / Q362E (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution set S364K / E357L:L368D / K370S (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each contain the amino acid substitution set S364K / E357Q:K370S (according to EU numbering).

[0102]

[0112] In some embodiments, each of the first Fc domain and / or the second Fc domain independently further comprises amino acid substitutions selected from the group consisting of Q295E, N384D, Q418E, and N421D, or combinations thereof (as assigned by EU numbering). In some embodiments, the first Fc domain further comprises amino acid substitutions selected from the group consisting of Q295E, N384D, Q418E, and N421D, or combinations thereof (as assigned by EU numbering). In some embodiments, the second Fc domain further comprises amino acid substitutions selected from the group consisting of Q295E, N384D, Q418E, and N421D, or combinations thereof (as assigned by EU numbering). In some embodiments, each of the first Fc domain and the second Fc domain further comprises amino acid substitutions selected from the group consisting of Q295E, N384D, Q418E, and N421D, or combinations thereof (as assigned by EU numbering). In some embodiments, the first Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E, and N421D (according to EU numbering). In some embodiments, the second Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E, and N421D (according to EU numbering). In some embodiments, each of the first and second Fc domains further comprises amino acid substitutions Q295E, N384D, Q418E, and N421D (according to EU numbering).

[0103]

[0113] In some embodiments, the first Fc domain does not contain free cysteine ​​at position 220. In some embodiments, the first Fc domain contains the amino acid substitution C220S (according to EU numbering). In some embodiments, the second Fc domain does not contain free cysteine ​​at position 220. In some embodiments, the second Fc domain contains the amino acid substitution C220S (according to EU numbering). In some embodiments, neither the first nor the second Fc domain contains free cysteine ​​at position 220. In some embodiments, both the first and second Fc domains contain the amino acid substitution C220S (according to EU numbering).

[0104]

[0114] In some embodiments, the first Fc domain further comprises one of the amino acid substitutions selected from the group consisting of E233P, L234V, L235A, G236del, G236R, S239K, S267K, A327G, and L328R or combinations thereof (as assigned by EU numbering). In some embodiments, the first Fc domain further comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (as assigned by EU numbering). In some embodiments, the second Fc domain further comprises one of the amino acid substitutions selected from the group consisting of E233P, L234V, L235A, G236del, G236R, S239K, S267K, A327G, and L328R or combinations thereof (as assigned by EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering). In some embodiments, the first Fc domain and the second Fc domain each comprise the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering).

[0105]

[0115] The locations of various Fc domain substitutions are based on the corresponding locations in the Fc domain of wild-type IgG1 (SEQ ID NO: 12). The amino acid sequence of the Fc domain of wild-type IgG1 (SEQ ID NO: 12) is an exemplary sequence provided for comparison purposes, and the Fc domain of the heterodimeric protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the Fc domain of wild-type IgG1 (SEQ ID NO: 12). For example, the Fc domain of the heterodimeric protein may be derived from different wild-type human IgG1 alleles. In some embodiments, the Fc domain of the heterodimeric protein does not include any additional amino acid modifications relative to the Fc domain of wild-type IgG1 (SEQ ID NO: 12). Those skilled in the art will be able to determine the corresponding substitutions in the Fc domain derived from the Fc domain of IgG2, IgG3, or IgG4. For example, those skilled in the art will be able to see that residues E233, L234, L235, and G236 are present in the Fc domain derived from the Fc domain of IgG1 or IgG3. In some embodiments, the locations of various Fc domain substitutions are relative to the corresponding locations in the Fc domain of wild-type IgG3 (SEQ ID NO: 14). The amino acid sequence of the Fc domain of wild-type IgG3 (SEQ ID NO: 14) is an exemplary sequence provided for comparison purposes, and the Fc domain of the heterodimer protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the Fc domain of wild-type IgG3 (SEQ ID NO: 14). For example, the Fc domain of the heterodimer protein may be derived from different wild-type human IgG3 alleles. In some embodiments, the Fc domain of the heterodimer protein does not include any additional amino acid modifications relative to the Fc domain of wild-type IgG3 (SEQ ID NO: 14).

[0106]

[0116] In some embodiments, each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / L234V / L235A / G236del / S239K;E233P / L234V / L235A / G236del / S267K;E233P / L234V / L235A / G236del / S239K / A327G;E233P / L234V / L235A / G236del / S267K / A327G; and E233P / L234V / L235A / G236del (as assigned by EU numbering), wherein the Fc domain is derived from the Fc domain of IgG1 or IgG3. In some embodiments, the first second Fc domain further comprises amino acid substitutions selected from the group consisting of G236R / L328R;E233P / L234V / L235A / G236del / S239K;E233P / L234V / L235A / G236del / S267K;E233P / L234V / L235A / G236del / S239K / A327G;E233P / L234V / L235A / G236del / S267K / A327G; and E233P / L234V / L235A / G236del (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG1 or IgG3. In some embodiments, the second Fc domain further comprises amino acid substitutions selected from the group consisting of G236R / L328R;E233P / L234V / L235A / G236del / S239K;E233P / L234V / L235A / G236del / S267K;E233P / L234V / L235A / G236del / S239K / A327G;E233P / L234V / L235A / G236del / S267K / A327G; and E233P / L234V / L235A / G236del (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG1 or IgG3.In some embodiments, the first and second Fc domains further include amino acid substitutions selected from the group consisting of G236R / L328R;E233P / L234V / L235A / G236del / S239K;E233P / L234V / L235A / G236del / S267K;E233P / L234V / L235A / G236del / S239K / A327G;E233P / L234V / L235A / G236del / S267K / A327G; and E233P / L234V / L235A / G236del (according to EU numbering), wherein the Fc domains are derived from the Fc domains of IgG1 or IgG3.

[0107]

[0117] Those skilled in the art will understand that the corresponding residues in the Fc domain derived from the Fc domain of IgG2 are P233, V234, and A235, and that the Fc domain derived from IgG2 lacks the residue corresponding to G236. Therefore, those skilled in the art will understand that if the Fc domain is the Fc domain of IgG2 (i.e., the PVA-sequence present in wild-type IgG2), then references to E233P, L234V, L235A, and G236del herein refer to P233, V234, A235, and -236. In some embodiments, the locations of various Fc domain substitutions are based on the corresponding locations in the Fc domain of wild-type IgG2 (SEQ ID NO: 13). The amino acid sequence of the Fc domain of wild-type IgG2 (SEQ ID NO: 13) is an exemplary sequence provided for comparative purposes, and the Fc portion of the heterodimeric protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the Fc domain of wild-type IgG2 (SEQ ID NO: 13). For example, the Fc domain of the heterodimer protein may be derived from different wild-type human IgG2 alleles. In some embodiments, the Fc domain of the heterodimer protein does not involve any additional amino acid modifications to the Fc domain of wild-type IgG2 (SEQ ID NO: 13).

[0108]

[0118] In some embodiments, each of the first Fc domain and / or the second Fc domain independently further comprises an amino acid substitution selected from the group consisting of L328R;S239K;S267K;S239K / A327G; and S267K / A327G (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG2. In some embodiments, the first Fc domain further comprises an amino acid substitution selected from the group consisting of L328R;S239K;S267K;S239K / A327G; and S267K / A327G (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG2. In some embodiments, the second Fc domain further comprises amino acid substitutions selected from the group consisting of L328R;S239K;S267K;S239K / A327G; and S267K / A327G (according to EU numbering), and the Fc domain is derived from the Fc domain of IgG2. In some embodiments, the first and second Fc domains further comprise amino acid substitutions selected from the group consisting of L328R;S239K;S267K;S239K / A327G; and S267K / A327G (according to EU numbering), and the Fc domain is derived from the Fc domain of IgG2.

[0109]

[0119] Those skilled in the art will also understand that in the Fc domain derived from IgG4, residue 234 is phenylalanine. Therefore, those skilled in the art will understand that if the Fc domain is derived from the Fc domain of IgG4, then references to L234 (e.g., L234V) herein refer to F234 (e.g., F234V). In some embodiments, the locations of various Fc domain substitutions are relative to the corresponding locations in the Fc domain of wild-type IgG4 (SEQ ID NO: 15). The amino acid sequence of the Fc domain of wild-type IgG4 (SEQ ID NO: 15) is an exemplary sequence provided for comparative purposes, and the Fc domain of the heterodimeric protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) to the Fc domain of wild-type IgG4 (SEQ ID NO: 15). For example, the Fc domain of the heterodimeric protein may be derived from different alleles of wild-type human IgG4. In some embodiments, the Fc domain of the heterodimer protein does not involve any additional amino acid modifications to the Fc domain of wild-type IgG4 (SEQ ID NO: 15).

[0110]

[0120] In some embodiments, each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K;E233P / F234V / L235A / G236del / S239K / A327G;E233P / F234V / L235A / G236del / S267K / A327G; and E233P / F234V / L235A / G236del (as assigned by EU numbering), wherein the Fc domain is derived from the Fc domain of IgG4. In some embodiments, the first Fc domain further comprises amino acid substitutions selected from the group consisting of G236R / L328R;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K;E233P / F234V / L235A / G236del / S239K / A327G;E233P / F234V / L235A / G236del / S267K / A327G; and E233P / F234V / L235A / G236del (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG4 or IgG3. In some embodiments, the second Fc domain further comprises amino acid substitutions selected from the group consisting of G236R / L328R;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K;E233P / F234V / L235A / G236del / S239K / A327G;E233P / F234V / L235A / G236del / S267K / A327G; and E233P / F234V / L235A / G236del (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG4.In some embodiments, the first and second Fc domains further comprise amino acid substitutions selected from the group consisting of G236R / L328R;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K;E233P / F234V / L235A / G236del / S239K / A327G;E233P / F234V / L235A / G236del / S267K / A327G; and E233P / F234V / L235A / G236del (according to EU numbering), wherein the Fc domains are derived from the Fc domain of IgG4.

[0111]

[0121] In some embodiments, the first Fc domain further comprises the amino acid substitution M428L or N434S (according to EU numbering). In some embodiments, the first Fc domain further comprises the amino acid substitution M428L (according to EU numbering). In some embodiments, the first Fc domain further comprises the amino acid substitution N434S (according to EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitution M428L or N434S (according to EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitution M428L (according to EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitution N434S (according to EU numbering). In some embodiments, the first Fc domain further comprises the amino acid substitutions M428L and N434S (according to EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitutions M428L and N434S (according to EU numbering). In some embodiments, each of the first and second Fc domains further comprises amino acid substitutions M428L and N434S (according to EU numbering).

[0112]

[0122] In some embodiments, the first Fc domain and / or the second Fc domain further comprises the amino acid substitution K246T (according to EU numbering). In some embodiments, the first Fc domain further comprises the amino acid substitution K246T (according to EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitution K246T (according to EU numbering). When the K246T substitution appears in the second Fc domain, it is also called the K100T mutation based on the amino acid numbering of the second monomer (see, for example, SEQ ID NOs: 10 and 16). In some embodiments, the first Fc domain and the second Fc domain further comprise the amino acid substitution K246T (according to EU numbering).

[0113]

[0123] In some embodiments, the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain comprises amino acid substitutions S364K and E357Q; and each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering). In some embodiments, the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions L368D and K370S; and each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering).

[0114]

[0124] In some embodiments, the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain comprises amino acid substitutions K246T, S364K, and E357Q; and each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L, and N434S (according to EU numbering). In some embodiments, the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions K246T, K246T and K370S; and each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering).

[0115]

[0125] In some embodiments, the first Fc domain of the heterodimer protein contains the sequence shown in SEQ ID NO: 6. In some embodiments, the second Fc domain of the heterodimer protein contains the sequence shown in SEQ ID NO: 7. In some embodiments, the second Fc domain of the heterodimer protein contains the sequence shown in SEQ ID NO: 8.

[0116]

[0126] In some embodiments, any one of the amino acid substitutions of the Fc variant domain described herein is located on one or both monomers (e.g., on the first Fc domain; on the second Fc domain; or on both Fc domains).

[0117]

[0127] In some embodiments, the Fc domain of the first monomer is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain of the first monomer is derived from IgG1. In some embodiments, the Fc domain of the first monomer is derived from IgG2. In some embodiments, the Fc domain of the first monomer is derived from IgG3. In some embodiments, the Fc domain of the first monomer is derived from IgG4. In some embodiments, the Fc domain of the second monomer is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain of the second monomer is derived from IgG1. In some embodiments, the Fc domain of the second monomer is derived from IgG2. In some embodiments, the Fc domain of the second monomer is derived from IgG3. In some embodiments, the Fc domain of the second monomer is derived from IgG4.

[0118]

[0128] As used herein, “IL-15,” “IL15,” or “interleukin-15” are interchangeable and may refer to a 4-α-helix protein belonging to a family of cytokines. IL-15 signals through a receptor complex composed of the IL-2 / IL-15 receptor β (IL-15Rβ) (CD122) subunit. In some embodiments, the IL-15 protein comprises the polypeptide sequence shown in SEQ ID NO: 2 (full-length human IL-15). In some embodiments, the IL-15 protein comprises the polypeptide sequence shown in SEQ ID NO: 1 (cleaved or mature human IL-15).

[0119]

[0129] In some embodiments, the first monomeric IL-15 protein is an IL-15 protein variant having a different amino acid sequence from the wild-type IL-15 protein (SEQ ID NO: 1). In some embodiments, the IL-15 variant has reduced binding affinity to the IL-2 / IL-15βγ receptor complex (compared to wild-type IL-15) and CD8, with the aim of improving tolerability and expanding pharmacokinetics by reducing acute toxicity. +It is engineered to ultimately promote anti-tumor immunity via IL-15-mediated signaling on T cells and NK cells. In some embodiments, the sequence of the first monomeric IL-15 protein variant has at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid substitutions compared to the wild-type IL-15 sequence protein (SEQ ID NO: 1). In some embodiments, the amino acid substitutions may include one or more amino acid substitutions or deletions in the domain of IL-15 that interacts with IL-15R and / or the IL-2 / IL-15βγ receptor complex. In some embodiments, the amino acid substitutions may include one or more amino acid substitutions or deletions in the domain of the IL-15 protein that result in a reduced binding affinity to the IL-2 / IL-15βγ receptor complex compared to the affinity of wild-type IL-15. In some embodiments, the IL-15 protein includes one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E. In some embodiments, the IL-15 protein includes one or more amino acid substitutions selected from the group consisting of E87C, V49C, L52C, E89C, Q48C, E53C, C42S, and L45C. The amino acid substitutions of the IL-15 protein disclosed herein are relative to wild-type IL-15 (mature form; SEQ ID NO: 1). The amino acid sequence of wild-type IL-15 (mature form; SEQ ID NO: 1) is an exemplary sequence provided for comparison purposes, and the heterodimeric IL-15 protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to wild-type IL-15. For example, the heterodimeric IL-15 protein may be derived from different wild-type human IL-15 alleles. In some embodiments, the heterodimeric IL-15 protein does not contain any additional amino acid modifications compared to wild-type IL-15. In some embodiments, the IL-15 protein variant present in the first monomer contains the amino acid sequence shown in SEQ ID NO: 5 (XENP24306 / XENP32803).

[0120]

[0130] In some embodiments, the IL-15 protein includes the amino acid substitutions D30N, E64Q, and N65D. In some embodiments, the IL-15 protein includes the following amino acid substitutions: N4D and N65D. In some embodiments, the IL-15 protein includes the following amino acid substitutions: D30N and N65D. In some embodiments, the IL-15 protein present in the first monomer includes the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q. In some embodiments, the IL-15 protein present in the first monomer includes the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q. In some embodiments, the IL-15 protein present in the first monomer includes the N65D amino acid substitution and consists of the amino acid substitutions N4D, D30N, and E64Q. The amino acid substitutions of the IL-15 protein disclosed herein are relative to wild-type IL-15 (SEQ ID NO: 1). The amino acid sequence of wild-type IL-15 (SEQ ID NO: 1) is an exemplary sequence provided for comparison purposes, and the heterodimeric IL-15 protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to wild-type IL-15. For example, the heterodimeric IL-15 protein may be derived from different wild-type human IL-15 alleles. In some embodiments, the heterodimeric IL-15 protein does not include any additional amino acid modifications compared to wild-type IL-15.

[0121]

[0131] The IL-15Rα protein is a transmembrane protein with extremely high affinity for IL-15 that facilitates the transport of IL-15 from the endoplasmic reticulum (ER) through the cytoplasm and the presentation of the IL-15 / IL-15Rα complex on the cell surface. As used herein, the term "success domain of IL-15Rα" refers to the cleaved extracellular domain of IL-15Rα or recombinant human IL-15 receptor α. In some embodiments, the IL-15Rα protein contains the polypeptide sequence of SEQ ID NO: 3 (full-length human IL-15Rα). In some embodiments, the IL-15Rα protein contains the polypeptide sequence of SEQ ID NO: 4 (success domain of human IL-15Rα).

[0122]

[0132] In some embodiments, the IL-15Rα protein includes one or more amino acid modifications selected from the group consisting of DPC or DCA insertions after residues 65 (65DPC or D96 / P97 / C98, 65DCA or D96 / C97 / A98), S40C, K34C, G38C, L42C, and A37C. The numbering of these amino acid substitutions in the IL-15Rα protein is relative to the sucoid domain of human IL-15Rα (SEQ ID NO: 4). The amino acid sequence of the sucoid domain of human IL-15Rα (SEQ ID NO: 4) is an exemplary sequence provided for comparison purposes, and the heterodimeric IL-15Rα protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the sucoid domain of human IL-15Rα (SEQ ID NO: 4). For example, the heterodimeric IL-15Rα protein may be derived from different wild-type human IL-15Rα alleles. In some embodiments, the heterodimeric IL-15Rα protein does not involve any additional amino acid modifications to the sucrose domain (SEQ ID NO: 4) of human IL-15Rα.

[0123]

[0133] In some embodiments, the IL-15 protein and the IL-15Rα protein each contain a set of amino acid substitutions or additions selected from the group consisting of E87C:65DPC (DPC insertion after residue 65 or D96 / P97 / C98); E87C:65DCA (DCA insertion after residue 65 or D96 / C97 / A98); V49C:S40C; L52C:S40C; E89C:K34C; Q48C:G38C; E53C:L42C; C42S:A37C; and L45C:A37C. The numbering of these amino acid substitutions in the IL-15Rα protein is relative to the sucoid domain (SEQ ID NO: 4) of human IL-15Rα. The amino acid sequence of the human IL-15Rα sucoid domain (SEQ ID NO: 4) is provided as an exemplary sequence for comparison purposes, and the heterodimeric IL-15Rα protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) to the human IL-15Rα sucoid domain (SEQ ID NO: 4). For example, the heterodimeric IL-15Rα protein may be derived from different wild-type human IL-15Rα alleles. In some embodiments, the heterodimeric IL-15Rα protein does not include any additional amino acid modifications to the human IL-15Rα sucoid domain (SEQ ID NO: 4).

[0124]

[0134] In some embodiments, the IL-15Rα protein contains the amino acid sequence of SEQ ID NO: 3 (full-length human IL-15Rα). In some embodiments, the IL-15Rα protein contains the amino acid sequence of SEQ ID NO: 4 (sucrose domain of human IL-15Rα). In some embodiments, the IL-15 protein contains the amino acid substitutions D30N, E64Q and N65D; the IL-15Rα protein contains SEQ ID NO: 4 (sucrose domain of human IL-15Rα).

[0125]

[0135] The heterodimer protein of this disclosure is an IL-15 / IL-15Rα-Fc heterodimer fusion protein. One N-terminus of the heterodimer Fc domain is covalently bound to the C-terminus of the IL-15 protein, and the other N-terminus is covalently bound to the sucrose domain (cleaved extracellular region) of IL-15Rα. In some embodiments, the IL-15 protein and IL-15Rα (sucrose domain) may have a variable-length linker between the C-terminuses of IL-15 and IL-15Rα and the N-terminus of each Fc domain. In some embodiments, the IL-15 protein is covalently bound to the N-terminus of the first Fc domain via a first linker. In some embodiments, the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain using a second linker. As used herein, the term “linker” refers to a polypeptide sequence that links two or more domains. The properties of linkers and their stability for particular purposes are known in the art. For example, refer to Chen et al. Adv Drug Deliv Rev. October 15;65(10):1357-1369 (2013) (disclosing various types of linkers, their properties, and related linker design tools and databases), which is incorporated herein by reference. In some embodiments, the linker is flexible, rigid, or cleavable in vivo. In some embodiments, the linker is flexible. Flexible linkers typically contain small nonpolar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr). Examples of flexible linkers that can be used in this disclosure are sequences consisting mainly of stretches of Gly and Ser residues ("GS" linkers). In some embodiments, the flexible linker contains repeats of four Gly and Ser residues. In some embodiments, the flexible linker contains 1-5 repeats of five Gly and Ser residues.Non-limiting examples of flexible linkers include (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 39), (Ser-Ser-Ser-Ser-Gly)n (SEQ ID NO: 40), (Gly-Ser-Ser-Gly-Gly)n (SEQ ID NO: 41), and (Gly-Gly-Ser-Gly-Gly)n (SEQ ID NO: 42) (wherein n can be any integer between 1 and 5). In some embodiments, the linker has an amino acid residue length of 5 to 25. In some embodiments, the flexible linker contains 5, 10, 15, 20, or 25 residues. Other suitable linkers may be selected from the group consisting of AS (SEQ ID NO: 43), AST (SEQ ID NO: 44), TVAAPS (SEQ ID NO: 45), TVA (SEQ ID NO: 46), ASTSGPS (SEQ ID NO: 47), KESGSVSSEQLAQFRSLD (SEQ ID NO: 48), EGKSSGSGSESKST (SEQ ID NO: 49), (Gly)6 (SEQ ID NO: 50), (Gly)8 (SEQ ID NO: 51), and GSAGSAAGSGEF (SEQ ID NO: 52). Generally, flexible linkers can provide good flexibility and solubility and function as passive linkers to maintain distance between functional domains. The length of the flexible linker can be adjusted to enable proper folding or to achieve optimal biological activity of the fusion protein. In some embodiments, the linker contains the sequence (Gly-Gly-Gly-Gly-Ser; SEQ ID NO: 53). In some embodiments, the first and second linkers contain different sequences. In some embodiments, the first and second linkers contain the same sequence. In some embodiments, the first and second linkers include the sequence shown in Sequence ID No. 53.

[0126]

[0136] In some embodiments, the heterodimer protein useful in the methods disclosed herein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain, wherein the sucoid domain of the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; each of the first and second Fc domains independently comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises one or more amino acid substitutions selected from the group consisting of the N65D amino acid substitution and N4D, D30N, and E64Q. The various Fc domain substitution locations are based on the corresponding locations in the Fc domain of wild-type IgG1 (SEQ ID NO: 12). The amino acid sequence of the Fc domain of wild-type IgG1 (SEQ ID NO: 12) is an exemplary sequence provided for comparison purposes, and the heterodimeric IL-15Rα protein may contain additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the Fc domain of wild-type IgG1 (SEQ ID NO: 12). For example, the Fc domain of the heterodimeric protein may be derived from different wild-type human IgG1 alleles. In some embodiments, the Fc domain of the heterodimeric protein does not contain any additional amino acid modifications relative to the Fc domain of wild-type IgG1 (SEQ ID NO: 12). The amino acid substitutions of the IL-15 protein disclosed herein are relative to wild-type IL-15 (mature form; SEQ ID NO: 1). The amino acid sequence of wild-type IL-15 (mature form; SEQ ID NO: 1) is provided as an illustrative sequence for comparison purposes, and heterodimeric IL-15 proteins may contain additional amino acid modifications (e.g., substitutions, insertions, and deletions) compared to wild-type IL-15. For example, heterodimeric IL-15 proteins may be derived from different wild-type human IL-15 alleles.In some embodiments, the heterodimeric IL-15 protein does not contain any additional amino acid modifications compared to wild-type IL-15.

[0127]

[0137] Those skilled in the art will be able to determine the corresponding substitutions in the Fc domain derived from the Fc domain of IgG2, IgG3, or IgG4. For example, those skilled in the art will be able to see that residues E233, L234, L235, G236, and A327 are present in the Fc domain derived from the Fc domain of IgG1 or IgG3. In some embodiments, the locations of various Fc domain substitutions are based on the corresponding locations in the Fc domain of wild-type IgG3 (SEQ ID NO: 14). The amino acid sequence of the Fc domain of wild-type IgG3 (SEQ ID NO: 14) is an exemplary sequence provided for comparison purposes, and the IL-15Rα protein of the heterodimeric protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the Fc domain of wild-type IgG3 (SEQ ID NO: 14). For example, the Fc domain of the heterodimeric protein may be derived from different alleles of wild-type human IgG3. In some embodiments, the Fc domain of the heterodimeric protein does not include any additional amino acid modifications relative to the Fc domain of wild-type IgG3 (SEQ ID NO: 14). Therefore, those skilled in the art will understand that each of the first and second Fc domains independently comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering), when the Fc domain is derived from the Fc domain of IgG1 or IgG3.

[0128]

[0138] Those skilled in the art will understand that the corresponding residues in the Fc domain derived from the Fc domain of IgG2 are P233, V234, A235, and G327, and that the Fc domain derived from IgG2 lacks the residue corresponding to residue G236. Therefore, those skilled in the art will understand that if the Fc domain is the Fc domain of IgG2 (i.e., the PVA-sequence present in wild-type IgG2), then the references to E233P, L234V, L235A, G236del, and A327G herein refer to P233, V234, A235, and -236, and not to the substitution at residue 327. In some embodiments, the locations of various Fc domain substitutions are based on the corresponding locations in the Fc domain of wild-type IgG2 (SEQ ID NO: 13). The amino acid sequence of the Fc domain of wild-type IgG2 (SEQ ID NO: 13) is provided as an exemplary sequence for comparative purposes, and the IL-15Rα protein of the heterodimeric protein may contain additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the Fc domain of wild-type IgG2 (SEQ ID NO: 13). For example, the Fc domain of the heterodimeric protein may be derived from different wild-type human IgG2 alleles. In some embodiments, the Fc domain of the heterodimeric protein does not contain any additional amino acid modifications relative to the Fc domain of wild-type IgG2 (SEQ ID NO: 13). Therefore, those skilled in the art will understand that each of the first and second Fc domains independently contains the amino acid substitution S267K (according to EU numbering) when the Fc domain is derived from the Fc domain of IgG2.

[0129]

[0139] Those skilled in the art will understand that in the Fc domain derived from IgG4, residue 234 is phenylalanine and residue 327 is glycine. Therefore, those skilled in the art will understand that, when the Fc domain is derived from the IgG4Fc domain, references to L234 (e.g., L234V) and A327 (e.g., A327G) herein refer to F234 (e.g., F234V), respectively, and not to substitutions at residue 327. In some embodiments, the locations of various Fc domain substitutions are relative to the corresponding locations in the Fc domain of wild-type IgG4 (SEQ ID NO: 15). The amino acid sequence of the Fc domain of wild-type IgG4 (SEQ ID NO: 15) is an exemplary sequence provided for comparative purposes, and the heterodimeric IL-15Rα protein may include additional amino acid modifications (e.g., substitutions, insertions, and deletions) relative to the Fc domain of wild-type IgG4 (SEQ ID NO: 15). For example, the Fc domain of the heterodimeric protein may be derived from different wild-type human IgG4 alleles. In some embodiments, the Fc domain of the heterodimeric protein does not involve any additional amino acid modifications to the Fc domain of wild-type IgG4 (SEQ ID NO: 15). Therefore, those skilled in the art will understand that when the Fc domain is derived from the Fc domain of IgG4, each of the first and second Fc domains independently contains the amino acid substitutions E233P, F234V, L235A, G236del, and S267K (according to EU numbering).

[0130]

[0140] In some embodiments, the first Fc domain and / or the second Fc domain are independently manipulated to further extend systemic exposure at lower pH (6.0) and to lengthen the half-life through enhanced FcRn binding. In some embodiments, additional manipulation of the Fc region results in the heterodimer protein of this disclosure becoming effector-less (i.e., inactivating binding to the Fcγ receptor) and eliminating antibody-mediated CL on T cells and NK cells. In some embodiments, the first Fc domain and / or the second Fc domain are independently manipulated to promote heterodimerization over homodimerization. In some embodiments, the first Fc domain and / or the second Fc domain are independently manipulated to have an improved PK. In some embodiments, the first Fc domain and / or the second Fc domain are independently manipulated to allow purification of homodimers from the heterodimer by increasing the pI difference between the two monomers. In some embodiments, the Fc variant domain may further include molecules or sequences lacking one or more naive Fc amino acid residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) terminal heterogeneity (N-terminal heterogeneity) upon expression in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than neonatal receptors, (7) antibody-dependent cell-mediated cytotoxicity (ADCC), or (8) antibody-dependent phagocytosis (ADCP). The Fc variants are described in more detail below.

[0131]

[0141] In some embodiments, the first or second Fc domain of this disclosure may contain a “skew” variant (e.g., a set of amino acid substitutions shown in Figure 1A-1C of U.S. Patent No. 10,259,887; all of these are incorporated herein by reference in their entirety). The skew variant promotes the formation of heterodimers rather than homodimers. In some embodiments, the scuba ant is selected from the group consisting of S364K / E357Q (of the first Fc domain):L368D / K370S (of the second Fc domain);L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K370S:S364K,E357L,K370S S364K / E357Q, T366S / L368A / Y407V:T366W and T366S / L368A / Y407V / Y349C:T366W / S354C (according to EU numbering). In some embodiments, the first Fc domain further comprises amino acid substitutions L368D and K370S, and the second Fc domain further comprises amino acid substitutions S364K and E357Q (according to EU numbering).

[0132]

[0142] In some embodiments, the first Fc domain further comprises an amino acid substitution selected from the group consisting of Q295E, N384D, Q418E, and N421D, or combinations thereof (as assigned by EU numbering). In some embodiments, the second Fc domain further comprises any one of the amino acid substitutions selected from the group consisting of Q295E, N384D, Q418E, and N421D, or combinations thereof (as assigned by EU numbering). In some embodiments, the first Fc domain further comprises the amino acid substitutions Q295E, N384D, Q418E, and N421D (as assigned by EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitutions Q295E, N384D, Q418E, and N421D (as assigned by EU numbering). In some embodiments, the first and second Fc domains further comprise amino acid substitutions Q295E, N384D, Q418E, and N421D (according to EU numbering).

[0133]

[0143] In some embodiments, the first Fc domain does not contain free cysteine ​​at position 220. In some embodiments, the first Fc domain contains the amino acid substitution C220S (according to EU numbering). In some embodiments, the second Fc domain does not contain free cysteine ​​at position 220. In some embodiments, the second Fc domain contains the amino acid substitution C220S (according to EU numbering). In some embodiments, neither the first nor the second Fc domain contains free cysteine ​​at position 220. In some embodiments, neither the first nor the second Fc domain contains the amino acid substitution C220S (according to EU numbering).

[0134]

[0144] In some embodiments, the first or second Fc domain of the Disclosure may include an amino acid substitution (Xtend substitution) for an improved PK. In some embodiments, the first and / or second Fc domains of the Disclosure independently include the amino acid substitutions M428L and / or N434S (as assigned by EU numbering). In some embodiments, the first Fc domain includes the amino acid substitutions M428L or N434S. In some embodiments, the first Fc domain includes the amino acid substitutions M428L and N434S. In some embodiments, the first Fc domain includes the amino acid substitution M428L. In some embodiments, the first Fc domain includes the amino acid substitution N434S. In some embodiments, the second Fc domain includes the amino acid substitutions M428L or N434S. In some embodiments, the second Fc domain includes the amino acid substitutions M428L and N434S. In some embodiments, the second Fc domain includes the amino acid substitution M428L. In some embodiments, the second Fc domain includes the amino acid substitution N434S.

[0135]

[0145] In some embodiments, the first Fc domain and / or the second Fc domain further comprises the amino acid substitution K246T (according to EU numbering). In some embodiments, the first Fc domain further comprises the amino acid substitution K246T (according to EU numbering). In some embodiments, the second Fc domain further comprises the amino acid substitution K246T (according to EU numbering). When the K246T substitution appears in the second Fc domain, it is also called the K100T mutation based on the amino acid numbering of the second monomer (see, for example, SEQ ID NOs: 10 and 16). In some embodiments, the first Fc domain and the second Fc domain further comprise the amino acid substitution K246T (according to EU numbering).

[0136]

[0146] In some embodiments, the first Fc domain of the heterodimer protein contains the sequence shown in SEQ ID NO: 6. In some embodiments, the second Fc domain of the heterodimer protein contains the sequence shown in SEQ ID NO: 7. In some embodiments, the second Fc domain of the heterodimer protein contains the sequence shown in SEQ ID NO: 8.

[0137]

[0147] In some embodiments, any one of the amino acid substitutions of the Fc variant domain described herein is located on one or both monomers (e.g., on the first Fc domain; on the second Fc domain; or on both Fc domains).

[0138]

[0148] In some embodiments, the Fc domain of the first monomer is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain of the first monomer is derived from IgG1. In some embodiments, the Fc domain of the first monomer is derived from IgG2. In some embodiments, the Fc domain of the first monomer is derived from IgG3. In some embodiments, the Fc domain of the first monomer is derived from IgG4. In some embodiments, the Fc domain of the second monomer is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain of the second monomer is derived from IgG1. In some embodiments, the Fc domain of the second monomer is derived from IgG2. In some embodiments, the Fc domain of the second monomer is derived from IgG3. In some embodiments, the Fc domain of the second monomer is derived from IgG4.

[0139]

[0149] In some embodiments, the first Fc domain includes the following amino acid substitutions: C220S, E233P, L234V, L235A, G236del, S267K, L368D, K370S, M428L and N434S (according to EU numbering).

[0149] In some embodiments, the second Fc domain includes the following amino acid substitutions: C220S, E233P, L234V, L235A, G236del, S267K, S364K, E357Q, M428L and N434S (according to EU numbering). In some embodiments, the second Fc domain includes the following amino acid substitutions: C220S, E233P, L234V, L235A, G236del, S267K, L368D, K370S, M428L, and N434S (as assigned by EU numbering). In some embodiments, the first Fc domain includes the following amino acid substitutions: C220S, E233P, L234V, L235A, G236del, S267K, S364K, E357Q, M428L, and N434S (as assigned by EU numbering). In some embodiments, the first Fc domain does not include any additional amino acid changes compared to the Fc domain of wild-type IgG. In some embodiments, the first Fc domain does not include any additional amino acid changes compared to the Fc domain of wild-type IgG1. In some embodiments, the first Fc domain does not include any additional amino acid changes compared to SEQ ID NO: 12. In some embodiments, the second Fc domain does not include any additional amino acid modifications compared to the Fc domain of wild-type IgG. In some embodiments, the second Fc domain does not include any additional amino acid modifications compared to the Fc domain of wild-type IgG1. In some embodiments, the second Fc domain does not include any additional amino acid modifications compared to SEQ ID NO: 12.

[0140]

[0150] In some embodiments, each of the first Fc domain and the second Fc domain independently includes an additional set of amino acid substitutions selected from the group consisting of G236R, S239K, L328R, and A327G (according to EU numbering).

[0141]

[0151] In some embodiments, the Fc domain of the first monomer is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain of the first monomer is derived from IgG1. In some embodiments, the Fc domain of the first monomer is derived from IgG2. In some embodiments, the Fc domain of the first monomer is derived from IgG3. In some embodiments, the Fc domain of the first monomer is derived from IgG4. In some embodiments, the Fc domain of the second monomer is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain of the second monomer is derived from IgG1. In some embodiments, the Fc domain of the second monomer is derived from IgG2. In some embodiments, the Fc domain of the second monomer is derived from IgG3. In some embodiments, the Fc domain of the second monomer is derived from IgG4.

[0142]

[0152] In some embodiments, the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising a wild-type sucoid domain of an IL-15Rα protein and a second Fc domain, wherein the sucoid domain of the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first Fc domain is substituted with amino acid C220S, E2 The IL-15 protein contains 33P, L234V, L235A, G236del, S267K, Q295E, L368D, K370S, N384D, Q418E, N421D, M428L, and N434S, and the second Fc domain contains amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, E357Q, S364K, M428L, and N434S (according to EU numbering); the IL-15 protein contains amino acid substitutions D30N, E64Q, and N65D compared to the wild-type IL-15 protein (SEQ ID NO: 1).

[0143]

[0153] In some embodiments, the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising a sucoid domain of a wild-type IL-15Rα protein and a second Fc domain, wherein the sucoid domain of the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first Fc domain is substituted with amino acid C220S, E2 The IL-15 protein contains 33P, L234V, L235A, G236del, S267K, Q295E, E357Q, S364K, N384D, Q418E, N421D, M428L, and N434S; the second Fc domain contains amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, L368D, K370S, M428L, and N434S (according to EU numbering); the IL-15 protein contains amino acid substitutions D30N, E64Q, and N65D compared to the wild-type IL-15 protein (SEQ ID NO: 1).

[0144]

[0154] In some embodiments, the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising a sucoid domain of a wild-type IL-15Rα protein and a second Fc domain, wherein the sucoid domain of the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first Fc domain is substituted with amino acids C220S, E233P, L2 The IL-15 protein contains 34V, L235A, G236del, S267K, Q295E, L368D, K370S, N384D, Q418E, N421D, M428L, and N434S, and the second Fc domain contains amino acid substitutions C220S, C220S, E233P, L234V, L235A, G236del, K246T, S267K, E357Q, S364K, M428L, and N434S (according to EU numbering); the IL-15 protein contains amino acid substitutions D30N, E64Q, and N65D compared to the wild-type IL-15 protein (SEQ ID NO: 1).

[0145]

[0155] In some embodiments, the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising a sucoid domain of a wild-type IL-15Rα protein and a second Fc domain, wherein the sucoid domain of the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first Fc domain is substituted with amino acids C220S, E233P, L2 The IL-15 protein contains 34V, L235A, G236del, S267K, Q295E, E357Q, S364K, N384D, Q418E, N421D, M428L, and N434S; the second Fc domain contains amino acid substitutions C220S, C220S, E233P, L234V, L235A, G236del, K246T, S267K, L368D, K370S, M428L, and N434S (according to EU numbering); the IL-15 protein contains amino acid substitutions D30N, E64Q, and N65D compared to the wild-type IL-15 protein (SEQ ID NO: 1).

[0146]

[0156] In some embodiments, the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 16.

[0147]

[0157] In some embodiments, the first monomer comprises (1) IL-15 and (2) a first Fc domain containing the sequence shown in SEQ ID NO: 6. In some embodiments, the second monomer comprises (1) IL-15Rα and (2) a second Fc domain containing the sequence shown in SEQ ID NO: 7.

[0148]

[0158] In some embodiments, amino acid substitutions present in heterodimeric proteins are disclosed in U.S. Patent Application Publication No. 2018 / 0118805, which is incorporated herein by reference in its entirety.

[0149]

[0159] The sequences referenced herein are shown in Table 1 below. TIFF2026062701000002.tif221170TIFF2026062701000003.tif234170TIFF2026062701000004.tif220170TIFF2026062701000005.tif118170

[0150]

[0160] In some embodiments, the heterodimer proteins of this disclosure are XENP20818, XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21475, XENP21476, XENP21477, XENP21988, XENP21989, XENP21990, XENP21991, XENP219 92, XENP22013, XENP22014, XENP22015, XENP22017, XENP22815, XENP22816, XENP22817, XENP22818, XENP22819, XENP22820, XENP22821, XENP22822, XENP22823, XENP22824, XENP22825, XENP22826, XEN P22827, XENP22828, XENP22829, XENP22830, XENP22831, XENP22832, XENP22833, XENP22834, XENP23 343, XENP23472, XENP23504, XENP23554, XENP23555, XENP23557, XENP23559, XENP23560, XENP23561 A heterodimer protein is selected from the group consisting of XENP24017, XENP24018, XENP24019, XENP24020, XENP24043, XENP24044, XENP24046, XENP24051, XENP24052, XENP24113, XENP24301, XENP24306, XENP24341, and XENP32803. Its sequence is disclosed in Figures 104A-104AY of U.S. Patent No. 10,501,543, which are incorporated herein by reference.

[0151]

[0161] In some embodiments, the heterodimer proteins of this disclosure are selected from the group consisting of the heterodimer proteins XENP22822, XENP23504, XENP24045, XENP24306, XENP22821, XENP23343, XENP23557, XENP24113, XENP24051, XENP24341, XENP24052, XENP24301, and XENP32803. These are listed in Table 2 below. The sequences of XENP22822, XENP23504, XENP24045, XENP24306, XENP22821, XENP23343, XENP23557, XENP24113, XENP24051, XENP24341, XENP24052, and XENP24301 are also shown in U.S. Patent Application Publication No. 2018 / 0118805, which is incorporated herein by reference. In some embodiments, the heterodimer protein of the Disclosure is XENP24306. In some embodiments, the heterodimer protein of the Disclosure is XENP32803. In some embodiments, a combination of two or more heterodimer proteins of the Disclosure (e.g., 2, 3, 4, 5, etc.) is used in the methods disclosed herein. In some embodiments, a combination of two heterodimer proteins of the Disclosure is used in the methods disclosed herein. In some embodiments, a combination of XENP24306 and XENP32803 is used in the methods disclosed herein.

[0152]

[0162] In some embodiments, the XENP24306 protein accounts for approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 85% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 84% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 83% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 82% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 81% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 80% of the combined heterodimer protein.

[0153]

[0163] In some embodiments, the XENP32803 protein accounts for approximately 95%, 90%, 85%, 80%, 75%, 70%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for approximately 15% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for approximately 16% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for approximately 17% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for approximately 18% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for approximately 19% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for approximately 20% of the combined heterodimer protein.

[0154]

[0164] In some embodiments, the XENP24306 protein accounts for approximately 50–100%, approximately 70–95%, approximately 80–90%, or approximately 80–85% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP32803 protein accounts for approximately 1–50%, approximately 5–30%, approximately 10–20%, or approximately 15–20% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 85% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 15% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 84% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 16% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 83% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 17% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 82% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 18% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 81% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 19% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for approximately 80% of the combined heterodimer protein, and the XENP32803 protein accounts for approximately 20% of the combined heterodimer protein.

[0155]

[0165] In some embodiments, the XENP24306 protein accounts for 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 85% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 84% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 83% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 82% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 81% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 80% of the combined heterodimer protein.

[0156]

[0166] In some embodiments, the XENP32803 protein accounts for 95%, 90%, 85%, 80%, 75%, 70%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for 15% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for 16% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for 17% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for 18% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for 19% of the combined heterodimer protein. In some embodiments, the XENP32803 protein accounts for 20% of the combined heterodimer protein.

[0157]

[0167] In some embodiments, the XENP24306 protein accounts for 50-100%, 70-95%, 80-90%, or 80-85% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the XENP32803 protein accounts for 1-50%, 5-30%, 10-20%, or 15-20% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 85% of the combined heterodimer protein, and the XENP32803 protein accounts for 15% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 84% of the combined heterodimer protein, and the XENP32803 protein accounts for 16% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 83% of the combined heterodimer protein, and the XENP32803 protein accounts for 17% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 82% of the combined heterodimer protein, and the XENP32803 protein accounts for 18% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 81% of the combined heterodimer protein, and the XENP32803 protein accounts for 19% of the combined heterodimer protein. In some embodiments, the XENP24306 protein accounts for 80% of the combined heterodimer protein, and the XENP32803 protein accounts for 20% of the combined heterodimer protein. TIFF2026062701000006.tif248170TIFF2026062701000007.tif240170TIFF2026062701000008.tif240170 TIFF2026062701000009.tif239170TIFF2026062701000010.tif240170TIFF2026062701000011.tif122170

[0158] Therapeutic methods using IL15-IL15Rα heterodimer Fc fusion proteins

[0168] In one embodiment, the present disclosure provides a method for treating a solid tumor in a subject requiring treatment of a solid tumor, the method comprising administering to the subject a therapeutically effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein.

[0159]

[0169] In another aspect, the Disclosure provides any one or any combination thereof of the heterodimeric proteins disclosed herein for use in the treatment of solid tumors in subjects requiring treatment of solid tumors.

[0160]

[0170] In another aspect, the Disclosure provides the use of any one or any combination thereof of the heterodimeric proteins disclosed herein in the manufacture of a pharmaceutical for the treatment of solid tumors in subjects requiring treatment of solid tumors.

[0161]

[0171] In some embodiments, a combination of two or more heterodimer proteins (e.g., 2, 3, 4, 5, 6, etc.) is used in the method described herein. In some embodiments, a combination of a first heterodimer protein and a second heterodimer protein is administered to a subject.

[0162]

[0172] In some embodiments, the first heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 10; the second heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 16.

[0163]

[0173] In some embodiments, the first heterodimer protein accounts for approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 85% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 84% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 83% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 82% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 81% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 80% of the combined heterodimer protein.

[0164]

[0174] In some embodiments, the second heterodimer protein accounts for approximately 95%, 90%, 85%, 80%, 75%, 70%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the combination. In some embodiments, the second heterodimer protein accounts for approximately 15% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for approximately 16% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for approximately 17% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for approximately 18% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for approximately 19% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for approximately 20% of the combined heterodimer protein.

[0165]

[0175] In some embodiments, the first heterodimer protein accounts for about 50–100%, 70–95%, 80–90%, or 80–85% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the second heterodimer protein accounts for about 1–50%, 5–30%, 10–20%, or 15–20% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for about 85% of the combined heterodimer protein, and the second heterodimer protein accounts for about 15% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for about 84% of the combined heterodimer protein, and the second heterodimer protein accounts for about 16% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 83% of the combined heterodimer protein, and the second heterodimer protein accounts for approximately 17% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 82% of the combined heterodimer protein, and the second heterodimer protein accounts for approximately 18% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 81% of the combined heterodimer protein, and the second heterodimer protein accounts for approximately 19% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for approximately 80% of the combined heterodimer protein, and the second heterodimer protein accounts for approximately 20% of the combined heterodimer protein.

[0166]

[0176] In some embodiments, the first heterodimer protein accounts for 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 85% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 84% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 83% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 82% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 81% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 80% of the combined heterodimer protein.

[0167]

[0177] In some embodiments, the second heterodimer protein accounts for 95%, 90%, 85%, 80%, 75%, 70%, 75%, 70%, 65%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the combination. In some embodiments, the second heterodimer protein accounts for 15% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for 16% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for 17% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for 18% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for 19% of the combined heterodimer protein. In some embodiments, the second heterodimer protein accounts for 20% of the combined heterodimer protein.

[0168]

[0178] In some embodiments, the first heterodimer protein accounts for 50-100%, 70-95%, 80-90%, or 80-85% of the combined heterodimer protein. In some embodiments of any of the methods disclosed herein, the second heterodimer protein accounts for 1-50%, 5-30%, 10-20%, or 15-20% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 85% of the combined heterodimer protein, and the second heterodimer protein accounts for 15% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 84% of the combined heterodimer protein, and the second heterodimer protein accounts for 16% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 83% of the combined heterodimer protein, and the second heterodimer protein accounts for 17% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 82% of the combined heterodimer protein, and the second heterodimer protein accounts for 18% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 81% of the combined heterodimer protein, and the second heterodimer protein accounts for 19% of the combined heterodimer protein. In some embodiments, the first heterodimer protein accounts for 80% of the combined heterodimer protein, and the second heterodimer protein accounts for 20% of the combined heterodimer protein.

[0169]

[0179] In some embodiments, the first heterodimer protein and the second heterodimer protein are administered simultaneously. In some embodiments, the first heterodimer protein and the second heterodimer protein are administered sequentially. In some embodiments, the first heterodimer protein is administered before the second heterodimer protein. In some embodiments, the second heterodimer protein is administered before the first heterodimer protein. In some embodiments, the first heterodimer protein and the second heterodimer protein are administered in the same composition. In some embodiments, the first heterodimer protein and the second heterodimer protein are administered in separate compositions.

[0170]

[0180] A solid tumor refers to an abnormal mass of tissue that does not typically contain a cyst or fluid area. Different types of solid tumors are named according to the type of cells that form them. Solid tumors treated by the methods and uses disclosed herein include, but are not limited to, carcinomas, lymphomas, blastomas, and sarcomas. Further detailed examples of such tumors include squamous cell carcinoma, cutaneous squamous cell carcinoma (cSCC), small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), gastrointestinal cancer, stomach cancer (GC), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liposarcoma, soft tissue sarcoma, urothelial carcinoma (UCC), ureter and renal pelvis, multiple myeloma, osteosarcoma, hepatocellular carcinoma, melanoma, stomach cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cell carcinoma (RCC), liver cancer, esophageal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, Merkel cell carcinoma (MCC), germ cell carcinoma, high microsatellite instability-H (MSI-H) cancer, and head and neck cancer. In some embodiments, solid tumors are locally progressive, recurrent, or metastatic incurable solid tumors. In some embodiments, the solid tumor is selected from the group consisting of melanoma, NSCLC, head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), UCC, RCC, SCLC, GC, MCC, cSCC, and MSI-H cancer. In some embodiments, the solid tumor is selected from melanoma, RCC, NSCLC, HNSCC, and TNBC. In some embodiments, the solid tumor is melanoma. In some embodiments, the solid tumor is RCC. In some embodiments, the solid tumor is selected from melanoma, RCC, and NSCLC. In some embodiments, the solid tumor is selected from melanoma, NSCLC, HNSCC, and TNBC. In some embodiments, the solid tumor is NSCLC. In some embodiments, the solid tumor is HNSCC. In some embodiments, the solid tumor is TNBC. In some embodiments, the solid tumor is a solid tumor for which standard treatment is nonexistent, ineffective, unacceptable, or considered inappropriate, or for which clinical trials of an investigational drug have been recognized as standard treatment.

[0171]

[0181] The methods and uses described herein involve administering to a subject a therapeutically effective amount of any or a combination thereof of the heterodimeric proteins described herein, or a composition described herein, in order to produce such an effect. The identification of a subject requiring such treatment may be made at the discretion of the subject or a medical professional, and may be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method). Such treatment would be appropriately administered to subjects who have cancer, have cancer, are susceptible to cancer, or are at risk of cancer.

[0172]

[0182] In another aspect, this disclosure applies to CD8 + A method is provided for inducing the proliferation of effector memory T cells, the method comprising administering to a subject an effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein.

[0173]

[0183] In another aspect, the present disclosure provides a method for inducing the proliferation of NK cells in a subject, the method comprising administering to the subject an effective amount of any one or any combination thereof of the heterodimeric proteins disclosed herein.

[0174]

[0184] In another aspect, the Disclosure provides a method for inducing NK cell proliferation in a subject, the method comprising administering to the subject an effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein, wherein upon administration of an effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein, the NK cell proliferation response is CD8 + This response is stronger than the proliferative response of effector memory T cells.

[0175]

[0185] In another aspect, this disclosure applies to CD8 +The present invention provides a method for inducing the proliferation of effector memory T cells and NK cells, the method comprising administering to a subject an effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein. In some embodiments, upon administration of an effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein, the proliferation response of NK cells is induced by CD8 + This response is stronger than the proliferative response of effector memory T cells.

[0176]

[0186] In another aspect, this disclosure applies to CD4 + A method is provided for inducing the proliferation of effector memory T cells, the method comprising administering to a subject an effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein.

[0177]

[0187] In another aspect, the Disclosure provides a method for inducing IFNγ production in a subject, the method comprising administering to the subject an effective amount of any or any combination thereof of the heterodimeric proteins disclosed herein.

[0178]

[0188] Routes of administration include, but are not limited to, parenteral, oral, or nasal administration, intravenous infusion into the bladder, or via a suitable delivery device or implant containing a conventional non-toxic, pharmaceutically acceptable carrier and adjuvant. In some embodiments, parenteral administration is by injection, infusion, or implantation. In some embodiments, parenteral administration is subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, intradermal, intrathecal, intraosseous, intracardiac, intravesical, intradural, epidural, intracerebral, intraventricular, intrapleural, inhalation, or percutaneous administration. In some embodiments, parenteral administration is subcutaneous. In some embodiments, parenteral administration is intravenous. In some embodiments, parenteral administration is intramuscular. In some embodiments, parenteral administration is intraperitoneal.

[0179]

[0189] In some embodiments, the heterodimer proteins of this disclosure are administered systemically. In some embodiments, the heterodimer proteins are administered topically. In some embodiments, the heterodimer proteins are administered as a composition comprising a pharmaceutically acceptable buffer. Suitable carriers and formulations thereof are described, for example, in Remington's Pharmaceutical Sciences by EW Martin. In some embodiments, the heterodimer proteins are provided in dosage forms suitable for parenteral administration routes.

[0180]

[0190] Compositions containing heterodimer proteins may be provided in unit dose forms (e.g., single-dose ampoules, syringes, or bags). In some embodiments, the heterodimer proteins are provided in vials containing multiple doses. Appropriate preservatives may be added to the composition (see below). The composition may be presented in the form of a solution, suspension, emulsion, infusion device, or delivery device for implantation, or as a dry powder that is reconstituted with water or another suitable vehicle before use. Apart from the heterodimer proteins disclosed herein, the composition may contain suitable carriers and / or additives that are acceptable. In some embodiments, the composition is suitable for parenteral administration. One or more heterodimer proteins may be incorporated into microspheres, microcapsules, nanoparticles, or liposomes, etc., for release control. Furthermore, the composition may contain suspending agents, solubilizers, stabilizers, pH adjusters, isotonic adjusters, and / or dispersants.

[0181]

[0191] Pharmaceutical compositions containing heterodimeric proteins may also be in a form suitable for sterile injection. To prepare such compositions, the proteins are dissolved or suspended in a parenterally acceptable liquid vehicle. Acceptable vehicles and solvents that can be used include water, water adjusted to a suitable pH by the addition of a suitable amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic saline and dextrose solution. Aqueous formulations may contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate).

[0182]

[0192] In some embodiments, the heterodimer proteins of this disclosure are administered orally. Methods for the oral administration of biologically active proteins and peptides are known in the art. Numerous strategies for preventing degradation of orally administered proteins have been demonstrated. Examples of methods for the oral administration of heterodimer proteins include, but are not limited to, the use of core-shell particles (U.S. Patent No. 7,090,868) and nanotubes (U.S. Patent No. 7,195,780); liposomes and aqueous emulsions and suspensions (U.S. Patent No. 7,316,818; International Publication No. 06 / 062544; U.S. Patent No. 6,071,535; and U.S. Patent No. 5,874,105); gas-filled liposomes (U.S. Patent No. 6,551,576; U.S. Patent No. 6,808,720; and U.S. Patent No. 7,083,572); and aqueous media. This includes nanodroplets dispersed in a matrix (U.S. Patent Application Publication 2007 / 0184076); matrix carriers containing peptide effectors that provide penetration across biological barriers for the administration of hydrophobic proteins (International Publications 06 / 097793, 05 / 094785, and 03 / 066859); the use of non-covalent protein-polysaccharide complexes (EP0491114B1); the use of the pharmaceutical composition described in U.S. Patent No. 8,936,786; and the use of the Peptelligence® system (Enteris Biopharma) (International Publications 2014 / 138241, 2016 / 115082, and 2004 / 064758). All of these patent publications and patents are specifically incorporated herein by reference.

[0183]

[0193] The amount of heterodimeric protein of this disclosure administered will vary depending on the form of administration, the patient's age and weight, and the clinical stage of the cancer being treated. Doses for humans can be determined by estimation from the amount of protein used in mice or non-human primates. In some embodiments, the dose may vary by body weight from about 0.0001 mg of protein / kg to about 5 mg / kg of compound; or about 0.001 mg / kg to about 4 mg / kg; or about 0.005 mg / kg to about 1 mg / kg of body weight; or about 0.005 mg / kg to about 0.3 mg / kg; or about 0.005 mg / kg to about 0.2 mg / kg; or about 0.005 mg / kg to about 0.02 mg / kg. In some embodiments, this dose is approximately 0.0001, approximately 0.00025, approximately 0.0003, approximately 0.0005, approximately 0.001, approximately 0.003, approximately 0.005, approximately 0.008, approximately 0.01, approximately 0.015, approximately 0.02, approximately 0.03, approximately 0.04, approximately 0.05, approximately 0.06, approximately 0.07, approximately 0.08, approximately 0.09, approximately 0.1, approximately 0.12, approximately 0.135, approximately 0.15, approximately 0.16, approximately 0.2, approximately 0.2025, approximately 0.24 per kg of body weight. It may be approximately 0.25, 0.3, 0.32, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mg. In some embodiments, the dose is approximately 0.0025 mg, approximately 0.005 mg, approximately 0.01 mg, approximately 0.015 mg / kg, approximately 0.02 mg / kg, approximately 0.025 mg / kg, approximately 0.03 mg / kg, approximately 0.04 mg / kg, approximately 0.05 mg / kg, approximately 0.06 mg / kg, approximately 0.08 mg / kg, approximately 0.1 mg / kg, approximately 0.12 mg / kg, approximately 0.16 mg / kg, approximately 0.2 mg / kg, approximately 0.24 mg / kg, and approximately 0.32 mg / kg by body weight. In some embodiments, the dose is approximately 0.0025 mg / kg by body weight. In some embodiments, the dose is approximately 0.01 mg / kg by body weight. In some embodiments, the dose is approximately 0.015 mg / kg by body weight.In some embodiments, the dose is approximately 0.02 mg / kg of body weight. In some embodiments, the dose is approximately 0.03 mg / kg of body weight. In some embodiments, the dose is approximately 0.04 mg / kg of body weight. In some embodiments, the dose is approximately 0.06 mg / kg of body weight. In some embodiments, the dose is approximately 0.08 mg / kg of body weight. In some embodiments, the dose is approximately 0.09 mg / kg of body weight. In some embodiments, the dose is approximately 0.12 mg / kg of body weight. In some embodiments, the dose is approximately 0.135 mg / kg of body weight. In some embodiments, the dose is approximately 0.16 mg / kg of body weight. In some embodiments, the dose is approximately 0.2025 mg / kg of body weight. In some embodiments, the dose is approximately 0.24 mg / kg of body weight. In some embodiments, the dose is approximately 0.32 mg / kg of body weight. In some embodiments, the heterodimer protein of this disclosure is administered by IV infusion according to these doses.

[0184]

[0194] In some embodiments, the dose may vary by body weight from 0.0001 mg of protein / kg to 5 mg of compound / kg; or from 0.001 mg / kg to 4 mg / kg; or from 0.005 mg / kg to 1 mg / kg; or from 0.005 mg / kg to 0.3 mg / kg; or from 0.005 mg / kg to 0.2 mg / kg; or from 0.005 mg / kg to 0.02 mg / kg. In some embodiments, this dose may vary by body weight from 0.0001, 0.0003, 0.0005, 0.001, 0.003, 0.005, 0.008, 0.01, 0.015, 0.02, 0.03, 0.05, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, It may be 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mg / kg. In some embodiments, the dose is selected from the group consisting of 0.0025 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.08 mg / kg, 0.09 mg / kg, 0.10 mg / kg, 0.12 mg / kg, 0.135 mg / kg, 0.16 mg / kg, 0.20 mg / kg, 0.2025 mg / kg, 0.24 mg / kg, and 0.32 mg / kg in body weight. In some embodiments, the dose is 0.0025 mg / kg in body weight. In some embodiments, the dose is 0.01 mg / kg in body weight. In some embodiments, the dose is 0.015 mg / kg in body weight. In some embodiments, the dose is 0.02 mg / kg of body weight. In some embodiments, the dose is 0.03 mg / kg of body weight. In some embodiments, the dose is 0.04 mg / kg of body weight. In some embodiments, the dose is 0.06 mg / kg of body weight. In some embodiments, the dose is 0.08 mg / kg of body weight. In some embodiments, the dose is 0.09 mg / kg of body weight.In some embodiments, the dose is 0.12 mg / kg of body weight. In some embodiments, the dose is 0.135 mg / kg of body weight. In some embodiments, the dose is 0.16 mg / kg of body weight. In some embodiments, the dose is 0.2025 mg / kg of body weight. In some embodiments, the dose is 0.24 mg / kg of body weight. In some embodiments, the dose is 0.32 mg / kg of body weight. In some embodiments, the heterodimer protein of this disclosure is administered by IV infusion according to these doses.

[0185]

[0195] In some embodiments, the dose of the heterodimeric protein combination may vary by body weight from about 0.0001 mg of protein / kg to about 5 mg of compound / kg; or from about 0.001 mg / kg to about 4 mg / kg; or from about 0.005 mg / kg to about 1 mg / kg; or from about 0.005 mg / kg to about 0.3 mg / kg; or from about 0.005 mg / kg to about 0.2 mg / kg; or from about 0.005 mg / kg to about 0.02 mg / kg. In some embodiments, this dose is approximately 0.0001, approximately 0.0003, approximately 0.0005, approximately 0.001, approximately 0.003, approximately 0.005, approximately 0.008, approximately 0.01, approximately 0.015, approximately 0.02, approximately 0.03, approximately 0.05, approximately 0.08, approximately 0.1, approximately 0.15, approximately 0.2, approximately 0.25, approximately 0.3, approximately 0.35, approximately 0.4, approximately 0.45, approximately 0.5, It may be approximately 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mg / kg. In some embodiments, the dose is approximately 0.0025 mg, approximately 0.005 mg, approximately 0.01 mg, approximately 0.015 mg / kg, approximately 0.02 mg / kg, approximately 0.025 mg / kg, approximately 0.03 mg / kg, approximately 0.04 mg / kg, approximately 0.05 mg / kg, approximately 0.06 mg / kg, approximately 0.08 mg / kg, approximately 0.10 mg / kg, approximately 0.12 mg / kg, approximately 0.16 mg / kg, approximately 0.20 mg / kg, approximately 0.24 mg / kg, and approximately 0.32 mg / kg by body weight. In some embodiments, the dose is approximately 0.0025 mg / kg by body weight. In some embodiments, the dose is approximately 0.01 mg / kg by body weight. In some embodiments, the dose is approximately 0.015 mg / kg by body weight. In some embodiments, the dose is approximately 0.02 mg / kg by body weight. In some embodiments, the dose is approximately 0.03 mg / kg of body weight. In some embodiments, the dose is approximately 0.04 mg / kg of body weight. In some embodiments, the dose is approximately 0.06 mg / kg of body weight. In some embodiments, the dose is approximately 0.08 mg / kg of body weight.In some embodiments, the dose is approximately 0.12 mg / kg of body weight. In some embodiments, the dose is approximately 0.16 mg / kg of body weight. In some embodiments, the dose is approximately 0.24 mg / kg of body weight. In some embodiments, the dose is approximately 0.32 mg / kg of body weight. In some embodiments, the heterodimeric protein combinations of the present disclosure are administered by IV infusion according to these doses.

[0186]

[0196] In some embodiments, the dose of the heterodimeric protein combination may vary by body weight from 0.0001 mg of protein / kg to 5 mg of compound / kg; or 0.001 mg / kg to 4 mg / kg; or 0.005 mg / kg to 1 mg / kg; or 0.005 mg / kg to 0.3 mg / kg; or 0.005 mg / kg to 0.2 mg / kg; or 0.005 mg / kg to 0.02 mg / kg. In some embodiments, this dose may vary by body weight from 0.0001, 0.0003, 0.0005, 0.001, 0.003, 0.005, 0.008, 0.01, 0.015, 0.02, 0.03, 0.05, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, It may be 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mg / kg. In some embodiments, the doses are 0.0025 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.08 mg / kg, 0.10 mg / kg, 0.12 mg / kg, 0.16 mg / kg, 0.20 mg / kg, 0.24 mg / kg, and 0.32 mg / kg in body weight. In some embodiments, the dose is 0.0025 mg / kg in body weight. In some embodiments, the dose is 0.01 mg / kg in body weight. In some embodiments, the dose is 0.015 mg / kg in body weight. In some embodiments, the dose is 0.02 mg / kg in body weight. In some embodiments, the dose is 0.03 mg / kg in body weight. In some embodiments, the dose is 0.04 mg / kg of body weight. In some embodiments, the dose is 0.06 mg / kg of body weight. In some embodiments, the dose is 0.08 mg / kg of body weight. In some embodiments, the dose is 0.12 mg / kg of body weight. In some embodiments, the dose is 0.16 mg / kg of body weight.In some embodiments, the dose is 0.24 mg / kg of body weight. In some embodiments, the dose is 0.32 mg / kg of body weight. In some embodiments, the heterodimeric protein combinations of this disclosure are administered by IV infusion according to these doses.

[0187]

[0197] In some embodiments, the heterodimer proteins of this disclosure, or combinations thereof, are administered daily, i.e., every 24 hours. In some embodiments, the heterodimer proteins, or combinations thereof, are administered weekly, i.e., once a week (Q1W). In some embodiments, the heterodimer proteins, or combinations thereof, are administered every two weeks, i.e., once every 14 days (Q2W). In some embodiments, the heterodimer proteins, or combinations thereof, are administered every three weeks, i.e., once every 21 days (Q3W). In some embodiments, the heterodimer proteins, or combinations thereof, are administered every four weeks, i.e., once every 28 days (Q4W). In some embodiments, the heterodimer proteins, or combinations thereof, are administered every five weeks (Q5W). In some embodiments, the heterodimer proteins, or combinations thereof, are administered every six weeks (Q6W). In some embodiments, the heterodimer proteins, or combinations thereof, are administered every seven weeks (Q7W). In some embodiments, the heterodimer protein or a combination thereof is administered once every 8 weeks (Q8W). In some embodiments, the heterodimer protein or a combination thereof is administered once every 9 weeks (Q9W). In some embodiments, the heterodimer protein or a combination thereof is administered once every 10 weeks (Q10W). In some embodiments, the heterodimer protein or a combination thereof is administered once every 11 weeks (Q11W). In some embodiments, the heterodimer protein or a combination thereof is administered once every 12 weeks (Q12W). In some embodiments, the heterodimer protein or a combination thereof is administered once every month. In some embodiments, the heterodimer protein or a combination thereof is administered once every two months. In some embodiments, the heterodimer protein or a combination thereof is administered once every three months. In some embodiments, the heterodimer protein or a combination thereof is administered once every four months. In some embodiments, the heterodimer protein or a combination thereof is administered once every five months.In some embodiments, the heterodimer protein or a combination thereof is administered once every six months. In some embodiments, the heterodimer protein or a combination thereof is administered once every seven months. In some embodiments, the heterodimer protein or a combination thereof is administered once every eight months. In some embodiments, the heterodimer protein or a combination thereof is administered once every nine months. In some embodiments, the heterodimer protein or a combination thereof is administered once every ten months. In some embodiments, the heterodimer protein or a combination thereof is administered once every eleven months. In some embodiments, the heterodimer protein or a combination thereof is administered once every twelve months. In some embodiments, the heterodimer protein or a combination thereof is administered once a year. In some embodiments, the heterodimer protein or a combination thereof of this disclosure is administered by intravenous infusion according to the frequencies disclosed herein.

[0188]

[0198] In some embodiments, the subject has not previously received any medication for the treatment of the condition. In some embodiments, the subject is currently receiving a checkpoint inhibitor. In some embodiments, the subject has previously received a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor targets PD-1. In some embodiments, the checkpoint inhibitor targets PD-L1. In some embodiments, the checkpoint inhibitor targets CTLA-4. In some embodiments, the checkpoint inhibitor targeting PD-1 is an anti-PD-1 antibody. Antibodies that specifically bind to PD-1 are known in the art, for example, Naidoo et al. Ann Oncol. 2015;26(12):2375-2391, Philips et al. Int Immunol. 2015;27(1):39-46, Tunger et al. J Clin Med. 2019;8(10) and Sunshine et al. Curr Opin This is described in Pharmacol.2015;32-8; and in U.S. Patent No. 8008449, U.S. Patent No. 8168757, U.S. Patent Publication No. 20110008369, U.S. Patent Publication No. 20130017199, U.S. Patent Publication No. 20130022595, and International Publication No. 2006121168, International Publication No. 20091154335, International Publication No. 2012145493, International Publication No. 2013014668, International Publication No. 2009101611, EP2262837, and EP2504028. Examples of anti-PD-1 antibodies include, but are not limited to, nivolumab (BMS-936558), pembrolizumab (brand name Keytruda, formerly known as lambrolizumab; also known as Merck3475 and SCH-900475), pidilizumab (CT-011), semiprimab, spartalizumab (PDR001), camrelizumab (SHR1210), cintilimab (IBI308), tislerizumab (BGB-A317), tripalimab (JS 001), MDX-1106, AMP-514 (Amplimmune), and AMP-224 (Amplimmune).Nivolumab is an anti-PD-1 antibody described in International Publication No. 2006 / 121168. Pembrolizumab is an anti-PD-1 antibody described in International Publication No. 2009 / 114335 and Hamid et al. (2013). New England Journal of Medicine 369(2):134-44. Pidilizumab is a humanized IgGk monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD1 monoclonal antibodies are disclosed in International Publication No. 2009 / 101611. AMP-224 is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1, and is disclosed in International Publication No. 2010 / 027827 and International Publication No. 2011 / 066342. Other anti-PD-1 antibodies include, among others, AMP514, for example, the anti-PD-1 antibody disclosed in U.S. Patent No. 8,609,089, U.S. Patent Application Publication No. 2010028330 and / or U.S. Patent Application Publication No. 20120114649. In some embodiments, the anti-PD-1 antibody is nivolumab.

[0189]

[0199] In some embodiments, the PD-L1-targeting checkpoint inhibitor is an anti-PD-L1 antibody. Antibodies that specifically bind to PD-L1 are known in the art and are described, for example, in Naidoo et al. Ann Oncol. 2015 Dec;26(12):2375-2391, Philips et al. Int Immunol. 2015 Jan;27(1):39-46, Tunger et al. J Clin Med. 2019 Sep 25;8(10), Sunshine et al. Curr Opin Pharmacol. 2015:32-8, and in U.S. Patent No. 7,943,743 and U.S. Patent Application Publication No. 2012,003,9906. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559 (also known as MSB-0010718C and MDX-1105), BMS-39886, atezolizumab (MDPL3280A; Tecentriq), avelumab (Bavencio), durvalumab (MEDI4736; Imfinzi), KN035, CK-301 (Checkpoint Therapeutics), and MSB0010718C. BMS-936559 is an anti-PD-L1 antibody described in International Publication No. 2007 / 005874. Atezolizumab is a humanized monoclonal antibody containing human Fc-optimized IgG1 that binds to PD-L1. BMS-39886 is an anti-PD-L1 antibody described in Brahmer JR et al. N Engl J Med 2012;366:2455-2465. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0190]

[0200] In some embodiments, CTLA-4-targeting checkpoint inhibitors are anti-CTLA-4 antibodies. Antibodies that specifically bind to CTLA-4 are known in the art and are described, for example, in Callahan MK et al. Semin Oncol. 2010;37(5):473-484. Examples of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab and tremelimumab. Both ipilimumab and tremelimumab are fully human antibodies against CTLA-4. Ipilimumab (also known as MDX-010 or Yervoy; Bristol-Myers Squibb, Princeton, NJ) is an IgG1 with a plasma half-life of 12-14 days (Hodi, FS et al. The New England Journal of Medicine. 2010;363(8):711-723). Tremelimumab (also known as CP-675, 206, or tisilimmab; Pfizer, New York, NY) is an IgG2 with a plasma half-life of approximately 22 days (Reuben, JM et al. Cancer. 2006; 106(11): 2437-44).

[0191] A treatment method using IL15-IL15Rα heterodimer Fc fusion protein and PD-L1 / PD-1 inhibitors as combination therapy.

[0201] Another aspect of this disclosure provides a method for treating solid tumors in subjects requiring treatment of solid tumors, the method comprising administering to a subject an effective amount of (a) any heterodimer protein disclosed herein (i.e., IL15-IL15Rα heterodimer Fc fusion protein) or a combination thereof, and (b) a PD-L1 / PD-1 axis-targeting agent. The heterodimer protein may be administered according to any of the methods disclosed herein. The heterodimer protein may be administered in any of the compositions disclosed herein.

[0192]

[0202] In some embodiments, two or more of the heterodimer proteins disclosed herein may be administered to a subject. In some embodiments, three or more of the heterodimer proteins disclosed herein may be administered to a subject. In some embodiments, four or more of the heterodimer proteins disclosed herein may be administered to a subject. In some embodiments, five or more of the heterodimer proteins disclosed herein may be administered to a subject.

[0193]

[0203] In some embodiments, a combination of a first heterodimer protein and a second heterodimer protein is administered to the subject. In some embodiments, the first heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 10; the second heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 16.

[0194]

[0204] Programmed death-ligand-1 (PD-L1) is a cell surface protein widely expressed by tumor cells and tumor-infiltrating immune cells in many human cancers. Overexpression of PD-L1 is associated with a poor prognosis in patients with multiple cancers. PD-L1 binds to two known receptors, PD-1 and B7.1, whose expression on activated T cells is maintained under prolonged stimuli such as chronic infection or cancer. Ligation of PD-L1 by PD-1 or B7.1 inhibits T cell proliferation, cytokine production, and cytolytic reactions, thereby leading to functional inactivation or inhibition of T cells. Abnormal expression of PD-L1 on tumor cells has been reported to interfere with anti-tumor immunity, resulting in immune evasion. Interference with the PD-L1 / PD-1 and PD-L1 / B7.1 pathways is an attractive strategy for reactivating tumor-specific T-cell immunity, and indeed, multiple inhibitors of PD-L1 or PD-1 have shown clinical efficacy or promising antitumor activity in a wide range of tumor types, including melanoma, RCC, NSCLC, SCLC, urothelial bladder cancer, HNSCC, ovarian cancer, and TNBC. The demonstrated benefits have led to the approval of multiple anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, and durvalumab) and anti-PD-1 antibodies (e.g., nivolumab, pembrolizumab, and semiprimab-rwlc) in selected indications to date.

[0195]

[0205] In some embodiments, the drug targeting the PD-L1 / PD-1 axis is a PD-1 inhibitor.

[0196]

[0206] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. Antibodies that specifically bind to PD-1 are known in the art, for example, Naidoo et al. Ann Oncol. 2015;26(12):2375-2391, Philips et al. Int Immunol. 2015;27(1):39-46, Tunger et al. J Clin Med. 2019;8(10), and Sunshine et al. Curr Opin This is described in Pharmacol.2015;32-8; and in U.S. Patent No. 8008449, U.S. Patent No. 8168757, U.S. Patent Publication No. 20110008369, U.S. Patent Publication No. 20130017199, U.S. Patent Publication No. 20130022595, and International Publication No. 2006121168, International Publication No. 20091154335, International Publication No. 2012145493, International Publication No. 2013014668, International Publication No. 2009101611, EP2262837, and EP2504028. Examples of anti-PD-1 antibodies include, but are not limited to, nivolumab (BMS-936558), pembrolizumab (brand name Keytruda, formerly known as lambrolizumab; also known as Merck3475 and SCH-900475), pidilizumab (CT-011), semiprimab, spartalizumab (PDR001), camrelizumab (SHR1210), cintilimab (IBI308), tislerizumab (BGB-A317), tripalimab (JS 001), MDX-1106, AMP-514 (Amplimmune), and AMP-224 (Amplimmune). Nivolumab is an anti-PD-1 antibody described in International Publication No. 2006 / 121168. Pembrolizumab is an anti-PD-1 antibody described in International Publication No. 2009 / 114335 and Hamid et al. (2013). New England Journal of Medicine 369(2):134-44. Pidilizumab is a humanized IgGk monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD1 monoclonal antibodies are disclosed in International Publication No. 2009 / 101611.AMP-224 is a PD-L2Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1, and is disclosed in International Publication No. 2010 / 027827 and International Publication No. 2011 / 066342. Other anti-PD-1 antibodies include, among others, AMP514, for example, the anti-PD-1 antibody disclosed in U.S. Patent No. 8609089, U.S. Patent Application Publication No. 2010028330 and / or U.S. Patent Application Publication No. 20120114649. In some embodiments, the anti-PD-1 antibody is nivolumab. In some embodiments, the anti-PD-1 antibody is administered in combination with XENP24306. In some embodiments, the anti-PD-1 antibody is administered in combination with XENP32803. In some embodiments, the anti-PD-1 antibody is administered in combination with both XENP24306 and XENP32803. In some embodiments, nivolumab is administered in combination with XENP24306. In some embodiments, nivolumab is administered in combination with XENP32803. In some embodiments, nivolumab is administered in combination with both XENP24306 and XENP32803.

[0197]

[0207] In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. Antibodies that specifically bind to PD-L1 are known in the art and are described, for example, in Naidoo et al. Ann Oncol. 2015 Dec;26(12):2375-2391, Philips et al. Int Immunol. 2015 Jan;27(1):39-46, Tunger et al. J Clin Med. 2019 Sep 25;8(10), Sunshine et al. Curr Opin Pharmacol. 2015:32-8, and in U.S. Patent No. 7,943,743 and U.S. Patent Application Publication No. 2012,003,9906. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559 (also known as MSB-0010718C and MDX-1105), BMS-39886, atezolizumab (MDPL3280A; Tecentriq), avelumab (Bavencio), durvalumab (MEDI4736; Imfinzi), KN035, CK-301 (Checkpoint Therapeutics), and MSB0010718C. BMS-936559 is an anti-PD-L1 antibody described in International Publication No. 2007 / 005874. Atezolizumab is a humanized monoclonal antibody containing human Fc-optimized IgG1 that binds to PD-L1. BMS-39886 is an anti-PD-L1 antibody described in Brahmer JR et al. N Engl J Med 2012;366:2455-2465. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody is administered in combination with XENP24306. In some embodiments, the anti-PD-L1 antibody is administered in combination with XENP32803. In some embodiments, the anti-PD-L1 antibody is administered in combination with both XENP24306 and XENP32803. In some embodiments, atezolizumab is administered in combination with XENP24306. In some embodiments, atezolizumab is administered in combination with XENP32803. In some embodiments, atezolizumab is administered in combination with both XENP24306 and XENP32803.

[0198]

[0208] The amount of PD-L1 / PD-1 axis-targeting agents administered in combination with the heterodimeric proteins (or combinations thereof) of this disclosure will vary depending on the method of administration, the patient's age and weight, and the clinical stage of the cancer being treated. In some embodiments, the anti-PD-1 antibody or anti-PD-L1 antibody is administered at its approved dose. A physician will be able to determine the appropriate dose for administration in combination with the proteins of this disclosure. In some embodiments, the PD-L1 / PD-1 axis-targeting agents are administered using approved drug regimens. In some embodiments, the dose may vary by body weight from approximately 0.5 mg of protein / kg to approximately 100 mg of compound / kg; or approximately 1 mg of protein / kg to approximately 100 mg of compound / kg; or approximately 2 mg of protein / kg to approximately 50 mg of compound / kg; or approximately 2.5 mg of protein / kg to approximately 10 mg of compound / kg; or approximately 3 mg of protein / kg to approximately 5 mg of compound / kg. In some embodiments, this dose may be about 0.1, about 0.3, about 0.5, about 1, about 3, about 5, about 7.5, about 10, about 15, about 25, about 50, about 75, or about 100 mg / kg by body weight. In some embodiments, the dose of anti-PD-1 antibody is 3 mg / kg. In some embodiments, the dose of nivolumab is about 3 mg / kg. In some embodiments, the dose of nivolumab is about 3 mg / kg every two weeks. In some embodiments, the dose of nivolumab is about 1 mg / kg. In some embodiments, the dose of nivolumab is about 240 mg. In some embodiments, the dose of nivolumab is about 480 mg. In some embodiments, the dose of nivolumab is about 240 mg every two weeks. In some embodiments, the dose of nivolumab is about 480 mg every four weeks. In some embodiments, the dose of anti-PD-L1 antibody is about 3 mg / kg. In some embodiments, the dose of anti-PD-L1 antibody is approximately 840 mg. In some embodiments, the dose of atezolizumab is approximately 840 mg. In some embodiments, the dose of atezolizumab is approximately 1200 mg. In some embodiments, the dose of atezolizumab is approximately 1680 mg.In some embodiments, the dose of atezolizumab is approximately 840 mg every two weeks. In some embodiments, the dose of atezolizumab is approximately 1200 mg every three weeks. In some embodiments, the dose of atezolizumab is approximately 1680 mg every four weeks. In some embodiments, the dose of pembrolizumab is approximately 200 mg. In some embodiments, the dose of pembrolizumab is approximately 200 mg every three weeks. In some embodiments, the dose of pembrolizumab is approximately 200 mg every two weeks. In some embodiments, the dose of pembrolizumab is approximately 200 mg every week.

[0199]

[0209] In some embodiments, the dose may vary by body weight from 0.5 mg of protein / kg to 100 mg of compound / kg; or 1 mg of protein / kg to 100 mg of compound / kg; or 2 mg of protein / kg to 50 mg of compound / kg; or 2.5 mg of protein / kg to 10 mg of compound / kg; or 3 mg of protein / kg to 5 mg of compound / kg. In some embodiments, this dose may be 0.1, 0.3, 0.5, 1, 3, 5, 7.5, 10, 15, 25, 50, 75, or 100 mg / kg by body weight. In some embodiments, the dose of anti-PD-1 antibody is 3 mg / kg. In some embodiments, the dose of nivolumab is 3 mg / kg. In some embodiments, the dose of nivolumab is 3 mg / kg every two weeks. In some embodiments, the dose of nivolumab is 1 mg / kg. In some embodiments, the dose of nivolumab is 240 mg. In some embodiments, the dose of nivolumab is 480 mg. In some embodiments, the dose of nivolumab is 240 mg every two weeks. In some embodiments, the dose of nivolumab is 480 mg every four weeks. In some embodiments, the dose of anti-PD-L1 antibody is 3 mg / kg. In some embodiments, the dose of anti-PD-L1 antibody is 840 mg. In some embodiments, the dose of atezolizumab is 840 mg. In some embodiments, the dose of atezolizumab is 1200 mg. In some embodiments, the dose of atezolizumab is 1680 mg. In some embodiments, the dose of atezolizumab is 840 mg every two weeks. In some embodiments, the dose of atezolizumab is 1200 mg every three weeks. In some embodiments, the dose of atezolizumab is 1680 mg every four weeks. In some embodiments, the dose of pembrolizumab is 200 mg. In some embodiments, the dose of pembrolizumab is 200 mg every 3 weeks. In some embodiments, the dose of pembrolizumab is 200 mg every 2 weeks. In some embodiments, the dose of pembrolizumab is 200 mg every week.

[0200]

[0210] The heterodimer proteins, or combinations thereof, disclosed herein may be administered simultaneously with or sequentially with PD-L1 / PD-1 axis-targeting agents (e.g., anti-PD1 or anti-PD-L1 antibodies). In some embodiments, the PD-L1 / PD-1 axis-targeting agents are administered after the administration of the heterodimer proteins. In some embodiments, the PD-L1 / PD-1 axis-targeting agents are administered before the administration of the heterodimer proteins. In some embodiments, the heterodimer proteins, or combinations thereof, disclosed herein and the PD-L1 / PD-1 axis-targeting agents (e.g., anti-PD1 or anti-PD-L1 antibodies) are administered in the same composition. In some embodiments, the heterodimer proteins, or combinations thereof, disclosed herein are administered in a different composition from the PD-L1 / PD-1 axis-targeting agents (e.g., anti-PD1 or anti-PD-L1 antibodies).

[0201]

[0211] In some embodiments, therapy with PD-L1 / PD-1 axis-targeting agents is an established treatment for cancer, and the addition of heterodimeric protein therapy to this regimen improves the therapeutic effect on patients. Such improvements could be measured as an increase in response per patient or an increase in response in a patient population. The heterodimeric proteins or combinations thereof disclosed herein and PD-L1 / PD-1 axis-targeting agents may act synergistically. In some embodiments, the heterodimeric proteins or combinations thereof disclosed herein may be administered in doses lower than their therapeutically effective dose when administered as monotherapy. In some embodiments, PD-L1 / PD-1 axis-targeting agents may be administered in doses lower than their therapeutically effective dose when administered as monotherapy.

[0202]

[0212] In some embodiments, the PD-L1 / PD-1 axis-targeting agent is administered by IV infusion. In some embodiments, the PD-L1 / PD-1 axis-targeting agent is administered in a fixed dose by IV infusion on day 1 of each 14-day cycle in combination with the heterodimer protein of the Disclosure. In some embodiments, atezolizumab is administered in a dose of approximately 840 mg on day 1 of each 14-day cycle in combination with the heterodimer protein of the Disclosure. In some embodiments, atezolizumab is administered in a dose of 840 mg on day 1 of each 14-day cycle in combination with the heterodimer protein of the Disclosure. In some embodiments, atezolizumab is administered using an approved dosing regimen. In some embodiments, nivolumab is administered using an approved dosing regimen. In some embodiments, pembrolizumab is administered using an approved dosing regimen.

[0203]

[0213] In some embodiments, the subject has not previously received any medication for the treatment of the condition. In some embodiments, the subject is currently receiving a checkpoint inhibitor. In some embodiments, the subject has previously received a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor targets PD-1. In some embodiments, the checkpoint inhibitor targets PD-L1. In some embodiments, the checkpoint inhibitor targets CTLA-4.

[0204]

[0214] Examples of solid tumors treated with a combination of the heterodimeric protein of this disclosure and a PD-L1 / PD-1 axis-targeting agent (e.g., an anti-PD1 or anti-PD-L1 antibody) include, but are not limited to, carcinomas, lymphomas, blastomas, and sarcomas. Further detailed examples of such solid tumors include squamous cell carcinoma, cutaneous squamous cell carcinoma (cSCC), small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), gastrointestinal cancer, stomach cancer (GC), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liposarcoma, soft tissue sarcoma, urothelial carcinoma (UCC), ureter and renal pelvis, multiple myeloma, osteosarcoma, hepatocellular carcinoma, melanoma, stomach cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cell carcinoma (RCC), liver cancer, esophageal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, Merkel cell carcinoma (MCC), germ cell carcinoma, high microsatellite instability-H (MSI-H) cancer, and head and neck cancer. In some embodiments, the solid tumor is a locally progressive, recurrent, or metastatic solid tumor that is incurable. In some embodiments, the solid tumor is selected from the group consisting of melanoma, NSCLC, head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), UCC, RCC, SCLC, GC, MCC, cSCC, and MSI-H cancer. In some embodiments, the solid tumor is selected from melanoma, renal cell carcinoma (RCC), NSCLC, head and neck squamous cell carcinoma (HNSCC), and triple-negative breast cancer. In some embodiments, the solid tumor is selected from melanoma, RCC, NSCLC, HNSCC, and TNBC. In some embodiments, the solid tumor is selected from melanoma, RCC, and NSCLC. In some embodiments, the solid tumor is selected from melanoma, NSCLC, HNSCC, and TNBC. In some embodiments, the solid tumor is melanoma. In some embodiments, the solid tumor is RCC. In some embodiments, the cancer is NSCLC. In some embodiments, the solid tumor is HNSCC. In some embodiments, the solid tumor is TNBC. In some embodiments, a solid tumor is a solid tumor for which standard treatment is nonexistent, ineffective, unbearable, or considered inappropriate, or for which clinical trials of an investigational drug have been recognized as standard treatment.

[0205]

[0215] Combination therapy may also provide an improved response with lower doses or less frequent administration of PD-L1 / PD-1 axis-targeting agents (e.g., anti-PD1 or anti-PD-L1 antibodies), leading to more tolerable treatment regimens. For example, combination therapy with one or more heterodimeric proteins and PD-L1 / PD-1 axis-targeting agents (e.g., anti-PD1 or anti-PD-L1 antibodies) may provide enhanced clinical activity through various mechanisms, including increased levels of ADCC, ADCP, and / or NK cells, T cells, neutrophils, or mononuclear cells or immune responses.

[0206] Numbered Embodiments

[0216] Specific embodiments of this disclosure are shown in the following numbered embodiments. 1. A method for treating a solid tumor in a subject requiring treatment of a solid tumor, comprising administering to the subject a therapeutically effective amount of a heterodimer protein, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L3 68D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D A method comprising a set of amino acid substitutions selected from the group consisting of / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering). 2. CD8 +A method for inducing the proliferation of effector memory T cells, comprising administering an effective amount of heterodimer protein to a subject, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357 Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D / K A method comprising a set of amino acid substitutions selected from the group consisting of 370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering). 3. A method for inducing the proliferation of NK cells, comprising administering an effective amount of heterodimer protein to a subject, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains comprising S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357Q:L3 68D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K3 70S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D / K37 A method comprising a set of amino acid substitutions selected from the group consisting of 0S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering). 4. CD8 +A method for inducing the proliferation of effector memory T cells and NK cells, comprising administering an effective amount of heterodimeric protein to a subject, the heterodimeric protein comprising (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E 357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L36 8D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D A method comprising a set of amino acid substitutions selected from the group consisting of / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering). 5. A method for inducing IFNγ production in a subject, comprising administering an effective amount of heterodimer protein to the subject, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains comprising S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357Q:L 368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K 370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D / K3 A method comprising a set of amino acid substitutions selected from the group consisting of 70S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering). 6. The method according to any one of Embodiments 1 to 5, wherein each of the first Fc domain and / or the second Fc domain independently further comprises the amino acid substitutions Q295E, N384D, Q418E and N421D (as assigned by EU numbering). 7. The method according to any one of Embodiments 1 to 6, wherein each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / L234V / L235A / G236del / S239K;E233P / L234V / L235A / G236del / S239K / A327G;E233P / L234V / L235A / G236del / S267K / A327G; and E233P / L234V / L235A / G236del (as assigned by EU numbering), and the Fc domain is derived from the Fc domain of IgG1 or IgG3. 8. The method according to any one of Embodiments 1 to 6, wherein each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of L328R;S239K; and S267K (as assigned by EU numbering), and the Fc domain is derived from the Fc domain of IgG2. 9. The method according to any one of Embodiments 1 to 6, wherein each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K; and E233P / F234V / L235A / G236del (as assigned by EU numbering), and the Fc domain is derived from the Fc domain of IgG4. 10. The method according to any one of Embodiments 1 to 9, wherein the IL-15 protein comprises one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E. 11. The method according to any one of Embodiments 1 to 9, wherein the IL-15 protein and the IL-15Rα protein include a set of amino acid substitutions or additions selected from :65DPC;E87C:65DCA;V49C:S40C;L52C:S40C;E89C:K34C;Q48C:G38C;E53C:L42C;C42S:A37C and L45C:A37C, respectively. 12. The method according to any one of Embodiments 1 to 11, wherein the IL-15 protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. 13. The method according to any one of Embodiments 1 to 12, wherein the IL-15Rα protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4. 14. The method according to any one of Embodiments 1 to 5, wherein the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain further comprises amino acid substitutions S364K and E357Q; each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4. 15. The method according to any one of Embodiments 1 to 5, wherein the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions L368D and K370S; each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4. 16. The method according to any one of Embodiments 1 to 5, wherein the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain comprises amino acid substitutions K246T, S364K and E357Q; each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4. 17. The method according to any one of Embodiments 1 to 5, wherein the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions K246T, L368D and K370S; each of the first and second Fc domains further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (according to EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO: 4. 18. The method according to any one of Embodiments 1 to 17, wherein the IL-15 protein is covalently bonded to the N-terminus of a first Fc domain via a first linker. 19. The method according to any one of Embodiments 1 to 18, wherein the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker. 20. The method according to any one of Embodiments 1 to 19, wherein the IL-15 protein is covalently bound to the N-terminus of a first Fc domain via a first linker, and the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker. 21. The method according to any one of embodiments 18 to 20, wherein the first linker and / or the second linker are independently variable-length Gly-Ser linkers. 22. The method according to Embodiment 21, wherein the first linker and / or the second linker independently include a linker selected from the group consisting of (Gly-Gly-Gly-Gly-Ser)n (Sequence ID 39), (Ser-Ser-Ser-Ser-Gly)n (Sequence ID 40), (Gly-Ser-Ser-Gly-Gly)n (Sequence ID 41), and (Gly-Gly-Ser-Gly-Gly)n (Sequence ID 42), where n is an integer between 1 and 5. 23. The method according to any one of Embodiments 1 to 22, wherein the heterodimer protein is selected from the group consisting of XENP22822, XENP23504, XENP24045, XENP24306, XENP22821, XENP23343, XENP23557, XENP24113, XENP24051, XENP24341, XENP24052, XENP24301, and XENP32803 proteins. 24. A method for treating a solid tumor in a subject requiring treatment of a solid tumor, comprising administering to the subject a therapeutically effective amount of a heterodimer protein, wherein the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucrose domain of an IL-15Rα protein and a second Fc domain. A method comprising: a second monomer wherein the sucrose domain of the IL-15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q. 25. CD8 +A method for inducing the proliferation of effector memory T cells, comprising administering an effective amount of heterodimeric protein to a subject, wherein the heterodimeric protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain, A method comprising: a second monomer in which the sucoid domain of the -15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q. 26. A method for inducing the proliferation of NK cells, comprising administering an effective amount of heterodimeric protein to a subject, wherein the heterodimeric protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain and a second Fc domain of an IL-15Rα protein, wherein IL-15 A method comprising: a second monomer in which the sucoid domain of the Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q. 27. CD8 +A method for inducing the proliferation of effector memory T cells and NK cells, comprising administering an effective amount of heterodimeric protein to a subject, wherein the heterodimeric protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain. A method comprising: a first monomer of the IL-15Rα protein having the sucoid domain covalently bonded to the N-terminus of the second Fc domain; each of the first and second Fc domains comprising the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprising the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q. 28. A method for inducing IFNγ in a subject, comprising administering an effective amount of a heterodimer protein to the subject, wherein the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bonded to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucrose domain and a second Fc domain of an IL-15Rα protein, wherein IL-15 A method comprising: a second monomer in which the sucoid domain of the Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q. 29. The method according to any one of Embodiments 24 to 28, wherein the first Fc domain further comprises amino acid substitutions L368D and K370S, and the second Fc domain further comprises amino acid substitutions S364K and E357Q (according to EU numbering). 30. The method according to any one of Embodiments 24 to 28, wherein the first Fc domain further comprises amino acid substitutions S364K and E357Q, and the second Fc domain further comprises amino acid substitutions L368D and K370S (according to EU numbering). 31. The method according to any one of Embodiments 24 to 30, wherein the first Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E and N421D (according to EU numbering). 32. The method according to any one of Embodiments 24 to 30, wherein the second Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E and N421D (according to EU numbering). 33. The method according to any one of embodiments 24 to 32, wherein the second Fc domain further comprises the amino acid substitution K246T (according to EU numbering). 34. The method according to any one of Embodiments 24 to 33, wherein the IL-15 protein comprises amino acid substitutions D30N, E64Q, and N65D. 35. The method according to any one of Embodiments 24 to 34, wherein the IL-15 protein comprises the amino acid sequence shown in Sequence ID No. 5. 36. The method according to any one of Embodiments 24 to 35, wherein the sucrose domain of the IL-15Rα protein comprises the amino acid sequence shown in SEQ ID NO: 4. 37. The method according to any one of embodiments 24 to 36, wherein the IL-15 protein is covalently bound to the N-terminus of a first Fc domain via a first linker. 38. The method according to any one of embodiments 24 to 37, wherein the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker. 39. The method according to any one of embodiments 24 to 38, wherein the IL-15 protein is covalently bound to the N-terminus of a first Fc domain via a first linker, and the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker. 40. The method according to any one of embodiments 37 to 39, wherein the first linker and / or the second linker are independently variable-length Gly-Ser linkers. 41. The method according to Embodiment 40, wherein the first linker and / or the second linker independently include a linker selected from the group consisting of (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 39), (Ser-Ser-Ser-Ser-Gly)n (SEQ ID NO: 40), (Gly-Ser-Ser-Gly-Gly)n (SEQ ID NO: 41), and (Gly-Gly-Ser-Gly-Gly)n (SEQ ID NO: 42), where n is an integer between 1 and 5. 42. The method according to any one of embodiments 1 to 5 and 24 to 28, wherein the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 10. 43. The method according to any one of embodiments 1 to 5 and 24 to 28, wherein the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO: 16. 44. The method according to any one of embodiments 1 to 5 and 24 to 28, wherein the heterodimer protein is XENP24306, XENP32803, or a combination thereof. 45. The method according to any one of Embodiments 1 to 44, wherein a combination of a first heterodimer protein and a second heterodimer protein is administered to a subject. 46. ​​The method according to Embodiment 45, wherein the first heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 10; and the second heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 16. 47. The method according to Embodiment 45 or 46, wherein the first heterodimer protein and the second heterodimer protein are administered simultaneously. 48. The method according to embodiment 45 or 46, wherein the first heterodimer protein and the second heterodimer protein are administered sequentially. 49. The method according to any one of Embodiments 1, 6-24 and 29-48, wherein the solid tumor is locally progressive, recurrent, or metastatic. 50. The method according to any one of Embodiments 1, 6-24 and 29-48, wherein the solid tumor is selected from the group consisting of squamous cell carcinoma, cutaneous squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liposarcoma, soft tissue sarcoma, urothelial carcinoma, ureter and renal pelvis, multiple myeloma, osteosarcoma, hepatocellular carcinoma, melanoma, stomach cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cell carcinoma, liver cancer, esophageal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, Merkel cell carcinoma, germ cell carcinoma, high-frequency microsatellite instability cancer, and head and neck squamous cell carcinoma. 51. The method according to Embodiment 50, wherein the solid tumor is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and triple-negative breast cancer. 52. The method according to Embodiment 51, wherein the solid tumor is selected from melanoma, renal cell carcinoma, and non-small cell lung cancer. 53. The method according to Embodiment 51, wherein the solid tumor is selected from melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and triple-negative breast cancer. 54. The method according to any one of Embodiments 1, 6-24 and 29-53, wherein the subject has not previously received any drugs for the treatment of solid tumors. 55. The method according to any one of Embodiments 1, 6-24 and 29-53, wherein the subject is currently receiving a checkpoint inhibitor. 56. The method according to any one of Embodiments 1, 6-24 and 29-53, wherein the subject has previously been administered a checkpoint inhibitor. 57. The method according to Embodiment 55 or 56, wherein the checkpoint inhibitor targets PD-1. 58. The method according to Embodiment 55 or 56, wherein the checkpoint inhibitor targets PD-L1. 59. The method according to embodiment 55 or 56, wherein the checkpoint inhibitor targets CTLA-4. 60. The method according to any one of Embodiments 1 to 59, wherein the heterodimer protein or combination of heterodimer proteins is administered in doses selected from the group consisting of approximately 0.0025 mg / kg, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.015 mg / kg, approximately 0.02 mg / kg, approximately 0.025 mg / kg, approximately 0.03 mg / kg, approximately 0.04 mg / kg, approximately 0.05 mg / kg, approximately 0.06 mg / kg, approximately 0.08 mg / kg, approximately 0.1 mg / kg, approximately 0.12 mg / kg, approximately 0.16 mg / kg, approximately 0.2 mg / kg, approximately 0.24 mg / kg, and approximately 0.32 mg / kg by body weight. 61. The method according to Embodiment 60, wherein the heterodimer protein or combination of heterodimer proteins is administered in a dose selected from the group consisting of approximately 0.01 mg / kg, approximately 0.02 mg / kg, approximately 0.04 mg / kg, and approximately 0.06 mg / kg by body weight. 62. The method according to any one of Embodiments 1 to 60, wherein the heterodimer protein or combination of heterodimer proteins is administered in doses selected from the group consisting of 0.0025 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.08 mg / kg, 0.10 mg / kg, 0.16 mg / kg, 0.20 mg / kg, 0.24 mg / kg, and 0.32 mg / kg by body weight. 63. The method according to Embodiment 62, wherein the heterodimer protein or combination of heterodimer proteins is administered in a dose selected from the group consisting of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, and 0.06 mg / kg by body weight. 64. The method according to any one of Embodiments 1 to 63, wherein the heterodimer protein is administered at a frequency selected from the group consisting of Q1W, Q2W, Q3W, Q4W, Q5W, and Q6W. 65. The method according to Embodiment 64, wherein the heterodimer protein is administered at a frequency of Q2W. 66. The method according to any one of Embodiments 1 to 65, further comprising administering a drug that targets the PD-L1 / PD-1 axis to a subject. 67. The method according to Embodiment 66, wherein the agent targeting the PD-L1 / PD-1 axis is an anti-PD-1 antibody. 68. The method according to Embodiment 67, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, pidilizumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, MDX-1106, AMP-514, and AMP-224. 69. The method according to Embodiment 68, wherein the agent targeting the PD-L1 / PD-1 axis is an anti-PD-L1 antibody. 70. The method according to Embodiment 69, wherein the anti-PD-L1 antibody is selected from avelumab, durvalumab, atezolizumab, BMS-936559, BMS-39886, KN035, CK-301, and MSB0010718C. [Examples]

[0207] Example 1: Non-clinical pharmacology of XmAb24306

[0217] As detailed below, the combination of IL15 / IL15Rα heterodimer proteins (XENP24306 (~82%) and XENP32803 (~18%) ("XENP24306+XENP32803")) was evaluated in multiple in vitro and in vivo studies to characterize its nonclinical pharmacological properties. In vitro studies showed that the IL15 / IL15Rα heterodimer protein combination bound to the human and cynomolgus monkey IL-2 / IL-15βγ receptor complex (CD122 / CD132), and also to human and cynomolgus monkey CD8 +It was demonstrated that it is active on T cells and NK cells, but inactive in rodent cells (mouse and rat). XENP24306+XENP32803 showed increased neonatal Fc receptor (FcRn) binding (pH 6.0), but did not have effector function in terms of mediating antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cell-mediated cytotoxicity (CDC). Both in vitro and in vivo studies showed that XENP24306+XENP32803 preferably mediates CD8 + T cells and NK cells are enlarged, CD4 + It has a moderate effect on the expansion of T helper lymphocytes, but minimal effect on the expansion of the Treg population and cytokine release syndrome (CRS)-related cytokines.

[0208] in vitro test

[0218] The IL-15 components of XENP24306 and XENP32803 contain three amino acid substitutions (D30N, E64Q, and N65D). These substitutions result in reduced IL-15 potency. The binding affinity of XENP24306+XENP32803 to the IL-2 / IL-15βγ receptor complex (CD122 / CD132) in humans and cynomolgus monkeys was determined by surface plasmon resonance. Similar binding kinetics and affinities were observed between the two species, establishing the relevance of cynomolgus monkeys as a preclinical animal species for pharmacological and toxicity studies.

[0209]

[0219] XENP24306 and XENP32803 are effectorless, as indicated by the lack of binding to FcγR and human complement component 1q (C1q), and are not expected to induce target cell death via ADCC or CDC mechanisms. Specifically, the Fc regions of XENP24306 and XENP32803 were manipulated to remove binding to human, cynomolgus monkey, and mouse FcγR; no binding interaction was detected by biolayer interferometry (BLI). Furthermore, the binding of XENP24306+XENP32803 to human C1q, a key component of the C1 complex that initiates the complement system, was evaluated using BLI, and no binding was observed.

[0210]

[0220] Furthermore, with the goal of extending the half-life of XmAb24306, the Fc regions of XENP24306 and XENP32803 were manipulated to enhance binding to FcRn at a lower pH (6.0). Binding interactions with human, cynomolgus monkey, and mouse FcRn were determined by BLI methods, and the affinity of XENP24306+XENP32803 to these receptors was significantly enhanced at pH 6.0, a pH physiologically relevant to endosomal transport.

[0211]

[0221] The selectivity of the XENP24306+XENP32803 species was evaluated using a phospho-STAT5 assay. Binding of the IL-15 / IL-15Rα receptor complex to CD122 / CD132-expressing lymphocytes led to activation of Janus kinase signal transducers and transcriptional signaling pathways, resulting in STAT5 phosphorylation and subsequent cell proliferation. XENP24306+XENP32803 was found in mouse or rat CD8 + It did not induce STAT5 phosphorylation in T cells, thereby eliminating the need for rodents for toxicity testing or syngeneic mouse models for evaluating the antitumor activity of XENP24306+XENP32803.

[0212]

[0222] The efficacy of XENP24306+XENP32803 was evaluated in an in vitro cell proliferation assay. Human CD8 + T cells and NK cells showed a strong proliferative response to XENP24306+XENP32803 treatment. Among these two target cell populations, XENP24306+XENP32803 showed a strong proliferative response to CD8 + T cells (half effective concentration [EC 50 ]:12.7μg / mL) Proliferation of NK cells (median effect concentration [EC2) 50 It showed relatively high efficacy compared to 1.2 μg / mL (Figures 1A and 1B). CD8 +In addition to T cell and NK cell proliferation, XENP24306+XENP32803 also induced IFNγ production in human PBMCs. XENP24306+XENP32803 also induced NK cell (EC) production in cynomolgus monkey PBMCs. 50 :0.5μg / mL) and CD8 + T cells (EC 50 It promoted growth at a concentration of 3.8 μg / mL, thus confirming the suitability of cynomolgus monkeys as a nonclinical animal species for pharmacological and toxicity studies.

[0213]

[0223] XENP24306 and XENP32803 are reduced-potency recombinant human IL-15 proteins designed as IL-15 / IL-15Rα heterodimer Fc fusion proteins. CD8 + As shown on terminal effector T cells, XENP24306+XENP32803 demonstrated approximately 1 / 900th the potency of recombinant wild-type IL-15 and approximately 1 / 400th the potency of recombinant wild-type IL-15 (rIL15) in a similar format (wild-type IL-15 / wild-type IL-15Rα heterodimer Fc fusion; nominal XENP22853; SEQ ID NO: 11 (first monomer of wild-type IL-15-Fc) and SEQ ID NO: 7 (second monomer of IL-15Rα-Fc)) (Figure 2). The potency of XENP24306+XENP32803 was evaluated in different human immune cell subsets. Specifically, human PBMCs were treated for 4 days with gradually increasing concentrations of XENP24306+XENP32803, recombinant wild-type IL-15, or wild-type IL-15 / wild-type IL-15Rα heterodimer Fc fusion (XENP22853), and proliferation was assayed by flow cytometry using intracellular staining for the cell cycle protein Ki67. Figure 2 shows CD3 + CD8 + CD45RA + CCR7 - CD28 - CD95 + CD8 defined by group gating + The results for terminal effector T cells are shown. Curve fitting was generated using the least squares method. EC 50The values ​​were determined by nonlinear regression analysis using agonist-versus-response and variable gradient (4-parameter) equations. XENP24306+XENP32803 is indicated by an increased frequency of these cell subsets expressing the cell proliferation marker Ki67 and the cell activation markers CD69 and CD25, as well as effector memory CD8 + and CD4 + It enhanced the activation of T cells and NK cells. XmAb24306 is naive CD8 + or CD4 + It had minimal effect on T cells.

[0214]

[0224] Two additional in vitro toxicity studies were conducted: (1) evaluation of the binding profile of XENP24306+XENP32803 using a human plasma membrane protein cell array, and (2) evaluation of cytokine release induced by XENP24306+XENP32803, comparing the ability of soluble and immobilized XENP24306+XENP32803 to induce cytokine production. Data from multiple experiments using an optimized concentration of XENP24306+XENP32803 (20 μg / mL) showed no compelling off-target binding interactions identified for XENP24306+XENP32803. The potential risk of cytokine release syndrome (CRS) induced by XENP24306+XENP32803 was investigated in vitro using unstimulated human PBMCs. To evaluate the potential of XENP24306+XENP32803 to induce the production of cytokines associated with CRS, in vitro stimulation of human PBMCs was performed with XENP24306+XENP32803 at the recommended FIH dose (0.01 mg / kg) concentrations of 10 and 20 μg / mL in the blood (43- and 87-fold folds of the expected Cmax (0.23 μg / mL)). Both immobilized and soluble formats of XENP24306+XENP32803 induced IFNγ production. The magnitude of IFNγ induction by XmAb24306 (9-14-fold compared to vehicle control) was a fraction of that observed with anti-CD28 antibody (393-fold compared to vehicle control) or anti-CD3 antibody (1605-fold compared to vehicle control), which were used as positive controls. No induction of any other cytokines, such as IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, or TNF, was observed. XENP24306+XENP32803 did not induce inflammatory cytokines known to be involved in CRS, such as IL-6 and TNF, indicating that XENP24306+XENP32803 carries a low risk of inducing CRS.

[0215] in vivo testing

[0225] The immune response was evaluated in cynomolgus monkeys after single or repeated administration of XENP24306+XENP32803. No significant increases in inflammatory cytokines such as IL-6, tumor necrosis factor-α (TNFα), and IFNγ were observed after IV administration of XENP24306+XENP32803. Transient increases in other cytokines and chemokines such as IP-10, MCP-1 (monocyte chemoattractant protein-1), MIP-1α (macrophage inflammatory protein-1α), MIP-1β (macrophage inflammatory protein-1β), TARC (thymic and activation-modulating chemokine), and eotaxin were observed, indicating PD activity. These cytokines and chemokines reached peak serum concentrations within 1 day of administration and returned to pre-treatment levels by day 15. Serum concentrations of soluble CD25 peaked approximately 4 days after treatment and returned to pre-treatment levels by 15 days.

[0216]

[0226] The XENP24306+XENP32803 treatment involves the extraction of CD8 in peripheral blood. + This increased the number of T cells and NK cells, confirming the validity of the expected targeting of the immune cell population. This was likely followed by an initial decrease in hematopoietic lymphocytes due to marginal trend, and CD8 + T cells and NK cells showed a dose-dependent expansion beyond pre-treatment levels. A peak response in the blood was achieved one week after administration, and cell counts appeared to return to near pre-treatment levels after two weeks. CD8 cells, including central and effector memory, terminal effector, and stem cell memory cells, were also affected. + The memory T cell subset expanded, but naive CD8 + T cells did not expand. CD4 +T cells, Tregs, B cells, and granulocytes showed minimal expansion or did not respond to XENP24306+XENP32803. Transient and dose-dependent increases in the frequency of Ki67 expression (a cell proliferation marker) were also observed in these target cell populations and were consistent with the expansion of absolute cell numbers. Repeated administration of XENP24306+XENP32803 (0.03, 0.2, and 0.6 mg / kg, Q2W) showed transient increases in cytokine and chemokine responses after each administration. The response to XENP24306+XENP32803 was dose-dependent and reversible at the cytokine, chemokine, and sCD25 levels. Repeated dose toxicity studies showed peripheral blood CD8 + T cell and NK cell enlargements (approximately 6-fold at medium doses and 14-17-fold at high doses) were transient after each administration, and a decrease in peak numbers was observed after repeated XENP24306+XENP32803 treatment (Figure 3). Peripheral CD8 + The number of T cells and NK cells returned to pre-treatment levels after a 4-week recovery period.

[0217]

[0227] The ability of XENP24306+XENP32803 to enhance leukocyte proliferation and effector activity was tested in a repeated dose study in a mouse graft-versus-host disease (GVHD) model. XENP24306+XENP32803 (four dose levels administered on days 0, 7, 14, and 21: 0.01, 0.03, 0.1, or 0.3 mg / kg) was evaluated as monotherapy in non-obese diabetic / severely immunodeficient gamma (NSG) mice transplanted with human PBMCs. This study assessed the immune response to the mouse host, measurable by clinical signs of GVHD (i.e., weight loss and mortality), and immune monitoring, e.g., peripheral human CD8 + Increases in T cell and NK cell counts and elevated serum IFNγ concentrations were monitored. Dose-dependent GVHD-inducing activity was observed in mice treated with 0.3 mg / kg of XENP24306 + XENP32803, resulting in significant weight loss and CD8 + A significant increase in the number of T cells and NK cells, and a significant increase in serum IFNγ concentration were observed at lower doses. CD8 +A time-dependent and dose-dependent increase in the number of T cells and NK cells was observed (days 7, 14, and 21). CD4 + T cell expansion was observed only on day 14 at the two highest dose levels tested. The minimum pharmacologically active dose that demonstrated NK cell expansion enhancement was 0.01 mg / kg, and CD8 + Higher doses were required to show significant enhancement of T cells and serum IFNγ. Therefore, XENP24306+XENP32803 contributes to CD8 in GVHD. + It promoted the proliferation of T cells and NK cells, as well as the enhancement of effectors.

[0218]

[0228] XENP24306+XENP32803 (three dose levels administered on days 0, 7, 14, and 21: 0.1, 0.3, or 1.0 mg / kg) was evaluated as a monotherapy for its antitumor efficacy in mice. Using NSG mice transplanted with MCF-7 human breast cancer cells and human PBMCs, it was determined whether XENP24306+XENP32803 promoted an antitumor response. Significant antitumor activity, demonstrated by a reduction in tumor growth, was observed at all XENP24306+XENP32803 dose levels (0.1, 0.3, and 1.0 mg / kg) when administered as a monotherapy. Time and dose-dependent peripheral CD8 + T cells, CD4 + Increases in the number of T cells and NK cells, as well as elevated serum IFNγ concentrations, were measured, demonstrating that XENP24306+XENP32803 promotes an antitumor response.

[0219] Example 2: Pharmacokinetics and drug metabolism in animals

[0229] The combination of XENP24306 (~82%) and XENP32803 (~18%) ("XENP24306+XENP32803") binds to the IL-2 / IL-15βγ heterodimer receptor complex in humans and cynomolgus monkeys with equivalent affinity, and is effective against CD8 in both humans and cynomolgus monkeys. +It is active in T cells and NK cells. Therefore, the pharmacokinetics (PK) of XENP24306 + XENP32803 was investigated in cynomolgus monkeys to support dose selection for good laboratory practice (GLP) toxicity testing and to support dose selection and dosing regimens in the first-in-human (FIH) trial. To support GLP toxicity testing, an electrochemiluminescence assay was developed and validated to quantify XENP24306 + XENP32803 in cynomolgus monkey serum samples. Goat anti-human IL-15Rα antibody was used as capture, and mouse anti-human / primate IL-15 biotinylated antibody and sulfo-tagged streptavidin were used as primary and secondary detection reagents. The lower limit of quantification (LLOQ) was 30.0 ng / mL.

[0220]

[0230] Time-resolved fluorescence method was developed to quantify XENP24306 + XENP32803 concentration in non-GLP PK / PD trials in cynomolgus monkey serum samples. The LLOQ of this assay was 1.4 ng / mL.

[0221] Pharmacokinetics of a single dose in cynomolgus monkeys

[0231] A preliminary pilot study design was performed to evaluate the effectiveness of the GLP test design and to define the maximum tolerated dose. The single-dose pharmacokinetics of XENP24306 + XENP32803 was characterized in two independent PK / PD trials in male cynomolgus monkeys at 3.0 mg / kg and in female cynomolgus monkeys at 0.6 mg / kg. After a single 3.0 mg / kg IV administration to male cynomolgus monkeys, XENP24306 + XENP32803 showed a multiphasic profile with an average clearance (CL) of 66.4 mL / day / kg and an average volume of distribution at steady state (V ss ) of 107 mL / kg. The average C max and exposure (area under the concentration-time curve from time 0 to infinity [AUC 0-∞ ) were 69.6 μg / mL and 45.4 day μg / mL, respectively. After a single IV administration of 0.6 mg / kg of XENP24306 + XENP32803 to female cynomolgus monkeys, the average C maxThe concentration was 11.9 μg / mL, and the exposure (AUC) 0-∞ ) was 11.7 μg / mL per day, CL was 52.6 mL / day / kg, V ss The value was 89.0 mL / kg. See Table 3. TIFF2026062701000012.tif71170

[0222] Repeated-dose pharmacokinetics in cynomolgus monkeys

[0232] The toxicological kinetics (TK) of XENP24306+XENP32803 were characterized in a 5-week GLP repeated dose toxicity study in cynomolgus monkeys. Three dose levels (0.03, 0.2, and 0.6 mg / kg of XENP24306+XENP32803) were administered at 14-day intervals for a total of three doses. Systemic exposure was confirmed in all animals, and no sex-related differences were observed in XENP24306+XENP32803 exposure in cynomolgus monkeys (Figure 4). max The effect was proportional to the dose after the initial dose. For repeated administration, C max There was a slight tendency for it to decrease; however, the range (mean ± SD) was C after the first, second, and third doses. max There was overlap regarding AUC. 0-14 The exposure (AUC) after the initial dose was slightly below dose-proportional. In addition, exposure (AUC) decreased with repeated administration of XENP24306+XENP32803, particularly at doses of 0.2 mg / kg (7.74–5.96 days μg / mL, a 22% decrease) and 0.6 mg / kg (21.1–14.9 days μg / mL, a 30% decrease; Table 4). Such a decrease in systemic exposure (AUC) with repeated administration was thought to be due to increased TMDD as a result of the increase in the target cell population. The CL of XENP24306+XENP32803 after the initial dose ranged from 18–28 mL / day / kg, and V ssThe IgG clearance ranged from 52 to 86 mL / kg. The higher-than-normal IgG clearance observed in these studies with XENP24306+XENP32803 (<10 mL / day / kg for typical IgG) was thought to be a result of TMDD. For XENP24306+XENP32803, the CL increased with dose increases after the initial dose, and the AUC after repeated administration was also observed. 0-14 Time-varying, nonlinear pharmacokinetic behavior was observed across multiple dose levels, as indicated by a further increase smaller than the dose-proportionality observed at the initial dose level. Similar pharmacokinetic behavior is expected in human XENP24306+XENP32803. The increase in the target cell population in response to XENP24306+XENP32803 administration is thought to enhance the TMDD effect, resulting in the time-varying pharmacokinetics observed in this study. No accumulation was observed after repeated administration, as indicated by the decrease in AUC values, and the AUC ratio between the first and second doses was 0.704–0.991 times (Table 4). TIFF2026062701000013.tif118170

[0223] Example 3: Pharmacodynamic effects Effects on cytokines, chemokines, and soluble CD25

[0233] Cytokines were evaluated after a single dose of 0.6 or 3.0 mg / kg of IL15 / IL15Rα heterodimer protein (XENP24306 (~82%) and XENP32803 (~18%) in two independent cynomolgus monkey PK / PD studies) in combination ("XENP24306+XENP32803"). With both the 0.6 mg / kg and 3.0 mg / kg XENP24306+XENP32803 doses, serum markers, as well as cytokines and chemokines, peaked within 8–16 hours post-administration and returned to approximately pre-treatment levels by day 15. Serum markers elevated after XENP24306+XENP32803 treatment included eotaxin, eotaxin-3, IL-8, IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β, and TARC. The increased expression of these cytokines and chemokines may further contribute to the lymphocyte expansion induced by XENP24306+XENP32803.

[0224]

[0234] In two independent PK / PD trials, sCD25 / IL-2Rα was evaluated after a single dose of 0.6 or 3.0 mg / kg of XENP24306+XENP32803. In both the 0.6 mg / kg and 3.0 mg / kg XENP24306+XENP32803 dose groups, the sCD25 pattern showed a gradual increase over 3-4 days post-administration, which was consistent with CD25 expression on T cells.

[0225] Effects on lymphocytes

[0235] Following a single dose of 0.6 mg / kg or 3.0 mg / kg of XENP24306+XENP32803, lymphocytes showed a mild to moderate decrease for up to 3 days post-administration. Subsequently, a fluctuating, dose-dependent moderate to significant increase occurred, peaking at 7–9 days post-administration. Lymphocytes then recovered or partially recovered towards pre-treatment levels by the end of the study. Monocytes tended to reflect lymphocytes more faithfully, but to a much smaller extent. Blood smear examinations performed on animals receiving the 0.6 mg / kg dose revealed that a large proportion of lymphocytes were atypical / reactive. Mononuclear cell infiltration Following a single dose of 0.6 mg / kg of XENP24306+XENP32803, minimal to mild mononuclear cell infiltration was observed in the sinusoidal capillaries of the liver. With a single dose of 3.0 mg / kg of XENP24306+XENP32803, mononuclear cell infiltration was observed in the liver, kidneys, lungs, jejunum, bladder, and skin.

[0226] Example 4: Repeated dose toxicity

[0236] Two repeated-dose GLP studies were conducted: (1) a 5-week toxicity study with a 4-week recovery period as described in this example, and (2) a dedicated cardiovascular safety pharmacology study as described in Example 5.

[0227]

[0237] A 5-week repeated-dose GLP toxicity study was conducted in male and female cynomolgus monkeys to evaluate the toxicity, pharmacology, and TK of the combination of IL15 / IL15Rα heterodimer protein (XENP24306 (~82%) and XENP32803 (~18%)) ("XENP24306+XENP32803"). Animals were administered either a vehicle (control group) via IV bolus on days 1, 15, and 29, or 0.03, 0.2, or 0.6 mg / kg of XENP24306+XENP32803, and were necropsy on day 34 (primary study cohort) or day 64 (recovery cohort; control and 0.6 mg / kg of XmAb24306). A 30-day recovery period was designed to assess the reversibility or persistence of the effects associated with XENP24306+XENP32803.

[0228]

[0238] Toxicity assessments were based on clinical observations, body weight, quantitative food testing, ophthalmology, ECG, clinicopathological parameters (hematology, coagulation, clinical chemistry, urinalysis, and urinalysis), bioanalytical and TK parameters, ADA, cytokines, flow cytometry analysis, significant autopsy findings, organ weights, and histopathological examinations.

[0229]

[0239] TK analysis confirmed systemic exposure to XENP24306+XENP32803 at all tested dose levels. There were no gender-based differences in exposure. max The AUC after the initial dose was proportional to the dose. 0-14 The exposure (AUC) increased with dose, but less proportionally to the dose, and decreased with repeated administration. XENP24306+XENP32803 appeared to have nonlinear dynamics in cynomolgus monkeys due to TMDD at the tested dose levels (Example 2).

[0230]

[0240] All findings in the repeated-dose GLP toxicity studies were consistent with the expected pharmacological response of T cell and NK cell expansion and activation accompanied by associated pro-inflammatory responses. The NOAEL determined from the dedicated repeated-dose GLP toxicity studies was determined to be 0.03 mg / kg of XENP24306+XENP32803. The corresponding safety margin for the proposed FIH dose of XENP24306+XENP32803 at 0.01 mg / kg, IV Q2W relative to the NOAEL is described in Example 5.

[0231] Example 5: Safety Pharmacology

[0241] A single, dedicated GLP safety pharmacology study was conducted in telemetry-equipped male cynomolgus monkeys (4 monkeys per group, including a vehicle control group) to evaluate the potential cardiovascular effects of the IL15 / IL15Rα heterodimer protein combination (XENP24306 (~82%) and XENP32803 (~18%) ("XENP24306+XENP32803")). XENP24306+XENP32803 was administered by IV bolus injection at doses of 0.03, 0.2, and 0.6 mg / kg (the same dose as the GLP toxicity study) on days 1 and 15, and the animals were returned to their colonies on day 23. The following parameters and endpoints were evaluated: clinical signs, food intake (quantitative assessment), body weight, cardiovascular assessment (cardiac systolic, diastolic, and MAP, heart rate, and ECG (quantitative assessment, including measurement of RR interval, PR interval, QRS interval, and QT interval, and induced heart rate-corrected QT [QTca] interval)), body temperature, serum albumin concentration, and XENP24306+XENP32803 exposure and ADA incidence.

[0232]

[0242] XENP24306+XENP32803 was clinically well-tolerated at all doses (0.03, 0.2, and 0.6 mg / kg), and all animals survived the study period without veterinary intervention. No clinical signs, changes in food intake, changes in body weight, or ECG abnormalities related to the test substance were observed at any dose. ECG was qualitatively normal for cynomolgus monkeys, and there were no changes in PR interval, QRS interval, or QTca interval related to the treatment.

[0233]

[0243] Systemic exposure to XENP24306+XENP32803 was demonstrated at all dose levels. No treatment-related changes in body weight or qualitative food intake occurred during the study period.

[0234]

[0244] Based on the overall findings of the GLP trial in cynomolgus monkeys, the no-observed-adverse-effect level (NOAEL) dose was considered to be 0.03 mg / kg of XENP24306+XENP32803. Due to the immunoagonist properties of XENP24306+XENP32803, the determination of the first-in-the-day (FIH) dose was based on the estimated minimum pharmacokinetic level (MABEL) method. A monotherapy dose of 0.01 mg / kg of XENP24306+XENP32803, IV, is proposed as the FIH dose of XENP24306+XENP32803. This FIH dose is EC 20 Based on (0.23 μg / mL; geometric mean of 20 donors), this is the most sensitive in vitro assay for XENP24306+XENP32803 in human PBMCs (in vitro NK cells (CD3)). - CD56 + )Obtained using proliferation (percentage of cells expressing Ki67). See Figure 1. The recommended FIH dose of 0.01 mg / kg of XENP24306+XENP32803 is presumed to be safe, provide minimal biological impact, and have a minimal risk of treatment-mediated reactions in humans. C max This is this EC 20It is not expected to exceed the level. The starting dose of XENP24306+XENP32803 in humans, 0.01 mg / kg, has a 3-fold safety margin compared to the NOAEL dose (0.03 mg / kg of XENP24306+XENP32803, Q2W) in a 5-week GLP toxicity study in cynomolgus monkeys. C max This is observed in cynomolgus monkeys at NOAEL doses. max The AUC is expected to be 3.3 times lower than (0.75 ± 0.04 μg / mL; initial dose). See Table 5. Furthermore, the AUC of 0.01 mg / kg of XENP24306 + XENP32803 in humans is expected to be 1.8 times lower than the AUC observed at the NOAEL dose in cynomolgus monkeys (Table 5). That is, the C2 max The AUC further supports the MABEL-based starting dose of XENP24306+XENP32803 IV at 0.01 mg / kg and provides a sufficient safety margin for the study (Table 5).

[0235]

[0245] The frequency of administration of XENP24306+XENP32803 to humans is Q2W, and there was no significant acute toxicity when administered Q2W, supported by a 5-week GLP toxicity study in cynomolgus monkeys in which XENP24306+XENP32803 showed generally good tolerability. Peak peripheral PD response (NK and CD8 +Expansion of target cells such as T cells was achieved 1 week after administration, and these peripheral target cell counts decreased towards their baseline by the end of 2 weeks after XENP24306 + XENP32803 administration. Furthermore, cytokines and chemokines showing PD activity reached their peak between 8 and 16 hours after administration and returned to baseline within 14 days of administration (see Example 3). Therefore, the initial dosing frequency Q2W is considered appropriate in the dose escalation study of monotherapy using XENP24306 + XENP32803 during the dose-limiting toxicity observation period including the first cycle of the test treatment. TIFF2026062701000014.tif114170

[0236] Example 6: Open-label, multicenter, global dose escalation study of a combination of IL15 / IL15Rα heterodimeric proteins as monotherapy

[0246] An open-label, multicenter, global dose escalation study of monotherapy will be conducted to evaluate the safety, tolerability, pharmacokinetics and activity of the IL15 / IL15Rα heterodimeric protein (combination of XENP24306 (~82%) and XENP32803 (~18%) (「XENP24306 + XENP32803」)).

[0237]

[0247] The study consists of a screening period of up to 28 days, a treatment period, and a minimum follow-up period of 90 days after treatment.

[0238]

[0248] Patients will be enrolled in two stages: a dose escalation stage and an expansion stage.

[0239]

[0249] Approximately 21–54 patients with locally progressive, recurrent, or metastatic, incurable solid tumors will be enrolled in a dose-escalation trial. The initial dose of XENP24306+XENP32803 is 0.01 mg / kg Q2W. XENP24306+XENP32803 will be administered by IV infusion. The dose of XENP24306+XENP32803 will be increased up to 100% of the preceding dose level in each consecutive cohort until a safety threshold (defined as a dose-limiting toxicity (DLT) in one patient or a major organ adverse event of grade ≥ 2 not attributable to another clearly identifiable cause in at least two patients during the DLT evaluation period in a given cohort) is observed. Subsequently, to determine the maximum tolerated dose (MTD) or maximum dose (MAD) of the monotherapy XENP24306 + XENP32803, each cohort of 3 to 9 patients was evaluated at escalating dose levels according to a 3+3+3 design. Figure 7.

[0240]

[0250] Patients in this trial will first be assessed for eligibility during a screening period (duration ≤ 28 days). Following eligibility confirmation, patients will receive 0.01 mg / kg of XENP24306 + XENP32803 via IV infusion on the first day of each 14-day cycle (Q2W). The pharmacokinetics (PK) of XENP24306 + XENP32803 will be evaluated. Patients will be evaluated weekly by physical examination and blood sampling for routine hematological and metabolic laboratory assessments for the first 8 cycles of XENP24306 + XENP32803 treatment during dose escalation, for the first 2 cycles during expansion, and thereafter at a lower frequency. Tumor assessments will be performed at baseline and after the start of the trial.

[0241]

[0251] Patients enrolled in the cleared cohort (i.e., backfill cohort) of the monotherapy dose-escalation cohort must have one of the following PD-L1 selective tumor types: melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC) urothelial carcinoma (UCC), renal cell carcinoma (RCC), small cell lung cancer (SCLC), GC, Merkel cell carcinoma (MCC), cutaneous squamous cell carcinoma (cSCC), or highly microsatellite instability-H cancer.

[0242]

[0252] Approximately 185–240 patients with locally advanced, recurrent, or metastatic incurable malignancies that have progressed after available standard treatment, or who have demonstrated a lack of response to or intolerance of standard treatment, or for whom clinical trials of the investigational drug are recognized as standard treatment, will be enrolled in the study's expanded cohort. Such an expanded phase will consist of a defined patient cohort to better characterize the safety, pharmacokinetics, PD activity, and preliminary antitumor activity of XENP24306+XENP32803 as monotherapy. XENP24306+XENP32803 will be administered intravenously during the expanded phase. The provisional recommended expanded dose (RED) of XENP24306+XENP32803 will be proposed to be below the MTD / MAD established in dose escalation. If RED (Reduction Therapy) for XENP24306+XENP32803 is proposed, additional patients will be enrolled in the expansion phase and treated with RED.

[0243]

[0253] All patients will be closely monitored for adverse events throughout the study and for at least 90 days after the final dose of the study treatment or until the initiation of another systemic anticancer therapy, whichever comes first. Adverse events will be classified according to NCI CTCAE v5.0.

[0244]

[0254] To characterize the pharmacokinetics, immunogenic response, and PD characteristics of XENP24306+XENP32803 as monotherapies, blood samples were collected at various time points before and after administration.

[0245]

[0255] Patients will be subjected to tumor assessment at screening (baseline) and at regular intervals throughout the trial, measured according to the Criteria for Efficacy of Solid Tumors (RECIST) v1.1. The revised RECIST v1.1, based on immunology-based therapies (iRECIST), may also be used in this study to better characterize the different patterns of response associated with cancer immunotherapy (CIT) and to enable a better understanding of the preliminary activity profile of XENP24306+XENP32803. iRECIST is intended to supplement the standard RECIST v1.1 in this trial, enabling investigators to comprehensively assess the benefits and risks for patients.

[0246]

[0256] The objective of this study is to conduct a preliminary evaluation of the activity of XENP24306 + XENP32803 when administered as monotherapy, based on the following endpoints: • Serum concentrations of XENP24306 + XENP32803; • Percentage of participants experiencing adverse events; Objective response rate (ORR) is defined as the percentage of patients who achieve complete remission (CR) or partial remission (PR). Duration of response (DOR) is defined as the time from the first documented objective response to disease progression or death due to either cause (whichever occurs first). • Progression-free survival (PFS) after registration, defined as the time from registration to disease progression or death due to any cause (whichever occurs first); and • Overall survival (OS) after registration, defined as the time from registration to death from any cause.

[0247]

[0257] The safety objective of this study is to evaluate the safety of XENP24306+XENP32803 when administered as monotherapy, based on the occurrence and severity of adverse events, changes from baseline in targeted vital signs, or laboratory results or ECG parameters.

[0248]

[0258] The objective of this pharmacokinetic (PK) study is to characterize the PK profile of XENP24306+XENP32803 when administered as monotherapy, based on serum concentrations of XENP24306+XENP32803 at specific time points.

[0249]

[0259] The purpose of this study for immunogenicity is to evaluate the immune response (Ia) to XENP24306+XENP32803 when administered as a monotherapy, based on the occurrence of baseline adverse reactions (ADA) to XENP24306+XENP32803 and the occurrence of ADA during the study.

[0250] Example 7: Open-label, multicenter, global dose-escalation study of XENP24306 monotherapy.

[0260] A monotherapy, open-label, multicenter, global dose-escalation study will be conducted to evaluate the safety, tolerability, pharmacokinetics, and activity of XENP24306.

[0251]

[0261] The trial consists of a screening period of up to 28 days, a treatment period, and a minimum follow-up period of 90 days after treatment.

[0252]

[0262] Patients are enrolled in two stages: a dose escalation stage and a dose expansion stage.

[0253]

[0263] Approximately 21–54 patients with locally progressive, recurrent, or metastatic, incurable solid tumors are enrolled in a dose-escalation trial. The initial dose of XENP24306 is 0.01 mg / kg Q2W. XENP24306 is administered by IV infusion. The dose of XENP24306 is increased up to 100% of the preceding dose level in each consecutive cohort until a safety threshold (defined as a dose-limiting toxicity (DLT) in one patient or a grade ≥ 2 major organ adverse event not attributable to another clearly identifiable cause in at least two patients during the DLT evaluation period in a given cohort) is observed. Subsequently, each cohort of 3–9 patients is evaluated at escalating dose levels according to a 3+3+3 design to determine the maximum tolerated dose (MTD) or maximum dose (MAD) of monotherapy XENP24306. Figure 7.

[0254]

[0264] Patients in this trial will first be assessed for eligibility during a screening period (duration ≤ 28 days). Following eligibility confirmation, patients will receive 0.01 mg / kg of XENP24306 via IV infusion on the first day of each 14-day cycle (Q2W). The pharmacokinetics (PK) of XENP24306 will be evaluated. Patients will be evaluated weekly by physical examination and blood concentration for routine hematological and metabolic laboratory assessments during the first 8 cycles of XENP24306 treatment during dose escalation, the first 2 cycles during expansion, and thereafter at a lower frequency. Tumor assessments will be performed at baseline and after the start of the trial.

[0255]

[0265] Patients enrolled in the cleared cohort (i.e., backfill cohort) of the monotherapy dose-escalation cohort must have one of the following PD-L1 selective tumor types: melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC) urothelial carcinoma (UCC), renal cell carcinoma (RCC), small cell lung cancer (SCLC), GC, Merkel cell carcinoma (MCC), cutaneous squamous cell carcinoma (cSCC), or highly microsatellite instability-H cancer.

[0256]

[0266] Approximately 185–240 patients with locally progressive, recurrent, or metastatic incurable malignancies that have progressed after available standard treatment, or who have demonstrated a lack of response to or intolerance of standard treatment, or for whom clinical trials of the investigational drug are recognized as standard treatment, will be enrolled in the study's expanded cohort. Such an expanded phase will consist of a defined patient cohort to better characterize the safety, pharmacokinetics, PD activity, and preliminary antitumor activity of XENP24306 as a monotherapy. XENP24306 will be administered intravenously via IV infusion during the expanded phase. A provisional recommended expanded dose (RED) of XENP24306 will be proposed, below the MTD / MAD established in dose escalation. Once a RED for XENP24306 is proposed, additional patients will be enrolled in the expanded phase and treated with the RED.

[0257]

[0267] All patients will be closely monitored for adverse events throughout the study and for at least 90 days after the final dose of the study treatment or until the initiation of another systemic anticancer therapy, whichever comes first. Adverse events will be classified according to NCI CTCAE v5.0.

[0258]

[0268] To characterize the pharmacokinetics, immunogenic response, and PD characteristics of XENP24306 as a monotherapy agent, blood samples were collected at various time points before and after administration.

[0259]

[0269] Patients will be subjected to tumor assessment at screening (baseline) and at regular intervals throughout the trial, measured according to the Criteria for Efficacy of Solid Tumors (RECIST) v1.1. The revised RECIST v1.1, based on immunology-based therapies (iRECIST), will also be used in this trial to better characterize the different patterns of response associated with cancer immunotherapy (CIT) and to enable a better understanding of the preliminary activity profile of XENP24306. iRECIST is intended to supplement the standard RECIST v1.1 in this trial, enabling investigators to comprehensively assess the benefits and risks for patients.

[0260]

[0270] The objective of this study is to conduct a preliminary evaluation of the activity of XENP24306 when administered as a monotherapy, based on the following endpoints: • Serum concentration of XENP24306; • Percentage of participants experiencing adverse events; Objective response rate (ORR) is defined as the percentage of patients who achieve complete remission (CR) or partial remission (PR). Duration of response (DOR) is defined as the time from the first documented objective response to disease progression or death due to either cause (whichever occurs first). • Progression-free survival (PFS) after registration, defined as the time from registration to disease progression or death due to any cause (whichever occurs first); and • Overall survival (OS) after registration, defined as the time from registration to death from any cause.

[0261]

[0271] The safety objective of this study is to evaluate the safety of XENP24306 when administered as a monotherapy, based on the occurrence and severity of adverse events, changes from baseline in targeted vital signs, or clinical laboratory results or ECG parameters.

[0262]

[0272] The objective of this pharmacokinetic (PK) study is to characterize the PK profile of XENP24306 when administered as a monotherapy, based on the serum concentration of XENP24306 at specific time points.

[0263]

[0273] The purpose of this immunogenicity study is to evaluate the immune response (Ia) to XENP24306 when administered as a monotherapy, based on the occurrence of baseline adverse reactions (ADAs) to XENP24306 and during the study.

[0264] Example 8: Open-label, multicenter, global dose-escalation study of XENP32803 monotherapy.

[0274] A monotherapy, open-label, multicenter, global dose-escalation study will be conducted to evaluate the safety, tolerability, pharmacokinetics, and activity of XENP32803.

[0265]

[0275] The trial consists of a screening period of up to 28 days, a treatment period, and a minimum follow-up period of 90 days after treatment.

[0266]

[0276] Patients are enrolled in two stages: a dose escalation stage and a dose expansion stage.

[0267]

[0277] Approximately 21–54 patients with locally progressive, recurrent, or metastatic, incurable solid tumors are enrolled in a dose-escalation trial. The initial dose of XENP32803 is 0.01 mg / kg Q2W. XENP32803 is administered by IV infusion. The dose of XENP32803 is increased up to 100% of the preceding dose level in each consecutive cohort until a safety threshold (defined as a dose-limiting toxicity (DLT) in one patient or a major organ adverse event of grade ≥ 2 not attributable to another clearly identifiable cause in at least two patients during the DLT evaluation period of a given cohort) is observed. Subsequently, each cohort of 3–9 patients is evaluated at escalating dose levels according to a 3+3+3 design to determine the maximum tolerated dose (MTD) or maximum dose (MAD) of monotherapy XENP32803. Figure 7.

[0268]

[0278] Patients in this trial will first be assessed for eligibility during a screening period (duration ≤ 28 days). Following eligibility confirmation, patients will receive 0.01 mg / kg of XENP32803 via IV infusion on the first day of each 14-day cycle (Q2W). The pharmacokinetics (PK) of XENP32803 will be evaluated. Patients will be evaluated weekly by physical examination and blood concentration for routine hematological and metabolic laboratory assessments during the first 8 cycles of XENP32803 treatment during dose escalation, the first 2 cycles during expansion, and thereafter at a lower frequency. Tumor assessments will be performed at baseline and after the start of the trial.

[0269]

[0279] Patients enrolled in the cleared cohort (i.e., backfill cohort) of the monotherapy dose-escalation cohort must have one of the following PD-L1 selective tumor types: melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC) urothelial carcinoma (UCC), renal cell carcinoma (RCC), small cell lung cancer (SCLC), GC, Merkel cell carcinoma (MCC), cutaneous squamous cell carcinoma (cSCC), or highly microsatellite instability-H cancer.

[0270]

[0280] Approximately 185–240 patients with locally progressive, recurrent, or metastatic incurable malignancies that have progressed after available standard treatment, or who have demonstrated a lack of response to or intolerance of standard treatment, or for whom clinical trials of the investigational drug are recognized as standard treatment, will be enrolled in the study's expanded cohort. Such an expanded phase will consist of a defined patient cohort to better characterize the safety, pharmacokinetics, PD activity, and preliminary antitumor activity of XENP32803 as a monotherapy. XENP32803 will be administered intravenously via IV infusion during the expanded phase. A provisional recommended expanded dose (RED) of XENP32803 will be proposed, below the MTD / MAD established in dose escalation. Once a RED for XENP32803 is proposed, additional patients will be enrolled in the expanded phase and treated with the RED.

[0271]

[0281] All patients will be closely monitored for adverse events throughout the study and for at least 90 days after the final dose of the study treatment or until the initiation of another systemic anticancer therapy, whichever comes first. Adverse events will be classified according to NCI CTCAE v5.0.

[0272]

[0282] To characterize the pharmacokinetics, immunogenic response, and PD characteristics of XENP32803 as a monotherapy agent, blood samples were collected at various time points before and after administration.

[0273]

[0283] Patients will be subjected to tumor assessment at screening (baseline) and at regular intervals throughout the trial, measured according to the Criteria for Efficacy of Solid Tumors (RECIST) v1.1. The revised RECIST v1.1, based on immunology-based therapies (iRECIST), will also be used in this trial to better characterize the different patterns of response associated with cancer immunotherapy (CIT) and to enable a better understanding of the preliminary activity profile of XENP32803. iRECIST is intended to supplement the standard RECIST v1.1 in this trial, enabling investigators to comprehensively assess the benefits and risks for patients.

[0274]

[0284] The objective of this study is to conduct a preliminary evaluation of the activity of XENP32803 when administered as a monotherapy, based on the following endpoints: • Serum concentration of XENP32803; • Percentage of participants experiencing adverse events; Objective response rate (ORR) is defined as the percentage of patients who achieve complete remission (CR) or partial remission (PR). Duration of response (DOR) is defined as the time from the first documented objective response to disease progression or death due to either cause (whichever occurs first). • Progression-free survival (PFS) after registration, defined as the time from registration to disease progression or death due to any cause (whichever occurs first); and • Overall survival (OS) after registration, defined as the time from registration to death from any cause.

[0275]

[0285] The safety objective of this study is to evaluate the safety of XENP32803 when administered as a monotherapy, based on the occurrence and severity of adverse events, changes from baseline in targeted vital signs, or laboratory results or ECG parameters.

[0276]

[0286] The objective of this pharmacokinetic (PK) study is to characterize the PK profile of XENP32803 when administered as a monotherapy, based on the serum concentration of XENP32803 at specific time points.

[0277]

[0287] The purpose of this study for immunogenicity is to evaluate the immune response (Ia) to XENP32803 when administered as a monotherapy, based on the occurrence of baseline adverse reactions (ADA) to XENP32803 and the occurrence of ADA during the study.

[0278] Example 9: Non-clinical pharmacological in vivo study of XENP24306 + XENP32803 in combination with an anti-PD-L1 / PD-1 inhibitor.

[0288] The ability of the IL15 / IL15Rα heterodimer protein (XENP24306 (~82%) and XENP32803 (~18%) combination ("XENP24306+XENP32803")) to enhance leukocyte proliferation and effector activity was tested in repeated dose studies in a mouse graft-versus-host disease (GVHD) model. XENP24306+XENP32803 (four dose levels administered on days 0, 7, 14, and 21: 0.01, 0.03, 0.1, or 0.3 mg / kg) in combination with the anti-PD-1 inhibitor XENP16432 administered at a fixed dose of 3.0 mg / kg was evaluated in non-obese diabetic / severely immunodeficient gamma (NSG) mice transplanted with human PBMCs. This study assesses the immune response to a mouse host, measurable by clinical signs of GVHD (i.e., weight loss and mortality), and immune monitoring, such as peripheral human CD8 + Increases in T cell and NK cell counts and elevated serum IFNγ concentrations were monitored. Dose-dependent GVHD-inducing activity was observed in mice treated with 0.3 mg / kg of XENP24306 + XENP32803, resulting in significant weight loss and CD8 + A significant increase in the number of T cells and NK cells, and a significant increase in serum IFNγ concentration were detected at lower doses (Figure 5). CD8 +An increase in the number of T cells and NK cells was observed in a time- (days 7, 14, 21) and dose-dependent manner. CD4 + T cell expansion was observed only on day 14 at the two highest dose levels tested. The minimum pharmacologically active dose revealed by the enhanced expansion of NK cells was 0.01 mg / kg, and higher doses were required to show significant enhancement of CD8 + T cells and serum IFNγ. Therefore, XENP24306 + XENP32803 promoted the proliferation and effector enhancement of CD8 + T cells and NK cells that contribute to GVHD. The combination groups of XENP24306 + XENP32803 (at doses of 0.1 and 0.3 mg / kg) and anti-PD-1 antibody showed significantly superior GVHD-inducing activity compared to the anti-PD-1 antibody alone.

[0279]

[0289] This study demonstrates the immune activation activity of XENP24306 + XENP32803, an IL15 / IL15Rα-Fc fusion protein, on human immune cells. Importantly, this study demonstrates the utility of combination therapy using XENP24306 + XENP32803 and XENP16432 / anti-PD1, a bivalent antibody of anti-PD1, to enhance immune responses that exceed those of anti-PD1 treatment alone, suggesting the potential to improve clinical utility by combining an approved anti-PD-L1 agent with XENP24306 + XENP32803.

[0280]

[0290] When only XENP24306 + XENP32803 was administered, the minimum pharmacologically active dose (MPAD) revealed by the increased expansion of NK cells compared to untreated controls was 0.01 mg / kg. Higher doses were required to demonstrate significant enhancement of T cells and serum IFNγ, as well as exacerbation of GVHD.

[0281]

[0291] Combination therapy with XENP24306+XENP32803 and XENP16432 / anti-PD1 also promoted a significant enhancement of leukocyte count and IFNγ production compared to anti-PD1 monotherapy. In particular, as leukocyte count increased in response to the amplification effect of XENP24306+XENP32803, the measured trough serum concentration of XENP24306+XENP32803 decreased, likely due to targeted pharmacokinetics on a progressively expanding leukocyte population.

[0282]

[0292] The antitumor efficacy of XENP24306 + XENP32803 (three dose levels administered on days 0, 7, 14, and 21: 0.1, 0.3, or 1.0 mg / kg) in combination with the anti-PD-1 inhibitor XENP16432, administered at a fixed dose of 3.0 mg / kg, was evaluated in mice. Using NSG mice transplanted with MCF-7 human breast cancer cells and human PBMCs, it was determined whether XENP24306 + XENP32803 in combination with anti-PD-1 promoted the antitumor response. Time and dose-dependent peripheral CD8 + T cells, CD4 + Increases in T cell and NK cell counts and elevated serum IFNγ concentrations were measured, demonstrating that XENP24306+XENP32803 promotes an antitumor response. Figure 6.

[0283]

[0293] Animals treated with PBS (Group A) showed stable tumor growth throughout the study. No animals in Group A were euthanized or died during the study. Animals treated with XENP16432 / anti-PD1 (Group B) initially showed similar tumor growth dynamics to the PBS-treated animals (Group A) until day 13. However, at day 15,

[0284]

[0294] Animals treated with XENP16432 / anti-PD1 showed statistically significant inhibition of tumor growth compared to mice treated with PBS. The reduction in tumor volume observed in animals treated with XENP16432 / anti-PD1 is consistent with a typical allogeneic antitumor response. No mice treated with XENP16432 / anti-PD1 were euthanized or died during the study. Treatment with 0.1 mg / kg of XENP24306+XENP32803 (group E) induced a significant reduction in tumor size compared to animals treated with PBS, as early as day 8. By day 13, all three dose levels of XENP24306+XENP32803 (1.0, 0.3, and 0.1 mg / kg; groups C, D, and E) showed a significant and dose-dependent reduction in tumor growth compared to mice treated with PBS. Tumor volume remained reduced until the end of the study. Treatment with XENP24306+XENP32803 monotherapy also showed significant inhibition of tumor growth as early as day 8 in animals treated with 0.1 mg / kg of XENP24306+XENP32803 (Group E) compared to treatment with XENP16432 / anti-PD1 monotherapy (Group B). By day 13, 1.0 mg / kg of XENP24306+XENP32803 (Group C) achieved a significant advantage over XENP16432 / anti-PD1 in terms of tumor volume reduction, and 0.3 mg / kg of XENP24306+XENP32803 (Group D) showed a significant advantage over XENP16432 / anti-PD1 by day 19.

[0285]

[0295] In addition, compared to the anti-PD-1 (monotherapy) group, higher doses of XENP24306 + XENP32803 (0.3 and 1.0 mg / kg) in combination with an anti-PD-1 inhibitor showed a significantly greater reduction in tumor growth and more pronounced peripheral CD8 +The study showed an increase in T cells and NK cells, as well as an increase in IFNγ. In particular, when administered in combination with XENP16432 / anti-PD1, 0.3 and 0.1 mg / kg of XENP24306 + XENP32803 (groups G and H) resulted in a dose-dependent statistically significant reduction in tumor volume as early as day 8, compared to both the PBS control and the XENP16432 / anti-PD1 monotherapy groups. All three combination dose groups of XENP24306 + XENP32803 and XEN16432 showed a dose-dependent statistically significant reduction in tumor size on day 11, compared to both the PBS and the XENP16432 / anti-PD1 monotherapy groups.

[0286]

[0296] This study describes the antitumor activity of the IL15 / IL15Rα-Fc fusion protein XENP24306+XENP32803. Importantly, this study also demonstrates the further utility of combination therapy using XENP24306+XENP32803 and the anti-PD1 bivalent antibody XENP16432, administered together to enhance the antitumor immune response beyond anti-PD1 treatment alone, suggesting the potential to improve clinical utility by combining approved anti-PD-L1 agents with XENP24306+XENP32803. The dose-dependent antitumor activity of XENP24306+XENP32803 correlated with a dose-dependent increase in peripheral blood leukocyte counts and elevated IFNγ production.

[0287]

[0297] All dose levels of XENP24306+XENP32803, including the lowest level of 0.1 mg / kg, were active in this antitumor model. All dose levels of XENP24306+XENP32803 promoted leukocytosis and increased IFNγ production, with doses of up to 1 mg / kg mediating the greatest effect. Combination therapy with XENP24306+XENP32803 and XENP16432 / anti-PD1 also increased leukocytosis and IFNγ production compared to anti-PD1 monotherapy.

[0288] Example 10: Open-label, multicenter, global dose-escalation study of combination therapy with atezolizumab and XENP24306 + XENP32803

[0298] An open-label, multicenter, global dose-escalation study will likely be conducted to evaluate the safety, tolerability, pharmacokinetics, and activity of XENP24306 (e.g., ~82%) + XENP32803 (e.g., ~18%) in combination with an anti-PD-L1 / PD-1 antibody such as atezolizumab.

[0289]

[0299] The trial consists of a screening period of up to 28 days, a treatment period, and a minimum follow-up period of 90 days post-treatment. Patients considering enrollment in the expanded cohort of combination therapy with PD-L1-selected tumors can undergo a tissue pre-screening for PD-L1 status, which will be conducted prior to the 28-day screening period.

[0290]

[0300] Patients are enrolled in two stages: a dose escalation stage and a dose expansion stage.

[0291]

[0301] Approximately 21 to 54 patients with locally advanced, recurrent, or metastatic, incurable solid tumors will be enrolled in the dose-escalation phase of the combination therapy portion of the study. XENP24306+XENP32803 and atezolizumab will be administered by IV infusion. Following eligibility confirmation, patients will receive XENP24306+XENP32803 in combination with atezolizumab by IV infusion on the first day of each 14-day cycle. The starting dose of the XENP24306+XENP32803 combination therapy is 0.01 mg / kg IV every two weeks. Atezolizumab will be administered by IV infusion at a fixed dose of 840 mg in combination with XENP24306+XENP32803 on the first day of each 14-day cycle. Atezolizumab is administered after XENP24306 + XENP32803 and the subsequent observation period.

[0292]

[0302] The dose of XENP24306+XENP32803 is increased up to 100% of the preceding dose level for each consecutive cohort until a safety threshold (defined as a dose-limiting dose-limiting time (DLT) in one patient) or another clearly identifiable cause-free grade ≥ 2 major organ adverse event in at least two patients during the DLT evaluation period for a given cohort is observed. Subsequently, each cohort of 3–9 patients is evaluated at escalating dose levels according to a 3+3+3 design to determine the MTD (or MAD) of XENP24306+XENP32803 in combination with atezolizumab. Figure 8.

[0293]

[0303] Patients enrolled in the cleared cohort (i.e., backfill cohort) of the combination therapy dose escalation cohort must have one of the following PD-L1 selective tumor types: melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC) urothelial carcinoma (UCC), renal cell carcinoma (RCC), small cell lung cancer (SCLC), gastric cancer (GC), Merkel cell carcinoma (MCC), cutaneous squamous cell carcinoma (cSCC), or highly microsatellite instability (MSI-H) cancer.

[0294]

[0304] Overall, approximately 225 to 350 patients may be enrolled in this trial across approximately 25 to 35 trial sites worldwide. Patients in this trial will first be evaluated for eligibility during a screening period (duration ≤ 28 days). The starting dose of XENP24306 + XENP32803 in combination with atezolizumab will not be higher than one dose level below the dose of XENP24306 + XENP32803 that shows PD activity in the monotherapy portion of the trial (Example 6). If the initial dose level of 0.01 mg / kg of XENP24306 + XENP32803 in monotherapy shows PD activity, the starting dose of XENP24306 + XENP32803 will not be higher than 0.005 mg / kg in the initial atezolizumab combination cohort. XENP24306 + XENP32803 and atezolizumab are administered by IV infusion during the dose escalation phase. The provisional recommended dose escalation (RED) for XENP24306 + XENP32803 will be proposed to be below the MTD / MAD established during dose escalation.

[0295]

[0305] If the RED regimen of XENP24306+XENP32803 in combination with atezolizumab is proposed, further patients will be enrolled in the expansion phase and treated with RED.

[0296]

[0306] The pharmacokinetics (PK) of XENP24306+XENP32803 will be evaluated. Patients will be evaluated weekly by physical examination and blood sampling for routine hematological and metabolic laboratory assessments during the first eight cycles of XENP24306+XENP32803 in combination with atezolizumab treatment during dose escalation, during the first two cycles during expansion, and thereafter at a less frequent frequency. Tumor assessments will be performed at baseline and after the start of the trial.

[0297]

[0307] All patients will be closely monitored for adverse events throughout the study and for at least 90 days after the final dose of the study treatment or until the initiation of another systemic anticancer therapy, whichever comes first. Adverse events will be classified according to NCI CTCAE v5.0.

[0298]

[0308] To characterize the pharmacokinetics, immunogenic response, and PD characteristics of XENP24306+XENP32803 in combination with atezolizumab, blood samples were collected at various time points before and after administration.

[0299]

[0309] Patients will be subjected to tumor assessments at screening (baseline) and periodically throughout the trial, as measured by RECIST v1.1. iRECIST may also be used in this study to better characterize different patterns of response associated with cancer immunotherapy (CIT) and to enable a better understanding of the preliminary activity profile of XENP24306+XENP32803 in combination with atezolizumab. iRECIST is intended to supplement standard RECIST v1.1 in this trial and enable investigators to comprehensively assess the benefits and risks for patients.

[0300]

[0310] The objective of this study is to conduct a preliminary evaluation of the activity of XENP24306+XENP32803 when administered in combination with atezolizumab, based on the following endpoints: • Serum concentrations of XENP24306 + XENP32803; • Percentage of participants experiencing adverse events; Objective response rate (ORR) is defined as the percentage of patients who achieve complete remission (CR) or partial remission (PR) in two consecutive opportunities separated by ≥ 4 weeks. Duration of response (DOR) is defined as the time from the first documented objective response to disease progression or death due to either cause (whichever occurs first). • Progression-free survival (PFS) after registration, defined as the time from registration to disease progression or death due to any cause (whichever occurs first); and • Overall survival (OS) after registration, defined as the time from registration to death from any cause.

[0301]

[0311] The safety objective of this study is to evaluate the safety of XENP24306+XENP32803 when administered in combination with atezolizumab, based on the occurrence and severity of adverse events, changes from baseline in targeted vital signs, or laboratory results or ECG parameters.

[0302]

[0312] The objective of this study's pharmacokinetic (PK) analysis is to characterize the PK profile of XENP24306+XENP32803 when administered in combination with atezolizumab, based on serum concentrations of XENP24306+XENP32803 at specific time points.

[0303]

[0313] The purpose of this study for immunogenicity is to evaluate the immune response to XENP24306+XENP32803 when administered in combination with atezolizumab, based on the ADA for XENP24306+XENP32803 and the ADA for XENP24306+XENP32803 and atezolizumab during the study.

[0304] Example 11: Open-label, multicenter, global dose-escalation study of XENP24306 in combination with atezolizumab.

[0314] An open-label, multicenter, global dose-escalation study will likely be conducted to evaluate the safety, tolerability, pharmacokinetics, and activity of XENP24306 in combination with anti-PD-L1 / PD-1 antibodies such as atezolizumab.

[0305]

[0315] The trial consists of a screening period of up to 28 days, a treatment period, and a minimum follow-up period of 90 days post-treatment. Patients considering enrollment in the expanded cohort of combination therapy with PD-L1-selected tumors can undergo a tissue pre-screening for PD-L1 status, which will be conducted prior to the 28-day screening period.

[0306]

[0316] Patients are enrolled in two stages: a dose escalation stage and a dose expansion stage.

[0307]

[0317] Approximately 21 to 54 patients with locally progressive, recurrent, or metastatic, incurable solid tumors will be enrolled in the dose-escalation phase of the combination therapy portion of the study. XENP24306 and atezolizumab will be administered by IV infusion. Following eligibility confirmation, patients will receive XENP24306 in combination with atezolizumab by IV infusion on the first day of each 14-day cycle. The starting dose of XENP24306 in combination therapy is 0.01 mg / kg IV every two weeks. Atezolizumab will be administered by IV infusion at a fixed dose of 840 mg in combination with XENP24306 on the first day of each 14-day cycle. Atezolizumab will be administered after XENP24306 and the subsequent observation period.

[0308]

[0318] The dose of XENP24306 is increased up to 100% of the preceding dose level for each consecutive cohort until a safety threshold (defined as a dose-limiting DLT in one patient or a grade ≥ 2 major organ adverse event in at least two patients during the DLT evaluation period for a given cohort) is observed. Subsequently, each cohort of 3 to 9 patients is evaluated at escalating dose levels according to a 3+3+3 design to determine the MTD (or MAD) of XENP24306 in combination with atezolizumab. Figure 8.

[0309]

[0319] Patients enrolled in the cleared cohort (i.e., backfill cohort) of the combination therapy dose escalation cohort must have one of the following PD-L1 selective tumor types: melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC) urothelial carcinoma (UCC), renal cell carcinoma (RCC), small cell lung cancer (SCLC), gastric cancer (GC), Merkel cell carcinoma (MCC), cutaneous squamous cell carcinoma (cSCC), or highly microsatellite instability (MSI-H) cancer.

[0310]

[0320] In total, approximately 225 to 350 patients may be enrolled in this trial across approximately 25 to 35 trial sites worldwide. Patients in this trial will first be evaluated for eligibility during a screening period (duration ≤ 28 days). The starting dose of XENP24306 in combination with atezolizumab will not be higher than one dose level lower than the dose of XENP24306 that shows PD activity in the monotherapy portion of the trial (Example 6). If the initial dose level of 0.01 mg / kg of XENP24306 as monotherapy shows PD activity, the starting dose of XENP24306 will not be higher than 0.005 mg / kg in the initial atezolizumab combination cohort. XENP24306 and atezolizumab will be administered by IV infusion during the expansion phase. The provisional recommended dose escalation (RED) for XENP24306 will be proposed below the established MTD / MAD in dose escalation.

[0311]

[0321] If RED, a combination of XENP24306 and atezolizumab, is proposed, further patients will be enrolled in the expansion phase and treated with RED.

[0312]

[0322] The pharmacokinetics (PK) of XENP24306 will be evaluated. Patients will be evaluated weekly by physical examination and blood sampling for routine hematological and metabolic laboratory assessments during the first eight cycles of XENP24306 treatment in combination with atezolizumab during dose escalation, during the first two cycles during expansion, and thereafter at a less frequent frequency. Tumor assessments will be performed at baseline and after the start of the trial.

[0313]

[0323] All patients will be closely monitored for adverse events throughout the study and for at least 90 days after the final dose of the study treatment or until the initiation of another systemic anticancer therapy, whichever comes first. Adverse events will be classified according to NCI CTCAE v5.0.

[0314]

[0324] To characterize the pharmacokinetics, immunogenic response, and PD characteristics of XENP24306 in combination with atezolizumab, blood samples will be collected at various time points before and after administration.

[0315]

[0325] Patients will be subjected to tumor assessments at screening (baseline) and periodically throughout the trial, as measured by RECIST v1.1. iRECIST will also be used in this study to better characterize the different patterns of response associated with cancer immunotherapy (CIT) and to enable a better understanding of the preliminary activity profile of XENP24306 in combination with atezolizumab. iRECIST is intended to supplement the standard RECIST v1.1 in this trial and enable investigators to comprehensively assess the benefits and risks for patients.

[0316]

[0326] The objective of this study is to conduct a preliminary evaluation of the activity of XENP24306 when administered in combination with atezolizumab, based on the following endpoints: • Serum concentration of XENP24306; • Percentage of participants experiencing adverse events; Objective response rate (ORR) is defined as the percentage of patients who achieve complete remission (CR) or partial remission (PR) in two consecutive opportunities separated by ≥ 4 weeks. Duration of response (DOR) is defined as the time from the first documented objective response to disease progression or death due to either cause (whichever occurs first). • Progression-free survival (PFS) after registration, defined as the time from registration to disease progression or death due to any cause (whichever occurs first); and • Overall survival (OS) after registration, defined as the time from registration to death from any cause.

[0317]

[0327] The safety objective of this study is to evaluate the safety of XENP24306 when administered in combination with atezolizumab, based on the occurrence and severity of adverse events, changes from baseline in targeted vital signs, or clinical laboratory results or ECG parameters.

[0318]

[0328] The objective of this pharmacokinetic (PK) study is to characterize the PK profile of XENP24306 when administered in combination with atezolizumab, based on the serum concentration of XENP24306 at specific time points.

[0319]

[0329] The purpose of this immunogenicity study is to evaluate the immune response to XENP24306 when administered in combination with atezolizumab, based on the ADA for XENP24306 and the ADA for XENP24306 and atezolizumab during the study.

[0320] Example 12: Open-label, multicenter, global dose-escalation study of XENP32803 in combination with atezolizumab.

[0330] An open-label, multicenter, global dose-escalation study will likely be conducted to evaluate the safety, tolerability, pharmacokinetics, and activity of XENP32803 in combination with anti-PD-L1 / PD-1 antibodies such as atezolizumab.

[0321]

[0331] The trial consists of a screening period of up to 28 days, a treatment period, and a minimum follow-up period of 90 days post-treatment. Patients considering enrollment in the expanded cohort of combination therapy with PD-L1-selected tumors can undergo a tissue pre-screening for PD-L1 status, which will be conducted prior to the 28-day screening period.

[0322]

[0332] Patients are enrolled in two stages: a dose escalation stage and a dose expansion stage.

[0323]

[0333] Approximately 21 to 54 patients with locally progressive, recurrent, or metastatic, incurable solid tumors will be enrolled in the dose-escalation phase of the combination therapy portion of the study. XENP32803 and atezolizumab will be administered by IV infusion. Following eligibility confirmation, patients will receive XENP32803 in combination with atezolizumab by IV infusion on the first day of each 14-day cycle. The starting dose of XENP32803 in combination therapy is 0.01 mg / kg IV every two weeks. Atezolizumab will be administered by IV infusion at a fixed dose of 840 mg in combination with XENP32803 on the first day of each 14-day cycle. Atezolizumab will be administered after XENP32803 and the subsequent observation period.

[0324]

[0334] The dose of XENP32803 is increased up to 100% of the preceding dose level for each consecutive cohort until a safety threshold (defined as a dose-limiting DLT in one patient or a grade ≥ 2 major organ adverse event in at least two patients during the DLT evaluation period for a given cohort) is observed. Subsequently, each cohort of 3 to 9 patients is evaluated at escalating dose levels according to a 3+3+3 design to determine the MTD (or MAD) of XENP32803 in combination with atezolizumab. Figure 8.

[0325]

[0335] Patients enrolled in the cleared cohort (i.e., backfill cohort) of the combination therapy dose escalation cohort must have one of the following PD-L1 selective tumor types: melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC) urothelial carcinoma (UCC), renal cell carcinoma (RCC), small cell lung cancer (SCLC), gastric cancer (GC), Merkel cell carcinoma (MCC), cutaneous squamous cell carcinoma (cSCC), or highly microsatellite instability (MSI-H) cancer.

[0326]

[0336] In total, approximately 225 to 350 patients may be enrolled in this trial across approximately 25 to 35 trial sites worldwide. Patients in this trial will first be evaluated for eligibility during a screening period (duration ≤ 28 days). The starting dose of XENP32803 in combination with atezolizumab will not be higher than one dose level lower than the dose of XENP32803 that shows PD activity in the monotherapy portion of the trial (Example 6). If the initial dose level of 0.01 mg / kg of XENP32803 in monotherapy shows PD activity, the starting dose of XENP32803 will not be higher than 0.005 mg / kg in the initial atezolizumab combination cohort. XENP32803 and atezolizumab will be administered by IV infusion during the expansion phase. The provisional recommended dose escalation (RED) for XENP32803 will be proposed below the established MTD / MAD in dose escalation.

[0327]

[0337] If RED, a combination of XENP32803 and atezolizumab, is proposed, further patients will be enrolled in the expansion phase and treated with RED.

[0328]

[0338] The pharmacokinetics (PK) of XENP32803 will be evaluated. Patients will be evaluated weekly by physical examination and blood sampling for routine hematological and metabolic laboratory assessments during the first eight cycles of XENP32803 treatment in combination with atezolizumab during dose escalation, during the first two cycles during expansion, and thereafter at a less frequent frequency. Tumor assessments will be performed at baseline and after the start of the trial.

[0329]

[0339] All patients will be closely monitored for adverse events throughout the study and for at least 90 days after the final dose of the study treatment or until the initiation of another systemic anticancer therapy, whichever comes first. Adverse events will be classified according to NCI CTCAE v5.0.

[0330]

[0340] To characterize the pharmacokinetics, immunogenic response, and PD characteristics of XENP32803 in combination with atezolizumab, blood samples will be collected at various time points before and after administration.

[0331]

[0341] Patients will be subjected to tumor assessments at screening (baseline) and periodically throughout the trial, as measured by RECIST v1.1. iRECIST will also be used in this study to better characterize the different patterns of response associated with cancer immunotherapy (CIT) and to enable a better understanding of the preliminary activity profile of XENP32803 in combination with atezolizumab. iRECIST is intended to supplement standard RECIST v1.1 in this trial and enable investigators to comprehensively assess the benefits and risks for patients.

[0332]

[0342] The objective of this study is to conduct a preliminary evaluation of the activity of XENP32803 when administered in combination with atezolizumab, based on the following endpoints: • Serum concentration of XENP32803; • Percentage of participants experiencing adverse events; Objective response rate (ORR) is defined as the percentage of patients who achieve complete remission (CR) or partial remission (PR) in two consecutive opportunities separated by ≥ 4 weeks. Duration of response (DOR) is defined as the time from the first documented objective response to disease progression or death due to either cause (whichever occurs first). • Progression-free survival (PFS) after registration, defined as the time from registration to disease progression or death due to any cause (whichever occurs first); and • Overall survival (OS) after registration, defined as the time from registration to death from any cause.

[0333]

[0343] The safety objective of this study is to evaluate the safety of XENP32803 when administered in combination with atezolizumab, based on the occurrence and severity of adverse events, changes from baseline in targeted vital signs, or clinical laboratory results or ECG parameters.

[0334]

[0344] The objective of this pharmacokinetic (PK) study is to characterize the PK profile of XENP32803 when administered in combination with atezolizumab, based on the serum concentration of XENP32803 at specific time points.

[0335]

[0345] The purpose of this immunogenicity study is to evaluate the immune response to XENP32803 when administered in combination with atezolizumab, based on the ADA for XENP32803 and the ADA for XENP32803 and atezolizumab during the study.

[0336]

[0346] While this disclosure is provided in some detail through descriptions and examples for the purpose of clear understanding, it will be obvious to those skilled in the art that various modifications and alterations can be implemented without departing from the spirit or scope of this disclosure. Therefore, the above description and examples should not be construed as limiting.

[0337] Example 13: An open-label, multicenter, global dose-escalation study of the IL15 / IL15Rα heterodimer protein combination alone or in combination with atezolizumab.

[0347] A monotherapy open-label, multicenter, global dose-escalation study was conducted to evaluate the safety, tolerability, pharmacokinetics, and activity of the IL15 / IL15Rα heterodimer protein combination (XENP24306 (~82%) and XENP32803 (~18%) ("XENP24306+XENP32803")) according to Example 6. A combination therapy open-label, multicenter, global dose-escalation study was also conducted to evaluate the safety, tolerability, pharmacokinetics, and activity of XENP24306+XENP32803 in combination with an anti-PD-L1 / PD-1 antibody such as atezolizumab according to Example 10.

[0338]

[0348] Twelve patients with solid tumors were enrolled in this study. In the dose-escalation study group (Phase 1a), on the first day of each 14-day cycle (Q2W), one patient received 0.01 mg / ml of XENP24306+XENP32803 via IV infusion; three patients received 0.02 mg / ml of XENP24306+XENP32803; three patients received 0.04 mg / ml of XENP24306+XENP32803; and two patients received 0.06 mg / ml of XENP24306+XENP32803. See Example 6 and Figure 7. The pharmacodynamic (PD) activity of these patients was monitored by CD8+ T cell and / or NK cell expansion.

[0339]

[0349] A dose-dependent increase in CD3-CD16+ / CD56+ NK cells was observed with XENP24306+XENP32803 in a Phase 1a dose-escalation study. The starting dose of XENP24306+XENP32803 in the combination therapy group (Phase 1b) of the study was set at 0.01 mg / kg of XENP24306+XENP32803, and three patients received 0.01 mg / ml of XENP24306+XENP32803 in combination with 840 mg of atezolizumab intravenously over Q2W. See Example 10 and Figure 8.

Claims

1. A method for treating a solid tumor in a subject requiring treatment of a solid tumor, comprising administering to the subject a therapeutically effective amount of a heterodimer protein, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E 357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L36 8D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D A method comprising a set of amino acid substitutions selected from the group consisting of / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

2. CD8 ＋ A method for inducing the proliferation of effector memory T cells, comprising administering an effective amount of heterodimer protein to a subject, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357 Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D / K A method comprising a set of amino acid substitutions selected from the group consisting of 370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

3. A method for inducing proliferation of NK cells, comprising administering an effective amount of heterodimer protein to a subject, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains comprising S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357Q:L36 8D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K37 0S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D / K37 A method comprising a set of amino acid substitutions selected from the group consisting of 0S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

4. CD8 ＋ A method for inducing proliferation of effector memory T cells and NK cells, comprising administering an effective amount of heterodimer protein to a subject, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains are S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E 357Q:L368D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L36 8D / K370S:S364K / E357L;K370S:S364K / E357Q;S267K / S364K / E357Q:S267K / L368D / K370S;L368D A method comprising a set of amino acid substitutions selected from the group consisting of / K370S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

5. A method for inducing IFNγ production in a subject, comprising administering an effective amount of heterodimer protein to the subject, the heterodimer protein comprising: (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain; and (ii) a second monomer comprising an IL-15Rα protein and a second Fc domain, wherein the IL-15Rα protein is covalently bound to the N-terminus of the second Fc domain; the first and second Fc domains comprising S267K / L368D / K370S:S267K / S364K / E357Q;S364K / E357Q:L3 68D / K370S;L368D / K370S:S364K;L368E / K370S:S364K;T411E / K360E / Q362E:D401K;L368D / K3 70S: S364K / E357L; K370S: S364K / E357Q; S267K / S364K / E357Q: S267K / L368D / K370S; L368D / K37 A method comprising a set of amino acid substitutions selected from the group consisting of 0S:S364K / E357Q;S364K:L368D / K370S;S364K:L368E / K370S;D401K:T411E / K360E / Q362E;S364K / E357L:L368D / K370S; and S364K / E357Q:K370S (according to EU numbering).

6. The method according to any one of claims 1 to 5, wherein each of the first Fc domain and / or the second Fc domain independently comprises the amino acid substitutions Q295E, N384D, Q418E and N421D (as assigned by EU numbering).

7. The method according to any one of claims 1 to 6, wherein each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / L234V / L235A / G236del / S239K;E233P / L234V / L235A / G236del / S267K;E233P / L234V / L235A / G236del / S239K / A327G;E233P / L234V / L235A / G236del / S267K / A327G; and E233P / L234V / L235A / G236del (as assigned by EU numbering), and the Fc domain is derived from the Fc domain of IgG1 or IgG3.

8. The method according to any one of claims 1 to 6, wherein each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of L328R; S239K; and S267K (as assigned by EU numbering), and the Fc domain is derived from the Fc domain of IgG2.

9. Each of the first Fc domain and / or the second Fc domain independently comprises an amino acid substitution selected from the group consisting of G236R / L328R;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K;E233P / F234V / L235A / G236del / S239K;E233P / F234V / L235A / G236del / S267K; and E233P / F234V / L235A / G236del (according to EU numbering), wherein the Fc domain is derived from the Fc domain of IgG4. The method according to any one of claims 1 to 6.

10. The method according to any one of claims 1 to 9, wherein the IL-15 protein comprises one or more amino acid substitutions selected from the group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E.

11. The method according to any one of claims 1 to 9, wherein the IL-15 protein and the IL-15Rα protein include a set of amino acid substitutions or additions selected from:65DPC;E87C:65DCA;V49C:S40C;L52C:S40C;E89C:K34C;Q48C:G38C;E53C:L42C;C42S:A37C and L45C:A37C, respectively.

12. The method according to any one of claims 1 to 11, wherein the IL-15 protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:

2.

13. The method according to any one of claims 1 to 12, wherein the IL-15Rα protein comprises a polypeptide sequence selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO:

4.

14. The method according to any one of claims 1 to 5, wherein the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain further comprises amino acid substitutions S364K and E357Q; each of the first Fc domain and the second Fc domain further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (as assigned by EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO:

4.

15. The method according to any one of claims 1 to 5, wherein the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions L368D and K370S; each of the first Fc domain and the second Fc domain further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (as assigned by EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO:

4.

16. The method according to any one of claims 1 to 5, wherein the first Fc domain comprises amino acid substitutions L368D and K370S; the second Fc domain comprises amino acid substitutions K246T, S364K and E357Q; each of the first Fc domain and the second Fc domain further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (as assigned by EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO:

4.

17. The method according to any one of claims 1 to 5, wherein the first Fc domain comprises amino acid substitutions S364K and E357Q; the second Fc domain comprises amino acid substitutions K246T, L368D and K370S; each of the first Fc domain and the second Fc domain further comprises amino acid substitutions C220S, E233P, L234V, L235A, G236del, S267K, M428L and N434S (as assigned by EU numbering); the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D; and the IL-15Rα protein comprises SEQ ID NO:

4.

18. The method according to any one of claims 1 to 17, wherein the IL-15 protein is covalently bound to the N-terminus of a first Fc domain via a first linker.

19. The method according to any one of claims 1 to 18, wherein the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker.

20. The method according to any one of claims 1 to 19, wherein the IL-15 protein is covalently bound to the N-terminus of a first Fc domain via a first linker, and the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker.

21. The method according to any one of claims 18 to 20, wherein the first linker and / or the second linker is independently a variable-length Gly-Ser linker.

22. The method according to claim 21, wherein the first linker and / or the second linker independently includes a linker selected from the group consisting of (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 39), (Ser-Ser-Ser-Ser-Gly)n (SEQ ID NO: 40), (Gly-Ser-Ser-Gly-Gly)n (SEQ ID NO: 41), and (Gly-Gly-Ser-Gly-Gly)n (SEQ ID NO: 42), where n is an integer between 1 and 5.

23. The method according to any one of claims 1 to 22, wherein the heterodimer protein is selected from the group consisting of XENP22822, XENP23504, XENP24045, XENP24306, XENP22821, XENP23343, XENP23557, XENP24113, XENP24051, XENP24341, XENP24052, XENP24301, and XENP32803 proteins.

24. A method for treating a solid tumor in a subject requiring treatment of a solid tumor, comprising administering a therapeutically effective amount of a heterodimer protein to the subject, wherein the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain, A method comprising: a second monomer in which the sucoid domain of the IL-15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

25. CD8 ＋ A method for inducing the proliferation of effector memory T cells, comprising administering an effective amount of heterodimeric protein to a subject, wherein the heterodimeric protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain, A method comprising: a second monomer in which the sucoid domain of the IL-15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

26. A method for inducing the proliferation of NK cells, comprising administering an effective amount of heterodimer protein to a subject, wherein the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain and a second Fc domain of an IL-15Rα protein. A method comprising: a second monomer in which the sucoid domain of the protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

27. CD8 ＋ A method for inducing the proliferation of effector memory T cells and NK cells, comprising administering an effective amount of heterodimeric protein to a subject, wherein the heterodimeric protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain of an IL-15Rα protein and a second Fc domain. A method comprising: a second monomer in which the sucoid domain of the IL-15Rα protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

28. A method for inducing IFNγ in a subject, comprising administering an effective amount of heterodimer protein to the subject, wherein the heterodimer protein comprises (i) a first monomer comprising an IL-15 protein and a first Fc domain, wherein the IL-15 protein is covalently bound to the N-terminus of the first Fc domain, and (ii) a second monomer comprising a sucoid domain and a second Fc domain of an IL-15Rα protein. A method comprising: a second monomer in which the sucoid domain of the protein is covalently bonded to the N-terminus of the second Fc domain; each of the first Fc domain and the second Fc domain comprises the amino acid substitutions E233P, L234V, L235A, G236del, and S267K (according to EU numbering); and the IL-15 protein comprises the N65D amino acid substitution and one or more amino acid substitutions selected from the group consisting of N4D, D30N, and E64Q.

29. The method according to any one of claims 24 to 28, wherein the first Fc domain further comprises amino acid substitutions L368D and K370S, and the second Fc domain further comprises amino acid substitutions S364K and E357Q (as assigned by EU numbering).

30. The method according to any one of claims 24 to 28, wherein the first Fc domain further comprises amino acid substitutions S364K and E357Q, and the second Fc domain further comprises amino acid substitutions L368D and K370S (as assigned by EU numbering).

31. The method according to any one of claims 24 to 30, wherein the first Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E and N421D (as assigned by EU numbering).

32. The method according to any one of claims 24 to 30, wherein the second Fc domain further comprises amino acid substitutions Q295E, N384D, Q418E and N421D (as assigned by EU numbering).

33. The method according to any one of claims 24 to 32, wherein the second Fc domain further comprises the amino acid substitution K246T (as assigned by EU numbering).

34. The method according to any one of claims 24 to 33, wherein the IL-15 protein comprises amino acid substitutions D30N, E64Q and N65D.

35. The method according to any one of claims 24 to 34, wherein the IL-15 protein comprises the amino acid sequence shown in SEQ ID NO:

5.

36. The method according to any one of claims 24 to 35, wherein the sucoid domain of the IL-15Rα protein comprises the amino acid sequence shown in SEQ ID NO:

4.

37. The method according to any one of claims 24 to 36, wherein the IL-15 protein is covalently bound to the N-terminus of a first Fc domain via a first linker.

38. The method according to any one of claims 24 to 37, wherein the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker.

39. The method according to any one of claims 24 to 38, wherein the IL-15 protein is covalently bound to the N-terminus of a first Fc domain via a first linker, and the IL-15Rα protein is covalently bound to the N-terminus of a second Fc domain via a second linker.

40. The method according to any one of claims 37 to 39, wherein the first linker and / or the second linker is independently a variable-length Gly-Ser linker.

41. The method according to claim 40, wherein the first linker and / or the second linker independently includes a linker selected from the group consisting of (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 39), (Ser-Ser-Ser-Ser-Gly)n (SEQ ID NO: 40), (Gly-Ser-Ser-Gly-Gly)n (SEQ ID NO: 41), and (Gly-Gly-Ser-Gly-Gly)n (SEQ ID NO: 42), where n is an integer between 1 and 5.

42. The method according to any one of claims 1 to 5 and 24 to 28, wherein the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO:

10.

43. The method according to any one of claims 1 to 5 and 24 to 28, wherein the first monomer comprises the amino acid sequence shown in SEQ ID NO: 9, and the second monomer comprises the amino acid sequence shown in SEQ ID NO:

16.

44. The method according to any one of claims 1 to 5 and 24 to 28, wherein the heterodimer protein is XENP24306, XENP32803, or a combination thereof.

45. The method according to any one of claims 1 to 44, wherein a combination of a first heterodimer protein and a second heterodimer protein is administered to a subject.

46. The method according to claim 45, wherein the first heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO: 10; and the second heterodimer protein comprises a first monomer having the amino acid sequence shown in SEQ ID NO: 9 and a second monomer having the amino acid sequence shown in SEQ ID NO:

16.

47. The method according to claim 45 or 46, wherein the first heterodimer protein and the second heterodimer protein are administered simultaneously.

48. The method according to claim 45 or 46, wherein the first heterodimer protein and the second heterodimer protein are administered in succession.

49. The method according to any one of claims 1, 6 to 24 and 29 to 48, wherein the solid tumor is locally progressive, recurrent, or metastatic.

50. The method according to any one of claims 1, 6 to 24 and 29 to 48, wherein the solid tumor is selected from the group consisting of squamous cell carcinoma, cutaneous squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liposarcoma, soft tissue sarcoma, urothelial carcinoma, ureter and renal pelvis, multiple myeloma, osteosarcoma, hepatocellular carcinoma, melanoma, stomach cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, renal cell carcinoma, liver cancer, esophageal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, Merkel cell carcinoma, germ cell carcinoma, high-frequency microsatellite instability cancer, and head and neck squamous cell carcinoma.

51. The method according to claim 50, wherein the solid tumor is selected from melanoma, renal cell carcinoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and triple-negative breast cancer.

52. The method according to claim 51, wherein the solid tumor is selected from melanoma, renal cell carcinoma, and non-small cell lung cancer.

53. The method according to claim 51, wherein the solid tumor is selected from melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and triple-negative breast cancer.

54. The method according to any one of claims 1, 6 to 24 and 29 to 53, wherein the subject has not previously been administered any drug for the treatment of a solid tumor.

55. The method according to any one of claims 1, 6 to 24 and 29 to 53, wherein the subject is currently being administered a checkpoint inhibitor.

56. The method according to any one of claims 1, 6 to 24 and 29 to 53, wherein the subject has previously been administered a checkpoint inhibitor.

57. The method according to claim 55 or 56, wherein the checkpoint inhibitor targets PD-1.

58. The method according to claim 55 or 56, wherein the checkpoint inhibitor targets PD-L1.

59. The method according to claim 55 or 56, wherein the checkpoint inhibitor targets CTLA-4.

60. The method according to any one of claims 1 to 59, wherein the heterodimer protein or combination of heterodimer proteins is administered in doses selected from the group consisting of approximately 0.0025 mg / kg, approximately 0.005 mg / kg, approximately 0.01 mg / kg, approximately 0.015 mg / kg, approximately 0.02 mg / kg, approximately 0.025 mg / kg, approximately 0.03 mg / kg, approximately 0.04 mg / kg, approximately 0.05 mg / kg, approximately 0.06 mg / kg, approximately 0.08 mg / kg, approximately 0.1 mg / kg, approximately 0.12 mg / kg, approximately 0.16 mg / kg, approximately 0.2 mg / kg, approximately 0.24 mg / kg, and approximately 0.32 mg / kg by body weight.

61. The method according to claim 62, wherein the heterodimer protein or combination of heterodimer proteins is administered in a dose selected from the group consisting of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, and 0.06 mg / kg by body weight.

62. The method according to any one of claims 1 to 60, wherein the heterodimer protein or combination of heterodimer proteins is administered in doses selected from the group consisting of 0.0025 mg / kg, 0.005 mg / kg, 0.01 mg / kg, 0.015 mg / kg, 0.02 mg / kg, 0.025 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.06 mg / kg, 0.08 mg / kg, 0.10 mg / kg, 0.16 mg / kg, 0.20 mg / kg, 0.24 mg / kg, and 0.32 mg / kg by body weight.

63. The method according to claim 62, wherein the heterodimer protein or combination of heterodimer proteins is administered in a dose selected from the group consisting of 0.01 mg / kg, 0.02 mg / kg, 0.04 mg / kg, and 0.06 mg / kg by body weight.

64. The method according to any one of claims 1 to 63, wherein the heterodimer protein is administered at a frequency selected from the group consisting of Q1W, Q2W, Q3W, Q4W, Q5W, and Q6W.

65. The method according to claim 64, wherein the heterodimer protein is administered at a frequency of Q2W.

66. The method according to any one of claims 1 to 65, further comprising administering a drug that targets the PD-L1 / PD-1 axis to a subject.

67. The method according to claim 66, wherein the agent targeting the PD-L1 / PD-1 axis is an anti-PD-1 antibody.

68. The method according to claim 67, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, pidilizumab, semiprimab, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, MDX-1106, AMP-514, and AMP-224.

69. The method according to claim 68, wherein the agent targeting the PD-L1 / PD-1 axis is an anti-PD-L1 antibody.

70. The method according to claim 69, wherein the anti-PD-L1 antibody is selected from avelumab, durvalumab, atezolizumab, BMS-936559, BMS-39886, KN035, CK-301, and MSB0010718C.

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