Il-15 / il-15ra heterodimeric fc fusion proteins and uses thereof

Abstract

To provide cytokine-based cancer treatments with enhanced pharmacokinetics and pharmacodynamics, synergistically combined with checkpoint blockade antibodies.SOLUTION: A novel IL-15 / IL-15Rα heterodimeric Fc fusion protein comprises a first monomer, comprising an IL-15 receptor alpha (IL-15Rα) sushi domain, a first domain linker and a first variant Fc domain comprising CH2-CH3, and a second monomer, comprising a variant IL-15 domain comprising an amino acid sequence, a second domain linker, and a second variant Fc domain comprising CH2-CH3.SELECTED DRAWING: Figure 9A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 659,563, filed April 18, 2018. , U.S. Provisional Application No. 62 / 684,143, filed June 12, 2018, 2018 U.S. Provisional Application No. 62 / 724,396, filed August 29, 2018, and November 2018 This application claims priority to and incorporates the disclosures of U.S. Provisional Application No. 62 / 756,800, filed on the 7th. are incorporated by reference in their entirety. [Background technology]

[0002] Two very promising approaches in cancer immunotherapy include cytokine-based therapy and These include blocking immune checkpoint proteins such as PD-1.

[0003] Cytokines such as IL-2 and IL-15 stimulate the proliferation of B cells, T cells, and NK cells. Both cytokines function in supporting proliferation and differentiation. receptor, common gamma chain (γc, CD132), and IL-2 receptor beta chain (IL-2 Rβ, CD122), and alpha chain receptors specific to each cytokine: IL-2 receptor Interleukin-2 receptor alpha (IL-2Rα, CD25) or IL-15 receptor alpha (IL-15R α, CD215) and their cell signaling functions through binding to a trimeric complex Both cytokines are considered potentially valuable therapeutic agents in oncology. IL-2 is approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. Currently, several clinical trials are underway, but there are no approved recombinant IL-15 treatments. However, as a promising drug, both cytokines are measured within minutes. IL-2 immunotherapy has a long half-life and suffers from very rapid clearance. It is associated with systemic toxicity when administered at high doses to overcome clearance. Such systemic toxicity has also been reported in recent clinical trials of IL-15 immunotherapy (Guo et al., J Immunol, 2015, 195(5):2353-64).

[0004] Immune checkpoint proteins such as PD-1 are upregulated following T cell activation. When it binds to immune checkpoint ligands such as PD-L1, it exhausts activated T cells. However, immune checkpoint proteins Qualitatively, thymocytes are also upregulated in tumor-infiltrating lymphocytes (TILs), and immune checkpoint ligands It is overexpressed in tumor cells and contributes to immune escape by tumor cells. Opdivo® ) (nivolumab) and Keytruda® (pembrolizumab) De-suppression of TILs by blocking immune checkpoint interactions is a promising approach for cancer treatment. Chemotherapy such as nivolumab and pembrolizumab has proven effective. Despite the promise of checkpoint blockade therapy, many patients still require checkpoint blockade therapy. Blockade alone has failed to achieve an adequate response.

[0005] Therefore, tumor-targeted subcutaneous administration is required to avoid high doses and systemic toxicity. There remains an unmet need in tumor treatment for cytokine-based therapeutic strategies. Furthermore, additional therapies in conjunction with checkpoint blockade may increase patient response rates. The present invention provides a method for the treatment of rheumatoid arthritis with enhanced pharmacokinetics and pharmacodynamics, and for the treatment of rheumatoid arthritis with improved pharmacokinetics and pharmacodynamics. Reduced potency IL-15 / Rα-Fc fusion synergistically combined with an intracellular blocking antibody Providing protein addresses these needs and warnings. Summary of the Invention

[0006] The present invention provides a novel IL-15 / IL-15Rα target in combination with a checkpoint blocking antibody. In some embodiments, the present invention relates to administering a heterodimeric Fc-fusion protein. Checkpoint blocking antibodies include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIM3 antibodies, and anti selected from the group consisting of TIGIT antibody, anti-LAG3 antibody, and anti-CTLA-4 antibody .

[0007] In some aspects, a method of treating cancer in a patient in need thereof is provided herein. provided to a therapeutically effective amount of an IL-15 / IL-15Rα heterodimeric Fc fusion protein, a) From the N-terminus to the C-terminus, i) IL-15 receptor alpha (IL-15Rα) sushi domain, ii) a first domain linker, and iii) a first monomer comprising a first variant Fc domain comprising CH2-CH3; And, b) From the N-terminus to the C-terminus, i) the amino acid sequence of SEQ ID NO: 2, as well as N4D / N65D, D30N / N65D, and any of the amino acid substitutions selected from the group consisting of D30N / E64Q / N65D a variant IL-15 domain comprising one of: ii) a second domain linker, and iii) a second monomer comprising a second variant Fc domain comprising CH2-CH3; and, The first and second variant Fc domains are S267K / L368 according to EU numbering. D / K370S:S267K / S364K / E357Q, S364K / E357Q:L3 68D / K370S, L368D / K370S:S364K, L368E / K370S: S364K, T411E / K360E / Q362E:D401K, L368D / K370 From the group consisting of S:S364K / E357L and K370S:S364K / E357Q a therapeutically effective amount of IL-15 / IL-15Rα having a set of amino acid substitutions selected from a heterodimeric Fc fusion protein; a therapeutically effective amount of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, a checkpoint antibody selected from the group consisting of an antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody. and a vasopressin-blocking antibody.

[0008] In some embodiments, the variant IL-15 domain has the amino acid sequence of SEQ ID NO:2. In some embodiments, the amino acid sequence includes the amino acid substitutions D30N / E64Q / N65D. The variant IL-15 domain has the amino acid sequence of SEQ ID NO: 2 and the amino acid substitution D30 In some embodiments, the variant IL-15 domain comprises the sequence It contains the amino acid sequence of number 2 and the amino acid substitutions N4D / N65D.

[0009] In some embodiments, the IL-15Rα sushi domain has the amino acid sequence of SEQ ID NO:4. Includes.

[0010] In some embodiments, the first and second variant Fc domains comprise S364K / E357Q:L368D / K370S substitutions. The variant Fc domain contains the S364K / E357Q substitutions and a second variant Fc domain. It has L368D / K370S substitutions.

[0011] In some embodiments, the first and second mutated Fc domains each comprise: M428L / Contains the N434S substitution.

[0012] In some embodiments, the first and second mutated Fc domains each comprise E233P / Contains L234V / L235A / G236del / S267K substitutions.

[0013] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The protein and checkpoint blockade antibody are administered simultaneously or sequentially.

[0014] In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, or It is pidilizumab.

[0015] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The protein comprises the amino acid sequence of XENP24306 or XENP24045.

[0016] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), and the anti-PD-1 antibody is In some embodiments, the IL-15 / IL-15Rα heterodimer is nivolumab. The Fc fusion protein comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), The PD-1 antibody is pembrolizumab. The -15Rα heterodimeric Fc fusion protein is XENP24306 (SEQ ID NO: XX) The anti-PD-1 antibody comprises the amino acid sequence pidilizumab.

[0017] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), and the anti-PD-1 antibody is In some embodiments, the IL-15 / IL-15Rα heterodimer is nivolumab. The Fc fusion protein comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), The PD-1 antibody is pembrolizumab. The -15Rα heterodimeric Fc fusion protein is The anti-PD-1 antibody comprises the amino acid sequence pidilizumab.

[0018] In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is breast cancer. cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, Kidney cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic from the group consisting of cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, and myeloma be selected.

[0019] In some embodiments, the methods of treating cancer outlined herein include administering the following to a patient: This results in a small level of vascular leakage.

[0020] In some embodiments, the level of vascular leakage is determined by measuring serum albumin in the patient after administration. The reduction range is less than 20%.

[0021] In some aspects, provided herein are methods of treating cancer in a patient, the methods comprising: IL-15 / IL-15Rα heterodimeric Fc fusion protein and checkpoint blockade administering to a patient a combination therapy comprising an IL-15 / IL-15Rα heterodimer; Dimeric Fc fusions are a) From the N-terminus to the C-terminus, i) IL-15 receptor alpha (IL-15Rα) sushi domain, ii) a first domain linker, and iii) a first monomer comprising a first variant Fc domain comprising CH2-CH3; And, b) From the N-terminus to the C-terminus, i) the amino acid sequence of SEQ ID NO: 2, as well as N4D / N65D, D30N / N65D, and any of the amino acid substitutions selected from the group consisting of D30N / E64Q / N65D a variant IL-15 domain comprising one of: ii) a second domain linker, and iii) a second monomer comprising a second variant Fc domain comprising CH2-CH3; and, The first and second variant Fc domains are S267K / L368 according to EU numbering. D / K370S:S267K / S364K / E357Q, S364K / E357Q:L3 68D / K370S, L368D / K370S:S364K, L368E / K370S: S364K, T411E / K360E / Q362E:D401K, L368D / K370 From the group consisting of S:S364K / E357L and K370S:S364K / E357Q and the checkpoint blockade antibody has a set of amino acid substitutions selected from the group consisting of an anti-PD-1 antibody and an anti-PD-1 antibody. antibodies, anti-PD-L1 antibodies, anti-TIM3 antibodies, anti-TIGIT antibodies, anti-LAG3 antibodies, and anti CTLA-4 antibodies.

[0022] In some embodiments, the variant IL-15 domain has the amino acid sequence of SEQ ID NO:2. In some embodiments, the amino acid sequence includes the amino acid substitutions D30N / E64Q / N65D. The variant IL-15 domain has the amino acid sequence of SEQ ID NO: 2 and the amino acid substitution D30 In some embodiments, the variant IL-15 domain comprises the sequence It contains the amino acid sequence of number 2 and the amino acid substitutions N4D / N65D.

[0023] In some embodiments, the IL-15Rα sushi domain has the amino acid sequence of SEQ ID NO:4. Includes:

[0024] In some embodiments, the first and second variant Fc domains comprise S364K / E357Q:L368D / K370S substitutions. The variant Fc domain contains the S364K / E357Q substitutions and a second variant Fc domain. It has L368D / K370S substitutions.

[0025] In some embodiments, the first and second mutated Fc domains each comprise: M428L / Contains the N434S substitution.

[0026] In some embodiments, the first and second mutated Fc domains each comprise E233P / Contains L234V / L235A / G236del / S267K substitutions.

[0027] In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, or It is pidilizumab.

[0028] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The quality is XENP24306 (sequence number XX) or XENP24045 (sequence number XX) It contains the amino acid sequence of

[0029] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), and the anti-PD-1 antibody is In some embodiments, the IL-15 / IL-15Rα heterodimer is nivolumab. The Fc fusion protein comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), The PD-1 antibody is pembrolizumab. The -15Rα heterodimeric Fc fusion protein is XENP24306 (SEQ ID NO: XX) The anti-PD-1 antibody comprises the amino acid sequence pidilizumab.

[0030] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), and the anti-PD-1 antibody is In some embodiments, the IL-15 / IL-15Rα heterodimer is nivolumab. The Fc fusion protein comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), The PD-1 antibody is pembrolizumab. The -15Rα heterodimeric Fc fusion protein is The anti-PD-1 antibody comprises the amino acid sequence pidilizumab.

[0031] In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is breast cancer. cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, Kidney cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic from the group consisting of cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, and myeloma be selected.

[0032] In some embodiments, the methods of treating cancer described herein include administering to a patient This results in a small level of vascular leakage.

[0033] In some embodiments, the level of vascular leakage is determined by measuring serum albumin in the patient after administration. The reduction range is less than 20%.

[0034] In some aspects, provided herein is a method of inducing T cell expansion in a patient, comprising: a therapeutically effective amount of an IL-15 / IL-15Rα heterodimeric Fc fusion protein, a) From the N-terminus to the C-terminus, i) IL-15 receptor alpha (IL-15Rα) sushi domain, ii) a first domain linker, and iii) a first monomer comprising a first variant Fc domain comprising CH2-CH3; And, b) From the N-terminus to the C-terminus, i) the amino acid sequence of SEQ ID NO: 2, as well as N4D / N65D, D30N / N65D, and any of the amino acid substitutions selected from the group consisting of D30N / E64Q / N65D a variant IL-15 domain comprising one of: ii) a second domain linker, and iii) a second monomer comprising a second variant Fc domain comprising CH2-CH3; and wherein the first and second variant Fc domains are S26 according to EU numbering. 7K / L368D / K370S: Including S267K / S364K / E35, 7Q, S36 4K / E357Q:L368D / K370S, L368D / K370S:S364K, L 368E / K370S:S364K, T411E / K360E / Q362E:D401K , L368D / K370S:S364K / E357L, and K370S:S364K / a therapeutically effective amount of IL having a set of amino acid substitutions selected from the group consisting of E357Q, E357Q, IL-15 / IL-15Rα heterodimeric Fc fusion protein, a therapeutically effective amount of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIM3 antibody, an anti-TIGIT antibody, a checkpoint antibody selected from the group consisting of an antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody. and a vasopressin-blocking antibody.

[0035] In some embodiments, the variant IL-15 domain has the amino acid sequence of SEQ ID NO:2. In some embodiments, the amino acid sequence includes the amino acid substitutions D30N / E64Q / N65D. The variant IL-15 domain has the amino acid sequence of SEQ ID NO: 2 and the amino acid substitution D30 In some embodiments, the variant IL-15 domain comprises the sequence It contains the amino acid sequence of number 2 and the amino acid substitutions N4D / N65D.

[0036] In some embodiments, the IL-15Rα sushi domain has the amino acid sequence of SEQ ID NO:4. Includes:

[0037] In some embodiments, the first and second variant Fc domains comprise S364K / E357Q:L368D / K370S substitutions. The variant Fc domain contains the S364K / E357Q substitutions and a second variant Fc domain. It has L368D / K370S substitutions.

[0038] In some embodiments, the first and second mutated Fc domains each comprise: M428L / Contains the N434S substitution.

[0039] In some embodiments, the first and second mutated Fc domains each comprise E233P / Contains L234V / L235A / G236del / S267K substitutions.

[0040] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The protein and checkpoint blockade antibody are administered simultaneously or sequentially.

[0041] In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, or In some embodiments, the IL-15 / IL-15Rα heterodimer is pidilizumab. The Fc fusion protein is XENP24306 (SEQ ID NO: XX) or XENP2404 5 (SEQ ID NO: XX).

[0042] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), and the anti-PD-1 antibody is In some embodiments, the IL-15 / IL-15Rα heterodimer is nivolumab. The Fc fusion protein comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), The PD-1 antibody is pembrolizumab. The -15Rα heterodimeric Fc fusion protein is XENP24306 (SEQ ID NO: XX) The anti-PD-1 antibody comprises the amino acid sequence pidilizumab.

[0043] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), and the anti-PD-1 antibody is In some embodiments, the IL-15 / IL-15Rα heterodimer is nivolumab. The Fc fusion protein comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), The PD-1 antibody is pembrolizumab. The -15Rα heterodimeric Fc fusion protein is The anti-PD-1 antibody comprises the amino acid sequence pidilizumab.

[0044] In some embodiments, the patient has cancer. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, Bladder cancer, renal cancer, kidney cancer , liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, The cancer is selected from the group consisting of leukemia, lymphoma, and myeloma.

[0045] In some embodiments, T cell expansion is at least a two-fold increase in T cells. In some embodiments, T cell expansion ranges from a 2-fold to a 15-fold increase in T cells.

[0046] In some embodiments, the method does not increase the likelihood of inducing hypoalbuminemia. .

[0047] In some embodiments, the T cells comprise tumor-infiltrating lymphocytes.

[0048] In some embodiments, the present invention provides an IL-15 / IL-15Rα heterodimer Fc fusion protein. The fusion protein and anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIM3 antibody, anti-TIGIT antibody a checkpoint blockade antibody selected from an antibody, an anti-LAG3 antibody, and an anti-CTLA-4 antibody; In some embodiments, the present invention provides a combination therapy comprising an IL-15 / IL-15Rα Heterodimeric Fc fusion proteins are a) From the N-terminus to the C-terminus, i) IL-15 receptor alpha (IL-15Rα) sushi domain, ii) a first domain linker, and iii) a first monomer comprising a first variant Fc domain comprising CH2-CH3; And, b) From the N-terminus to the C-terminus, i) the amino acid sequence of SEQ ID NO: 2, as well as N4D / N65D, D30N / N65D, and any of the amino acid substitutions selected from the group consisting of D30N / E64Q / N65D a variant IL-15 domain comprising one of: ii) a second domain linker, and iii) a second monomer comprising a second variant Fc domain comprising CH2-CH3; and, The first and second variant Fc domains are S267K / L368 according to EU numbering. D / K370S:S267K / S364K / E357Q, S364K / E357Q:L3 68D / K370S, L368D / K370S:S364K, L368E / K370S: S364K, T411E / K360E / Q362E:D401K, L368D / K370 From the group consisting of S:S364K / E357L and K370S:S364K / E357Q The amino acid sequence has a set of amino acid substitutions selected from:

[0049] In some embodiments, the variant IL-15 domain has the amino acid sequence of SEQ ID NO:2. In some embodiments, the amino acid sequence includes the amino acid substitutions D30N / E64Q / N65D. The variant IL-15 domain has the amino acid sequence of SEQ ID NO: 2 and the amino acid substitution D30 In some embodiments, the variant IL-15 domain comprises the sequence It contains the amino acid sequence of number 2 and the amino acid substitutions N4D / N65D.

[0050] In some embodiments, the IL-15Rα sushi domain has the amino acid sequence of SEQ ID NO:4. Includes.

[0051] In some embodiments, the first and second variant Fc domains comprise S364K / E357Q:L368D / K370S substitutions. The variant Fc domain contains the S364K / E357Q substitutions and a second variant Fc domain. It has L368D / K370S substitutions.

[0052] In some embodiments, the first and second variant Fc domains each comprise M4 Includes 28L / N434S substitution.

[0053] In some embodiments, the first and second variant Fc domains each comprise an E2 Contains 33P / L234V / L235A / G236del / S267K substitutions.

[0054] In some embodiments, the anti-PD-1 antibody is nivolumab, pembrolizumab, or It is pidilizumab.

[0055] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The quality is XENP24306 (sequence number XX) or XENP24045 (sequence number XX) It contains the amino acid sequence of

[0056] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), and the anti-PD-1 antibody is It is nivolumab.

[0057] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), and the anti-PD-1 antibody is Pembrolizumab.

[0058] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24306 (SEQ ID NO: XX), and the anti-PD-1 antibody is It is pidilizumab.

[0059] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), and the anti-PD-1 antibody is It is nivolumab.

[0060] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), and the anti-PD-1 antibody is Pembrolizumab.

[0061] In some embodiments, the IL-15 / IL-15Rα heterodimeric Fc fusion protein The antibody comprises the amino acid sequence of XENP24045 (SEQ ID NO: XX), and the anti-PD-1 antibody is It is pidilizumab. [Brief explanation of the drawings]

[0062] [Figure 1] The structure of IL-15 in complex with its receptors IL-15RA (CD215), IL-15RB (CD122), and the common gamma chain (CD132) is shown. [Figure 2-2B] The sequences of IL-15 and its receptor are shown. [Figures 3A-3E] Useful pairs of Fc heterodimer variant sets (including skew and pI variants) are shown in Figures 3D and 3E. In Figures 3D and 3E, there are variants that do not have a corresponding "monomer 2" variant; these are pI variants that can be used alone in either monomer. [Figure 4] A list of isosteric variant antibody constant regions and their respective substitutions is provided below. pI_( ) indicates a lower pI variant and pI_(+) indicates a higher pI variant. These can be optionally and independently combined with other heterodimerization variants of the invention (and also with other variant types as outlined herein). [Figure 5] Useful truncation variants that ablate FcγR binding are shown (sometimes referred to as "knockout" or "KO" variants). Generally, truncation variants are found in both monomers, although in some cases they may be found in only one monomer. [Figures 6A-6E] 1 illustrates a particularly useful embodiment of the "non-cytokine" component of the present invention. [Figure 7] Some exemplary variable length linkers are shown. In some embodiments, these linkers find use in linking the C-terminus of IL-15 and / or IL-15Rα(sushi) to the N-terminus of an Fc region. In some embodiments, these linkers find use in fusing IL-15 to IL-15Rα(sushi). [Figures 8A-8D]

[0039] Figure 1 shows the sequences of several useful IL-15 / Rα-Fc format scaffolds based on human IgG1 that do not contain cytokine sequences (e.g., IL-15 and / or IL-15Rα (sushi)). Scaffold 1 is based on human IgG1 (356E / 358M allotype) and contains the following variants in both chains: C220S, S364K / E357Q:L368D / K370S scuba variants, the pI variant of Q295E / N384D / Q418E / N421D in the chain with the L368D / K370S scuba variant, and the truncation variants of E233P / L234V / L235A / G236del / S267K in both chains. Scaffold 2 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K:L368D / K370S scubariant in both chains, the Q295E / N384D / Q418E / N421D pI variant in the chain with the L368D / K370S scubariant, and the E233P / L234V / L235A / G236del / S267K truncation variant in both chains. Scaffold 3 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K:L368E / K370S scubariant in both chains, the Q295E / N384D / Q418E / N421D pI variant in the chain with the L368E / K370S scubariant, and the E233P / L234V / L235A / G236del / S267K truncation variants in both chains. Scaffold 4 is based on human IgG1 (356E / 358M allotype) and contains the C220S, D401K:K360E / Q362E / T411E scubariant in both chains, the Q295E / N384D / Q418E / N421D pI variant in the chain with the K360E / Q362E / T411E scubariant, and the E233P / L234V / L235A / G236del / S267K truncation variants in both chains.Scaffold 5 is based on human IgG1 (356D / 358L allotype) and contains C220S in both chains, S364K / E357Q:L368D / K370S scuba variants, Q295E / N384D / Q418E / N421D pI variants in the chain with the L368D / K370S scuba variant, and E233P / L234V / L235A / G236del / S267K truncation variants in both chains. Scaffold 6 is based on human IgG1 (356E / 358M allotype) and contains the C220S, S364K / E357Q:L368D / K370S scuba variants in both chains, the Q295E / N384D / Q418E / N421D pI variant in the chain with the L368D / K370S scuba variant, and the E233P / L234V / L235A / G236del / S267K truncation variants in both chains, and the N297A variant in both chains. Scaffold 7 is identical to 6 except for the N297S mutation. Alternative formats for scaffolds 6 and 7 can omit the truncation variant E233P / L234V / L235A / G236del / S267K in both chains. Scaffold 8 is based on human IgG4 and contains a scuba variant of S364K / E357Q:L368D / K370S, a pI variant of Q295E / N384D / Q418E / N421D in the chain with the L368D / K370S scuba variant, and an S228P (according to EU numbering, which is S241P in Kabat) variant in both chains, which abrogates Fab arm exchange as known in the art. Scaffold 9 is based on human IgG2 and contains a scuba variant of S364K / E357Q:L368D / K370S, a pI variant of Q295E / N384D / Q418E / N421D in the chain with the L368D / K370S scuba variant. Scaffold 10 is based on human IgG2 and contains the S364K / E357Q:L368D / K370S scuba variant, the Q295E / N384D / Q418E / N421D pI variant in the chain with the L368D / K370S scuba variant, and the S267K variant in both chains. Scaffold 11 is identical to Scaffold 1 except that it contains the M428L / N434S Xtend mutations.Scaffold 12 is based on human IgG1 (356E / 358M allotype) and contains C220S in both identical chains, E233P / L234V / L235A / G236del / S267K truncation variants in both identical chains, Scaffold 13 is based on human IgG1 (356E / 358M allotype) and contains C220S in both chains, S364K / E357Q:L368D / K370S scuba variants, P217R / P229R / N276K pI variants in the chain with S364K / E357Q scuba variants, and E233P / L234V / L235A / G236del / S267K truncation variants in both chains. As will be appreciated by those skilled in the art and outlined below, these sequences can be used with any of the IL-15 and IL-15Rα(sushi) pairs outlined herein, including, but not limited to, IL-15 / Rα-heteroFc, ncIL-15 / Rα, scIL-15 / Rα, and dsIL-15 / Rα, as shown schematically in Figures 9A-9G and 39. Additionally, any IL-15 and / or IL-15Rα(sushi) variants can be incorporated into these scaffolds of Figures 8A-8E in any combination. Included within each of these scaffolds are sequences that are 90, 95, 98, and 99% identical (as defined herein) to the listed sequence and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions (compared to the "parent" in the figures, which already contains some amino acid modifications compared to the parent human IgG1 (or IgG2 or IgG4 based on the scaffold), as will be appreciated by those skilled in the art). That is, the listed backbone may contain additional amino acid modifications (generally amino acid substitutions) in addition to the skew, pI, and truncation variants contained within this figure backbone. [Figures 9A-9G]Several formats of the IL-15 / Rα-Fc fusion proteins of the present invention are shown. The IL-15Rα heterodimeric Fc fusion or "IL-15 / Rα-hetero-Fc" (Figure 9A) contains IL-15 recombinantly fused to one side of the heterodimeric Fc and IL-15Rα (sushi) recombinantly fused to the other side of the heterodimeric Fc. IL-15 and IL-15Rα (sushi) can have variable-length Gly-Ser linkers between the C- and N-termini of the Fc region. The single-chain IL-15 / Rα-Fc fusion or "scIL-15 / Rα-Fc" (Figure 9B) contains IL-15Rα(sushi) fused to IL-15 by a variable-length linker (referred to as "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of the heterodimeric Fc region, with either "Fc-only" or "empty Fc" at the other end of the molecule. The noncovalent IL-15 / Rα-Fc or "ncIL-15 / Rα-Fc" (Figure 9C) contains IL-15Rα(sushi) fused to the heterodimeric Fc region, while IL-15 is transfected separately to form the noncovalent IL-15 / Rα complex, with either "Fc-only" or "empty Fc" at the other end of the molecule. The bivalent noncovalent IL-15 / Rα-Fc fusion or "bivalent ncIL-15 / Rα-Fc" (Figure 9D) contains IL-15Rα(sushi) fused to the N-terminus of the homodimeric Fc region, while IL-15 is transfected separately to form a noncovalent IL-15 / Rα complex. The bivalent single-chain IL-15 / Rα-Fc fusion or "bivalent scIL-15 / Rα-Fc" (Figure 9E) contains IL-15 fused to IL-15Rα(sushi) by a variable-length linker (referred to as "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the N-terminus of the homodimeric Fc region. The Fc noncovalent IL-15 / Rα fusion or “Fc-ncIL-15 / Rα” ( Figure 9F ) contains IL-15Rα (sushi) fused to the C-terminus of the heterodimeric Fc region, while IL-15 is transfected separately to form a noncovalent IL-15 / Rα complex, and the other side of the molecule is “Fc only” or “empty Fc.”The Fc-single-chain IL-15 / Rα fusion or "Fc-scIL-15 / Rα" (Figure 9G) contains IL-15 fused to IL-15Rα(sushi) by a variable-length linker (referred to as "single-chain" IL-15 / IL-15Rα(sushi) complex or "scIL-15 / Rα"), which is then fused to the C-terminus of a heterodimeric Fc region, with "Fc-only" or "empty Fc" at the other end of the molecule. [Figure 10] The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-heteroFc" format, XENP20818 and XENP21475, are shown; additional sequences for XENP20819, XENP21471, XENP21472, XENP21473, XENP21474, XENP21476, and XENP21477 are listed as SEQ ID NOs: 418-423, 424-429, 430-435, 436-441, 442-447, 454-459, and 460-465, respectively, in Figures 104A-104D of WO2018 / 071919. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 11]1 shows the sequence of XENP21478, an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format; additional sequences for XENP21993, XENP21994, XENP21995, XENP23174, XENP23175, XENP24477, and XENP24480 are listed as SEQ ID NOs: 514-518, 519-523, 524-528, 849-853, 1063-1067, and 1078-1082, respectively, in WO2018 / 071919 in Figures 104G, 104H, 104AG, 104AU, and 104AV, respectively. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 12A-12B] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP21479, XENP22366, and XENP24348, in the "ncIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 13] The sequence of XENP21978, an exemplary IL-15 / Rα-Fc fusion protein in the "bivalent ncIL-15 / Rα-Fc" format, is shown; additional sequences for XENP21979 are listed as SEQ ID NOS: 480-483 in WO2018 / 071919 in Figure 104E. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc region. [Figure 14]7 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "bivalent SCIL-15 / Rα-Fc" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 15] The sequence of XENP22637, an exemplary IL-15 / Rα-Fc fusion protein in the "Fc-ncIL-15 / Rα" format, is shown; additional sequence for XENP22638 is listed as SEQ ID NO: 668 in WO2018 / 071919 in Figure 104T. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc region. [Figure 16] 7 shows the sequence of an exemplary IL-15 / Rα-Fc fusion protein in the "FC-SCIL-15 / Rα" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, linker, and Fc region. [Figures 17A-17E] Figure 17 provides data for an exemplary IL-15 / Rα-Fc fusion protein format of XENP20818. Figure 17A shows the IL-15 / Rα-Fc fusion protein format of XENP20818. Figure 17B shows the purity and homogeneity of XENP20818 as determined by SEC. Figure 17C shows the purity and homogeneity of XENP20818 as determined by CEF. Figure 17D shows the affinity of XENP20818 for IL-2Rβ as determined by Octet. Figure 17E shows the stability of XENP20818 as determined by DSF. [Figures 18A-18E] Figure 18A shows the IL-15 / Rα-Fc fusion protein format of XENP21478. Figure 18B shows the purity and homogeneity of XENP21478 as determined by SEC. Figure 18C shows the purity and homogeneity of XENP21478 as determined by CEF. Figure 18D shows the affinity of XENP21478 for IL-2Rβ as determined by Octet. Figure 18E shows the stability of XENP21478 as determined by DSF. [Figures 19A-19E] Figure 19A shows the IL-15 / Rα-Fc fusion protein format of XENP21479. Figure 19B shows the purity and homogeneity of XENP21479 as determined by SEC. Figure 19C shows the purity and homogeneity of XENP21479 as determined by CEF. Figure 19D shows the affinity of XENP21479 for IL-2Rβ as determined by Octet. Figure 19E shows the stability of XENP21479 as determined by DSF. [Figures 20A-20C] Figure 20 shows the induction of proliferation of NK (CD56+ / CD16+) cells (Figure 20A), CD4+ T cells (Figure 20B), and CD8+ T cells (Figure 20C) by exemplary IL-15 / Rα-Fc fusion proteins in IL-15 / Rα-hetero-Fc formats with different linker lengths based on Ki67 expression as measured by FACS. [Figures 21A-21C] Figure 21A shows the induction of proliferation of NK (CD56+ / CD16+) cells (Figure 21A), CD4+ T cells (Figure 21B), and CD8+ T cells (Figure 21C) by exemplary IL-15 / Rα-Fc fusion proteins in scIL-15 / Rα-Fc format (XENP21478) and ncIL-15 / Rα-Fc format (XENP21479) based on Ki67 expression as measured by FACS. [Figure 22]1 shows the enhancement of IL-2 secretion by an exemplary IL-15 / Rα-Fc fusion protein, an isotype control, and a bivalent anti-PD-1 antibody relative to a PBS control in an SEB-stimulated PBMC assay. [Figure 23] 1 shows survival curves of PBMC-engrafted NSG mice after treatment with XENP20818 and recombinant IL-15.

[0063] [Figure 24] 1 shows the engraftment of human PBMCs and the concentration of IFNγ in the serum of NSG mice 7 days after treatment with the indicated concentrations of XENP20818. [Figures 25A-25C] Shown are the numbers of CD4+ T cells (FIG. 25A), CD8+ T cells (FIG. 25B), and CD45+ cells (FIG. 25C) in the whole blood of human PBMC-engrafted NSG mice 7 days after treatment with XENP20818 at the indicated concentrations. [Figure 26] 1 shows a structural model of the IL-15 / Rα heterodimer showing the location of the engineered disulfide bond pairs.

[0064] [Figure 27] 1 shows the sequence of an exemplary IL-15Rα (sushi) variant engineered with additional residues at the C-terminus to serve as a scaffold for engineering cysteine ​​residues. [Figure 28] 1 shows the sequence of an exemplary cysteine ​​engineered IL-15 variant to form a covalent disulfide bond with the cysteine ​​engineered IL-15Rα (sushi) variant. [Figure 29] 1 shows the sequence of an exemplary cysteine-engineered IL-15Rα (sushi) variant to form a covalent disulfide bond with a cysteine-engineered IL-15 variant. [Figures 30A-30C]IL-15 / Rα heterodimers with or without an engineered disulfide bond between IL-15 and IL-15Rα(sushi) are shown. The noncovalently linked IL-15 / Rα heterodimer or "ncIL-15 / Rα heterodimer" (Figure 30A) contains separately transfected, noncovalently linked IL-15Rα(sushi) and IL-15. The disulfide-linked IL-15 / Rα heterodimer or "dsIL-15 / Rα heterodimer" (Figure 30B) contains separately transfected, covalently linked IL-15Rα(sushi) and IL-15 as a result of engineered cysteines. The single-chain IL-15 / Rα heterodimer or "scIL-15 / Rα heterodimer" (Figure 30C) contains IL-15Rα(sushi) fused to IL-15 by a variable-length Gly-Ser linker. [Figure 31] The sequence of an exemplary ncIL-15 / Rα heterodimer, XENP21996, is shown. It is important to note that these sequences were generated using a polyhistidine (Hisx6 or HHHHHH (SEQ ID NO:XX)) C-terminal tag at the C-terminus of IL-15Rα (Sushi). [Figure 32] The sequences of exemplary dsIL-15 / Rα heterodimers XENP22004, XENP22005, XENP22006, XENP22008, and XENP22494 are shown, with additional sequences of XENP22007, XENP22009, XENP22010, XENP22011, XENP22012, and XENP22493 shown as SEQ ID NOs: 543-544, 545-546, 547-548, 551-552, 553-554, and 647-648, respectively, in Figures 104J, 104K, and 104I of WO2018 / 071919. It is important to note that these sequences were generated using a polyhistidine (Hisx6 or HHHHHH) C-terminal tag at the C-terminus of IL-15Rα(sushi). [Figure 33]The sequence of an exemplary scIL-15 / Rα heterodimer, XENP22049, is shown. It is important to note that these sequences were generated using a polyhistidine (Hisx6 or HHHHHH) C-terminal tag at the C-terminus of IL-15. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, and the linker. [Figure 34] 1 shows the purity and homogeneity of exemplary IL-15 / Rα heterodimers with and without engineered disulfide bonds as determined by CEF. [Figure 35] 1 shows the purity and homogeneity of exemplary IL-15 / Rα heterodimers with and without engineered disulfide bonds as determined by CEF. [Figure 36] 1 shows the stability and melting temperature of exemplary IL-15 / Rα heterodimers with and without engineered disulfide bonds as shown by melting curves from DSF. [Figure 37] 1 shows the stability and melting temperature of exemplary IL-15 / Rα heterodimers with and without engineered disulfide bonds as shown by melting curves from DSF. [Figure 38] Expression yield, molecular weight, predicted change in affinity between IL-15 and IL-15Rα (sushi) as calculated by MOE software, melting temperature, and affinity for IL-2Rβ for IL-15 / Rα heterodimers with and without engineered disulfide bonds are shown. Mutations are shown in parentheses after the relevant monomer. [Figures 39A-39D]

[0039] Figure 39A shows additional formats of the IL-15 / Rα-Fc fusion proteins of the invention with engineered disulfide bonds. The disulfide-bonded IL-15 heterodimer Fc fusion or "dsIL-15 / Rα-heterodimer Fc" (Figure 39A) is the same as "IL-15 / Rα-heterodimer Fc," but IL-15Rα(sushi) and IL-15 are additionally covalently linked as a result of an engineered cysteine. The disulfide-bonded IL-15 / RαFc fusion or "dsIL-15 / Rα-Fc" (Figure 39B) is the same as "ncIL-15 / Rα-Fc," but IL-15Rα(sushi) and IL-15 are additionally covalently linked as a result of an engineered cysteine. The bivalent disulfide-bonded IL-15 / Rα-Fc or "bivalent dsIL-15 / Rα-Fc" (Figure 39C) is the same as the "bivalent ncIL-15 / Rα-Fc," but IL-15Rα(sushi) and IL-15 are additionally covalently linked as a result of an engineered cysteine. The Fc-disulfide-bonded IL-15 / Rα fusion or "Fc-dsIL-15 / Rα" (Figure 39D) is the same as the "Fc-ncIL-15 / Rα," but IL-15Rα(sushi) and IL-15 are additionally covalently linked as a result of an engineered cysteine. [Figure 40A-40B] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "dsIL-15 / Rα-heteroFc" format: XENP22013, XENP22014, XENP22015, and XENP22017. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 41A-41B]The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "dsIL-15 / Rα-Fc" format, XENP22357, XENP22358, XENP22359, XENP22684, and XENP22361, are shown. Additional sequences for XENPs 22360, 22362, 22363, 22364, 22365, and 22366 are shown in Figures 104O, 104P, 104Q, and 104R of WO2018 / 071919, respectively, which are incorporated by reference in their entireties. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 42] The sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP22634, XENP22635, and XENP22636, in the "bivalent dsIL-15 / Rα-Fc" format are shown. An additional sequence for XENP22687 is shown as SEQ ID NOS: 685-688 in Figure 104V of WO 2018 / 071919, which is incorporated herein by reference in its entirety. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (though one of skill in the art will appreciate that portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc region. [Figure 43]7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP22639 and XENP22640, in the "Fc-dsIL-15 / Rα" format. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc region. [Figure 44] 1 shows the purity and homogeneity of exemplary IL-15 / Rα-Fc fusion proteins with and without engineered disulfide bonds as determined by CEF. [Figures 45A-45C] Figure 45A shows the induction of NK (CD56+ / CD16+) cell (Figure 45A), CD8+ T cell (Figure 45B), and CD4+ T cell (Figure 45C) proliferation by exemplary IL-15 / Rα-Fc fusion proteins with or without an engineered disulfide bond based on Ki67 expression as measured by FACS. [Figure 46] 1 shows the structure of IL-15 in complex with IL-15Rα, IL-2Rβ, and the common γ chain. The locations of substitutions engineered for reduced potency are indicated. [Figures 47A-47C]

[0023] Figures 1A-1C show sequences of exemplary IL-15 variants engineered for reduced potency. Included within each of these variant IL-15 sequences are sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) to the listed sequences, and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions. In a non-limiting example, the listed sequences may contain additional amino acid modifications, such as those that contribute to the formation of covalent disulfide bonds, as described in Example 2. [Fig. 48A-48H]The sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-R / R" format, engineered for lower potency, are shown: XENP22816, XENP22819, XENP22820, XENP22821, XENP22822, XENP22829, XENP22834, XENP23554, XENP23557, XENP23561, XENP24018, XENP24019, XENP24045, XENP24051, and XENP24052. Additional sequences for 22815, 22817, 22818, 22823, 22824, 22825, 22826, 22827, 22828, 22830, 22831, 22832, 22333, 23555, 23559, 23560, 24017, 24020, 24043, and 24048 are shown in Figures 104Z, 104AA, 104AC, 104AD, 104AE, 104AF, 104AJ, 104AK, 104A in WO2018 / 071919. M, 104AN, and 104AO are shown in sequence numbers 729-734, 741-746, 747-752, 777-782, 783-788, 789-794, 795-800, 801-806, 807-812, 819-824, 825-830, 831-836, 837-842, 887-892, 899-904, 905-910, 937-942, 955-960, 961-966, and 979-984, respectively. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, and the Fc region. [Figures 49A-49D]

[0023] Figures 1A-1C show the sequences of exemplary IL-15 / Rα-Fc fusion proteins XENP24015, XENP24050, XENP24475, XENP24476, XENP24478, XENP24479, and XENP24481 in the "SCIL-15 / Rα-Fc" format, which have been engineered for lower potency. Additional sequences for XENP 24013, 24014, and 24016 are shown in Figures 104AK and 104AL of WO2018 / 071919 as SEQ ID NOs: 914-921, 922-926, and 932-936. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in Figure 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, and the Fc region. [Figure 50A-50B] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP24349, XENP24890, and XENP25138, in the "ncIL-15 / Rα-Fc" format, engineered for lower potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc region. [Figure 51]

[0023] Figures 104V, 104W, 104X, 104Y, and 104Z of WO2018 / 071919 show the sequences of exemplary ncIL-15 / Rα heterodimers engineered for lower potency, XENP22801 and XENP22802. Additional sequences for XENPs 22791, 22792, 22793, 22794, 22795, 22796, 22803, 22804, 22805, 22806, 22807, 22808, 22809, 22810, 22811, 22812, 22813, and 22814 are shown in Figures 104V, 104W, 104X, 104Y, and 104Z of WO2018 / 071919. These sequences are shown as SEQ ID NOs: 689-690, 691-692, 693-694, 695-696, 697-698, 699-700, 705-706, 707-708, 709-710, 711-712, 713-714, 715-716, 717-718, 719-720, 721-722, 723-724, 725-726, and 727-728, respectively. It is important to note that these sequences were generated using a polyhistidine (Hisx6 or HHHHHH) C-terminal tag at the C-terminus of IL-15Rα (sushi). [Figure 52] 7 shows the sequence of XENP24342, an exemplary IL-15 / Rα-Fc fusion protein in the "bivalent ncIL-15 / Rα-Fc" format engineered for low potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 53] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP23472 and XENP23473, in the "dsIL-15 / Rα-Fc" format, engineered for lower potency. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc region. [Figures 54A-54C] Figure 54 shows the induction of NK cell (Figure 54A), CD8+ (CD45RA-) T cell (Figure 54B), and CD4+ (CD45RA-) T cell (Figure 54C) proliferation by variant IL-15 / Rα-Fc fusion proteins based on Ki67 expression as measured by FACS. [Figure 55] EC50 for induction of NK and CD8+ T cell proliferation by variant IL-15 / Rα-Fc fusion proteins and fold reduction of EC50 relative to XENP20818 are shown. [Figures 56A-56C] Gating of lymphocytes and subpopulations in the experiment shown in Figures 59A-59D is shown. Figure 56A shows the gated lymphocyte population. Figure 56B shows the CD3-negative and CD3-positive subpopulations. Figure 56C shows the CD16-negative and CD16-positive subpopulations of CD3-negative cells. [Figures 57A-57C] Figures 59A-59D show gating of CD3+ lymphocyte subpopulations in the experiment shown. Figure 57A shows CD4+, CD8+, and γδ T cell subpopulations of CD3+ T cells. Figure 57B shows CD45RA(-) and CD45RA(+) subpopulations of CD4+ T cells. Figure 57C shows CD45RA(-) and CD45RA(+) subpopulations of CD8+ T cells. [Figure 58A-58B] The expression of CD69 and CD25 before (FIG. 58A) and after (FIG. 58B) incubation of human PBMC with XENP22821 is shown. [Figures 59A-59D] Figures 59A-C show cell proliferation in human PBMCs incubated with the indicated variant IL-15 / Rα-Fc fusion proteins for 4 days. Figures 59A-C show the percentage of proliferating NK cells (CD3-CD16+) (Figure 59A), CD8+ T cells (CD3+CD8+CD45RA-) (Figure 59B), and CD4+ T cells (CD3+CD4+CD45RA-) (Figure 59C). Figure 59D shows the fold change in EC50 of various IL-15 / IL-15Rα Fc dimers relative to the control (XENP20818). [Figures 60A-60D]Figures 60A-C show cell proliferation in human PBMCs incubated for 3 days with the indicated variant IL-15 / Rα-Fc fusion proteins. Figures 60A-C show the percentage of proliferating CD8+ (CD45RA-) T cells (Figure 60A), CD4+ (CD45RA-) T cells (Figure 60B), γδ T cells (Figure 60C), and NK cells (Figure 60D). [Figures 61A-61C] The percentage of Ki67 expression on CD8+ T cells (Figure 61A), CD4+ T cells (Figure 61B), and NK cells (Figure 61C) after treatment with additional IL-15 / Rα variants is shown. [Figures 62A-62E] Shown are the percentages of Ki67 expression on (Figure 62A) CD8+ (CD45RA-) T cells, (Figure 62B) CD4+ (CD45RA-) T cells, (Figure 62C) γδ T cells, (Figure 62D) NK (CD16+CD8α-) cells, and (Figure 62E) NK (CD56+CD8α-) cells after treatment with IL-15 / Rα variants. [Figures 63A-63E] The percentage of Ki67 expression on CD8+ (CD45RA-) T cells (Figure 63A), CD4+ (CD45RA-) T cells (Figure 63B), γδ T cells (Figure 63C), NK (CD16+CD8α-) cells (Figure 63D), and NK (CD56+CD8α-) cells (Figure 63E) after treatment with IL-15 / Rα variants is shown. [Figure 64A-64D] The percentage of Ki67 expression on (Figure 64A) CD8+ T cells, (Figure 64B) CD4+ T cells, (Figure 64C) γδ T cells, and (Figure 64D) NK (CD16+) cells after treatment with additional IL-15 / Rα variants engineered for reduced potency with different linker lengths is shown. [Figure 65A-65D] Shown are the percentages of Ki67 expression on (Figure 65A) CD8+ T cells, (Figure 65B) CD4+ T cells, (Figure 65C) γδ T cells, and (Figure 65D) NK (CD16+) cells after treatment with additional IL-15 / Rα variants. [Figures 66A-66D]Figures 67A-67C show gating of lymphocytes and their subpopulations in the experiment shown. Figure 66A shows gating of lymphocyte populations. Figure 66B shows CD4+ and CD8+ T cells. Figure 66C shows CD45RA and CD27 expressing subpopulations of CD4+ T cells. Figure 66D shows CD45RA and CD27 expressing subpopulations of CD8+ T cells. [Figures 67A-67C] Figure 67A shows STAT5 phosphorylation in CD8+ T cells (CD45RA-CD27-) (Figure 67B) and CD4+ T cells (CD45RA-CD27-) (Figure 67B) after 4 days of incubation of PBMCs with the indicated IL-15 / IL-15Rα-Fc fusion proteins at the indicated concentrations. Figure 67C shows the fold change in EC50 of various IL15 / IL15RαFc dimers relative to the control (XENP20818). [Figure 68A-68B] STAT5 phosphorylation in CD8+CD45RA- T cells (FIG. 68A) and CD4+CD45RA- T cells (FIG. 68B) in mouse splenocytes after incubation with the indicated test substances is shown. [Figure 69] PK of various IL-15 / RαFc fusion proteins or control upon IV-TV administration in C57BL / 6 mice at a single dose of 0.1 mg / kg. [Figure 70] Correlation of half-life versus NK cell potency is shown. [Figure 71] Correlation of half-life of IL-15 / Rα-Fc affinity variants versus mouse STAT5 signaling. [Figure 72] Serum concentrations of test substances 8 days after administration in C57BL / 6 albino mice are shown. [Figure 73] 1 shows the number of CD8+ T cells in the spleens of C57BL / 6 albino mice following administration of the indicated test substances. [Figure 74] This shows that CD45+ cell levels are a predictor of disease. [Figure 75A-75B] 75A and 75B show enhanced engraftment by variant IL-15 / Rα-Fc fusion proteins as indicated by CD45+ cell counts on day 4 (FIG. 75A) and day 8 (FIG. 75B). [Figures 76A-76C] IFNγ levels are shown on days 4 (Figure 76A), 7 (Figure 76B), and 11 (Figure 76C) after treatment of NSG mice engrafted with human PBMCs with the indicated variant IL-15 / Rα-Fc fusion proteins or control. [Figures 77A-77C] CD45+ lymphocyte cell counts are shown on days 4 (Figure 77A), 7 (Figure 77B), and 11 (Figure 77C) following treatment of human PBMC-engrafted NSG mice with the indicated variant IL-15 / Rα-Fc fusion proteins or control. [Figures 78A-78C] NK cell (CD16+CD56+CD45RA+) numbers are shown on days 4 (Figure 78A), 7 (Figure 78B), and 11 (Figure 78C) after treatment of human PBMC-engrafted NSG mice with the indicated IL-15 / Rα-Fc fusion proteins or control. [Figure 79A-79B] CD8+ T cell (CD8+CD45RA+) counts are shown at days 7 (Figure 79A) and 11 (Figure 79B) after treatment of human PBMC-transferred NSG mice with the indicated IL-15 / Rα-Fc fusion proteins or control. [Figure 80A-80B] CD4+ T cell (CD4+CD45RA+) counts are shown at days 7 (Figure 80A) and 11 (Figure 80B) following treatment of human PBMC-engrafted NSG mice with the indicated IL-15 / Rα-Fc fusion proteins or control. [Figure 81] IFNγ levels on days 4, 7, and 11 in the serum of huPBMC-transplanted mice after treatment with additional variant IL-15 / Rα-Fc fusion proteins are shown. [Figures 82A-82C] Shown are CD8+ T cell counts in whole blood of huPBMC-engrafted mice after treatment with additional variant IL-15 / Rα-Fc fusion proteins on days 4 (FIG. 82A), 7 (FIG. 82B), and 11 (FIG. 82C). [Figures 83A-83C]Shown are CD4+ T cell counts on days 4 (Figure 83A), 7 (Figure 83B), and 11 (Figure 83C) in the whole blood of huPBMC-engrafted mice after treatment with additional variant IL-15 / Rα-Fc fusion proteins. [Figures 84A-84C] Shown are CD45+ cell counts in whole blood of huPBMC-engrafted mice after treatment with additional variant IL-15 / Rα-Fc fusion proteins on days 4 (Figure 84A), 7 (Figure 84B), and 11 (Figure 84C). [Figures 85A-85C] Body weight as a percentage of initial body weight of huPBMC-transplanted mice is shown on days 4 (Figure 85A), 7 (Figure 85B), and 11 (Figure 85C) after treatment with additional IL-15 / Rα variants. Each point represents a single NSG mouse. Mice whose body weight fell below 70% of their initial weight were euthanized. Mice that died are represented as 70%. [Figure 86A-86B] Shown are the percentages of cynomolgus CD8+ T cells (FIG. 86A) and cynomolgus NK cells (FIG. 86B) expressing Ki67 after incubation with the indicated test substances. [Figures 87A-87E] Lymphocyte counts after administration of XENP22819 to cynomolgus monkeys are shown in Figures 84A-E, which show the fold change in absolute numbers of CD56+ NK cells (Figure 87A), CD16+ NK cells (Figure 87B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 87C), CD8+ T cells (Figure 87D), and CD4+ T cells (Figure 87E), respectively. [Figures 88A-88E] Shown are the proliferation of CD56+ NK cells (Figure 88A), CD16+ NK cells (Figure 88B), CD8+ T cells (CD45RA+) (Figure 88C), CD8+ T cells (CD45RA-) (Figure 88D), and CD4+ T cells (CD45RA-) (Figure 88E) after administration of XENP22819 to cynomolgus monkeys. [Figures 89A-89E]Figures 89A-E show lymphocyte counts after administration of XENP22819 to cynomolgus monkeys. Figures 89A-E show the fold change in absolute numbers of CD56+ NK cells (Figure 89A), CD16+ NK cells (Figure 89B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 89C), CD8+ T cells (Figure 89D), and CD4+ T cells (Figure 89E). [Figures 90A-90E] Figure 90A shows the proliferation of CD56+ NK cells (Figure 90A), CD16+ NK cells (Figure 90B), CD8+ T cells (CD45RA+) (Figure 90C), CD8+ T cells (CD45RA-) (Figure 90D), and CD4+ T cells (CD45RA-) (Figure 90E) after administration of XENP22819 to cynomolgus monkeys. [Figures 91A-91E] Figures 91A-91E show lymphocyte counts after administration of XENP22821 to cynomolgus monkeys. Figures 91A-91E show the fold change in absolute numbers of CD56+ NK cells (Figure 91A), CD16+ NK cells (Figure 91B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 91C), CD8+ T cells (Figure 91D), and CD4+ T cells (Figure 91E). [Figures 92A-92E] Shown are the proliferation of CD56+ NK cells (Figure 92A), CD16+ NK cells (Figure 92B), CD8+ T cells (CD45RA+) (Figure 92C), CD8+ T cells (CD45RA-) (Figure 92D), and CD4+ T cells (CD45RA-) (Figure 92E) after administration of XENP22821 to cynomolgus monkeys. [Figures 93A-93E] Lymphocyte counts after administration of XENP22822 to cynomolgus monkeys are shown in Figures 93A-E, which show the fold change in absolute numbers of CD56+ NK cells (Figure 93A), CD16+ NK cells (Figure 93B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 93C), CD8+ T cells (Figure 93D), and CD4+ T cells (Figure 93E), respectively. [Figures 94A-94E] Shown are the proliferation of CD56+ NK cells (Figure 94A), CD16+ NK cells (Figure 94B), CD8+ T cells (CD45RA+) (Figure 94C), CD8+ T cells (CD45RA-) (Figure 94D), and CD4+ T cells (CD45RA-) (Figure 94E) after administration of XENP22822 to cynomolgus monkeys. [Figures 95A-95E] Lymphocyte counts after administration of XENP22834 to cynomolgus monkeys are shown in Figures 95A-E, which show the fold change in absolute numbers of CD56+ NK cells (Figure 95A), CD16+ NK cells (Figure 95B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 95C), CD8+ T cells (Figure 95D), and CD4+ T cells (Figure 95E), respectively. [Figures 96A-96E] Shown are the proliferation of CD56+ NK cells (Figure 96A), CD16+ NK cells (Figure 96B), CD8+ T cells (CD45RA+) (Figure 96C), CD8+ T cells (CD45RA-) (Figure 96D), and CD4+ T cells (CD45RA-) (Figure 96E) after administration of XENP22834 to cynomolgus monkeys. [Figures 97A-97E] Lymphocyte counts after administration of XENP23343 to cynomolgus monkeys are shown in Figures 97A-E, which show the fold change in absolute numbers of CD56+ NK cells (Figure 97A), CD16+ NK cells (Figure 97B), γδ T cells (CD45RA+CD3+CD4-CD8-) (Figure 97C), CD8+ T cells (Figure 97D), and CD4+ T cells (Figure 97E), respectively. [Figures 98A-98E] Shown are the proliferation of CD56+ NK cells (Figure 98A), CD16+ NK cells (Figure 98B), CD8+ T cells (CD45RA+) (Figure 98C), CD8+ T cells (CD45RA-) (Figure 98D), and CD4+ T cells (CD45RA-) (Figure 98E) after administration of XENP23343 to cynomolgus monkeys. [Figures 99A-99C] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins in the "IL-15 / Rα-hetero-Fc" format, XENP23343, XENP23504, XENP24113, XENP24301, XENP24306, and XENP24341, with M428L / N434S substitutions. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figure 100]1 shows the sequence of XENP25938 (also called XENP24294), an exemplary IL-15 / Rα-Fc fusion protein in the "scIL-15 / Rα-Fc" format with M428L / N434S substitutions. [Figure 101] 7 shows the sequence of XENP24383, an exemplary IL-15 / Rα-Fc fusion protein in the "ncIL-15 / Rα-Fc" format with M428L / N434S substitutions. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, linker, and Fc region. [Figure 102] 7 shows the sequences of exemplary IL-15 / Rα-Fc fusion proteins, XENP24346 and XENP24351, in the "bivalent ncIL-15 / Rα-Fc" format with M428L / N434S substitutions. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as will be understood by those skilled in the art, portions of the linker may be replaced with other linkers shown in FIG. 7), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and Fc regions. [Figures 103A-103C] The percentage of Ki67 expression on human CD8+ T cells (Figure 103A), human CD4+ T cells (Figure 103B), and human NK cells (Figure 103C) after treatment with an IL-15 / Rα variant with the M428L / N434S Fc mutation is shown. [Figures 104A-104D] Shown are the percentages of Ki67 expression on human CD8+ T cells (Figure 104A), human CD4+ T cells (Figure 104B), human NK cells (Figure 104C), and human γδ T cells (Figure 104D) after treatment with XmAb24306 (also called XENP24306). [Figures 105A-105C]The percentage of Ki67 expression on cynomolgus CD8+ T cells (Figure 105A), cynomolgus CD4+ T cells (Figure 105B), and cynomolgus NK cells (Figure 105C) after treatment with WT IL-15 / Rα Fc with the M428L / N434S mutation and a reduced-potency IL-15 / Rα variant is shown. [Figure 106] 1 shows the percentage of Ki67 expression on cynomolgus monkey CD8α+CD45RA− T cells after treatment with XENP20818 or XmAb24306. [Figures 107A-107C] Shown are CD4+ T cell counts in whole blood of huPBMC-engrafted mice on days 4 (Figure 107A) and 7 (Figure 107B), and in spleens on day 8 (Figure 107C), after treatment with additional variant IL-15 / Rα-Fc fusion proteins. [Figures 108A-108C] CD8+ T cell counts are shown in the whole blood of huPBMC-engrafted mice on days 4 (Figure 108C) and 7 (Figure 108B) and in the spleen on day 8 (Figure 108C) after treatment with additional variant IL-15 / Rα-Fc fusion proteins. [Figures 109A-109C] CD8+ T cell counts are shown in the whole blood of huPBMC-engrafted mice on days 4 (Figure 109A) and 7 (Figure 109B), and in the spleen on day 8 (Figure 109C), after treatment with additional variant IL-15 / Rα-Fc fusion proteins. [Figures 110A-110F] Body weight as a percentage of initial body weight of huPBMC-transplanted mice is shown on days -2 (Figure 110A), 1 (Figure 110B), 5 (Figure 110C), 8 (Figure 110D), and 11 (Figure 110E) after treatment with additional IL-15 / Rα variants. Each point represents a single NSG mouse. Figure 110F shows the time course of body weight in huPBMC-transplanted mice after treatment with IL-15 / Rα variants. [Figures 111A-111D] The percentages of Ki67-expressing CD8+CD45RA- T cells (Figure 111A), CD4+CD45RA- T cells (Figure 111B), γδ T cells (Figure 111C), and CD16+ NK cells (Figure 111D) are shown after incubation with the indicated test substances. [Figures 112A-112D] Shown are CD8+ T cells (Figure 112A), CD4+ T cells (Figure 112B), NK cells (Figure 112C), and γδ T cells (Figure 112D) in cynomolgus monkeys after treatment with IL-15 / Rα variants. [Figures 113A-113H] 1 shows serum concentrations over time and half-lives of the indicated test articles in cynomolgus monkeys. [Figures 114A-114F] Binding of AF647-labeled test substances (WT IL-15 / Rα-Fc and IL-15 / Rα-Fc affinity variants with and without Xtend domain linker) to CD8+CD45RA- T cells (Figure 114A), CD8+CD45RA+ T cells (Figure 114B), CD4+CD45RA- T cells (Figure 114C), CD4+CD45RA+ T cells (Figure 114D), CD16+ NK cells (Figure 114E), and γδ T cells (Figure 114F) in fresh and activated PBMCs is shown. [Figures 115A-115F] Figure 115A shows binding of AF647-labeled test articles (WT IL-15 / Rα-Fc and IL-15 / Rα-Fc affinity variants containing Xtend and domain linker) to (Figure 115A) CD8+CD45RA- T cells, (Figure 115B) CD8+CD45RA+ T cells, (Figure 115C) CD4+CD45RA- T cells, (Figure 115D) CD4+CD45RA+ T cells, (Figure 115E) CD16+ NK cells, and (Figure 115F) γδ T cells in fresh and activated PBMCs. [Figures 116A-116F] Figure 116A shows binding of AF647-labeled test articles (WT IL-15 / Rα-Fc and IL-15 / Rα-Fc fusion proteins, including Xtend variants, such as FcRn variants) to (Figure 116A) CD8+CD45RA- T cells, (Figure 116B) CD8+CD45RA+ T cells, (Figure 116C) CD4+CD45RA- T cells, (Figure 116D) CD4+CD45RA+ T cells, (Figure 116E) CD16+ NK cells, and (Figure 116F) γδ T cells in fresh and activated PBMCs. [Figure 117] Binding of AF647-labeled test substances (including XENP24341 and XENP24113) to CD8+CD45RA- T cells in activated PBMCs is shown. [Figures 118A-118B] (Figure 118A) Shows CD25 expression on CD8+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 118B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figure 119A-119B] (Figure 119A) Shows CD25 expression on CD4+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 119B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figure 120A-120B] (Figure 120A) Shows CD69 expression on CD8+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 120B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figure 121A-121B] (Figure 121A) Shows CD69 expression on CD4+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 121B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figure 122A-122B] (Figure 122A) Shows intracellular IFNγ expression in CD8+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 122B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figure 123A-123B](Figure 123A) Shows intracellular IFNγ expression in CD4+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 123B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figures 124A-124C] Shown are the (Figure 124A) Ki-67+ / IFNγ-, (Figure 124B) Ki-67+ / IFNγ+, and (Figure 124C) Ki-67- / IFNγ+ fraction percentages of CD8+ T cells in Group 1 (purified T cells incubated with parental MCF-7 tumor cells and the indicated test substances). [Figures 125A-125C] Shown are the (Figure 125A) Ki-67+ / IFNγ-, (Figure 125B) Ki-67+ / IFNγ+, and (Figure 125C) Ki-67- / IFNγ+ fraction percentages of CD4+ T cells in Group 1 (purified T cells incubated with parental MCF-7 tumor cells and the indicated test substances). [Figures 126A-126C] Shown are the (Figure 126A) Ki-67+ / IFNγ-, (Figure 126B) Ki-67+ / IFNγ+, and (Figure 126C) Ki-67- / IFNγ+ fraction percentages of CD8+ T cells in Group 1 (purified T cells incubated with pp65-expressing MCF-7 tumor cells and the indicated test substances). [Figures 127A-127C] Shown are the (Figure 127A) Ki-67+ / IFNγ-, (Figure 127B) Ki-67+ / IFNγ+, and (Figure 127C) Ki-67- / IFNγ+ fraction percentages of CD4+ T cells in Group 1 (purified T cells incubated with pp65-expressing MCF-7 tumor cells and the indicated test substances). [Figure 128A-128B] (Figure 128A) Shows CD107a expression on CD8+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 128B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figure 129A-129B](Figure 129A) Shows CD107a expression on CD4+ T cells in Group 1 (parental MCF-7 tumor cells and purified T cells incubated with the indicated test substances) and (Figure 129B) Group 2 (pp65-expressing MCF-7 tumor cells and purified T cells incubated with the indicated test substances). [Figure 130A-130B] Purified T cells and remaining target cells after incubation with the indicated test substances [Fig. 130A: parental MCF-7 tumor cells; Fig. 130B: MCF-7 tumor cells expressing pp65] are shown. [Figure 131A-131B] The number of dead cells [Figure 131A: parental MCF-7 tumor cells; Figure 131B: MCF-7 tumor cells expressing pp65] after incubation with purified T cells and the indicated test substances is shown. [Figure 132] The percentage of the Ki-67+ / IFNγ+ fraction of CD8+ T cells is shown after incubation of purified T cells with parental MCF-7 tumor cells or MCF-7 tumor cells expressing pp65, with or without anti-HLA-A antibodies. [Figure 133] The numbers of target cells (i.e., parental MCF-7 tumor cells or pp65-expressing MCF-7 tumor cells) are shown after incubating purified T cells with parental MCF-7 tumor cells or pp65-expressing MCF-7 tumor cells with or without anti-HLA-A antibodies. [Fig. 134A-134B] Shown is CD25 expression on (Figure 134A) CD8+ T cells and (Figure 134B) CD4+ T cells after incubation of purified T cells with pp65-expressing MCF-7 tumor cells and the indicated test substances. [Fig. 135A-135B] Shown is CD69 expression on (Figure 135A) CD8+ T cells and (Figure 135B) CD4+ T cells after incubation of purified T cells with pp65-expressing MCF-7 tumor cells and the indicated test substances. [Figures 136A-136B]Ki67 expression on (Figure 136A) CD8+ and (Figure 136B) CD4+ T cells is shown after incubation of purified T cells with pp65-expressing MCF-7 tumor cells and the indicated test substances. [Figure 137A-137B] The percentage of Ki-67+ / IFNγ+ fractions of (Figure 137A) CD8+ T cells and (Figure 137B) CD4+ T cells are shown after incubation of purified T cells with pp65-expressing MCF-7 tumor cells and the indicated test substances. [Figures 138A-138B] The percentage of CD69+ / IFNγ+ fraction of (Figure 138A) CD8+ T cells and (Figure 138B) CD4+ T cells is shown after incubation of purified T cells with pp65-expressing MCF-7 tumor cells and the indicated test substances. [Figure 139A-139B] The percentage of CD69+ / Ki67+ fraction of (Figure 139A) CD8+ T cells and (Figure 139B) CD4+ T cells is shown after incubation of purified T cells with pp65-expressing MCF-7 tumor cells and the indicated test substances. [Figure 140] Shown are the remaining target cells (MCF-7 tumor cells expressing pp65) after incubation with purified T cells and the indicated test substances. [Figure 141] The number of dead cells (MCF-7 tumor cells expressing pp65) after incubation with purified T cells and the indicated test substances is shown. [Figure 142A-142B] Shown are the mean tumor volume (Figure 142A) and change in tumor volume (Figure 142B) in mice implanted with pp65-expressing MCF-7 tumor cells and pp65-reactive human PBMCs after treatment with XENP24045, a non-Xtend analog of XmAb24306. [Figures 143A-143D]Shown are the numbers of CD45+ cells (Figure 143A), CD4+ cells (Figure 143B), CD8+ cells (Figure 143C), and NK cells (Figure 143D) in the whole blood of mice transplanted with pp65-expressing MCF-7 tumor cells and pp65-reactive human PBMCs after treatment with XENP24045, a non-Xtend analog of XmAb24306. [Figure 144] 1 shows the serum concentrations and half-lives over time of various test substances at various concentrations in cynomolgus monkeys. [Figure 145] 1 shows the Cmax normalized serum concentrations over time of XENP22821 at 1x and 3x doses in cynomolgus monkeys. [Figures 146A-146E] Shown are the mean fold changes in CD8+ T cell (Figure 146A), CD4+ T cell (Figure 146B), CD16+ NK cell (Figure 146C), CD56+ NK cell (Figure 146D), and γδ T cell (Figure 146E) numbers in cynomolgus monkeys after administration of the indicated test articles. [Figure 147A-147B] Fold changes in CD8+ T cells (FIG. 147A) and γδ T cells (FIG. 147B) in cynomolgus monkey whole blood over time following administration of either a 3x dose or a 0.6x dose of XENP24306 are shown. [Figure 148] 1 shows the percentage of CD8α+CD45RA+ T cells in cynomolgus monkey lymph nodes that express Ki67 after administration of a 0.3x dose of XENP22821 and a 0.6x dose of XENP24306. [Figure 149] The percentage (left axis) and fold change in cell number (right axis) of CD8α+CD45RA+ T cells in cynomolgus monkey whole blood expressing Ki67 after administration of a 0.3x dose of XENP22821 and a 0.6x dose of XENP24306 are shown. [Figure 150] 1 shows the percentage of various lymphocyte populations in cynomolgus monkey PBMCs that express Ki67 after administration of a 0.3 dose of XENP22821. [Figure 151] 1 shows the percentage of various lymphocyte populations in cynomolgus monkey PBMCs that express Ki67 after administration of a 0.6x dose of XmAb24306. [Figure 152]Shown is the percentage of CD8α+CD45RA+ T cells in cynomolgus monkey whole blood expressing Ki67 over time, following administration of a 0.6x dose of XmAb24306, and an overlay of the serum concentration of XmAb24306. [Figure 153] Figure 1 shows the fold change in the number of CD8+ T cells, CD4+ T cells, CD56+CD8α+ NK cells, and γδ T cells in cynomolgus monkey whole blood over time following administration of a 0.6 dose of XmAb24306. [Fig. 154] Figure 1 shows the fold change in the number of CD8+ T cells and Tregs in cynomolgus monkey whole blood over time following administration of a 0.6 dose of XmAb24306. [Figures 155A-155C] Shown is an overlay of CD8+ T cell (Figure 155A), CD4+ T cell (Figure 155B), and CD16+ NK cell (Figure 155C) counts over time in whole blood of cynomolgus monkeys after administration of either XENP20818 or XmAb24306. [Figures 156A-156C] Shown are fold changes in immune-related gene expression in (Figure 156A) total PBMCs, (Figure 156B) purified NK cells, and (Figure 156C) purified CD8+ T cells after 24 hours of treatment with either XENP20818 or XmAb24306 at their EC50 concentrations compared to untreated. [Figures 157A-157C] Shown are fold changes in gene expression in (Figure 157A) whole PBMCs, (Figure 157B) purified NK cells, and (Figure 157C) purified CD8+ T cells after 48 hours of treatment with either XENP20818 or XmAb24306 at their EC50 concentrations compared to untreated. [Figure 158A-158B] Shown are fold changes in gene expression in whole PBMCs after 48 hours of treatment with (Figure 158A) XmAb24306 or IL-15 and (Figure 158B) XmAb24306 or IL-2 at their EC50 concentrations compared to untreated. [Figure 159A-159B] Shown is the proliferation of (Fig. 159A) CD8+ and (Fig. 159B) CD4+ responder T cells in the presence of XmAb24306 and various concentrations of rapamycin-expanded Tregs. [Figure 160]

[0033] Figure 1 shows the sequence of XENP26842, a bivalent anti-PD-1 mAb with a truncation variant (E233P / L234V / L235A / G236del / S267K, "IgG1_PVA_ / S267k"). The CDRs are underlined. As is true for all sequences described herein and containing CDRs herein, the exact identification of the CDR positions may vary slightly depending on the numbering used as shown in Table 1; therefore, not only the underlined CDRs, but also CDRs contained within the VH and VL domains using other numbering systems are included herein. Furthermore, each CDR has its own unique SEQ ID NO or sequence identifier in the Sequence Listing, and each VH and VL domain has its own SEQ ID NO or sequence identifier in the Sequence Listing. [Figure 161A-161B] CD8+ T cells in whole blood of mice are shown on (Figure 161A) day 6 and (Figure 161B) day 10 after the first dose of the indicated test substance. [Figure 162A-162B] CD4+ T cells in whole blood of mice are shown on (Figure 162A) day 6 and (Figure 162B) day 10 after the first dose of the indicated test substance. [Figure 163A-163B] CD45+ T cells in whole blood of mice (Figure 163A) 6 days and (Figure 163B) 10 days after the first dose of the indicated test substance are shown. [Figure 164A-164B] NK cells in whole blood of mice are shown on (Figure 164A) day 6 and (Figure 164B) day 10 after the first administration of the indicated test substance. [Figure 165] IFNγ in serum of NSG mice 7 days after the first dose of the indicated test substance is shown. [Figures 166A-166D] Shown are the body weights of mice 7 (Figure 166A), 11 (Figure 166B), 14 (Figure 166C), and 18 (Figure 166D) days after the first administration of the indicated test substance. [Figure 167] The percentage of the indicated lymphocyte populations expressing Ki67 after incubation with XENP20818 is shown. [Figure 168]STAT5 phosphorylation on CD8+CD45RA+ T cells after incubation with XENP20818, XENP22821, XENP24050, and XENP24306 is shown. [Figure 169] STAT5 phosphorylation on CD8+CD45RA+ T cells after incubation with XENP20818, XENP22819, XENP22821, and XENP22834 is shown. [Figure 170] Shown is the percentage of CD8+CD45RA- T cells expressing Ki67 over time following incubation with XENP20818 or XENP24306 at their respective EC50s. [Figure 171] Figure 1 shows BCL2 expression on CD8+CD45RA- T cells over time after incubation with XENP20818 or XENP24306 at their respective EC50s. [Fig. 172] Shown is CD25 expression on CD8+CD45RA- T cells over time after incubation with XENP20818 or XENP24306 at their respective EC50s. [Figure 173] STAT5 phosphorylation of CD8+ T cells in fresh versus stimulated human PBMCs after incubation with XENP24045 or XENP20818 is shown. [Fig. 174] Shown is the percent CD8+CD45RA- proliferation (determined by CFSE dilution) following incubation of human PBMCs with the indicated doses of XENP24306 and plate-bound anti-CD3 (OKT3). [Figure 175] Serum IFNγ concentrations in huPBMC-engrafted NSG mice on day 10 after the first dose of the indicated test substance at the indicated concentration are shown. [Figure 176] Shown are CD45+ cell counts in the blood of huPBMC-engrafted NSG mice on day 17 after the first dose of the indicated test substance at the indicated concentration. [Figure 177]Shown are the body weights (as a percentage of initial body weight) of huPBMC-engrafted NSG mice on day 25 after the first administration of the indicated test substance at the indicated concentration. [Figure 178] Serum IFNγ concentrations in huPBMC-engrafted NSG mice on day 7 after the first dose of the indicated test substance at the indicated concentration are shown. [Figures 179A-179D] Shown are the numbers of A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, and D) CD8+ T cells in the blood of huPBMC-engrafted NSG mice on day 10 after the first dose of the indicated test substance at the indicated concentration. [Figure 180] Shown are the body weights (as a percentage of initial body weight) of huPBMC-engrafted NSG mice on day 11 after the first administration of the indicated test substance at the indicated concentration. [Figure 181] Shown is the time course of body weight (as a percentage of initial body weight) of huPBMC-engrafted NSG mice following the first administration of the indicated test substance at the indicated concentration. [Figure 182] 1 shows CD45+ cell counts on day 21 in pp65-MCF7 and huPBMC-transplanted NSG mice treated with XENP16432 and / or XENP24045. [Figure 183] 1 shows tumor volumes on day 31 in pp65-MCF7 and huPBMC-engrafted NSG mice administered XENP16432 and / or XENP24045. [Figure 184] 1 shows tumor volume over time (after huPBMC transplantation) in pp65-MCF7 and huPBMC (1.5x106) transplanted NSG mice treated with XENP16432 and / or XENP24045. [Figure 185] 1 shows tumor volume over time (after huPBMC transplantation) in pp65-MCF7 and huPBMC (5x106) transplanted NSG mice treated with XENP16432 and / or XENP24045. [Figure 186]1 shows the sequence of XENP21993, a scIL-15 / Rα-Fc fusion containing wild-type IL-15. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (those skilled in the art will understand that portions of the linker may be replaced with other linkers shown in the figure), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, and the constant / Fc region. [Figure 187] 1 shows the sequence of XENP22853, an IL-15 / Rα-hetero-Fc fusion containing wild-type IL-15 and the Xtend Fc (M428L / N434S) variant. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (though one of skill in the art will understand that portions of the linker may be replaced with other linkers shown in the figure), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, the linker, and the constant / Fc region. [Figure 188] 1 shows the sequence of XENP24294, a scIL-15 / Rα-Fc fusion containing the IL-15 (N4D / N65D) variant and Xtend Fc (M428L / N434S) substitutions. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as one of skill in the art will understand, portions of the linker may be replaced with other linkers shown in the figure), and a slash ( / ) indicates the boundary(s) between the IL-15, IL-15Rα, linker, and constant / Fc regions. [Figure 189] 1 shows serum concentrations of the indicated test substances over time in cynomolgus monkeys after the first dose at the indicated relative concentrations. [Figure 190] Relative serum concentrations of XENP22853 and corresponding WT non-Xtend XENP20818 over time are shown. [Figure 191]The sequences of exemplary IL-15 variants engineered to reduce potency and containing a D30N substitution are shown. Included within each of these variant IL-15 sequences are sequences that are 90%, 95%, 98%, and 99% identical (as defined herein) to the listed sequences and / or contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions. In a non-limiting example, the listed sequences may contain additional amino acid modifications, such as those that contribute to the formation of covalent disulfide bonds, as described in Example 2. [Figures 192A-192C] 1 shows an exemplary scIL-15 / Rα-Fc fusion with an IL-15 variant containing a D30N substitution. IL-15 and IL-15Rα (sushi) are underlined, the linker is double underlined (as one of skill in the art will understand, portions of the linker may be replaced with other linkers shown in the figure), and a slash ( / ) indicates the boundary(s) between IL-15, IL-15Rα, linker, and constant / Fc region. [Figures 193A-193G] Shown are the percentages of A) CD4+CD45RA-, B) CD4+CD45RA+, C) CD8+CD45RA-, D) CD8+CD45RA+, E) CD16+ NK cells, F) CD56+ NK cells, and G) γδ cells expressing Ki67 after incubation with the indicated test substances. [Figures 194A-194E] As discussed in Example 18, we demonstrate that the mechanistic PK / RO / TMDD model is indicative of relative PD efficacy. Surprisingly, the predicted RO-AUC values ​​correlate very well with the experimentally determined PD-AUC (pharmacodynamic AUC), indicating that RO-AUC is a target value for optimization, allowing prediction of optimal Kd values. [Figure 195] A schematic illustrating the hypothesis that receptor-mediated internalization is an important factor influencing the PK / PD relationship of various IL-15s is shown. kProliferation indicates the proliferation rate. kDeathCTL indicates the rate of cell death. Kd indicates the dissociation constant. kRemoval indicates the removal rate. kInfusion indicates the rate of infusion into central blood. kSynthesis indicates the rate of synthesis of IL-15 receptor. kDegradation indicates the rate of degradation of IL-15 receptor. [Figure 196]196 shows an overlay of PK data from multiple cynomolgus monkey studies that were input into a simulation based on the model shown in FIG. 195. [Figure 197] It is shown that the KD values ​​estimated based on the model shown in Figure 1 correlate linearly with the experimentally determined EC50 values ​​of binding. [Figure 198] The simulated PK profile, receptor occupancy (RO) curve, and pharmacodynamic profile are shown based on the model shown in Figure 195. The model indicates that reduced potency prolongs exposure and that an optimal KD exists for maximum pharmacodynamics (i.e., T cell expansion). [Figures 199A-199F] Correlations between A) predicted RO-AUC values ​​by experimentally determined PD-AUC (pharmacodynamic AUC), B) predicted RO-AUC values ​​by experimentally determined PD-Max (peak CD8+ T cell count), C) predicted RO-AUC values ​​by experimentally determined ALB-min (minimum serum albumin concentration), D) predicted RO-Max (maximum receptor occupancy) values ​​by experimentally determined PD-AUC, E) predicted RO-Max by experimentally determined PD-Max, and F) predicted RO-Max by experimentally determined ALB-Min are shown. Notably, predicted RO-AUC values ​​correlate highly with experimentally determined PD-AUC and PD-max, while predicted RO-Max values ​​correlate highly with experimentally determined ALB-Min. [Figure 200] Simulation scans (based on the model shown in Figure 195) for various KD values ​​performed on either the Xtend or non-Xtend version of the IL-15 / Rα-hetero-Fc fusion are shown, revealing that the predicted optimal KD is approximately 200 nM, similar to the estimated KD value of XmAb24306. [Figures 201A-201B]Figure 1 shows A) human CD8+ T cell counts and B) CD25 expression on human CD8+ T cells in pp65-MCF7 and huPBMC-transplanted NSG mice treated with XENP16432 and / or XENP24045. *** indicates p<0.001, and ** indicates p<0.01, as determined by unpaired t-test. Treatment with a combination of XENP24045 and anti-PD-1 mAb was found to significantly enhance CD8+ T cell expansion and induce early CD8+ T cell activation at later days. [Figures 202A-202C] Figure 1 shows the expansion of A) CD8+ T cells, B) CD16+ NK cells, and C) lymphocytes in cynomolgus monkeys after administration of a 0.3x dose of XENP20818, a 0.3x dose of XmAb24306, and a 0.6x dose of XmAb4306. The data demonstrate the improved pharmacodynamics conferred by the reduced potency of XmAb24306. [Figure 203] Figure 1 shows the percent change in serum albumin (as an indicator of vascular leakage) in cynomolgus monkeys following administration of a 0.3x dose of XENP20818, a 0.3x dose of XmAb24306, and a 0.6x dose of XmAb24306. The data demonstrate improved tolerability (as indicated by reduced albumin drop) conferred by the reduced efficacy of XmAb24306. [Figures 204A-204F] Shown are A) CD45+ cell, B) CD3+ T cell, C) CD4+ T cell, D) CD8+ T cell, E) CD16+CD56+ NK cell counts, and F) CD4 to CD8 ratio in the blood of huPBMC-engrafted NSG mice on day 7 after the first dose of the indicated test article at the indicated concentration. [Figures 205A-205F]Figure 1 shows A) CD45+ cell, B) CD3+ T cell, C) CD4+ T cell, D) CD8+ T cell, E) CD16+CD56+ NK cell counts, and F) CD4 to CD8 ratio in the blood of huPBMC-engrafted NSG mice on day 14 after the first dose of the indicated test substance at the indicated concentration. The data show that by day 14, treatment with a combination of 0.3 mg / kg or 0.1 mg / kg XENP24306 and 3 mg / kg XENP16432 significantly enhanced CD3+ T cell expansion over treatment with 3 mg / kg XENP16432 alone (statistics performed on log-transformed data using an unpaired t-test). [Figures 206A-206F] Shown are A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, D) CD8+ T cells, E) CD16+CD56+ NK cell counts, and F) CD4 to CD8 ratio in the blood of huPBMC-engrafted NSG mice on day 21 after the first dose of the indicated test article at the indicated concentration. [Figures 207A-207I] Figure 1 shows the change in body weight (as an indicator of GVHD) of huPBMC-engrafted NSG mice on A) day 3, B) day 6, C) day 10, D) day 13, E) day 17, F) day 20, G) day 25, H) day 28, and I) day 29 after the first administration of the indicated test substance at the indicated concentration. The data show that by day 10, treatment with a combination of 0.3 mg / kg, 0.1 mg / kg, or 0.01 mg / kg XENP24306 and 3 mg / kg XENP16432 significantly enhanced GVHD over treatment with 3 mg / kg XENP16432 alone (statistics performed using an unpaired t-test). [Figure 208] Shown is the change in body weight (as an indicator of GVHD) of huPBMC-engrafted NSG mice over time following administration of the indicated test substances at the concentrations indicated. [Figures 209A-209C] Serum IFNγ concentrations are shown in huPBMC-engrafted NSG mice at A) day 7, B) day 14, and C) day 21 after the first dose of the indicated test substance at the indicated concentration. [Figure 210A-201F]Figure 1 shows the number of A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, D) CD8+ T cells, E) CD16+CD56+ NK cells, and F) CD4 to CD8 ratios in the blood of pp65-MCF-7 and huPBMC-engrafted NSG mice on day 14 after the first administration of the indicated test substance at the indicated concentration. * indicates P<0.05, unpaired t-test, for the indicated group compared to the PBS-treated group; † indicates P<0.04, unpaired t-test, for the indicated group compared to the XENP16432-treated group. Data were log-transformed before statistical analysis. The data show that by day 14, groups treated with XENP24306 alone or XENP24306 in combination with XENP16432 each significantly enhanced lymphocyte expansion over PBS treatment. Notably, by day 14, the combination of XENP24306 and XENP16432, as well as the high concentration of XENP24306 alone, significantly enhanced lymphocyte expansion beyond that of XENP16432 treatment, regardless of the XENP24306 concentration. [Figures 211A-211F] Figure 1 shows the number of A) CD45+ cells, B) CD3+ T cells, C) CD4+ T cells, D) CD8+ T cells, E) CD16+CD56+ NK cells, and F) CD4 to CD8 ratio in the blood of pp65-MCF-7 and huPBMC-engrafted NSG mice on day 21 after the first dose of the indicated test substance at the indicated concentration. * indicates P<0.05, unpaired t-test, for the indicated group compared to the PBS-treated group; † indicates P<0.04, unpaired t-test, for the indicated group compared to the XENP16432-treated group. Data were log-transformed before statistical analysis. [Figures 212A-212I]Figure 1 shows the change in tumor volume (baseline corrected) in pp65-MCF-7 and huPBMC-implanted NSG mice after the first administration of the indicated test substance at the indicated concentration on A) day 4, B) day 6, C) day 8, D) day 11, E) day 13, F) day 15, G) day 19, H) day 22, and I) day 25. The data show that by day 11, treatment with a combination of 1.0 mg / kg, 0.3 mg / kg, or 0.1 mg / kg XENP24306 and 3 mg / kg XENP16432 significantly reduced tumor volume compared to treatment with XENP16432 alone (statistics performed using an unpaired t-test on baseline-corrected tumor measurements). [Figures 213A-213B] Figure 1 shows A) the change in tumor volume and B) the change in body weight (as an indicator of GVHD) in pp65-MCF-7 and huPBMC-implanted NSG mice over time after administration of the indicated test substances at the concentrations indicated. The data show that all mice in groups C, F, and G died, which corresponded to GVHD (as indicated by the change in body weight). [Figures 214A-214C] Serum IFNγ concentrations in pp65-MCF-7 and huPBMC-engrafted NSG mice are shown on A) day 7, B) day 14, and C) day 21 after the first administration of the indicated test substance at the indicated concentration. * indicates P<0.05, unpaired t-test, for the indicated group compared to the PBS-treated group; † indicates P<0.04, unpaired t-test, for the indicated group compared to the XENP16432-treated group. Data were log-transformed before statistical analysis. The data show that by day 7, treatment with a combination of 1.0 mg / kg, 0.3 mg / kg, or 0.1 mg / kg XENP24306 and 3 mg / kg XENP16432 significantly enhanced IFNγ secretion compared to treatment with XENP16432 alone. DETAILED DESCRIPTION OF THE INVENTION

[0065] I. Definition In order that the present invention may be more fully understood, some definitions are provided below. Such definitions are intended to encompass grammatical equivalents.

[0066] As used herein, "cleavage" means reduction or elimination of binding and / or activity. Thus, for example, "aborts FcγR binding" refers to an Fc region amino acid variant. has less than 50% starting binding compared to an Fc region that does not contain that particular variant This means that a bond loss of 70 to 80 to 90 to 95 to less than 98% is preferred, and generally, In some cases, the level of binding is below detectable in a Biacore assay. Particularly useful in truncating the nucleotide sequence are the variants shown in Figure 5. However, other Unless otherwise specified, the Fc monomers of the present invention retain binding to FcRn.

[0067] As used herein, "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to the Nonspecific cytotoxic cells expressing cγR recognize the bound antibody on the target cell and subsequently target ADCC refers to a cell-mediated reaction that results in the lysis of cells. Increased binding to FcγRIIIa correlates with increased ADCC activity, and increased binding to FcγRIIIa results in increased ADCC activity. As discussed herein, many embodiments of the present invention completely ablate ADCC activity.

[0068] As used herein, "ADCP" or antibody-dependent cell-mediated phagocytosis refers to the process by which FcγRs are activated. Nonspecific cytotoxic cells expressing the cytotoxic antibody recognize the ligated antibody on the target cell and subsequently phagocytose the target cell. It refers to a cell-mediated reaction that causes an effect.

[0069] As used herein, "modification" refers to an amino acid substitution, insertion, or deletion in a polypeptide sequence. This refers to modifications and / or deletions, or alterations to moieties chemically linked to a protein. For example, the modification may be an altered carbohydrate or PEG structure attached to the protein. As used herein, "amino acid modification" refers to the substitution or insertion of an amino acid in a polypeptide sequence. For clarity, unless otherwise stated, amino Acid modifications are always present in DNA-encoded amino acids, e.g. There are 20 amino acids for which there are codons.

[0070] As used herein, an "amino acid substitution" or "substitution" refers to a specific amino acid in a parent polypeptide sequence. This means substituting an amino acid at a position with a different amino acid. In this case, the substitution is non-naturally occurring (not occurring naturally in organisms or For example, substitutions for amino acids that are not present in any organism. E272Y or 272Y is a variant in which glutamic acid at position 272 is replaced with tyrosine. For clarity, the nucleic acid coding Altering the sequence but not changing the starting amino acids (e.g., increasing expression levels in the host organism) To convert CGG (which encodes arginine) to CGA (which also encodes arginine), A protein engineered to replace a nucleotide with a nucleotide is not an "amino acid substitution." Despite the creation of new genes that code for the same protein, the protein If it has the same amino acid at a particular starting position, it is not an amino acid substitution. can include naturally occurring amino acids and possibly synthetic amino acids. are disclosed in U.S. Patent No. 6,586,207, WO98 / 48032, WO03 / 073238 , US2004-0214988A1, WO05 / 35727A2, WO05 / 7452 4A2, JWChin et al.,(2002),Journal of th e American Chemical Society 124:9026-902 7, J. W. Chin, & P. ​​G. Schultz, (2002), ChemBioCh em 11:1135-1137, JWCin,et al.,(2002),P ICAS United States of America 99:11020-1 1024, and L. Wang, & P. ​​G. Schultz, (2002), Chem. 1-10, all of which are incorporated by reference in their entirety.

[0071] As used herein, an "amino acid insertion" or "insertion" refers to a specific amino acid in a parent polypeptide sequence. This means adding an amino acid residue or an amino acid sequence at a specific position. For example, -23 3E indicates the insertion of glutamic acid after position 233 and before position 234. 3ADE or A233ADE is an AlaAspGl residue after position 233 and before position 234. Indicates the insertion of u.

[0072] As used herein, an "amino acid deletion" or "deletion" refers to a specific amino acid deletion in a parent polypeptide sequence. This means removing an amino acid residue or amino acid sequence at a specific position. For example, E23 3. E233#, E233(), or E233del indicates a deletion of glutamic acid at position 233. Furthermore, EDA233- or EDA233# indicates a deletion of the sequence G1 starting at position 233. The deletion of uAspAla is shown.

[0073] As used herein, a "variant protein," "protein variant," or A "variant" is a protein that differs from that of a parent protein based on at least one amino acid modification. A protein variant refers to a protein itself, a protein Compositions comprising the amino acid sequence encoding the same, or DNA or nucleic acid encoding the same Preferably, a protein variant has less than at least one amino acid modification, e.g., from about 1 to about 70 amino acid modifications relative to the parent, and Preferably, the amino acid sequence has about 1 to about 5 amino acid modifications. The modifications may be additions, deletions, or substitutions. As described below, in some embodiments, the parent polypeptide, e.g., Fc The parent polypeptide may be an Fc region from IgG1, IgG2, IgG3, or IgG4. Human wild-type sequences, but with mutations, can also serve as "parent polypeptides." For example, IgG1 / 2 hybrids can be included. The sequences of the protein variants described herein preferably have at least about 8 amino acids different from the parent protein sequence. 0% identity, most preferably at least about 90% identity, more preferably at least They share approximately 95-98-99% identity.

[0074] Thus, as used herein, an "antibody variant" or "variant antibody" refers to means an antibody that differs from a parent antibody by virtue of at least one amino acid modification, as used herein As used herein, "IgG variant" or "variant IgG" refers to an IgG variant that contains at least one antigen. They differ from the parent IgG (again, often derived from human IgG sequences) based on amino acid modifications. As used herein, "immunoglobulin variant" or "barrier" refers to an antibody A "parent immunoglobulin" is a compound that is different from a parent immunoglobulin based on at least one amino acid modification. As used herein, "Fc variant" refers to an immunoglobulin sequence that is different from that of the Fc variant. "Ant" or "variant Fc" refers to a protein containing amino acid modifications in the Fc domain. The modifications may be additions, deletions, or substitutions. The Fc variants of the present invention comprise They are defined according to the amino acid modifications that make them up. or 434S, an Fc polypeptide with a substitution of a serine residue at position 434 relative to the parent Fc polypeptide. Variants, numbering follows the EU index. Similarly, M428L / N434S is , defining an Fc mutant having substitutions M428L and N434S relative to the parent Fc polypeptide The identity of the WT amino acid may not be specified, in which case the variants described above may be used. The substitutions may be provided in any order, i.e., For example, 428L / 434S is the same Fc variant as 434S / 428L. It is noted that all of the information discussed in this invention relating to antibodies or derivatives and fragments thereof For positions , unless otherwise specified, the numbering of amino acid positions follows the EU index. EU index or an EU index similar to Kabat or EU numbering schemes The numbering refers to the EU antibody numbering (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, the entire contents of which are incorporated herein by reference. (The text is incorporated herein by reference.) Modifications can be additions, deletions, or substitutions. Substitutions include additions, deletions, or substitutions of naturally occurring amino acids. It may contain naturally occurring and possibly synthetic amino acids.

[0075] As used herein, "protein" includes proteins, polypeptides, oligonucleotides, and the like. "peptide" means at least two covalently attached amino acids, including peptides, peptides, and peptides. The butidyl group may be a naturally occurring amino acid and peptide bond, or a synthetic peptidomimetic structure. peptoid structures (Simon et al., 2004, incorporated by reference in its entirety) PNAS USA 89(20):9367 (1992)). Amino acids may be naturally occurring or synthetic, as will be appreciated by those skilled in the art. can be either a For example, homophenylalanine, citrulline, ornithine, and noreoleucine For the purposes of this invention, amino acids are considered synthetic amino acids and include those in the D- and L- (R or S) configurations. The variants of the present invention are not limited to, for example, Cropp & Shultz, 2004, all of which are incorporated by reference in their entirety. Trends Genet.20(12):625-30, Anderson et a l.,2004,Proc Natl Acad Sci USA 101(2):75 66-71, Zhang et al.,2003,303(5656):371-3, and Chin et al., 2003, Science 301(5635):96 4-7, including the method developed by Schultz and coworkers. Modifications may include the use of synthetic amino acids incorporated using conventional techniques. Polypeptides may contain one or more side chain or terminal synthetic derivatizations, glycosylation, PEGylation , circular permutation, cyclization, linkers to other molecules, fusion to proteins or protein domains and the addition of peptide tags or labels. When including the above proteins, each protein is called a "monomer" or a "subunit" or They may be called "domains" and biologically functional molecules may be called "complexes." .

[0076] "Residue," as used herein, refers to a position in a protein and its associated For example, asparagine 297 (Asn297 or N297) (also referred to as ) is the residue at position 297 in the human antibody IgG1.

[0077] As used herein, "IgG subclass modification" or "isotype modification" refers to One amino acid of each IgG isotype is compared with a different, matched pair of IgG isotypes. For example, an amino acid modification that converts an I amino acid at EU position 296 into a corresponding amino acid. Since IgG1 contains tyrosine and IgG2 contains phenylalanine, the F The 296Y substitution is thought to be an IgG subclass modification.

[0078] As used herein, a "naturally occurring modification" refers to a non-isotypic amino acid sequence. For example, none of the IgGs contain serine at position 434, In IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) The substitution 434S is considered to be a non-naturally occurring modification.

[0079] As used herein, "amino acid" and "amino acid identity" refer to DNA and RNA The amino acid α refers to one of the 20 naturally occurring amino acids encoded by

[0080] As used herein, "effector function" refers to the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include the biochemical events that result from the interaction of ADCs with other molecules. C, ADCP, and CDC.

[0081] As used herein, "IgG Fc ligand" or "Fc ligand" refers to an IgG any biologically derived molecule that binds to the Fc region of an antibody to form an F / Fc ligand complex; Preferably, it refers to a polypeptide. Fc ligands include FcγRI, FcγRII ... cγRIII, FcRn, C1q, C3, mannan-binding lectin, mannose receptor, s taphylococcal protein A, streptococcal protein G, Fc ligands also include, but are not limited to, FcγRs ... It contains a family of Fc receptors homologous to FcγR, the Fc receptor homologs (FcRH). (Davis et al., 2002, Immunotherapy and Immunotherapy, vol. 1, pp. 111-114, which are incorporated by reference in their entirety) (Norological Reviews 190:123-136). Fc ligands are Fc Specific IgG Fc ligands may include molecules that bind to FcRn and FcRn. c gamma receptor.

[0082] As used herein, "Fc gamma receptor," "FcγR," or "Fc gamma R" It is a protein that binds to the Fc region of an IgG antibody and is encoded by the FcγR gene. This refers to any member of the isoform family. In humans, this family includes isoforms FcγRI (C) including the following forms: FcγRIa, FcγRIb, and FcγRIc D64); isoform FcγRIIa (allotypes H131 and R1 31), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2) , as well as FcγRII (CD32), including FcγRIIc; and isoforms FcγRIIIa (including allotypes V158 and F158), and F cγRIIIb (allotypes FcγRIIb-NA1 and FcγRIIb-NA 2) (Jef Ferris et al., 2002, Immunol Lett 82:57-65) and any undiscovered human FcγR or FcγR isoform or FcγRs include, but are not limited to, human, mouse, rat, It can be from any organism, including, but not limited to, rabbit, and monkey. FcγR includes FcγRI (CD64), FcγRII (CD32), and FcγRIII ( CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse These include, but are not limited to, FcγR or FcγR isoforms or allotypes. Not determined.

[0083] As used herein, "FcRn" or "neonatal Fc receptor" refers to the FcRn of IgG antibodies. c region and binds to the protein encoded, at least in part, by the FcRn gene. FcRn is a protein found in various species of FcRn, including humans, mice, rats, rabbits, and monkeys. As known in the art, functional FcRn proteins can be derived from any organism, without limitation. Proteins contain two polypeptides, often called heavy and light chains. The light chains are β- The heavy chain is expressed by the FcRn gene. Unless otherwise specified herein, FcRn or FcRn protein is encoded by Protein refers to the complex of FcRn heavy chain and β2-microglobulin. The use of antibodies to increase binding to FcRn and, in some cases, serum half-life Generally, unless otherwise specified, the Fc monomers of the present invention can bind to FcRn. (and, as described below, amino acids to increase binding to FcRn) (may contain variants).

[0084] As used herein, a "parent polypeptide" refers to a polypeptide that is subsequently modified to produce a variant. A parent polypeptide refers to a starting polypeptide that is to be modified. or a variant or modified version of a naturally occurring polypeptide. The parent polypeptide may be the polypeptide itself, a composition containing the parent polypeptide, or The term "parenteral immune" as used herein can refer to the amino acid sequence that encodes it. "immunoglobulin" refers to an unmodified immunoglobulin polypeptide that is modified to generate a variant. "Parent antibody" as used herein means a peptide that is used to generate a variant antibody. "Parent antibody" refers to an unmodified antibody that is modified in a manner similar to that described above. It should be noted that commercially available recombinantly produced antibodies are included.

[0085] As used herein, "Fc" or "Fc region" or "Fc domain" means In some cases, the first constant region immunoglobulin domain (e.g., CH1) or In some cases, the hinge may be removed entirely or partially. Fc refers to the polypeptides comprising the constant region of an antibody, excluding IgA, IgD, and the last two constant region immunoglobulin domains of IgG (e.g., CH2 and CH3), the last three constant region immunoglobulin domains of IgE and IgM; and optionally all or part of a flexible hinge N-terminal to these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, Fc The domains are immunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and CH The Fc region contains the lower hinge region between CH1 (Cγ1) and CH2 (Cγ2). However, the human IgG heavy chain Fc region typically contains residues E216, C22 at its carboxy terminus. 6, or A231, where the numbering is the same as in Kabat. In some embodiments, as described in more detail below, e.g. and / or FcγR or FcRn. Amino acid modifications are carried out.

[0086] As will be appreciated by those skilled in the art, the exact numbering and arrangement of the heavy constant region domains may vary. Numbering systems may differ. A useful comparison of EU and Kabat heavy constant region numbering is See Edelman et al., which are incorporated by reference in their entireties. 1969,Proc Natl Acad Sci USA 63:78-85, and Kabat et al., 1991, Sequences of Proteins of Immunological Interest,5th Ed.,United States Public Health Service,National I See Institutes of Health, Bethesda. [Table 1]

[0087] As used herein, a "fusion protein" refers to a covalent bond between at least two proteins. As described herein, a fusion protein may comprise an artificial sequence, e.g., a domain. As used herein, the term "Fc fusion protein" or "immunoadjuvant" may include an interlinker. "Hesin" generally refers to IL-15 and / or IL-16, as described herein. 15R (optionally linked to one or more different proteins, such as Fc domains (e.g., via a domain linker) are proteins containing an Fc region. Some examples are: In this study, two Fc fusion proteins were synthesized as homodimeric Fc fusion proteins or heterodimeric Fc fusion proteins. A heterodimer-Fc fusion protein can be formed, the latter being preferred. One monomer of the dimeric Fc fusion protein contains only the Fc domain (e.g., an empty Fc domain). The other monomer contains a variant Fc domain and a receptor, ligand, or or other binding partners.

[0088] As used herein, "position" means a location in the sequence of a protein. numbering or following established formats, e.g., EU International for antibody numbering. It may be numbered by index.

[0089] As used herein, "chain ( "Strandedness" is a property of "matching" double-stranded DNA, and Heterodimerization to preserve, generate, and / or enhance the ability to form heterodimers This means incorporating variants into each monomer, e.g., several pI variants. If is engineered into monomer A (e.g., to have a higher pI), it can be similarly utilized. The "charge pair" steric variants do not interfere with the pI variants, e.g., the pI-increasing charge variants The cargo variants are placed on the same "chain" or "monomer" and retain both functions. , as outlined in more detail below, for "skew" variants that result in paired sets. Thus, one skilled in the art can determine which chain or monomer incorporates one set of pairs. Consider pI when selecting a protein, and use skew pI to maximize pI separation. .

[0090] As used herein, "wild type or WT" refers to a naturally occurring gene, including allelic variations. The WT protein refers to the amino acid sequence or nucleotide sequence of a protein that has not been intentionally modified. It has an amino acid sequence or a nucleotide sequence that is not known.

[0091] The heterodimeric proteins of the present invention are generally isolated or recombinant. "Isolated" when used to describe the various polypeptides disclosed herein. "Identified and isolated from the cell or cell culture in which it was expressed" means that it has been identified and isolated from the cell or cell culture in which it was expressed and / or An isolated polypeptide generally refers to a polypeptide that has been isolated from at least one An "isolated protein" may be prepared by a purification step of the host cell. It refers to a protein that is substantially free of other proteins derived from cell culture, such as proteins. "Recombinant" means that a protein is produced in an exogenous host cell using recombinant nucleic acid technology. This means that

[0092] "Percent (%) amino acid sequence identity" for protein sequences refers to the maximum sequence identity. After aligning the sequences and introducing gaps if necessary to achieve a percent identity, the percentage of amino acid residues in the candidate sequence that are identical to those in the (parent) sequence; and and does not take into account any conservative substitutions as part of the sequence identity. Alignment to determine percent sequence identity can be performed using, for example, BLAST, BL AST-2, ALIGN, or Megalign (DNASTAR) software, etc. using publicly available computer software within the skill of the art. This can be achieved in a variety of ways. Those skilled in the art will find that the best possible match is achieved over the entire length of the sequences being compared. The appropriate parameters for measuring the match, including any algorithms required for One particular program is incorporated herein by reference. The ALIGN-2 program is outlined in U.S. Patent Application Publication No. 20160244525, paragraphs

[0279] to

[0280] .

[0093] The degree of identity between the amino acid sequences of the present invention ("sequences of the present invention") and the parent amino acid sequence is the integral of the two sequences divided by the shorter of the length of the "sequence of the invention" or the length of the parent sequence. The result is expressed as percent identity.

[0094] In some embodiments, the two or more amino acid sequences are at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 28 In some embodiments, the two or more amino acid sequences are at least are also 95%, 97%, 98%, 99%, and even 100% identical.

[0095] As used herein, an "antigen binding domain" or "ABD" refers to a domain that binds to an antigenic determinant. It refers to the part of an antigen-binding molecule that confers specificity.

[0096] As used herein, the term "antigen-binding molecule" refers to an antigen-binding molecule in its broadest sense. Antigen-binding molecules are any molecule that specifically binds to an antigenic determinant. Antigen-binding molecules include proteins, carbohydrates, and The antigen-binding molecule may be a peptide, a lipid, or other chemical compound. Examples of antigen-binding molecules include immunoglobulins. Antigen binding proteins include Fab and scFv, and their derivatives or fragments. An additional example of a combined molecule is a receptor and a ligand.

[0097] "Specific binding" to or "specifically binding to" a particular antigen or epitope "Specific for" a particular antigen or epitope refers to a specific interaction, not a non-specific interaction. Specific binding generally refers to binding that is measurably different from that of a specific molecule, e.g., a molecule that has binding activity. by determining the binding of a molecule relative to the binding of a control molecule, which is a molecule of similar structure that does not bind to the molecule. For example, specific binding can be measured by competition with a control molecule that is similar to the target. The decision can be made by the parties.

[0098] The strength or affinity of specific binding is determined by the dissociation constant (K D ) can be expressed as , K. D A smaller value indicates a greater affinity, and K DA larger value indicates a lower affinity. Binding properties can be determined using methods well known in the art, such as biolayer interferometry and surface plasmon resonance based methods. The antigen-binding site / antigen binding site can be determined by known methods. Alternatively, the method involves measuring the association and dissociation rates of a receptor / ligand complex, where the rates are The concentrations of the binding partners, the affinity of the interaction, and the geometry affect the rates in both directions equally. Therefore, both the association rate (ka) and dissociation rate (kd) depend on the biological parameters. The ratio kd / ka is the dissociation constant K D (e.g., Natu re 361:186-187 (1993) and Davies et al. (199 0)Annual Rev Biochem 59:439-473).

[0099] Specific binding to a particular antigen or epitope can be achieved by, for example, At least about 10-4M, at least about 10-5M, at least about 10-6M, at least about 10-7M, at least about 10-8M, at least about 10-8M, alternatively at least at least about 10-10M, at least about 10-11M, at least about 10-12M, or is more than K D Typically, the antibody is specific for an antigen. The antibody binding molecules bind to the antigen or epitope 20-fold more than the control molecule, 50x, 100x, 500x, 1000x, 5,000x, 10,000x, or Beyond the big K D This will result in the following:

[0100] Specific binding to a particular molecule or epitope can also be achieved by, for example, binding to an antigen or epitope. The Ka or association rate for the epitope is at least 20-fold higher than that of the control. , 50x, 100x, 500x, 1000x, 5,000x, 10,000x, or more This can be demonstrated by antigen-binding molecules that are larger than this.

[0101] "Epitope," as used herein, refers to a specific antigen-binding domain, e.g., a paratope. An epitope refers to a determinant that interacts with the variable region of a known antibody molecule. or sugar side chains, usually with specific structural characteristics as well as specific charge characteristics. A single molecule may have two or more epitopes. The amino acid residues directly involved (also called immunodominant components of the epitope) and Other amino acid residues not directly involved, e.g., those effectively blocked by specific antigen-binding peptides amino acid residues that are fragmented, in other words, within the footprint of the specific antigen-binding peptide It may comprise amino acid residues. Epitopes may be either conformational or linear. Conformational epitopes are composed of amino acids from different segments of a linear polypeptide chain. Linear epitopes are created by spatial juxtaposition of adjacent amino acids within a polypeptide chain. Conformational and non-conformational epitopes are those created by amino acid residues. The group loses its bond to the former but not to the latter in the presence of denaturing solvents. Epitopes can be distinguished by their ability to bind to at least one molecule within a unique spatial conformation. It contains three, more usually at least five, or eight to ten amino acids. An antigen-binding molecule that recognizes a target antigen of another antigen-binding molecule The ability to block the binding of β-glucan has been verified in simple immunoassays, e.g., "binning." As outlined below, the present invention relates to the antigen-binding molecules and antigens of the present invention. Not only do they contain the original binding domains, but also the antigen-binding molecules or antigen-binding domains listed The term also includes those that compete for binding to the epitope bound by the antibody.

[0102] "Fused" or "covalently linked" refers to the combination of components as outlined herein (e.g., IL-1 5 and Fc domains) are linked to the peptides either directly or via domain linkers. It means connected by a bond.

[0103] As used herein, the term "single chain" refers to a molecule linearly linked by peptide bonds. Refers to a molecule comprising linked amino acid monomers.

[0104] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such It should be understood that the scope of the present invention is set forth in the accompanying patents. The terms used herein refer to specific embodiments only as limited by the scope of the claims. It is also understood that the following is for illustrative purposes only and is not intended to be limiting. should be.

[0105] II. Antibodies As discussed below, the term "antibody" is used generically. The antibodies can take several formats, as described herein, Antibodies include conventional antibodies as described herein and shown in the figures, as well as antibody derivatives, fragments and mimetics. nothing.

[0106] A conventional antibody structural unit typically comprises a tetramer. Each tetramer typically comprises two identical It consists of a pair of polypeptide chains, each pair consisting of one "light" chain (typically about 25 kDa) molecular weight) and one "heavy" chain (typically about 50-70 kDa molecular weight). Human light chains are classified as kappa and lambda light chains. The present invention generally relates to IgG light chains. The present invention relates to a bispecific antibody based on IgG1, IgG2, IgG3, and It has several subclasses, including, but not limited to, IgG1 and IgG2. IgG1, IgG2, and IgG4 are used more frequently than IgG3. IgG1 is There are different allotypes with polymorphisms at 356 (D or E) and 358 (L or M). It should be noted that the sequences presented herein are of the 356E / 358M allotype. Although other allotypes are included herein, the I Any sequence containing the gG1 Fc domain is a 356E / 358M allotype. It can have 56D / 358L.

[0107] Additionally, many of the sequences herein have at least one cysteine ​​at position 220 substituted with serines. This is generally referred to as "sc" for most of the sequences described herein. The Fv monomer side is located on the Fab monomer side to reduce disulfide formation. Specifically included within the sequences herein are those one or both cysteines are substituted (C220S).

[0108] Therefore, as used herein, "isotype" refers to the structure of their constant regions. means any subclass of immunoglobulin defined by its biological and antigenic characteristics Therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses. See, for example, U.S. Publication No. 2009 / 0163699, which is incorporated by reference. As shown in No. 1, the present invention is the use of human IgG1 / G2 hybrids.

[0109] The hypervariable region generally consists of approximately amino acid residues 24 to 34 (LC DR1, "L" represents the light chain), 50-56 (LCDR2), and 89-97 (LCD R3), and approximately 31-35B in the heavy chain variable region (HCDR1, "H" indicates heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) amino acid residues (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST,5th Ed.Public Health Service,National Institutes of H Health, Bethesda, Md. (1991)) and / or hypervariable loops Residues forming the nucleotide sequence (e.g., residues 26-32 (LCDR1), 50-55 in the light chain variable region) 2 (LCDR2), and 91 to 96 (LCDR3), and 2 in the heavy chain variable region 6-32 (HCDR1), 53-55 (HCDR2), and 96-101 (HCDR3 ) (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917). Particular CDRs of the invention are described below.

[0110] As will be appreciated by those skilled in the art, the exact numbering and arrangement of the CDRs varies between different numbering systems. However, the disclosure of variable heavy and / or variable light chain sequences may be relevant (fixed). It should be understood that the disclosure of each variable heavy region includes the disclosure of the corresponding CDRs. The illustration shows disclosure of vhCDRs (e.g., vhCDR1, vhCDR2, and vhCDR3). and the disclosure of each variable light region includes a vlCDR (e.g., vlCDR1, vlCDR2, and A useful comparison of CDR numbering is as follows: franc et al.,Dev.Comp.Immunol.27(1):55-7 Please refer to 7(2003). [Table 2]

[0111] Throughout this specification, the Kabat numbering system generally refers to residues within a variable domain (approximately When referring to residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region The EU numbering system is for the Fc region (see, e.g., Kabat et al. L., supra (1991)).

[0112] Another type of Ig domain in the heavy chain is the hinge region. or "hinge region" or "antibody hinge region" or "hinge domain" refers to the first amino acid sequence of an antibody. It refers to a flexible polypeptide comprising the amino acids between the first and second constant domains. Generally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain The base begins at EU position 231. Thus, for IgG, the antibody hinge is referred to herein as The amino acid sequence is shown to include positions 216 (E216 in IgG1) to 230 (P230 in IgG1). and numbering is according to the EU index as in Kabat. A "hinge fragment" is used, which is either the N-terminal or C-terminal end of the hinge domain. As described herein, the pI barrier Contigs can be similarly fabricated in the hinge region.

[0113] The light chain generally comprises a variable light chain domain (which includes the light chain CDRs and, together with the variable heavy chain domain, the F The constant light region (often called CL or Cκ) forms the κ region. Includes the domain.

[0114] Another region of interest for additional substitutions, as outlined below, is the Fc region.

[0115] The present invention provides a number of different CDR sets. A "set" includes three variable light chain and three variable heavy chain CDRs, e.g., vlCDR1, vlCD R2, vlCDR3, vhCDR1, vhCDR2, and vhCDR3. may be part of a larger variable light or variable heavy chain domain, respectively. As outlined more fully in the literature, the variable heavy and variable light chain domains are On a separate polypeptide chain when a light chain is used (e.g., when a Fab is used). The sequences may be on separate polypeptide chains, or in the case of scFv sequences, on a single polypeptide chain.

[0116] CDRs are responsible for forming antigen binding, or more specifically, forming the epitope-binding site of an antibody. An "epitope" is a region within the variable region of an antibody molecule known as the paratope. An epitope is a determinant that interacts with a specific antigen-binding site on an antibody. A grouping of molecules, such as chains, that usually have specific structural characteristics as well as specific charge characteristics A single antigen may have more than one epitope.

[0117] An epitope is a molecule that contains amino acid residues that are directly involved in binding (also known as immunodominant components of the epitope). and other amino acid residues not directly involved in binding, e.g., specific antigen-binding peptides. The amino acid residues that are effectively blocked by the nucleotides, in other words, the amino acid residues of the specific antigen-binding peptide It may include amino acid residues that lie within the footprint.

[0118] Epitopes can be either conformational or linear. Conformational epitopes are created by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain Linear epitopes are created by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes are identified in the presence of denaturing solvents. They can be distinguished in that below, the bond to the former is lost but the bond to the latter is not.

[0119] An epitope typically has at least three, and more commonly, Contains at least 5 or 8-10 amino acids. Recognizes the same epitope. Antibodies can be characterized by the ability of one antibody to block the binding of another antibody to its target antigen, e.g., "binning" As outlined below, the present invention can be validated in a simple immunoassay showing The antigen-binding domains and antibodies of the present invention are not limited to the antigen-binding domains listed above. Also included are those that compete for binding to the epitope bound by the domain.

[0120] Thus, the present invention provides different antibody domains. As is known in the art, the heterodimeric antibodies of the present invention may contain different domains within the heavy and light chains. These domains include the Fc domain, CH1 domain, and the IgG domain, which may also overlap. In, CH2 domain, CH3 domain, hinge domain, heavy chain constant domain (CH1- hinge-Fc domain or CH1-hinge-CH2-CH3), variable heavy chain domain, It contains a variable light chain domain, a light chain constant domain, a Fab domain, and an scFv domain. , but not limited to these.

[0121] Thus, an "Fc domain" refers to a -CH2-CH3 domain, optionally a hinged In the embodiment herein, the scFv comprises an Fc domain (-H-CH2-CH3). When bound to the Fc domain, all or part of the hinge is bound to the s The C-terminus of the cFv construct, e.g., it generally contains the sequence EPKS, which is the beginning of the hinge The heavy chain comprises a variable heavy domain and a constant domain, which are CH1- The light chain comprises a variable light chain and a hinge-Fc domain comprising an optional CH2-CH3 domain. scFvs contain a variable heavy chain, an scFv linker, and a variable light chain constant domain. In most of the constructs and sequences outlined herein, the C of the variable heavy chain The C-terminus of the scFv linker is attached to the N-terminus of the variable light chain. (N-vh-linker-vl-C), which can be switched (N-vl- linker-vh-C).

[0122] Some embodiments of the present invention comprise at least one scFv domain, which is The variable heavy chain domain and the variable light chain domain are not naturally occurring but are linked together by an scFv linker. As outlined herein, an scFv domain generally comprises: The vh-scFv linker-vl is oriented from the N-terminus to the C-terminus. It is constructed using vh and vl sequences from an scFv domain (or Fab). For either the . Alternatively, the linker can be reversed to vl-scFv linker-vh.

[0123] As indicated herein, suitable linkers (domain linkers or scFv linkers) There are several methods for covalently binding (recombinantly) to the listed domains. It can be used to create peptide bonds (including conventional peptide bonds created by conventional techniques). In some embodiments, the linker peptide is composed primarily of the following amino acid residues: Gly, Ser, Linker peptides may contain , Ala, or Thr. Linker peptides may be selected so that they retain the desired activity. It is enough to bind two molecules together in such a way that they assume the correct conformation relative to each other so that In one embodiment, the linker is about 1 to 50 amino acids in length, preferably Preferably, it is about 1 to 30 amino acids in length. In one embodiment, it is about 1 to 20 amino acids in length. A carrier can be used, and in some embodiments, about 5 to about 10 amino acids are used. Useful linkers include, for example, those in which n is at least an integer (generally 3 to 4) (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, glycine-a Glycine polymers containing alanine, alanine-serine polymers, and other flexible linkers Alternatively, polyethylene glycol (PEG), polypropylene Pyrene glycol, polyoxyalkylene, or polyethylene glycol and polypropylene Various non-proteinaceous polymers, including but not limited to copolymers with ethylene glycol Mers can be useful as linkers.

[0124] Other linker sequences include any sequence of the CL / CH1 domain of any length, but It may not contain all residues of the CL / CH1 domain, e.g., The linker is the first 5 to 12 amino acid residues of an immunoglobulin light chain, e.g., Cκ or The linker may be derived from, for example, Cγ1, Cγ2, Cγ3, Cγ4, Cα1, derived from immunoglobulin heavy chains of any isotype, including Cα2, Cδ, Cε, and Cμ The linker sequence may also be used to identify Ig-like proteins (e.g., TCR, FcR, KIR). , sequences derived from hinge regions, and other naturally occurring sequences from other proteins. It may be derived from.

[0125] In some embodiments, the linker connects any two domains outlined herein. The "domain linker" used to link them together. For example, the CH1 of Fab There is a domain linker that connects the C-terminus of the domain to the N-terminus of the scFv, and another arbitrary domain The main linker connects the C-terminus of the scFv to the CH2 domain (although in most cases In embodiments, a hinge is used as this domain linker.) Any suitable linker can be used, but many embodiments prefer, for example, n to be at least 1 (generally 3 to 4). 4-5), (GS)n, (GSGGS)n, (GGGGS)n, and (G Glycine-serine polymers as domain linkers containing GGS)n, and It consists of two domains that are long and flexible enough to allow the enzyme to retain its biological function. Any peptide sequence that allows recombinant binding of the polypeptide may be used. The scFv linker is constructed with careful attention to the "strandedness" outlined. As used in some embodiments, charged domain linkers can be used. Cut.

[0126] In some embodiments, the linker comprises a linker comprising the vh and vl domains discussed herein. scFv linkers are often used to covalently link is a charged scFv linker.

[0127] Thus, the present invention further provides a method for enhancing the pI separation between the first and second monomers. The present invention provides a charged scFv linker for the purpose of synthesizing the scFv fragments. cFv linker (or both in the case of scaffolds that use scFvs for different monomers) By incorporating a monomer containing a charged linker without further altering the Fc domain, These charged linkers can be used in any combination, including standard linkers. As will be appreciated by those skilled in the art, substitutions can be made in any scFv. Depending on the desired variation, charged scFv linkers are used on the correct "chains" or monomers. For example, as discussed herein, triple F format heterodimeric antibodies can be generated. To determine the pI of the Fv region for each of the desired antigen-binding domains, the original pI is calculated. and one is selected to generate the scFv, and depending on the pI, either positive or negative phosphorylation is performed. One of the cars is selected.

[0128] Charged domain linkers may also be used to increase the pI separation of the monomers of the present invention. Therefore, what is included herein is the invention that utilizes a linker. It can be used in any embodiment of the book.

[0129] Specifically, some formats of antibodies are commonly referred to as "heterodimeric antibodies" The protein contains a heterodimeric Fc domain and either a Fab or scFv. and having at least two related Fc sequences self-assembled into at least two Fv regions. This means:

[0130] III. Chimeric and Humanized Antibodies In certain embodiments, the checkpoint blockade antibodies of the invention are directed to specific germline Heavy chain variable regions derived from heavy chain immunoglobulin genes and / or specific germline light chain immunoglobulins For example, such antibodies may contain light chain variable regions derived from immunoglobulin genes. Contains heavy or light chain variable regions that are "the product of" or "derived from" cell lineage sequences It may comprise or consist of a human antibody. A human antibody that is "the product of" or "derived from" a human antibody is one that has its amino acid sequence modified by human in vivo synthesis. Comparison with germline immunoglobulin amino acid sequences and the sequence closest to that of human antibodies Selecting human germline immunoglobulins that are sequences (i.e., highest % identity) and can be identified as such (using the methods outlined herein) by "A product of" or "derived from" a specific human germline immunoglobulin sequence. "Human antibodies" refer to antibodies that have been engineered to be human, e.g., through natural somatic mutations or site-specific mutations. Due to recombinant DNA technology, the sequence may contain amino acid differences compared to the germline sequence. However, humanized antibodies are typically encoded by human germline immunoglobulin genes. the amino acid sequence is at least 90% identical to that of the target gene, and When compared to cell line immunoglobulin amino acid sequences (e.g., mouse germ line sequences), In some cases, the antibody contains amino acid residues that identify it as derived from a human sequence. In some cases, humanized antibodies are antibodies encoded by germline immunoglobulin genes. At least 95%, 96%, 97%, 98%, or may be 99%, or even at least 96%, 97%, 98%, or 99% identical Typically, humanized antibodies derived from specific human germline sequences are derived from human germline antibodies. Amino acid sequences encoded by immunoglobulin genes differ by 10-20 amino acids or less. (Any scuba variants, pI variants, and truncation variants herein are not included.) Prior to the introduction of variants, i.e., the variants of the present invention, the number of variants was generally In certain cases, humanized antibodies are derived from germline immunoglobulin genes. The amino acid sequence encoded by the (Similarly, any scuba Before the introduction of the barriers, pI variants, and truncation variants, i.e., the barriers of the present invention (Before the introduction of a new agent, the number of variants is generally low.)

[0131] In one embodiment, the parent antibody has been affinity matured as known in the art. Structure-based methods, such as those described in USSN 11 / 004,590, , humanization and affinity maturation, including, but not limited to, all in their entirety. Wu et al., 1999, J. Mol. Biol. 29, incorporated by reference. 4:151-162, Baca et al., 1997, J. Biol. Chem. 2 72(16):10678-10684, Rosok et al., 1996, JB iol.Chem.271(37):22611-22618, Rader et al. .,1998,Proc.Natl.Acad.Sci.USA 95:8910-89 15, Krauss et al., 2003, Protein Engineerin Selection-based methods can be used to identify antibodies, including those described in U.S. Pat. No. 6,629,169, ... Other humanization methods can be used for humanization and / or affinity maturation of the variable region. This may include transplanting only a portion of the DR, including, but not limited to, transplanting the entire USSN 09 / 810,510, Tan et al., incorporated by reference in its entirety. .,2002,J.Immunol.169:1119-1125,De Pascal is et al., 2002, J. Immunol. 169:3076-3084 This includes the methods described.

[0132] IV. Heterodimeric Fc Fusion Proteins The present invention relates to the binding of IL-15 and IL-15 receptor alpha (IL-15Rα) in different ways. ) protein domains. The Fc domain is , an IgG Fc domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc The IgG1 Fc domain is of particular use in the present invention. can be.

[0133] The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Kabat et al. collected a large number of primary sequences of the heavy and light chain variable regions. Based on the degree of homology, Kabat et al. classified each primary sequence into CDR and framework. , which created the list (incorporated by reference in its entirety, SEQUENCES OF IMMUNOLOGICAL INTEREST,5th edition,NIH p Publication, No. 91-3242, E.A. Kabat et al. Throughout this specification, the Kabat numbering system generally refers to the number of residues in a variable domain. (approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) The EU numbering system is for the Fc region (e.g., Kaba see t et al., supra (1991).

[0134] The IgG subclass of immunoglobulins contains several immunoglobulin domains in the heavy chain. As used herein, "immunoglobulin (Ig) domains" refer to domains that have distinct tertiary structures. The term "antibody" refers to the region of an immunoglobulin that has the structure of a constant heavy (CH) domain and a hinge domain. Of interest in the present invention are heavy chain domains containing: Each IgG isotype has three CH regions. The "CH" domains in the According to the EU index, it is ranked 118-215. "Hinge" is a word that is used in Kabat. According to the EU index, it is ranked 216-230. According to the EU index, it is ranked 231-340, and "CH3" is the Kabat It refers to the rankings 341-447 according to the EU index as shown in Table 1. However, the exact numbering and arrangement of heavy chain domains may vary in different numbering systems. As shown in and described below, the pI variants are similar to the hinge region discussed below. Likewise, there may be one or more CH regions.

[0135] Another type of Ig domain in the heavy chain is the hinge region. or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" means a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody Structurally, the IgG CH1 domain ends at EU position 215, and the IgG CH2 domain The main chain begins at residue EU position 237. Thus, for IgG, the antibody hinge is to include positions 216 (E216 in IgG1) to 230 (P230 in IgG1) Definitions and numbering herein are according to the EU index as per Kabat. In some embodiments, for example, in relation to the Fc region, a hinge is included, generally at position 21. As described herein, pI variants are also created in the hinge region. It can be manufactured.

[0136] Thus, the present invention provides different antibody domains, as described herein, and As is known in the art, the heterodimeric proteins of the present invention may be composed of different domains. These domains include the Fc domain, CH1 domain, and the ATP domain, which may also overlap. domain, CH2 domain, CH3 domain, hinge domain, heavy constant domain (CH1-hinge di-Fc domain or CH1-hinge-CH2-CH3). do not have.

[0137] Thus, an "Fc domain" refers to a -CH2-CH3 domain and optionally a Hin domain. In some embodiments, the Fc domain also comprises one of the CH1 domains. In some embodiments, protein fragments, such as IL-15 or When IL-15Rα binds to the Fc domain, it binds to the hinge of the Fc domain. IL-15 or IL-15Rα constructs bound in whole or in part to the C-terminus of the construct. For example, it is generally attached to the sequence EPKS, which is the beginning of the hinge. In other embodiments, a protein fragment, such as IL-15 or IL- When 15Rα is bound to the Fc domain, it binds to the C-terminus of the CH3 domain. It is the N-terminus of the protein fragment.

[0138] Some of the Fc domain protein constructs and sequences outlined herein In this case, the C-terminus of IL-15 or IL-15Rα binds to the N-terminus of the domain linker. , its C-terminus linked to the N-terminus of the constant Fc domain (N-IL-15 or IL- 15Rα protein fragment-linker-Fc domain-C), which can be switched (N-Fc domain-linker-IL-15 or IL-15Rα protein domain) In other constructs and sequences outlined herein, the first protein fragment The C-terminus of the second protein fragment is linked, optionally via a domain linker, to the N-terminus of the second protein fragment. and the C-terminus of the second protein fragment is optionally linked to a constant Fc domain via a domain linker. In further constructs and sequences outlined herein, and a constant Fc domain that is not bound to the first protein fragment or the second protein fragment. Heterodimeric Fc fusion proteins are provided. Heterodimeric Fc fusion proteins comprise two or more of the exemplary Fc fusion proteins described herein. In yet another construct, the first protein may comprise a monomeric Fc domain protein. The N-terminus of the protein fragment is connected, optionally via a domain linker, to the N-terminus of a second protein fragment. The C-terminus of the second protein fragment is optionally linked to a constant Fc domain via a domain linker. Binds to the main N-terminus.

[0139] In some embodiments, the linker connects any two domains outlined herein. The "domain linkers" used to link them together are some of which are shown in Figure 87. While any suitable linker can be used, many embodiments involve linkers such as , where n is at least 1 (and generally 0-1-2-3-4-5), (GS)n, (G Glycine-serine polymers including (SGGS), (GGGGS), and (GGGS) - and sufficient length and flexibility to allow each domain to retain its biological function Any peptide sequence that allows recombinant binding of the two domains can be used. Depending on the situation, attention should be paid to the "strandedness" outlined below. The linker is a charged domain linker.

[0140] Thus, in some embodiments, the present invention provides a method for self-assembling heterodimeric Fc domains. This relies on the use of two different heavy chain variant Fc sequences to form a fusion polypeptide. The present invention provides heterodimeric Fc fusion proteins.

[0141] In one embodiment, the heterodimeric Fc fusion protein is a heterodimeric Fc fusion protein, which is characterized by heterodimeric Fc fusion proteins, such as pI engineering. It comprises at least two constant domains that can be engineered to form dimers. Other Fc domains that may be used include pI engineered CH1, CH2, CH3 and CH4 domains of the present invention. and one or more of the hinge domains. The formats shown in 9A-39D are heterodimeric Fc fusion proteins, which are This protein contains two linked Fc sequences and a heterodimeric Fc domain that self-assemble into a heterodimeric Fc domain. and at least one protein fragment (e.g., one, two, or more protein fragments In some cases, the first protein fragment has a first F In other cases, the first protein fragment is linked to a second Fc sequence, and the second protein fragment is linked to a second Fc sequence. wherein the first protein fragment is linked to a first Fc sequence, and the first protein fragment is an F c sequence is non-covalently attached to a second protein fragment that is not linked to the Thus, a heterodimeric Fc fusion protein comprises a second Fc sequence linked to a first Fc sequence. a first protein fragment linked to a first or second protein fragment; The antibody comprises a second Fc sequence that is not linked to either of the two protein fragments.

[0142] The present invention allows for binding to one or more binding partners, ligands, or receptors. The present invention relates to novel constructs for providing heterodimeric Fc fusion proteins. -Fc constructs are composed of two Fc domains of antibody heavy chains assembled into a "dimer," e.g. Heterodimeric Fc fusions are based on the self-assembly properties of two "monomers" that are Created by varying the amino acid sequence of each monomer, as explained in more detail below Thus, the present invention generally relates to a method for detecting a binding partner(s) or ligand(s). heterodimers that can co-bind the receptor(s) in some way For the production of Fc fusion proteins, to promote heterodimer formation, and / or To facilitate purification of heterodimers rather than homodimers, different constant region fragments were used in each chain. It depends on the amino acid variants.

[0143] There are several mechanisms that can be used to generate the heterodimers of the present invention. Furthermore, as will be appreciated by those skilled in the art, these mechanisms may be combined to enhance This ensures a stable heterodimerization, thus resulting in the production of heterodimers. Such amino acid variants are referred to as "heterodimerization variants." The heterodimerization variant is a steric variant that allows for the purification of homodimers from heterodimers. variants (e.g., "knob-and-hole" or "skew" variants and " These mutations may include "charge pair" mutations as well as "pI variants." and specifically below for the discussion of "heterodimerization variants." As cited in

[1999] , useful mechanisms of heterodimerization include "knob-and-hole" ("KI"). H”, sometimes referred to herein as the “skew” variant ( WO2014 / 145 806), as described in WO2014 / 145806 "Electrostatic steering" or "charge pairs", as described in WO2014 / 145806 pI variants such as those described in WO2014 / 145806 and outlined below. Additional common Fc variants are included, such as:

[0144] In the present invention, the purification of heterodimeric proteins and antibodies can be facilitated. There are several basic mechanisms, one of which is that each monomer and then each dimer species has a different pI By having Alternatively, several formats are available. The granules also allow for separation based on size. As further outlined below, It is also possible to "skew" the formation of heterodimers over homodimers. Therefore, steric heterodimerization variants and pI or charge pair variants are of particular use in the present invention.

[0145] Generally, the embodiments specifically used in the present invention are those between two monomers and each dimer. - In combination with pI variants that increase the pI difference between species, The heterodimerization depends on a set of variants, including a scuba variant that promotes heterodimerization. do.

[0146] Additionally, as outlined more fully below, heterodimeric Fc fusion proteins may be used. Depending on the format, the pI variants are located in the constant and / or Fc domains of the monomer. Either the domain can be included within the domain or a domain linker can be used. That is, the present invention provides a method for preparing a nucleotide sequence comprising: Charged domain linkers are also provided. Additionally, further amino acid manipulations for alternative functionality are possible. can also confer pI changes such as Fc, FcRn, and KO variants.

[0147] This invention utilizes pI as a separation mechanism to enable the purification of heterodimeric proteins. In particular, amino acid mutations can be introduced into one or both monomeric polypeptides. That is, the pI of one of the monomers (referred to herein as "monomer A" for simplicity) can be engineered to be different from monomer B, or both monomers A and B can be engineered to be different from monomer A's p As discussed, both can be modified to increase the pI of monomer B and decrease the pI of monomer B. The pI change of one or both monomers can be achieved by removing or adding charged residues (e.g. For example, neutral amino acids can be substituted with positively or negatively charged amino acid residues, e.g., glutamic acid. by changing the charged residue from positive or negative to the opposite charge (e.g., from carboxyl to glutamic acid); aspartic acid to lysine), or by changing a charged residue to a neutral residue (e.g. Loss of charge (lysine to serine) can be achieved. The figure shows the

[0148] Thus, this embodiment of the invention allows for the separation of heterodimers from homodimers. providing a sufficient pI change in at least one monomer to allow As will be appreciated by those skilled in the art, and as discussed further below, this is a "wild Increasing or decreasing the heavy chain constant region and its pI (wtA:+B or wtA:B-) By using variant regions engineered to enhance the expression of the gene, or by increasing the expression of one region. This is done by adding one area and decreasing the other (A+:B- or A-:B+). can be done.

[0149] Thus, in general, components of some embodiments of the present invention are dimeric proteins The isoelectric point (pI) of at least one, if not both, of the monomers is changed by the amino acid substitution ( By incorporating a pI variant or pI substitution into one or both of the monomers, These are amino acid variants in the constant region that are intended to alter the Separation of the heterodimer from the dimer was achieved when the pI of the two monomers differed by only 0.1 pH units. If the values ​​are different than 0.2, 0.3, 0.4, and 0.5, all find use in the present invention.

[0150] As will be appreciated by those skilled in the art, it is possible to separate each or both monomers ( The number of pI variants to be included (multiple variants allowed) depends in part on the starting pI of the constructs. That is, which monomers to manipulate or which "direction" (e.g., more positive or more negative), and in some cases, the sequence of the Fc domain. The protein domain(s) linked to the Fc domain are calculated, from which a determination can be made. As known in the art, different Fc domains and / or Protein domains will have different starting pIs to be utilized in the present invention. As outlined herein, pI is at least about 0.1 log of the total pI of each monomer. The difference in I is preferably between 0.2 and 0.5 as outlined herein. stomach.

[0151] Furthermore, as will be appreciated by those skilled in the art and as outlined herein, in some embodiments , heterodimers can be separated from homodimers based on size. For example, some of the formats are divided into heterodimers and homodimers based on size. Allows for separation of dimers.

[0152] By using the constant region(s) of the Fc domain(s), pI variants can be When heterodimerization is achieved using a fusion protein, heterodimeric Fc fusion proteins can be designed and This provides a more modular approach to the production and purification of In some embodiments, heterodimerization variants (skewed and purified heterodimerization variants) are used. Furthermore, in some embodiments, the pI barrier The potential for immunogenicity arising from the ant is reduced by altering the pI without introducing significant immunogenicity. By transferring pI variants from different IgG isotypes, significant Therefore, an additional problem to be solved is the ability to generate high human sequence content. Elucidation of low pI constant domains, e.g., minimization or deletion of non-human residues at any particular position or avoidance.

[0153] Potential side benefits of this pI engineering also include increased serum half-life and FcRn binding. Namely, USSN 13 / 194,904 (incorporated by reference in its entirety). As described herein, antibody constant domains (antibodies and Fc Lowering the pI of the nucleotides (including those found in fusions) results in longer serum retention in vivo. These pI variants for increased serum half-life may also be used to increase the pI for purification. Facilitate change.

[0154] Furthermore, the pI variants of heterodimerization variants are different when homodimers are present. Its remarkable ability to exclude, minimize, and differentiate makes it ideal for the analysis and characterization of Fc fusion proteins. This provides additional benefits to the quality control process. The ability to reliably test the reproducibility of the composition is important.

[0155] A. Heterodimerization variants The present invention provides a method for the preparation of heterodimers and / or their purification from homodimers. Heterodimeric Fc fusion proteins in various formats utilizing heterodimerization variants The heterodimeric fusion construct comprises two heterodimeric proteins. The self-assembly properties of the Fc domain, e.g., two "monomers" that assemble into a "dimer" Based on.

[0156] There are several suitable pairs of heterodimerization scubariant sets. The variants are "pairs" of "sets." That is, the pair of one set is the first monomer. One pair is incorporated into the body, and another pair is incorporated into a second monomer. The set of residues has a one-to-one correspondence between residues on one monomer and residues on the other monomer. The main difference is that it does not necessarily behave as a "knob-in-hole" variant, i.e. These pairs of sets form two monomers that promote heterodimer formation and prevent homodimer formation. The fraction of heterodimers that form spontaneously under biological conditions is predicted. 50% (25% homodimer A / A: 50% heterodimer A / B: 25% homodimer B / B) but above 90%.

[0157] B. Stereovariants In some embodiments, heterodimer formation is promoted by the addition of a stereovariant. That is, by changing the amino acids in each heavy chain, different heavy chains can be associated. Form heterodimeric structures rather than homodimers with the same Fc amino acid sequence A preferred stereovariant is included in Figure 29 of USSN 15 / 141,350. No. 6,299,793, all of which are incorporated herein by reference in their entireties, as well as Included in Figure 84.

[0158] One mechanism is described in detail in the following sections, all of which are incorporated herein by reference in their entirety. USSN61 / 596,846, Ridgway et al., Protein Engineering 9(7):617(1996), Atwell et al. , J.Mol.Biol.1997270:26, U.S. Patent No. 8,216,805. As described in the publication, the heterojunction is commonly referred to in the art as a "knob-and-hole" structure. Amino acid manipulations that create steric effects that favor dimer formation and disfavor homodimer formation These diagrams show several "monomer A - monomer B" structures that depend on "knob-and-hole" techniques. Furthermore, Merchant et al., Nature Biote As described in ch. 16:677 (1998), these "knob-and-hole" " Mutations combined with disulfide bond formation skew against heterodimerization It is possible.

[0159] Further mechanisms used to generate heterodimers are fully incorporated herein by reference. Gunasekaran et al., J. Biol. Chem. 285 (25):19637(2010), sometimes referred to as "electrostatic steering." This is sometimes referred to herein as a "charge pair." In this embodiment, , electrostatics are used to skew formation towards heterodimerization. As mentioned above, these may also affect the pI and therefore the purification. In some cases, they can also be considered pI variants. They were produced to force dimerization and were not used as a purification means, so they were These include, but are not limited to, D221R / P228R / K409R paired with D221E / P228E / L368E (e.g., These are the "corresponding sets of monomers"), and C220R / E224R / P22 These include the C220E / P228E / 368E paired with the 8R / K409R.

[0160] Further monomer A and monomer B variants may be used in accordance with the pI variants outlined herein. No. 6,201,212, which is expressly incorporated herein by reference in its entirety. Other variants, such as the other stereovariants shown in Figure 37 of 149876, and optionally They can be combined in any amount independently of one another.

[0161] In some embodiments, the steric variants outlined herein may be used to overcome any pI barrier. One or both of the FcRn variants (or other variants such as Fc variants, FcRn variants, etc.) can be optionally and independently incorporated into either monomer and independently incorporated into the protein of the invention. and can be optionally included or excluded.

[0162] A list of suitable scuba variants is shown in Figures 3A-3E. In many embodiments, Useful include, but are not limited to: S364K / E357Q:L368D / K3 70S, L368D / K370S:S364K, L368E / K370S:S364K, T411E / K360E / Q362E:D401K, L368D / K370S:S364 K / E357L, K370S:S364K / E357Q, and T366S / L368A / Y407V:T366W (optionally bridged disulfide, T366S / L368A / Y40 7V / Y349C:T366W / S354C) is a set of pairs. Regarding the pair "S364K / E357Q:L368D / K370S", one of the monomers is One has the heavy variant set S364K / E357Q, the other has the double variant set L36 8D / K370S, and as mentioned above, the "stratum" of these pairs The "boundness" depends on the starting pI.

[0163] C. Heterodimer pI (isoelectric point) variants Generally, as will be appreciated by those skilled in the art, there are two general categories of pI variants: : those that increase the pI of a protein (basic change) and those that decrease the pI of a protein As described herein, all combinations of these variants One monomer may be wild-type or significantly different from wild-type. The variant may not exhibit the same pI, while the other may be either more basic or more acidic. Alternatively, each monomer can be modified, one to become more basic and one to become more acidic. possible.

[0164] Preferred combinations of pI variants are shown in Figure 30 of USSN 15 / 141,350. All of which are incorporated herein by reference in their entirety. However, as shown in the figure, these changes are shown relative to IgG1, but not for all antibodies. Isotypes can be altered in this way, as can isotype hybrids. If the chain constant domain is derived from IgG2-4, R133E and R133Q are also used. The pI variants are shown in Figure 4.

[0165] In one embodiment, when one of the Fc monomers comprises a CH1 domain, the pI variant The preferred combination is the 208D / 295E / 384D / 418E / 421D variants (Compared to human IgG1: N208D / Q295E / N384D / Q418E / N4 21D). In some instances, the second monomer comprises (GK Optionally, the first monomer comprises a positively charged domain linker comprising a PGS (Polyphosphate Group I). It contains the CH1 domain including position 208. Therefore, the construct without the CH1 domain ( For example, a heterodimeric Fc fusion protein that does not utilize the CH1 domain as one of its domains In the case of quality, the preferred negative pI variant Fc set is 295E / 384D / 418E / 421D variant (Q295E / N384D / when compared to human IgG1) Q418E / N421D).

[0166] In some embodiments, the mutations are at positions 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 2 21, 222, 223, 224, 225, 226, 227, 228, 229, and 23 pI mutations, especially substitutions, occur in the hinge domain of the Fc domain, including pI 0. , and the 1, 2, 3, 4, or 5 mutations used in the present invention can be used to modify positions 216-230. Similarly, all possible combinations can be performed independently. or with other pI variants in other domains are contemplated.

[0167] Specific substitutions used to lower the pI of the hinge domain include: deletion, non-naturally occurring valine or threonine at position 222, deletion at position 223, non-naturally occurring guanine at position 224 glutamic acid, deletion at position 225, deletion at position 235, and deletion or non-naturally occurring mutation at position 236 In some cases, the hinge domain may contain p In other instances, only pI substitutions are made, and these substitution(s) may be made with other pI substitutions in other domains. Can be added to variants in any combination.

[0168] In some embodiments, positions 233, 234, 235, 236, 274, 296, 3 CH2 region including 00, 309, 320, 322, 326, 327, 334, and 339 Similarly, all possible combinations of these 14 positions can be mutated within the region. For example, antibodies with pIs of 1, 2, 3, 4, 5, 6, 7, 8, 9 Or it may have 10 CH2 pI substitutions.

[0169] Specific substitutions used to reduce the pI of the CH2 domain include: Unnatural glutamine or glutamic acid, unnatural phenylalanine at position 296, Unnatural phenylalanine, unnatural valine at position 309, unnatural glutamic acid at position 320, 3 Unnatural glutamic acid at position 22, unnatural glutamic acid at position 326, unnatural glycine at position 327 The natural glutamic acid at position 334, the unnatural threonine at position 339, and the amino acids in CH2 and This includes, but is not limited to, all possible combinations with other domains.

[0170] In this embodiment, the mutations are at positions 355, 359, 362, 384, 389, 392, 3 97, 418, 419, 444 and 447. Specific substitutions used to reduce the pI of the H3 domain include a non-natural substitution at position 355. Natural glutamine or glutamic acid, unnatural serine at position 384, unnatural asparagus at position 392 Glycine or glutamic acid, unnatural methionine at position 397, unnatural glutamic acid at position 419 , unnatural glutamic acid at position 359, unnatural glutamic acid at position 362, unnatural glutamic acid at position 389 glutamic acid, unnatural glutamic acid at position 418, unnatural glutamic acid at position 444, and 4 Examples include, but are not limited to, deletions or unnatural aspartic acid at position 47.

[0171] D. Isotype variants Additionally, many embodiments of the present invention provide for the detection of IgG from one IgG isotype to another. It depends on the "import" of pI amino acids at specific positions into the type, and therefore the desired Reduce or eliminate the possibility of introducing unwanted immunogenicity. U.S. Patent Application Publication No. 2014 / 0370013, which is incorporated herein by reference. The results are shown in Figure 21. IgG1 has various functions including high effector function. However, the heavy constant region of IgG1 is a common isotype for therapeutic antibodies. has a higher pI than that of IgG2 (8.10 vs. 7.31). By introducing G2 residues into the IgG1 backbone, the pI of the resulting monomer is decreased (or For example, IgG1 has a glycine ( IgG1 has glutamic acid (pI 3.22), and IgG2 has glutamic acid (pI 5.97). The introduction of β-glucan will affect the pI of the resulting protein. Several enzymes are generally required to significantly affect the pI of a variant Fc fusion protein. However, even changes in the IgG2 molecule can significantly reduce the serum half-life. It should be noted that allowing for increases is discussed below.

[0172] In other embodiments, non-isotypic amino acid changes are made to (e.g., amino acids with high pI). The overall improvement of the protein obtained by changing from a low pI amino acid to a low pI amino acid By reducing the charge state or adjusting the structure for stability, etc., as described in more detail below, This allows for adjustment.

[0173] Furthermore, by engineering the pI of both the heavy and light constant domains, heterologous Significant changes can be seen in each monomer of the dimer. The pI of the two monomers differs by at least 0.5, as determined by ion exchange chromatography. This allows separation by gel electrophoresis or isoelectric focusing, or other methods that are sensitive to the isoelectric point.

[0174] Calculation of E.pI The pI of each monomer depends on the pI of the variant heavy chain constant domain and the pI of the entire monomer. The heavy chain constant domains and fusion partners may vary. In some embodiments, the pI change is as described in U.S. Patent Application Publication No. 2014 / 0370013 The calculated values ​​are based on the variant heavy chain constant domain using the chart in Figure 19 of this issue. As discussed herein, the choice of which monomers to engineer generally depends on the unique pI of each monomer. Therefore, it is determined.

[0175] F. pI variants that also confer better FcRn binding in vivo If pI variants decrease the pI of the monomer, they may improve serum retention in vivo. This can have the added advantage of improving the

[0176] Although still under investigation, Fc is sequestered when it binds to FcRn at pH 6 in endosomes. Because of this, the Fc region is believed to have a longer half-life in vivo (see Overall integration, Ghetie and Ward, 1997 Immunol Today. 18(12):592-598). The endosomal compartment then expresses Fc on the cell surface. When the compartment opens to the extracellular space, a higher pH of about 7.4 is released into the bloodstream. In mice, Dall'Acqua et al. Fc mutations with increased FcRn binding at 7.4 actually reduced serum concentrations and wild-type FcRn binding. (Dall'A, incorporated by reference in its entirety) cqua et al.2002, J.Immunol.169:5171-5180) The increased affinity of Fc for FcRn at pH 7.4 prevents the release of Fc into the blood. Therefore, Fc mutations that increase the half-life of Fc in vivo are ideal. The purpose of this study was to prevent FcRn binding at lower pH while still allowing Fc release at higher pH. The amino acid histidine changes its charge state in the pH range of 6.0 to 7.4. Therefore, it is surprising to find His residues at key positions in the Fc / FcRn complex. This is not something that should be done.

[0177] G. Additional Fc variants for additional functions In addition to pI amino acid mutations, mutations that improve binding to one or more FcγRs include, but are not limited to: Several modifications can be performed for a variety of reasons, including altering the binding to FcRn, There are useful Fc amino acid modifications.

[0178] Thus, the proteins of the present invention include pI variants and conformational variants. It may contain amino acid modifications, including heterodimerization variants as outlined herein. Each set of variants can be independently and optionally included in a particular heterodimeric protein. or may be excluded from it.

[0179] H. FcγR variants Thus, modifications can be made to alter binding to one or more of the FcγR receptors. There are several useful Fc substitutions. Substitutions that result in increased binding as well as decreased binding are For example, increased binding to FcγRIIIa generally promotes ADCC (antibody-specific cell death). dependent cell-mediated cytotoxicity, i.e., nonspecific cytotoxic cells expressing FcγR A cell-mediated reaction that recognizes bound antibodies on target cells and subsequently causes lysis of the target cells Similarly, under some circumstances, FcγRIIb Decreased binding to inhibitory receptors (receptors) may also be beneficial. USSN11 / 124,620 (especially Figure 41), USSN11 / 174,287, U Includes those enumerated under SSN 11 / 396,495 and USSN 11 / 538,406. , all of which are expressly incorporated by reference in their entirety and specifically the variants disclosed therein. Specific variants used include 236A, 239 D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328 F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239 D, 332E / 330L, 243A, 243L, 264A, 264V, and 299T Includes but is not limited to:

[0180] Additionally, amino acid substitutions that increase affinity for FcγRIIc are also described herein. Fc domain variants as outlined in, for example, USSN 11 / 124,62 0 and the substitutions described in USSN 14 / 578,305 are useful.

[0181] Further, USSN 12 / 341,769, which is incorporated herein by reference in its entirety. 434S, 434A, 428L, 308F, 259I, 428L, as specifically disclosed in / 434S, 259I / 308F, 436I / 428L, 436I or V / 434S, Including but not limited to 436V / 428L, and 259I / 308F / 428L There are additional Fc substitutions that are used to increase binding to FcRn and increase serum half-life. do.

[0182] I. Truncated variants Similarly, another category of functional variants is called "FcγR-cleaving variants" or "F In these embodiments, the Fc knockout (FcKO or KO) variant is For some therapeutic uses, one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRII It is desirable to reduce or eliminate the normal binding of the Fc domain to antibodies (e.g., Ia). For example, in many embodiments, particularly in the use of immunomodulatory proteins, the Fc domain and FcγRI, such that one of the FcγRIs contains one or more Fcγ receptor truncation variants. It is desirable to cleave the IIa bond to eliminate or significantly reduce ADCC activity. Truncation variants of U are also included, all of which are incorporated herein by reference in their entirety. As shown in Figure 31 of SSN 15 / 141,350, and according to the EU index, G2 36R / L328R, E233P / L234V / L235A / G236del / S239 K, E233P / L234V / L235A / G236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L2 34V / L235A / G236del / S267K / A327G, and E233P / L Using a truncation variant selected from the group consisting of 234V / L235A / G236del Each can be independently optionally included or excluded in any preferred embodiment. The truncation variants referred to herein cleave FcγR binding, but generally do not bind to FcRn Note that the cleavage variants do not cleave FcγR binding. (sometimes called "knockout" or "KO" variants) are shown in Figure 5.

[0183] J. Heterodimer and Fc variant combinations As will be appreciated by those skilled in the art, the listed heterodimerization variants (skew and All of the nucleotides (including the nucleotides with different pI variants) retain their "twistness" or "monomer distribution." These can be combined independently in any way as long as they maintain their integrity. All variants can be combined in any of the heterodimerization formats.

[0184] In the case of pI variants, embodiments that find particular use are shown in the figures, but may also be used to facilitate purification. Other combinations follow the basic rule of changing the pI difference between the two monomers to promote Combinations can be generated.

[0185] Additionally, heterodimerization variants, skews, and pIs are all referred to herein. As generally outlined, Fc truncated variants, Fc variants, FcRn variants, and They can be independently combined in any way.

[0186] Furthermore, the monomeric Fc domain contains C220S / S267K / L368D / K370S or or a set of amino acid substitutions including C220S / S267K / S364K / E357Q It can be seen.

[0187] Additionally, heterodimeric Fc fusion proteins can be used in conjunction with scubariant (e.g., USSN The set of amino acid substitutions shown in Figures 1A-1C of 15 / 141,350, all of which (which are incorporated herein by reference in their entireties), and particularly useful skew -Variants are S364K / E357Q:L368D / K370S, L368D / K3 70S:S364K, L368E / K370S:S364K, T411E / K360E / Q362E:D401K, L368D / K370S:S364K / E357L, K370 S:S364K / E357Q, T366S / L368A / Y407V:T366W, and and T366S / L368A / Y407V:T366W (optionally, a bridging disulfide, T3 66S / L368A / Y407V / Y349C: including T366W / S354C) and optionally a truncation variant, an optionally charged domain linker, and an optional Optionally, pI variants may be included.

[0188] In some embodiments, the Fc domain is 236R, S239D according to EU numbering. , S239E, F243L, M252Y, V259I, S267D, S267E, S67 K, S298A, V308F, L328F, L328R, 330L, I332D, I33 2E, M428L, N434A, N434S, 236R / L328R, S239D / I3 32E, 236R / L328F, V259I / V308F, S267E / L328F, M 428L / N43S, Y436I / M428L, N436V / M428L, V436I / N434S, Y436V / N434S, S239D / I332E / 330L, M252Y / S54T / T256E, V259I / V308F / M428L, E233P / L234 V / L235A / G236del / S239K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G2 36del / S267K / A327G, E233P / L234V / L235A / G236 _, and E233P / L234V / L235A / G236del / S267K The amino acid sequence of the present invention comprises one or more amino acid substitutions selected from the group:

[0189] In one embodiment, specific skew and pI variants that find use in the present invention The combination is T366S / L368A / Y407V:T366W (optionally cross-linked disulfide). Fido, T366S / L368A / Y407V / Y349C:T366W / S354C One of the monomers contains Q295E / N384D / Q418E / N481D. and the other comprises a domain linker that is positively charged. The "knobs-in-holes" variant does not change the pI and therefore neither monomer It can also be used for

[0190] In certain embodiments, the Fc domain comprises amino acid substitutions including M428L / N434S. In some embodiments, a non-targeting IL-15 / Rα heterodimer Fc fusion tag is used. Each Fc domain of the protein contains amino acid substitutions including M428L / N434S.

[0191] Heterodimers comprising the IL-15 and IL-15Rα proteins described herein Useful embodiments of the Fc domain of the Fc fusion protein are provided in Figures 6A-6E. .

[0192] V. IL-15 and IL-15Rα Protein Domains The present invention provides a heterodimeric Fc fusion protein comprising IL-15 and IL-15Rα proteins. As shown in the figure, the IL-15 complex can take several forms. As mentioned above, the IL-15 protein itself can be expressed by the IL-15Rα protein. As is known in the art, The IL-15Rα protein contains a "sushi domain," which retains IL-15 binding activity. Therefore, the region containing the entire IL-15Rα protein is the shortest region of the receptor. Helodimeric fusion proteins can be made, but in the preferred embodiment herein The embodiment includes a complex that uses only the sushi domain, the sequence of which is shown in the figure.

[0193] Thus, the IL-15 complex generally consists of the IL-15 protein and IL-15Rα sushi domain of IL-15R (unless otherwise stated the full-length sequence is used) α,” “IL-15Rα(sushi),” and “sushi” are used interchangeably throughout. This complex can be used in three different formats: Figure 9A, As shown in Figures 9C, 9D, and 9F, IL-15 protein and IL-15Rα (Sushi) is not covalently bound, but rather is a typical ligand-ligand interaction. As explained more fully herein, the Fc domain is covalently linked to the ATP-binding domain. The conjugated linkage can be either the IL-15 domain or the sushi domain ( Generally, any domain linker is used). Alternatively, they may be generally as shown in Figure 9B, 9 As shown in Figures 9E and 9G, the domains can be covalently linked using a domain linker. Figure 9B shows the sushi domain as the N-terminal domain, but this can be reversed. Finally, the IL-15 domain and the sushi domain each contain a cysteine ​​amino acid. Similarly, disulfide bonds can be formed to form the nucleotides generally shown in Figures 39A-39C. 39D, covalently linked to the Fc domain (using any domain linker). It forms a complex with either the IL-15 domain or the sushi domain to which it is attached.

[0194] In some embodiments, the human IL-15 protein is identified by NCBI reference sequence number NP_ 000576.1 or the amino acid sequence set forth in SEQ ID NO: 1. The coding sequence of human IL-15 is shown in NCBI reference sequence number NM_000585. An exemplary Fc-fusion heterodimeric protein outlined herein is the IL-15 protein. The protein has the amino acid sequence of SEQ ID NO: 2 (mature IL-15) or amino acid 49 of SEQ ID NO: 1. In some embodiments, the IL-15 protein may have the sequence of SEQ ID NO: 2 to 162. At least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more sequence identity. In the present specification, the IL-15 protein has the amino acid sequence of SEQ ID NO: 2, as well as C42S, L45 From the group consisting of C, Q48C, V49C, L52C, E53C, E87C, and E89C The IL-15 protein of the Fc fusion protein has one or more amino acid substitutions selected from the group consisting of: The protein can have 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions.

[0195] The amino acid substitution(s) may be in the IL-15:IL-2β and IL-15:common gamma chains. In some embodiments, the human IL-15 protein may be an isosteric substitution at the interface of the Quality: N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E and any combination thereof. In some embodiments, the human IL-15 protein of the Fc fusion protein has It has the amino acid sequence of SEQ ID NO: 2 and the amino acid substitutions N4D / N65D. In this case, the human IL-15 protein in the Fc fusion protein contains the N4D / N65D substitutions. 2. In one embodiment, the human IL-15 protein of the Fc fusion protein has the amino acid sequence of SEQ ID NO: 2. and the amino acid substitutions D30N / N65D. In some cases, the Fc fusion protein The human IL-15 protein of interest is at least identical to SEQ ID NO: 2, which contains the D30N / N65D substitutions. In some embodiments, the Fc fusion proteins share 97% or 98% sequence identity with each other. The human IL-15 protein of interest has the amino acid sequence of SEQ ID NO: 2 and the amino acid substitutions D 30N / E64Q / N65D. In some cases, the human Fc fusion protein The IL-15 protein may have at least the sequence of SEQ ID NO: 2 containing the D30N / E64Q / N65D substitutions. Both have 96% or 97% sequence identity.

[0196] In some embodiments, the Fc fusion protein is a human mature IL-15 protein, such as The human IL-15 protein is identical to the amino acid sequence of SEQ ID NO: 2. In this study, human IL-15 proteins, such as human mature IL-15 proteins, contain amino acid substitutions. does not have.

[0197] In some embodiments, the human mature IL-15 variant protein comprises one or more amino acids. amino acid mutations (e.g., substitutions, insertions, and / or deletions). , which is a disulfide bond with the human IL-15 receptor alpha (IL-15Rα) protein due to the mutation. A cysteine ​​residue capable of forming a peptide bond is introduced.

[0198] In some embodiments, human IL-15 receptor alpha (IL-15Rα) protein The quality is determined by the amino acid sequence shown in NCBI reference sequence number NP_002180.1 or SEQ ID NO: 3. In some cases, the coding sequence of human IL-15Rα is available from NCBI. The Fc fusion heterologous antibody is shown in reference sequence number NM_002189.3. An exemplary IL-15Rα protein of a dimeric protein is the sushi domain of SEQ ID NO: 3 ( For example, amino acids 31 to 95 of SEQ ID NO: 3, or in other words, the amino acid sequence of SEQ ID NO: 4 In some embodiments, the IL-15Rα The protein has the amino acid sequence of SEQ ID NO: 4, as well as D96, P97, A98, D96 / P97, D96 / C97, D96 / P97 / A98, D96 / P97 / C98, and D 96 / C97 / A98, and the amino acid positions are: For example, D (e.g., Asp), P (e.g., Pro), A (e.g., Ala), DP (e.g., A sp-Pro), DC (e.g., Asp-Cys), DPA (e.g., Asp-Pro- Ala), DPC (e.g., Asp-Pro-Cys), or DCA (e.g., Asp -Cys-Ala) is an amino acid(s) that is present in the IL-15Rα protein of SEQ ID NO: 4. In some embodiments, the IL-15Rα protein may be added to the C-terminus of the protein. The amino acid sequence of sequence number 4, and K34C, A37C, G38C, S40C, and L 42C, and the amino acid positions are The IL-15Rα (sushi) protein of SEQ ID NO: 4 is , 3, 4, 5, 6, 7, 8, or more amino acid mutations (e.g., substitutions, insertions, and The chromosomes may have a nucleotide sequence (and / or deletion).

[0199] SEQ ID NO: 1 is MRISKPHLRSISIQCYLCLLLNSHFLTEAGIH VFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDAT LYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVE NLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS.

[0200] SEQ ID NO: 2 is NWVNVISDLKKIEDLIQSMHIDATLYTESDVH PSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANN SLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN It's TS.

[0201] SEQ ID NO: 3 is MAPRRARGCRTLGLPALLLLLLLRPPATRGIT CPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSL TECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPPSTVTT AGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQL MPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQ PPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQ TPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL.

[0202] SEQ ID NO: 4 is ITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR.

[0203] In some embodiments, the present invention provides a human IL-1-1 polypeptide having the amino acid variant D30N. In some embodiments, the protein comprises: The protein comprises the amino acid sequence of SEQ ID NO: 2 and a D30N substitution. The protein comprises the amino acid sequence of SEQ ID NO:2 and at least a D30N substitution.

[0204] In some embodiments, the present invention provides a method for producing human IL-1 having the amino acid variant N1D. In some embodiments, the protein comprises a 5 variant. In some embodiments, the protein comprises the amino acid sequence of sequence number 2 and the N1D substitution. comprises the amino acid sequence of SEQ ID NO:2 and at least an N1D substitution.

[0205] In some embodiments, the present invention provides a method for producing human IL-1 having the amino acid variant N4D. In some embodiments, the protein comprises a 5 variant. In some embodiments, the protein comprises the amino acid sequence of sequence number 2 and an N4D substitution. comprises the amino acid sequence of SEQ ID NO:2 and at least an N4D substitution.

[0206] In some embodiments, the present invention provides a human IL-1-1 polypeptide having the amino acid variant E64Q. In some embodiments, the protein comprises: The protein comprises the amino acid sequence of SEQ ID NO:2 and an E64Q substitution. The protein comprises the amino acid sequence of SEQ ID NO:2 and at least an E64Q substitution.

[0207] In some embodiments, the present invention provides a human IL-1 receptor agonist having the amino acid variant N65D. In some embodiments, the protein comprises: The protein comprises the amino acid sequence of SEQ ID NO:2 and an N65D substitution. The protein comprises the amino acid sequence of SEQ ID NO:2 and at least an N65D substitution.

[0208] In some embodiments, the present invention provides a method for the production of human IL-16 having the amino acid substitutions N1D / D30N. In some embodiments, the protein comprises a -15 variant. , comprising the amino acid sequence of SEQ ID NO:2 and N1D / D30N substitutions. The protein has the amino acid sequence of SEQ ID NO: 2 and at least N1D / D30N substitutions. include.

[0209] In some embodiments, the present invention provides a method for the production of human IL-16 having the amino acid substitutions N4D / D30N. In some embodiments, the protein comprises a -15 variant. , comprising the amino acid sequence of SEQ ID NO:2 and N4D / D30N substitutions. The protein has the amino acid sequence of SEQ ID NO: 2 and at least N4D / D30N substitutions. include.

[0210] In some embodiments, the present invention provides a human I having the amino acid substitutions D30N / E64Q. In some embodiments, the protein comprises an L-15 variant. comprises the amino acid sequence of SEQ ID NO: 2 and the D30N / E64Q substitutions. In this embodiment, the protein has the amino acid sequence of SEQ ID NO: 2 and at least D30N / E64Q Contains substitutions.

[0211] In some embodiments, the present invention provides a human IgE antibody having the amino acid substitutions D30N / N65D. In some embodiments, the protein comprises an L-15 variant. comprises the amino acid sequence of SEQ ID NO: 2 and the D30N / N65D substitutions. In this embodiment, the protein has the amino acid sequence of SEQ ID NO: 2 and at least D30N / N65D Contains substitutions.

[0212] In some embodiments, the present invention provides a method for the detection of HIV-1-associated ... In some embodiments, the protein comprises a human IL-15 variant that: The protein contains the amino acid sequence of SEQ ID NO:2 and the D30N / E64Q / N65D substitutions. In some embodiments, the protein has the amino acid sequence of SEQ ID NO:2 and at least also contains D30N / E64Q / N65D substitutions.

[0213] VI. Domain Linker In some embodiments, the IL-15 protein and the IL-15Rα protein are Optionally, the proteins are linked together via a linker. In other embodiments, the IL-15 protein and the IL-15Rα protein are It is non-covalently bound.

[0214] In some embodiments, the IL-15 protein is linked to the Fc domain via a linker. In some embodiments, the IL-15 protein is linked to a nucleotide sequence, e.g., a linker. In certain embodiments, the IL-15 protein is directly linked to the Fc domain. In some embodiments, the Fc domain is linked via a hinge region or a fragment thereof. In other studies, the IL-15Rα protein is linked to the Fc domain via a linker. In embodiments, the IL-15Rα protein may be, for example, a polypeptide directly linked to the Fc domain without a linker. In certain embodiments, the IL-15Rα protein is bound to the hinge region or In some cases, the linker is linked to the Fc domain via a fragment thereof. It is not used to bind the IL-15 protein or IL-15Rα protein to the Fc domain. Not used.

[0215] In some embodiments, the linker connects any two domains outlined herein. The "domain linker" used to link the domains together. Any suitable linker may be used. Although many embodiments may be used, for example, n is at least 0 (and generally 0 ~1~2~3~4~5), (GS)n, (GSGGS)n, (GGGGS)n, and and (GGGS)n-containing glycine-serine polymers, and the relationship between each domain and its biological Recombinant combination of two domains with sufficient length and flexibility to retain functional properties Any peptide sequence that allows for the following may be utilized. In certain cases, useful linkers include: Contains (GGGGS)0 or (GGGGS)1 or (GGGGS)2. The twist linker is shown in Figure 7. In some cases, attention is paid to the "twist level" as outlined below. It is noted that the charged domain linkers are shown in FIG. 7 and discussed herein. It can be used as follows.

[0216] VII. Useful Formats of the Invention As shown in Figures 9A to 9G and Figures 39A to 39D, the IL-15 / Rα- There are several useful formats of Fc fusion proteins. The dimeric fusion protein contains two functional components: IL-15 / IL-15Rα (IL-15Rα). ) component and Fc component, both of which may be of different shapes as outlined herein. Both may be combined with other components in any configuration.

[0217] The first and second Fc domains are: a) S267K / L368D / K370S:S267K / LS364K / E357Q, b)S364K / E357Q:L 368D / K370S, c)L368D / K370S:S364K, d)L368E / K 370S:S364K, e)T411E / K360E / Q362E:D401K, f)L 368D / K370S:S364K / E357L, and g)K370S:S364K / E357Q, E358Q, E359Q, E360Q, E361Q, E362Q, E363Q, E364Q, E365Q, E366Q, E367Q, E368Q, E369Q, E370Q, E371Q, E372Q, E373Q, E374Q, E375Q, E376Q, E377Q, E In one embodiment, the first Fc domain has a L368D / K370S substitution and the second Fc domain has a In some embodiments, the first Fc domain has S364K / E357Q substitutions. The main Fc domain has S364K / E357Q substitutions, and the second Fc domain has L368D / K It has a 370S substitution.

[0218] In some embodiments, the first and / or second Fc domains are numbered according to EU numbering. An additional set of amino acid substitutions, including Q295E / N384D / Q418E / N421D, was It has a

[0219] Optionally, the first and / or second Fc domain contains the amino acid sequence G236R / G236R according to EU numbering. L328R, E233P / L234V / L235A / G236del / S239K, E2 33P / L234V / L235A / G236del / S267K, E233P / L234 V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A327G, and E233P / L234V / L235A / G236del has an additional set of amino acid substitutions.

[0220] Optionally, the first and / or second Fc domains are 428L / 4 for half-life extension. Optionally, the first and / or second Fc domain has a half-life In some embodiments, the extension comprises the M428L / N434S variant. One Fc domain has M428L / N434S substitutions and the second Fc domain has M4 28L / N434S substitution

[0221] A. IL-15 / Rα-hetero Fc format In this embodiment, as shown in Figure 9B, the heterodimeric fusion protein comprises two The first monomer contains (from the N-terminus to the C-terminus) I L-15-any domain linker-CH2-CH3, The second monomer may contain all or part of the IL-15 / Rα (sushi) domain. The domain linker may include any domain linker -CH2-CH3, and may extend from the entire hinge to Sometimes includes parts of.

[0222] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is a skewed Use variants S364K / E357Q:L368D / K370S. In this case, the L368D / K370S variant is in the first monomer, and the S354K / E3 The 57Q variant is in the second monomer.

[0223] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Utilizing the D30N / N65D variant of the 5.

[0224] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 D30N / N65D variant and Scuba variant vs S364K / E357Q :L368D / K370S is used. In the IL-15 / Rα-hetero Fc format In this regard, a preferred embodiment is a D30N / N65D variant of IL-15, scubari Ant vs. S364K / E357Q:L368D / K370S, in each Fc monomer Utilizing the 428L / 434S variant, IL-15 / Rα-hetero Fc format In the present invention, a preferred embodiment is a skewed IL-15 D30N / N65D variant. The variant pair S364K / E357Q:L368D / K370S, in each Fc monomer It utilizes the M428L / N434S variant.

[0225] In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is a human mature IL-15 / Rα hetero-Fc fusion protein. The D30N / N65D variant of IL-15, the IL-15Rα (sushi) domain, and In some embodiments, the IL-1622 includes an Fc monomer containing the amino acid substitutions shown in FIG. The IL-15 / Rα-hetero Fc fusion protein is a D30N / N65D fragment of human mature IL-15. Variant; Human IL-15Rα (Sushi) domain; Amino acid substitution C220S, heterodimer mer pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q4 18E / N421D, cut and replace E233P / L234V / L235A / G236del / IL-15-Fc monomer containing S267K; and the amino acid substitution C220S, heterodimer Monomeric pI substitutions S364K / E357Q, and truncation substitutions E233P / L234V / L23 Contains IL-15Rα(sushi)-Fc monomer containing 5A / G236del / S267K. In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein comprises human mature IL-15. D30N / N65D variant of IL-15; human IL-15Rα (sushi) domain; acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236del / S267K, and FcRn substitution M428L / N434S IL-15-Fc monomer containing the amino acid substitution C220S, heterodimer pI substitution Replacement S364K / E357Q, cutting replacement E233P / L234V / L235A / G236d el / S267K, and IL-15Rα containing FcRn substitutions M428L / N434S ( Contains sushi)-Fc monomer.

[0226] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 Utilizes D30N / E64Q / N65D variants.

[0227] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 D30N / E64Q / N65D variants and Scuba variants vs S364K / Use E357Q:L368D / K370S.

[0228] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is D30N / E64Q / N65D variants of 5, Scuba liant vs S364K / E3 57Q:L368D / K370S, and 428L / 434S in each Fc monomer In the IL-15 / Rα-hetero Fc format, a preferred variant is used. Embodiments include the D30N / E64Q / N65D variant of IL-15, scubarian S364K / E357Q:L368D / K370S vs. S364K / E357Q:L368D / K370S, and in each Fc monomer In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein expresses the D30N / E64Q / N6 variant of human mature IL-15. The 5D variant, the IL-15Rα (sushi) domain, and the amino acid positions shown in Figure 6A In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein comprises an Fc monomer containing the IL-15 / Rα-hetero-Fc fusion protein. The fusion protein is the D30N / E64Q / N65D variant of human mature IL-15; IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L3 68D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421 D, containing the truncation substitutions E233P / L234V / L235A / G236del / S267K IL-15-Fc monomer; and amino acid substitution C220S, heterodimer pI substitution S3 64K / E357Q, and truncated replacements E233P / L234V / L235A / G236d IL-15Rα(sushi)-Fc monomer containing el / S267K. In this state, the IL-15 / Rα-hetero Fc fusion protein is expressed as D30 of human mature IL-15. N / E64Q / N65D variant; human IL-15Rα (sushi) domain; amino acid position C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q29 5E / N384D / Q418E / N421D, cutting replacement E233P / L234V / L23 5A / G236del / S267K, and FcRn substitutions M428L / N434S IL-15-Fc monomer; and amino acid substitution C220S, heterodimer pI substitution S3 64K / E357Q, cut and replace E233P / L234V / L235A / G236del / IL-15Rα (Sushi) containing S267K, and FcRn substitutions M428L / N434S -Contains Fc monomers.

[0229] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Utilizes the N4D variant of 5.

[0230] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 N4D variant and scubariant vs S364K / E357Q:L368D In the IL-15 / Rα-hetero Fc format, A preferred embodiment is the N4D variant of IL-15, S364K / E3 variant. 57Q:L368D / K370S, and 428L / 434S in each Fc monomer In the IL-15 / Rα-hetero Fc format, a preferred variant is used. Embodiments include the N4D variant of IL-15, Scubariant vs. S364K / E35 7Q:L368D / K370S, and M428L / N434 in each Fc monomer In some embodiments, the IL-15 / Rα-hetero-Fc fusion utilizes the S variant. The protein is the N4D variant of human mature IL-15, the IL-15Rα (sushi) domain. and Fc monomers containing the amino acid substitutions shown in Figure 6A. In this study, the IL-15 / Rα-hetero Fc fusion protein inhibited the N4D vector of human mature IL-15. Human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimer pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q41 8E / N421D, cut and replace E233P / L234V / L235A / G236del / S IL-15-Fc monomer containing 267K; and amino acid substitution C220S, heterodimer the nucleotide substitution S364K / E357Q, and the truncation substitutions E233P / L234V / L235 Contains IL-15Rα(sushi)-Fc monomer containing A / G236del / S267K. In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is a human mature IL-15 / Rα hetero-Fc fusion protein. N4D variant of -15; human IL-15Rα (sushi) domain; amino acid substitution C22 0S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N 384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G IL-1 containing 236del / S267K and FcRn substitutions M428L / N434S 5-Fc monomer; and the heterodimer with the amino acid substitution C220S and pI substitution S364K / E357Q, cut and replace E233P / L234V / L235A / G236del / S267 K, and IL-15Rα(sushi)-Fc singlet containing FcRn substitutions M428L / N434S Includes dimers.

[0231] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Utilizing the N65D variant of IL-15 / Rα-hetero Fc format Thus, a preferred embodiment is a combination of the N65D variant of IL-15 and a scubariant pair. Use S364K / E357Q:L368D / K370S.

[0232] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is N65D variant of 5, Scuba liant vs S364K / E357Q:L368D / Utilizing the K370S and 428L / 434S variants in each Fc monomer In the IL-15 / Rα-hetero Fc format, a preferred embodiment is N65D variant of 5, Scuba liant vs S364K / E357Q:L368D / Utilizes the K370S and M428L / N434S variants in each Fc monomer In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is human The N65D variant of mature IL-15, the IL-15Rα (sushi) domain, and Fig. 6A In some embodiments, the IL-15 Fc monomer comprises an Fc monomer comprising the amino acid substitutions shown in / Rα-hetero Fc fusion protein is the N65D variant of human mature IL-15; human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L3 68D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421 D, containing the truncation substitutions E233P / L234V / L235A / G236del / S267K IL-15-Fc monomer; and amino acid substitution C220S, heterodimer pI substitution S3 64K / E357Q, and truncated replacements E233P / L234V / L235A / G236d IL-15Rα(sushi)-Fc monomer containing el / S267K. In this state, the IL-15 / Rα-hetero Fc fusion protein has the N65 of human mature IL-15. D variant; human IL-15Rα (sushi) domain; amino acid substitution C220S, heterozygous Dimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q 418E / N421D, cutting replacement E233P / L234V / L235A / G236del / S267K, and IL-15-Fc monomer containing FcRn substitutions M428L / N434S as well as the amino acid substitution C220S, the heterodimeric pI substitution S364K / E357Q, truncation substitutions E233P / L234V / L235A / G236del / S267K, and F c) IL-15Rα(sushi)-Fc monomer containing the substitutions M428L / N434S.

[0233] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Utilizing the N4D / N65D variant of the 5.

[0234] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 N4D / N65D variants and Scuba variants vs S364K / E357Q: Use L368D / K370S.

[0235] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is N4D / N65D variant of 5, Scuba liant vs S364K / E357Q:L3 68D / K370S, and 428L / 434S variants in each Fc monomer. In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 15 N4D / N65D variants, Scuba liant vs S364K / E357Q:L 368D / K370S, and M428L / N434S variants in each Fc monomer. In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is N4D / N65D variant of human mature IL-15, IL-15Rα (sushi) domain, and an Fc monomer comprising the amino acid substitutions shown in Figure 6A. IL-15 / Rα-hetero Fc fusion protein is a fusion protein that binds N4D / N6 of human mature IL-15. 5D variant; human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimer Dimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236de IL-15-Fc monomer containing the amino acid substitution C220S, The dimeric pI substitution S364K / E357Q, and the truncation substitution E233P / L234V / L Contains IL-15Rα(sushi)-Fc monomer containing 235A / G236del / S267K In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is derived from a human adult human. N4D / N65D variant of mature IL-15; human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Replacement Q295E / N384D / Q418E / N421D, cutting replacement E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N434 IL-15-Fc monomer containing S; and the amino acid substitution C220S, heterodimer pI Replacement S364K / E357Q, cutting replacement E233P / L234V / L235A / G236 IL-15Rα containing del / S267K, and FcRn substitutions M428L / N434S (Sushi)-Containing Fc monomer.

[0236] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Utilizes the N1D / N65D variant.

[0237] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 N1D / N65D variant and Scuba variant vs S364K / E357Q: Use L368D / K370S.

[0238] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is N1D / N65D variant of 5, Scuba liant vs S364K / E357Q:L3 68D / K370S, and 428L / 434S variants in each Fc monomer. In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 15 N1D / N65D variants, Scuba liant vs S364K / E357Q:L 368D / K370S, and M428L / N434S variants in each Fc monomer. In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is N1D / N65D variant of human mature IL-15, IL-15Rα (sushi) domain, and an Fc monomer comprising the amino acid substitutions shown in Figure 6A. IL-15 / Rα-hetero Fc fusion protein is a fusion protein that binds N1D / N6 of human mature IL-15. 5D variant; human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimer Dimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236de IL-15-Fc monomer containing the amino acid substitution C220S, The dimeric pI substitution S364K / E357Q, and the truncation substitution E233P / L234V / L Contains IL-15Rα(sushi)-Fc monomer containing 235A / G236del / S267K In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is derived from a human adult human. N1D / N65D variant of mature IL-15; human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Replacement Q295E / N384D / Q418E / N421D, cutting replacement E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N434 IL-15-Fc monomer containing S; and the amino acid substitution C220S, heterodimer pI Replacement S364K / E357Q, cutting replacement E233P / L234V / L235A / G236 IL-15Rα containing del / S267K, and FcRn substitutions M428L / N434S (Sushi)-Containing Fc monomer.

[0239] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Utilizes the Q108E variant of the 5.

[0240] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 Q108E variant and scuba variant vs. S364K / E357Q:L36 Use 8D / K370S.

[0241] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 Q108E variant and scuba variant vs. S364K / E357Q:L36 Utilizes 8D / K370S and 428L / 434S variants in both monomers In the IL-15 / Rα-hetero Fc format, a preferred embodiment is -15 Q108E variant and scubariant vs. S364K / E357Q:L 368D / K370S and M428L / N434S variants in both monomers Use the

[0242] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 Q108E variant, Scuba liant vs S364K / E357Q:L368D / K370S, and 428L / 434S variants in each Fc monomer. In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 15 Q108E variant, Scuba liant vs. S364K / E357Q:L368 D / K370S, and M428L / N434S variants in each Fc monomer. In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is The Q108E variant of human mature IL-15, the IL-15Rα (sushi) domain, and In some embodiments, the IL-1622 includes an Fc monomer containing the amino acid substitutions shown in FIG. The IL-15 / Rα-hetero-Fc fusion protein is a Q108E variant of human mature IL-15. Human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI Substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236del / S267 IL-15-Fc monomer containing K; and amino acid substitution C220S, heterodimeric pI Replacement S364K / E357Q, and cut replacement E233P / L234V / L235A / G Contains IL-15Rα(sushi)-Fc monomer containing 236del / S267K. In one embodiment, the IL-15 / Rα-hetero-Fc fusion protein is a human mature IL-15 Q108E variant of human IL-15Rα (sushi) domain; amino acid substitution C220 S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N3 84D / Q418E / N421D, cut and replace E233P / L234V / L235A / G2 IL-15 containing 36del / S267K and FcRn substitutions M428L / N434S -Fc monomer; and amino acid substitution C220S, heterodimeric pI substitution S364K / E 357Q, cut and replace E233P / L234V / L235A / G236del / S267K , and IL-15Rα(sushi)-Fc monomer containing FcRn substitution M428L / N434S Including the body.

[0243] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Utilizing the wild-type variant of 5.

[0244] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 wild-type variant and scubariant vs. S364K / E357Q:L368D / Use K370S.

[0245] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is 5 wild-type variant, scavariant vs. S364K / E357Q:L368D / K 370S, and the 428L / 434S variant in each Fc monomer. In the IL-15 / Rα-hetero Fc format, a preferred embodiment is IL-15 wild-type variant, Scubariant vs. S364K / E357Q:L368D / K3 70S and utilizes the M428L / N434S variant in each Fc monomer In some embodiments, the IL-15 / Rα-hetero-Fc fusion protein is derived from human mature IL-15. The wild-type variant of IL-15, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6A In some embodiments, the Fc monomer comprises an Fc monomer that comprises an amino acid substitution selected from the group consisting of IL-15 / Rα. -Hetero-Fc fusion protein is a wild-type variant of human mature IL-15; human IL-1 5Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cleavage IL-1 containing substitutions E233P / L234V / L235A / G236del / S267K 5-Fc monomer; and the heterodimer with the amino acid substitution C220S and pI substitution S364K / E357Q, and truncation substitutions E233P / L234V / L235A / G236del / S In some embodiments, the IL-15Rα(sushi)-Fc monomer comprises 267K. The IL-15 / Rα-hetero Fc fusion protein is a wild-type variant of human mature IL-15. Human IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI Substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236del / S267 K, and IL-15-Fc monomer containing FcRn substitutions M428L / N434S; and The amino acid substitution C220S, heterodimeric pI substitutions S364K / E357Q, and truncation substitution E 233P / L234V / L235A / G236del / S267K and FcRn substitutions Contains IL-15Rα(sushi)-Fc monomer containing M428L / N434S.

[0246] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is Figure 48A of 18 / 071919 (X containing strand 1 (17693) and strand 2 (15908) ENP22822), Figure 94A (XENP23504 containing strand 1 and strand 2), Figure 104 AO (XENP24045 containing chain 1 and chain 2), Figure 104AQ (containing chain 1 and chain 2) XENP24306 containing strand 1 and strand 2), Figure 48A (XENP22821 containing strand 1 and strand 2), Figure 9 4A (XENP23343 containing strand 1 and strand 2), Fig. 104AJ (containing strand 1 and strand 2) XENP23557) Figure 104AP (XENP24113 including chain 1 and chain 2), Figure 104AP (XENP24051 containing strand 1 and strand 2), 104AR (strand 1 and strand XENP24341 containing strand 2), Figure 104AP (XENP2405 containing strand 1 and strand 2) 2), and Figure 104AP (XENP24301 containing strand 1 and strand 2), all of which are incorporated herein by reference in their entirety.

[0247] In the IL-15 / Rα-hetero Fc format, a preferred embodiment is shown in FIG. XENP22822) and (Strand 1) containing strand 1 (15902) and strand 2 (15908) (16479) and XENP2 (1475) containing strand 2 (16481).

[0248] B.scIL-15-Rα-Fc In this embodiment, as shown in Figure 9B, the heterodimeric fusion protein comprises two The first monomer comprises (from the N-terminus to the C-terminus) IL-15 / Rα (su shi)-domain linker-IL-15-optional domain linker-CH2-CH 3, and the domain linker often includes all or part of the hinge. includes an "empty" Fc, which contains all or part of the hinge-CH2-CH3. It is called "cIL-15 / Rα-Fc" and "sc" stands for "single chain" (e.g., IL-15 / R α represents the sushi complex.

[0249] In the scIL-15 / Rα-Fc format, a preferred embodiment is Take advantage of the N65D variant.

[0250] In the scIL-15 / Rα-Fc format, a preferred embodiment is D30N / E64Q / N65D variants and Scuba variants vs. S364K / E 357Q: Use L368D / K370S.

[0251] In the scIL-15 / Rα-Fc format, a preferred embodiment is D30N / E64Q / N65D variants of the Scuba variant vs. S364K / E35 7Q:L368D / K370S, and 428L / 434S balance in each Fc monomer In the scIL-15 / Rα-Fc format, a preferred embodiment The morphology is the D30N / E64Q / N65D variant of IL-15, scubariant vs. S364K / E357Q:L368D / K370S, and M in each Fc monomer Utilizes the 428L / N434S variant.

[0252] In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises human mature IL-15. Contains the D30N / N65D variant of IL-15 and the IL-15Rα (sushi) domain The Fc monomers include single chains, as well as Fc monomers containing the amino acid substitutions shown in Figure 6B. In embodiments, the scIL-15 / Rα-Fc fusion protein is the D of human mature IL-15. Single chain containing the 30N / N65D variant and the IL-15Rα (sushi) domain; acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, and truncated substitutions E233P / L23 scIL-15 / Rα-Fc monomer containing 4V / L235A / G236del / S267K as well as the amino acid substitution C220S, the heterodimeric pI substitution S364K / E357Q, and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises an empty Fc monomer. The protein is a D30N / N65D variant of human mature IL-15 and IL-15Rα Single chain containing the (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L36 8D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D , truncation substitutions E233P / L234V / L235A / G236del / S267K, and scIL-15 / Rα-Fc monomer containing FcRn substitutions M428L / N434S; and The amino acid substitution C220S, heterodimeric pI substitutions S364K / E357Q, and truncation substitution E 233P / L234V / L235A / G236del / S267K and FcRn substitutions Contains an empty Fc monomer containing M428L / N434S.

[0253] In the scIL-15 / Rα-Fc format, a preferred embodiment is Utilizes D30N / E64Q / N65D variants.

[0254] In the scIL-15 / Rα-Fc format, a preferred embodiment is D30N / E64Q / N65D variants and Scuba variants vs. S364K / E 357Q: Use L368D / K370S.

[0255] In the scIL-15 / Rα-Fc format, a preferred embodiment is D30N / E64Q / N65D variants of the Scuba variant vs. S364K / E35 7Q:L368D / K370S, and 428L / 434S balance in each Fc monomer In the scIL-15 / Rα-Fc format, a preferred embodiment The morphology is the D30N / E64Q / N65D variant of IL-15, scubariant vs. S364K / E357Q:L368D / K370S, and M in each Fc monomer Utilizes the 428L / N434S variant.

[0256] In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises human mature IL-15. D30N / E64Q / N65D variant of IL-15 and IL-15Rα (sushi) domain The Fc monomers include a single chain containing the amino acid substitutions shown in Figure 6B, as well as an Fc monomer containing the amino acid substitutions shown in Figure 6B. In some embodiments, the scIL-15 / Rα-Fc fusion protein is derived from human mature IL-15. D30N / E64Q / N65D variant of IL-15 and IL-15Rα (sushi) domain single-stranded containing the amino acid substitution C220S, heterodimeric pI substitution L368D / K370 S, isosteric pI substitutions Q295E / N384D / Q418E / N421D, and truncation substitutions scIL- containing recombinant E233P / L234V / L235A / G236del / S267K 15 / Rα-Fc monomer; and the amino acid substitution C220S, heterodimer pI substitution S3 64K / E357Q, and truncated replacements E233P / L234V / L235A / G236d In some embodiments, the scIL-1 The 5 / Rα-Fc fusion protein is a D30N / E64Q / N65D clone of human mature IL-15. Variant and single chain containing the IL-15Rα (sushi) domain; amino acid substitution C220 S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N3 84D / Q418E / N421D, cut and replace E233P / L234V / L235A / G2 scIL- 36del / S267K, and FcRn substitutions M428L / N434S 15 / Rα-Fc monomer; and the amino acid substitution C220S, heterodimer pI substitution S3 64K / E357Q, cut and replace E233P / L234V / L235A / G236del / S267K, and an empty Fc monomer containing the FcRn substitutions M428L / N434S.

[0257] In the scIL-15 / Rα-Fc format, a preferred embodiment is Take advantage of the N65D variant.

[0258] In the scIL-15 / Rα-Fc format, a preferred embodiment is N65D variant and scuba variant vs. S364K / E357Q:L368D / Use K370S.

[0259] In the scIL-15 / Rα-Fc format, a preferred embodiment is N65D variant of the Scuba liant vs. S364K / E357Q:L368D / K 370S, and the 428L / 434S variant in each Fc monomer. In the scIL-15 / Rα-Fc format, a preferred embodiment is N65D variant, Scuba liant vs. S364K / E357Q:L368D / K3 70S and utilizes the M428L / N434S variant in each Fc monomer .

[0260] In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises human mature IL-15. A single chain containing the N65D variant of IL-15 and the IL-15Rα (sushi) domain, and Fc monomers containing the amino acid substitutions shown in Figure 6B. The scIL-15 / Rα-Fc fusion protein mimics the N65D variant of human mature IL-15. A single chain containing the Ant and IL-15Rα (Sushi) domains; amino acid substitution C220S, Heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384 D / Q418E / N421D, and truncated substitutions E233P / L234V / L235A / G scIL-15 / Rα-Fc monomer containing 236del / S267K; and amino acid substitution C220S, heterodimeric pI substitution S364K / E357Q, and truncation substitution E23 Contains empty Fc monomer containing 3P / L234V / L235A / G236del / S267K In some embodiments, the scIL-15 / Rα-Fc fusion protein is derived from human mature IL-15. a single chain containing the N65D variant of IL-15 and the IL-15Rα (sushi) domain; Amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI Replacement Q295E / N384D / Q418E / N421D, cutting replacement E233P / L234 V / L235A / G236del / S267K, and FcRn substitution M428L / N43 scIL-15 / Rα-Fc monomer containing 4S; and the amino acid substitution C220S, Dimer pI substitution S364K / E357Q, truncation substitutions E233P / L234V / L235 A / G236del / S267K, and an empty space containing FcRn substitutions M428L / N434S Contains an Fc monomer.

[0261] In the scIL-15 / Rα-Fc format, a preferred embodiment is Utilizes the N4D / N65D variant.

[0262] In the scIL-15 / Rα-Fc format, a preferred embodiment is N4D / N65D variant and scuba variant vs. S364K / E357Q:L Use 368D / K370S.

[0263] In the scIL-15 / Rα-Fc format, a preferred embodiment is N4D / N65D variant of the Scuba liant vs. S364K / E357Q:L36 8D / K370S and 428L / 434S variants in each Fc monomer. In the scIL-15 / Rα-Fc format, a preferred embodiment is N4D / N65D variant of the -15, Scuba liant vs S364K / E357Q: L368D / K370S, and M428L / N434S variants in each Fc monomer Use Ant.

[0264] In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises human mature IL-15. The N4D / N65D variant of IL-15 and a gene encoding the IL-15Rα (sushi) domain The Fc monomers include two chains, as well as an Fc monomer containing the amino acid substitutions shown in Figure 6B. In embodiments, the scIL-15 / Rα-Fc fusion protein is N4 of human mature IL-15. Single chain containing the D / N65D variant and the IL-15Rα (sushi) domain; amino acids Substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q2 95E / N384D / Q418E / N421D, and truncated replacement E233P / L234V scIL-15 / Rα-Fc monomer containing / L235A / G236del / S267K; as well as the amino acid substitution C220S, the heterodimeric pI substitution S364K / E357Q, and and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises an Fc monomer of The protein expresses the N4D / N65D variant of human mature IL-15 and IL-15Rα (sucrose). ) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cleavage Substitutions E233P / L234V / L235A / G236del / S267K, and FcR scIL-15 / Rα-Fc monomer containing n-substitutions M428L / N434S; and acid substitution C220S, heterodimeric pI substitutions S364K / E357Q, truncation substitution E233 P / L234V / L235A / G236del / S267K, and FcRn substitution M42 Contains empty Fc monomers containing 8L / N434S.

[0265] In the scIL-15 / Rα-Fc format, a preferred embodiment is Utilizes the N1D / N65D variant.

[0266] In the scIL-15 / Rα-Fc format, a preferred embodiment is N1D / N65D variant and scuba variant vs. S364K / E357Q:L Use 368D / K370S.

[0267] In the scIL-15 / Rα-Fc format, a preferred embodiment is N1D / N65D variant of the Scuba liant vs. S364K / E357Q:L36 8D / K370S and 428L / 434S variants in each Fc monomer. In the scIL-15 / Rα-Fc format, a preferred embodiment is N1D / N65D variant of the -15, Scuba liant vs S364K / E357Q: L368D / K370S, and M428L / N434S variants in each Fc monomer Use Ant.

[0268] In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises human mature IL-15. The N1D / N65D variant of IL-15 and a gene encoding the IL-15Rα (sushi) domain The Fc monomers include two chains, as well as an Fc monomer containing the amino acid substitutions shown in Figure 6B. In embodiments, the scIL-15 / Rα-Fc fusion protein is a fusion protein that contains the N1 fragment of human mature IL-15. Single chain containing the D / N65D variant and the IL-15Rα (sushi) domain; amino acids Substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q2 95E / N384D / Q418E / N421D, and truncated replacement E233P / L234V scIL-15 / Rα-Fc monomer containing / L235A / G236del / S267K; as well as the amino acid substitution C220S, the heterodimeric pI substitution S364K / E357Q, and and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the scIL-15 / Rα-Fc fusion protein comprises an Fc monomer of The protein expresses the N1D / N65D variant of human mature IL-15 and IL-15Rα (success factor). ) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cleavage Substitutions E233P / L234V / L235A / G236del / S267K, and FcR scIL-15 / Rα-Fc monomer containing n-substitutions M428L / N434S; and acid substitution C220S, heterodimeric pI substitutions S364K / E357Q, truncation substitution E233 P / L234V / L235A / G236del / S267K, and FcRn substitution M42 Contains empty Fc monomers containing 8L / N434S.

[0269] In the scIL-15 / Rα-Fc format, preferred embodiments include chain 1 (1 XENP21478, which contains strand 2 (6478) and strand 2 (8924), is shown in Figure 11. Other embodiments of this format include XENP21993, XENP21994, XENP21995, XENP23174, XENP23175, XENP24477, and XENP24480, as shown in Figures 104G and 104 of WO2018 / 071919. The amino acid sequences are shown in Table 1. Row numbers 514~518, 519~523, 524~528, 849~853, 1063~ 1067, and 1078-1082.

[0270] C.ncIL-15 / Rα-Fc In this embodiment, as shown in Figure 9C, the heterodimeric fusion protein comprises three The first monomer comprises (from the N-terminus to the C-terminus) IL-15 / Rα (sucrose). )-domain linker-CH2-CH3, and the domain linker is the entire hinge or The second monomer sometimes contains all or part of the hinge -CH2-CH3. The third monomer is IL-15, which contains an "empty" Fc. This is called "ncIL-15 / Rα -Fc" and "nc" stands for "non-covalent."

[0271] In the ncIL-15 / Rα-Fc format, a preferred embodiment is scuba Use the Rianto vs. S364K / E357Q:L368D / K370S.

[0272] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Utilizes D30N / E64Q / N65D variants.

[0273] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 D30N / E64Q / N65D variants and Scuba variants vs. S364K / E 357Q: Use L368D / K370S.

[0274] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 D30N / E64Q / N65D variants of the Scuba variant vs. S364K / E35 7Q:L368D / K370S, and 428L / 434S balance in each Fc monomer In the ncIL-15 / Rα-Fc format, a preferred embodiment The morphology is the D30N / E64Q / N65D variant of IL-15, scubariant vs. S364K / E357Q:L368D / K370S, and M in each Fc monomer Utilizes the 428L / N434S variant.

[0275] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I D30N / E64Q / N65D variant of IL-15, IL-15Rα (sushi) domain and Fc monomers comprising the amino acid substitutions shown in Figure 6C. The ncIL-15 / Rα-Fc fusion protein expresses human mature IL-15 D30N / E. 64Q / N65D variant, IL-15Rα (sushi) domain; amino acid substitution C220 S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N3 84D / Q418E / N421D, and truncated replacements E233P / L234V / L235A / G236del / S267K; and the amino acid substitution C220S , heterodimeric pI substitution S364K / E357Q, and truncation substitution E233P / L234 IL-15Rα(sushi)-Fc monomer containing V / L235A / G236del / S267K In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises a human single chain containing the D30N / E64Q / N65D variant of mature IL-15; IL-15 Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K 370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cleavage position Conjugated E233P / L234V / L235A / G236del / S267K, and FcRn Empty Fc containing substitutions M428L / N434S; and amino acid substitution C220S, heterodimer Dimeric pI substitution S364K / E357Q, cleavage substitution E233P / L234V / L235A / G236del / S267K, and IL containing FcRn substitutions M428L / N434S -15Rα(sushi)-Fc monomer.

[0276] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Utilizes the N4D variant.

[0277] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 N4D variant, and scubariant vs. S364K / E357Q:L368D In the ncIL-15 / Rα-Fc format, Embodiments include IL-15 N4D variant, Scubariant vs. S364K / E35 7Q:L368D / K370S, and 428L / 434S balance in each Fc monomer In the ncIL-15 / Rα-Fc format, a preferred embodiment The morphology is the N4D variant of IL-15, scubariant vs. S364K / E357Q. : L368D / K370S, and M428L / N434S in each Fc monomer Use a riant.

[0278] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The N4D variant of IL-15, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6C In some embodiments, the ncIL-15 / R Fc monomer comprises an Fc monomer containing an amino acid substitution. The α-Fc fusion protein is composed of human mature IL-15 N4D variant, IL-15Rα ( Sushi domain: amino acid substitution C220S, heterodimeric pI substitution L368D / K370 S, isosteric pI substitutions Q295E / N384D / Q418E / N421D, and truncation substitutions Empty Fc single containing E233P / L234V / L235A / G236del / S267K heterodimer; and amino acid substitution C220S, heterodimer pI substitution S364K / E357Q , and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15Rα(sushi)-Fc monomer comprises an IL-15Rα(sushi)-Fc monomer. The 15 / Rα-Fc fusion protein contains the N4D variant of human mature IL-15. Chain; IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimer pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N4 21D, cut and replace E233P / L234V / L235A / G236del / S267K, and an empty Fc containing the FcRn substitution M428L / N434S; and the amino acid substitution C2 20S, heterodimeric pI substitution S364K / E357Q, truncation substitution E233P / L234 V / L235A / G236del / S267K, and FcRn substitution M428L / N43 Contains IL-15Rα(sushi)-Fc monomer containing 4S.

[0279] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Take advantage of the N65D variant.

[0280] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 N65D variant and scuba variant vs. S364K / E357Q:L368D In the ncIL-15 / Rα-Fc format, Embodiments include the N65D variant of IL-15, scubariant vs. S364K / E3 57Q:L368D / K370S, and 428L / 434S in each Fc monomer In the ncIL-15 / Rα-Fc format, a preferred embodiment is The embodiment is directed to the N65D variant of IL-15, Scubariant vs. S364K / E35 7Q:L368D / K370S, and M428L / N434 in each Fc monomer Use the S variant.

[0281] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The N65D variant of IL-15, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6C In some embodiments, the ncIL-15 / Fc monomer comprises an Fc monomer containing an amino acid substitution. The Rα-Fc fusion protein expresses the human mature IL-15 N65D variant, IL-15R α (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K3 70S, isosteric pI substitutions Q295E / N384D / Q418E / N421D, and Empty F containing E233P / L234V / L235A / G236del / S267K c monomer; and amino acid substitution C220S, heterodimeric pI substitution S364K / E35 7Q, and truncation substitutions E233P / L234V / L235A / G236del / S267 In some embodiments, the ncI The IL-15 / Rα-Fc fusion protein contains the N65D variant of human mature IL-15. Single chain containing IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimer p I substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236del / S26 7K, and an empty Fc containing FcRn substitutions M428L / N434S; and amino acid substitutions C220S substitution, heterodimer pI substitution S364K / E357Q, truncated substitution E233P / L 234V / L235A / G236del / S267K, and FcRn substitution M428L / Contains IL-15Rα(sushi)-Fc monomer containing N434S.

[0282] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Utilizes the N4D / N65D variant.

[0283] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 N4D / N65D variant and scuba variant vs. S364K / E357Q:L 368D / K370S. In the ncIL-15 / Rα-Fc format, A preferred embodiment is the N4D / N65D variant of IL-15, a scubariant vs. S364K / E357Q:L368D / K370S, and 4 in each Fc monomer Utilizing the 28L / 434S variant, the ncIL-15 / Rα-Fc format In this regard, a preferred embodiment is the N4D / N65D variant of IL-15, skew barrier S364K / E357Q:L368D / K370S vs. S364K / E357Q:L368D / K370S, and in each Fc monomer It utilizes the M428L / N434S variant.

[0284] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The N4D / N65D variant of L-15, the IL-15Rα (sushi) domain, and Fig. 6 In some embodiments, the ncIL-16 comprises an Fc monomer comprising the amino acid substitution shown in C. The -15 / Rα-Fc fusion protein expresses the human mature IL-15 N4D / N65D variant. IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N4 21D, and truncation substitutions E233P / L234V / L235A / G236del / S26 empty Fc monomer containing 7K; as well as the amino acid substitution C220S, heterodimer pI substitution S 364K / E357Q, and truncated replacements E233P / L234V / L235A / G236 IL-15Rα(sushi)-Fc monomer containing del / S267K. In this form, the ncIL-15 / Rα-Fc fusion protein is the N4D of human mature IL-15. Single chain containing the / N65D variant; IL-15Rα (sushi) domain; amino acid substitution C 220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236del / S267K, and empty containing FcRn substitutions M428L / N434S Fc; and IL-15Rα(sushi)-Fc monomer truncated amino acid substitution C220S, heterodimer Dimer pI substitution S364K / E357Q, truncation substitutions E233P / L234V / L235 A / G236del / S267K, and FcRn substitutions M428L / N434S.

[0285] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Utilizes the N1D / N65D variant.

[0286] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 N1D / N65D variant and scuba variant vs. S364K / E357Q:L 368D / K370S is used. In the ncIL-15 / Rα-hetero Fc format, In this regard, a preferred embodiment is the N1D / N65D variant of IL-15, skew barrier S364K / E357Q:L368D / K370S vs. S364K / E357Q:L368D / K370S, and in each Fc monomer ncIL-15 / Rα-hetero Fc phosphatase inhibitors utilize the 428L / 434S variant. In the present invention, a preferred embodiment is a human IL-15 N1D / N65D variant, Cubariant vs. S364K / E357Q:L368D / K370S, and their respective F c Utilizing the M428L / N434S variant in the monomer.

[0287] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The N1D / N65D variant of L-15, the IL-15Rα (sushi) domain, and Fig. 6 In some embodiments, the ncIL-16 comprises an Fc monomer comprising the amino acid substitution shown in C. The -15 / Rα-Fc fusion protein expresses the human mature IL-15 N1D / N65D variant. IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N4 21D, and truncation substitutions E233P / L234V / L235A / G236del / S26 empty Fc monomer containing 7K; as well as the amino acid substitution C220S, heterodimer pI substitution S 364K / E357Q, and truncated replacements E233P / L234V / L235A / G236 IL-15Rα(sushi)-Fc monomer containing del / S267K. In this form, the ncIL-15 / Rα-Fc fusion protein mimics the N1D of human mature IL-15. Single chain containing the / N65D variant; IL-15Rα (sushi) domain; amino acid substitution C 220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N421D, cut and replace E233P / L234V / L235A / G236del / S267K, and empty containing FcRn substitutions M428L / N434S Fc; and amino acid substitution C220S, heterodimer pI substitution S364K / E357Q , truncation substitutions E233P / L234V / L235A / G236del / S267K, and Contains IL-15Rα(sushi)-Fc monomer containing FcRn substitution M428L / N434S .

[0288] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Uses variant Q108E.

[0289] In the nIL-15 / Rα-Fc format, a preferred embodiment is liant Q108E and Scuba liant vs S364K / E357Q:L368D / Use the K370S.

[0290] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Variants Q108E and Scubariant vs. S364K / E357Q:L368 D / K370S, and 428L / 434S variants in both monomers. In the scIL-15 / Rα-Fc format, a preferred embodiment is 5 variant Q108E and scubariant vs. S364K / E357Q:L36 8D / K370S, as well as the M428L / N434S variant in both monomers. Use it.

[0291] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The Q108E variant of IL-15, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain are shown in Figure 6C. In some embodiments, the ncIL-15 Fc monomer comprises an Fc monomer containing an amino acid substitution that is The / Rα-Fc fusion protein contains human mature IL-15 Q108E variant, IL-1 5Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L368D / K370S, isosteric pI substitutions Q295E / N384D / Q418E / N421D, and and truncation substitutions E233P / L234V / L235A / G236del / S267K Fc monomer; and the amino acid substitution C220S, heterodimer pI substitution S364K / E 357Q, and truncated substitutions E233P / L234V / L235A / G236del / S2 In some embodiments, the IL-15Rα(sushi)-Fc monomer comprises 67K. The cIL-15 / Rα-Fc fusion protein is a Q108E variant of human mature IL-15. single chain containing the IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimer mer pI substitution L368D / K370S, isosteric pI substitution Q295E / N384D / Q4 18E / N421D, cut and replace E233P / L234V / L235A / G236del / S267K, and an empty Fc containing FcRn substitutions M428L / N434S; and acid substitution C220S, heterodimeric pI substitutions S364K / E357Q, truncation substitution E233 P / L234V / L235A / G236del / S267K, and FcRn substitution M42 Contains IL-15Rα(sushi)-Fc monomer containing 8L / N434S.

[0292] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Use a wild-type variant.

[0293] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 wild-type variant and scubariant vs. S364K / E357Q:L368D / In the ncIL-15 / Rα-hetero Fc format, A preferred embodiment is a wild-type variant of IL-15, S364K / E. 357Q:L368D / K370S, and 428L / 434 in each Fc monomer In the ncIL-15 / Rα-hetero Fc format, A preferred embodiment is a wild-type variant of IL-15, S364K / S404K. E357Q:L368D / K370S, and M428L / N in each Fc monomer Utilizes the 434S variant.

[0294] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The wild-type variant of IL-15, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6C In some embodiments, the ncIL-15 / R Fc monomer comprises an Fc monomer containing an amino acid substitution. The α-Fc fusion protein is a recombinant human mature IL-15 wild-type variant, IL-15Rα (SEQ ID NO: 1). C) domain: amino acid substitution C220S, heterodimeric pI substitution L368D / K370S , isosteric pI substitutions Q295E / N384D / Q418E / N421D, and truncation substitutions Empty Fc monomer containing E233P / L234V / L235A / G236del / S267K as well as the amino acid substitution C220S, the heterodimeric pI substitution S364K / E357Q, and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15Rα(sushi)-Fc monomer comprises ncIL-1 The 5 / Rα-Fc fusion protein is a single-chain fusion protein containing the wild-type variant of human mature IL-15. IL-15Rα (sushi) domain; amino acid substitution C220S, heterodimeric pI substitution L 368D / K370S, isosteric pI substitution Q295E / N384D / Q418E / N42 1D, truncated substitution E233P / L234V / L235A / G236del / S267K, and and an empty Fc containing the FcRn substitution M428L / N434S; and an amino acid substitution C22 0S, heterodimeric pI substitution S364K / E357Q, truncation substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N434 Contains IL-15Rα(sushi)-Fc monomer containing S.

[0295] In the ncIL-15 / Rα-hetero Fc format, a preferred embodiment is WO2018 / 07191, all of which are incorporated herein by reference in their entirety. 9 Figure 104AS (XENP24349 including chain 1 and chain 2) and Figure 104AT (chain 1 and XENP24383, which contains strand 2.

[0296] In the ncIL-15 / Rα-Fc format, a preferred embodiment is chain 1 (16 XENP21479, which contains strand 2 (8793) and strand 3 (16481); XENP22366, including strand 1 (16478) and strand 2 (8924) and strand 3 (), and X containing strand 1 (16484), strand 2 (8793), and strand 3 (15908). ENP22366, and XENP24348, as shown in Figures 12A and 12B. do.

[0297] D. Bivalent ncIL-15 / Rα-Fc In this embodiment, as shown in Figure 9D, the heterodimeric fusion protein comprises four The first and second monomers comprise (from the N-terminus to the C-terminus) IL-15 / Rα(sushi)-domain linker-CH2-CH3, where the domain linker is a hinge The third and fourth monomers contain IL-15. The antibody was designated "covalent ncIL-15 / Rα-Fc," where "nc" stands for "non-covalent."

[0298] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is - Use variant S364K / E357Q:L368D / K370S.

[0299] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 D30N / N65D variants are available.

[0300] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 D30N / E64Q / N65D variants and Scuba liant vs S364K / E357Q:L368D / K370S is used. In a preferred embodiment, the D30N / N65D variant of IL-15 is , Scubariant vs. S364K / E357Q:L368D / K370S, each Fc Utilizes the 428L / 434S variant in the monomer. Bivalent ncIL-15 / Rα- In the Fc format, a preferred embodiment is the D30N / N65D variant of IL-15. Ant, Scuba-Liant vs. S364K / E357Q:L368D / K370S, respectively Utilizes the M428L / N434S variant in the Fc monomer.

[0301] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein is derived from a human adult Mature IL-15 D30N / N65D variant, IL-15Rα (sushi) domain, and and two Fc monomers containing the amino acid substitutions shown in Figure 6C. The bivalent ncIL-15 / Rα-Fc fusion protein binds two human mature IL-15 D 30N / N65D variant; the first IL-15R containing the human IL-15Rα (sushi) domain 15Rα(sushi)-Fc monomer, and the amino acid substitution C220S and truncation substitution E23 Fc monomer containing 3P / L234V / L235A / G236del / S267K; and A second IL-15Rα(sushi)-Fc monomer containing the human IL-15Rα(sushi) domain and the amino acid substitution C220S and the truncation substitutions E233P / L234V / L235 In some embodiments, the Fc monomer comprises an Fc monomer comprising A / G236del / S267K. The ncIL-15 / Rα-Fc fusion protein contains two human mature IL-15 D30N / N65D variant; the first IL-15R containing the human IL-15Rα (sushi) domain α(sushi)-Fc monomer, with amino acid substitution C220S and truncation substitution E233P / L2 34V / L235A / G236del / S267K, and FcRn substitution M428L / N an Fc monomer containing 434S; and a second Fc monomer containing the human IL-15Rα (sushi) domain. IL-15Rα(sushi)-Fc monomer, and the amino acid substitution C220S and truncation substitutions E233P / L234V / L235A / G236del / S267K, and FcRn substitutions Contains an Fc monomer containing the recombinant M428L / N434S.

[0302] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 Utilizing D30N / E64Q / N65D variants.

[0303] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 D30N / E64Q / N65D variants and Scuba liant vs S364K / E357Q:L368D / K370S is used. In the format, a preferred embodiment is IL-15 D30N / E64Q / N65D Variant, Scuba-Variant vs. S364K / E357Q:L368D / K370S, and utilizes the 428L / 434S variant in each Fc monomer. In the IL-15 / Rα-Fc format, a preferred embodiment is the D30 of IL-15. N / E64Q / N65D variant, Scuba liant vs. S364K / E357Q:L 368D / K370S, and M428L / N434S barriers in each Fc monomer Use the account.

[0304] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 Utilizing the N65D variant.

[0305] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 N65D variants and scubariant vs. S364K / E357Q:L36 Utilizing 8D / K370S. In the bivalent ncIL-15 / Rα-Fc format, A preferred embodiment is the N65D variant of IL-15, Scubariant vs. S364 K / E357Q:L368D / K370S, and 428L / K370S in each Fc monomer. Utilizing the 434S variant in a bivalent ncIL-15 / Rα-Fc format , a preferred embodiment is the N65D variant of IL-15, Scubariant vs. S36 4K / E357Q:L368D / K370S, and M428 in each Fc monomer Utilizes the L / N434S variant.

[0306] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein is derived from a human adult Mature IL-15 N65D variant, IL-15Rα (sushi) domain, and Fig. 6C In some embodiments, the bivalent n The cIL-15 / Rα-Fc fusion protein contains two human mature IL-15 N65D variants. Ant; the first IL-15Rα(sushi)-containing human IL-15Rα(sushi) domain Fc monomer, and the amino acid substitution C220S and the truncation substitution E233P / L234V / Fc monomer containing L235A / G236del / S267K and human IL-15Rα (S A second IL-15Rα(sushi)-Fc monomer containing the IL-15Rα(sushi) domain, as well as amino acid substitutions C220S and truncation substitutions E233P / L234V / L235A / G236del / S2 and an Fc monomer comprising 67K. In some embodiments, the bivalent ncIL-15 / Rα The -Fc fusion protein contains two human mature IL-15 N65D variants; a first IL-15Rα(sushi)-Fc monomer containing the IL-15Rα(sushi) domain, and Amino acid substitution C220S, truncation substitutions E233P / L234V / L235A / G236d and an Fc monomer containing FcRn substitutions M428L / N434S; a second IL-15Rα(sushi)-Fc monomer containing a second IL-15Rα(sushi) domain; and the amino acid substitution C220S and the truncation substitutions E233P / L234V / L235A / FcRn subunits containing G236del / S267K and FcRn substitutions M428L / N434S and dimers.

[0307] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 Utilizing the N4D / N65D variant.

[0308] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 N4D / N65D variants and Scuba variants vs. S364K / E357Q :L368D / K370S is used for the bivalent ncIL-15 / Rα-Fc format. In this regard, a preferred embodiment is directed to the N4D / N65D variant of IL-15, Scubari Ant pair S364K / E357Q:L368D / K370S, and each Fc monomer The 428L / 434S variant is utilized in the bivalent ncIL-15 / Rα-Fc phosphatase. In the present invention, a preferred embodiment is the N4D / N65D variant of IL-15, Cubariant vs. S364K / E357Q:L368D / K370S, and their respective F c Utilizing the M428L / N434S variant in the monomer.

[0309] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 Utilizing the N1D / N65D variant.

[0310] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 N1D / N65D variants and scubariant vs. S364K / E357Q :L368D / K370S is used for the bivalent ncIL-15 / Rα-Fc format. In this regard, a preferred embodiment is directed to the N1D / N65D variant of IL-15, scubari Ant pair S364K / E357Q:L368D / K370S, and each Fc monomer The 428L / 434S variant is utilized in the bivalent ncIL-15 / Rα-Fc phosphatase. In the present invention, a preferred embodiment is a human IL-15 N1D / N65D variant, Cubariant vs. S364K / E357Q:L368D / K370S, and their respective F c Utilizing the M428L / N434S variant in the monomer.

[0311] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein is derived from a human adult Mature IL-15 N1D / N65D variant, IL-15Rα (sushi) domain, and In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in Figure 6C. The bivalent ncIL-15 / Rα-Fc fusion protein expresses two human mature IL-15 N1 The first IL-1 containing the D / N65D variant and the human IL-15Rα (sushi) domain 5Rα(sushi)-Fc monomer and the amino acid substitution C220S and truncation substitution E233 Fc monomer containing P / L234V / L235A / G236del / S267K; human I A second IL-15Rα(sushi)-Fc monomer containing the IL-15Rα(sushi) domain, and amino acid substitution C220S and truncation substitutions E233P / L234V / L235A / G2 and an Fc monomer comprising nc36del / S267K. In some embodiments, the bivalent nc The IL-15 / Rα-Fc fusion protein contains two human mature IL-15 N1D / N65 The first IL-15Rα (sushi) variant contains the human IL-15Rα (sushi) domain. C)-Fc monomer, and the amino acid substitution C220S, truncation substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N434 an Fc monomer containing the S; and a second IL-15R containing the human IL-15Rα (Sushi) domain α(sushi)-Fc monomer, and the amino acid substitution C220S and the truncation substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitution M428L / Fc monomer containing N434S.

[0312] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 Utilize the Q108E variant.

[0313] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 Q108E variant and scubariant vs. S364K / E357Q:L3 Utilizing 68D / K370S in a bivalent ncIL-15 / Rα-Fc format A preferred embodiment is a combination of the Q108E variant of IL-15 and a scubariant pair. S364K / E357Q:L368D / K370S, as well as 42 in both monomers. Utilizing the 8L / 434S variant in a bivalent ncIL-15 / Rα-Fc format In the present invention, a preferred embodiment is a Q108E variant of IL-15, a scubariant In the pair S364K / E357Q:L368D / K370S, and in each Fc monomer Utilizes the M428L / N434S variant.

[0314] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein is derived from a human adult Mature IL-15 Q108E variant, IL-15Rα (sushi) domain, and Fig. 6C In some embodiments, the bivalent Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in The ncIL-15 / Rα-Fc fusion protein contains two human mature IL-15 Q108E The first IL-15Rα (sushi) variant contains the human IL-15Rα (sushi) domain. )-Fc monomer, and the amino acid substitution C220S and the truncation substitution E233P / L234 Fc monomer containing V / L235A / G236del / S267K and human IL-15Rα a second IL-15Rα(sushi)-Fc monomer containing the (sushi) domain, as well as the amino acid Substitution C220S and truncation substitutions E233P / L234V / L235A / G236del / and an Fc monomer comprising S267K. In some embodiments, the bivalent ncIL / Rα- The Fc fusion protein contains two human mature IL-108E variants and human IL-15 The first IL-15Rα(sushi)-Fc monomer contains the Rα(sushi) domain, as well as No acid substitution C220S, cleavage substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N434S; and an Fc monomer containing human IL-1 a second IL-15Rα(sushi)-Fc monomer containing the IL-15Rα(sushi) domain, and Amino acid substitution C220S and truncation substitutions E233P / L234V / L235A / G23 6del / S267K, and FcRn substitutions M428L / N434S. Includes.

[0315] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 wild-type variants are utilized.

[0316] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 wild-type variants and the scubariant pair S364K / E357Q:L368 Use D / K370S.

[0317] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 wild-type variants, scubariant vs. S364K / E357Q:L368D / Utilizing the K370S and 428L / 434S variants in each Fc monomer In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is 15 wild-type variants, scubariant vs. S364K / E357Q:L368D / Utilizes the K370S and M428L / N434S variants in each Fc monomer do.

[0318] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein is derived from a human adult Mature IL-15 wild-type variant, IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6C. In some embodiments, the bivalent ncI comprises two Fc monomers containing the amino acid substitutions. The IL-15 / Rα-Fc fusion protein contains two wild-type variants of human mature IL-15. The first IL-15Rα(sushi)-Fc monomer contains the human IL-15Rα(sushi) domain. and the amino acid substitution C220S and the truncation substitutions E233P / L234V / L235 Fc monomer containing A / G236del / S267K and human IL-15Rα (sushi) domain A second IL-15Rα(sushi)-Fc monomer containing α-succinate and the amino acid substitution C220 S and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the bivalent ncIL / Rα-Fc fusion protein comprises an Fc monomer comprising the ncIL / Rα-Fc fusion protein. The protein expresses two human mature IL-15Rα wild-type variants and the human IL-15Rα (sushi) domain. The first IL-15Rα(sushi)-Fc monomer contains α-glucan, as well as the amino acid substitution C220 S, truncated substitutions E233P / L234V / L235A / G236del / S267K, and and Fc monomer containing FcRn substitutions M428L / N434S; human IL-15Rα (cleaved) A second IL-15Rα(sushi)-Fc monomer containing the IL-15Rα(sushi)-Fc domain, as well as the amino acid substitution C 220S and truncated substitutions E233P / L234V / L235A / G236del / S26 7K, and an Fc monomer containing the FcRn substitutions M428L / N434S.

[0319] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is WO2018 / 071919, all of which are incorporated herein by reference in their entireties. Figure 104E (XENP21979 containing strand 1 and strand 2) and SEQ ID NO: 48 in the sequence listing It is expressed as 0 to 483.

[0320] In the bivalent ncIL-15 / Rα-Fc format, a preferred embodiment is The forms include XENP21978, which contains strand 1 (17023) and strand 2 (16484). Shown in Figure 13.

[0321] E. Bivalent scIL-15 / Rα-Fc In this embodiment, as shown in Figure 9B, the heterodimeric fusion protein comprises two The first and second monomers comprise (from the N-terminus to the C-terminus) IL-15 / Rα(sushi)-domain linker-IL-15-any domain linker-CH2-CH 3, and the domain linker sometimes includes all or part of a hinge. IL-15 / Rα-Fc,” and “sc” stands for “single chain” (e.g., IL-15 / Rα Represents the sushi complex.

[0322] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is - Use variant S364K / E357Q:L368D / K370S.

[0323] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 Utilizing the N65D variant.

[0324] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 D30N / E64Q / N65D variants and Scuba variants vs. S364K / E357Q:L368D / K370S is used. In this format, a preferred embodiment is the IL-15 D30N / N65D variant. and Scuba liant vs. S364K / E357Q:L368D / K370S, and utilizes the 428L / 434S variant in both monomers.

[0325] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein comprises two Human mature IL-15 D30N / N65D variant, two IL-15Rα (sushi) The Fc monomers contain two Fc monomers containing the amino acid substitutions shown in Figure 6D. In one embodiment, the bivalent scIL-15 / Rα-Fc fusion protein comprises the amino acid substitution C2 20S and truncation substitutions E233P / L234V / L235A / G236del / S267 Human IL-15Rα (sushi) domain linked to an Fc monomer containing K The first IL-15 / Rα (Schizophrenia) containing the mature IL-15 D30N / N65D variant )-Fc monomer with amino acid substitution C220S and truncation substitutions E233P / L234V / L Human IL-15R linked to an Fc monomer containing 235A / G236del / S267K Human mature IL-15 D30N / N65D variant linked to the α(sushi) domain and a second IL-15 / Rα(sushi)-Fc monomer comprising: The bivalent scIL-15 / Rα-Fc fusion protein contains the amino acid substitution C220S, a truncation substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitutions Human IL-15Rα (sushi) linked to an Fc monomer containing recombinant M428L / N434S A first antibody comprising the human mature IL-15 D30N / N65D variant linked to the domain IL-15 / Rα(sushi)-Fc monomer; amino acid substitution C220S, truncation substitution E233 P / L234V / L235A / G236del / S267K, and FcRn substitution M42 Human IL-15Rα (sushi) domain linked to an Fc monomer containing 8L / N434S A second IL-15 comprising the linked human mature IL-15 D30N / N65D variant. / Rα(sushi)-Fc monomer. In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is 5 Utilizes D30N / E64Q / N65D variants.

[0326] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 D30N / E64Q / N65D variants and Scuba variants vs. S364K Use / E357Q:L368D / K370S.

[0327] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 D30N / E64Q / N65D variants, Scuba variants vs. S364K / E 357Q:L368D / K370S, and 428L / 434 in each Fc monomer In the bivalent scIL-15 / Rα-Fc format, the preferred A new embodiment is an IL-15 D30N / E64Q / N65D variant, scubari Ant pair S364K / E357Q:L368D / K370S, and each Fc monomer It utilizes the M428L / N434S variant.

[0328] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein comprises two Human mature IL-15 D30N / E64Q / N65D variant, two IL-15Rα (sushi) domain, and two Fc monomers containing the amino acid substitutions shown in Figure 6D In some embodiments, the bivalent scIL-15 / Rα-Fc fusion protein comprises an amino acid sequence similar to that of the IL-15 / Rα-Fc fusion protein. Acid substitution C220S and truncation substitutions E233P / L234V / L235A / G236del / S267K, linked to the human IL-15Rα (sushi) domain linked to an Fc monomer The first I containing the ligated human mature IL-15 D30N / E64Q / N65D variant L-15 / Rα(sushi)-Fc monomer with amino acid substitution C220S and truncation substitution E23 Linked to an Fc monomer containing 3P / L234V / L235A / G236del / S267K Human mature IL-15 D30N / α linked to the isolated human IL-15Rα (sushi) domain and a second IL-15 / Rα(sushi)-Fc monomer containing the E64Q / N65D variant. In some embodiments, the bivalent scIL-15 / Rα-Fc fusion protein comprises Amino acid substitution C220S, truncation substitutions E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N434S linked to an Fc monomer. Human mature IL-15 D30N / α linked to the isolated human IL-15Rα (sushi) domain a first IL-15 / Rα(sushi)-Fc monomer containing the E64Q / N65D variant; Amino acid substitution C220S, truncation substitutions E233P / L234V / L235A / G236de 1 / S267K, and FcRn substitutions M428L / N434S linked to an Fc monomer. Human mature IL-15 D30N / α linked to the isolated human IL-15Rα (sushi) domain and a second IL-15 / Rα(sushi)-Fc monomer containing the E64Q / N65D variant. include.

[0329] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 Utilizing the N65D variant.

[0330] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 N65D variant and scuba variant vs. S364K / E357Q:L36 8D / K370S. In the bivalent scIL-15 / Rα-Fc format, A preferred embodiment is the IL-15 N65D variant, scubariant vs. S364 K / E357Q:L368D / K370S, and 428L / 43 in each Fc monomer. In a bivalent scIL-15 / Rα-Fc format, the 4S variant is used. A preferred embodiment is the IL-15 N65D variant, scubariant vs. S364K / E357Q:L368D / K370S, and M428L / N4 in each Fc monomer Utilizes the 34S variant.

[0331] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein comprises two Human mature IL-15 N65D variant, two IL-15Rα (sushi) domains, and and two Fc monomers containing the amino acid substitutions shown in Figure 6D. Therefore, the bivalent scIL-15 / Rα-Fc fusion protein contains the amino acid substitutions C220S and and F containing truncation substitutions E233P / L234V / L235A / G236del / S267K Human mature IL-15Rα (sushi) domain linked to the c monomer The first IL-15 / Rα(sushi)-Fc monomer contains the N65D variant; Amino acid substitution C220S and truncation substitutions E233P / L234V / L235A / G236d Human IL-15Rα (sushi) domain linked to an Fc monomer containing el / S267K A second IL-15 / Rα (SEQ ID NO: 1) containing the linked human mature IL-15 N65D variant In some embodiments, the bivalent scIL-15 / Rα-Fc The fusion protein contains the amino acid substitution C220S, the truncation substitutions E233P / L234V / L23 5A / G236del / S267K, and FcRn substitutions M428L / N434S Human mature I linked to the human IL-15Rα (sushi) domain linked to an Fc monomer a first IL-15 / Rα(sushi)-Fc monomer containing the L-15 N65D variant; Amino acid substitution C220S, truncation substitutions E233P / L234V / L235A / G236de 1 / S267K, and FcRn substitutions M428L / N434S linked to an Fc monomer. Human mature IL-15 N65D vector linked to the isolated human IL-15Rα (sushi) domain and a second IL-15 / Rα(sushi)-Fc monomer containing a variant.

[0332] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 Utilizing the N4D / N65D variant.

[0333] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 N4D / N65D variant and Scuba variant vs. S364K / E357Q :L368D / K370S is used in the bivalent scIL-15 / Rα-Fc format In the present invention, a preferred embodiment is an IL-15 N4D / N65D variant, scubari Ant pair S364K / E357Q:L368D / K370S, and each Fc monomer The 428L / 434S variant is utilized in the bivalent scIL-15 / Rα-Fc phosphatase. In the present invention, a preferred embodiment is the IL-15 N4D / N65D variant, Cubariant vs. S364K / E357Q:L368D / K370S, and their respective F c Utilizing the M428L / N434S variant in the monomer.

[0334] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein comprises two Human mature IL-15 N4D / N65D variant, two IL-15Rα (sushi) domains and two Fc monomers containing the amino acid substitutions shown in Figure 6D. In embodiments, the bivalent scIL-15 / Rα-Fc fusion protein comprises the amino acid substitution C22 OS and truncated substitutions E233P / L234V / L235A / G236del / S267K Human IL-15Rα (sushi) domain linked to an Fc monomer containing The first, IL-15 / Rα (sushi), contains the mature IL-15 N4D / N65D variant. Fc monomer with amino acid substitution C220S and truncation substitutions E233P / L234V / L23 Human IL-15Rα ( A human mature IL-15 N4D / N65D variant linked to the sushi domain was identified. In some embodiments, the bivalent s The cIL-15 / Rα-Fc fusion protein contains the amino acid substitution C220S, the truncation substitution E23 3P / L234V / L235A / G236del / S267K, and FcRn substitution M4 Human IL-15Rα (sushi) domain linked to an Fc monomer containing 28L / N434S The first IL-15 antibody contains the human mature IL-15 N4D / N65D variant linked to a nucleotide. 5 / Rα(sushi)-Fc monomer; amino acid substitution C220S, truncation substitution E233P / L2 34V / L235A / G236del / S267K, and FcRn substitution M428L / N Linked to the human IL-15Rα (sushi) domain linked to an Fc monomer containing 434S A second IL-15 / Rα (SEQ ID NO: 1) containing the human mature IL-15 N4D / N65D variant (c)-Fc monomer.

[0335] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 Utilizing the N1D / N65D variant.

[0336] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 N1D / N65D variant and scuba variant vs. S364K / E357Q :L368D / K370S is used in the bivalent scIL-15 / Rα-Fc format In the present invention, a preferred embodiment is an IL-15 N1D / N65D variant, scubari Ant pair S364K / E357Q:L368D / K370S, and each Fc monomer The 428L / 434S variant is utilized in the bivalent scIL-15 / Rα-Fc phosphatase. In the present invention, a preferred embodiment is the IL-15 N1D / N65D variant, Cubariant vs. S364K / E357Q:L368D / K370S, and their respective F c Utilizing the M428L / N434S variant in the monomer.

[0337] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein comprises two Human mature IL-15 N1D / N65D variant, two IL-15Rα (sushi) domains and two Fc monomers containing the amino acid substitutions shown in Figure 6D. In embodiments, the bivalent scIL-15 / Rα-Fc fusion protein comprises the amino acid substitution C22 OS and truncated substitutions E233P / L234V / L235A / G236del / S267K Human IL-15Rα (sushi) domain linked to an Fc monomer containing The first, IL-15 / Rα (sushi), contains the mature IL-15 N1D / N65D variant. Fc monomer with amino acid substitution C220S and truncation substitutions E233P / L234V / L23 Human IL-15Rα ( A human mature IL-15 N1D / N65D variant linked to the sushi domain was identified. In some embodiments, the bivalent s The cIL-15 / Rα-Fc fusion protein contains the amino acid substitution C220S, the truncation substitution E23 3P / L234V / L235A / G236del / S267K, and FcRn substitution M4 Human IL-15Rα (sushi) domain linked to an Fc monomer containing 28L / N434S The first IL-15 antibody contains the human mature IL-15 N1D / N65D variant linked to a nucleotide. 5 / Rα(sushi)-Fc monomer; amino acid substitution C220S, truncation substitution E233P / L2 34V / L235A / G236del / S267K, and FcRn substitution M428L / N Linked to the human IL-15Rα (sushi) domain linked to an Fc monomer containing 434S A second IL-15 / Rα (SEQ ID NO: 1) containing the human mature IL-15 N1D / N65D variant (c)-Fc monomer.

[0338] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 Q108E variant and scuba variant vs. S364K / E357Q:L3 68D / K370S in a bivalent scIL-15 / Rα-Fc format A preferred embodiment is a combination of IL-15 Q108E variant and scubariant pair. S364K / E357Q:L368D / K370S, as well as 42 in both monomers. It utilizes the 8L / 434S variant.

[0339] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein comprises two Human mature IL-15 Q108E variant, two IL-15Rα (sushi) domains, and two Fc monomers containing the amino acid substitutions shown in Figure 6D. In this state, the bivalent scIL-15 / Rα-Fc fusion protein contains the amino acid substitutions C220S and and truncation substitutions E233P / L234V / L235A / G236del / S267K Human mature I linked to the human IL-15Rα (sushi) domain linked to an Fc monomer The first IL-15 / Rα(sushi)-Fc monomer contains the L-15 Q108E variant. ; amino acid substitution C220S and truncation substitutions E233P / L234V / L235A / G23 Human IL-15Rα (sushi) domain linked to Fc monomer containing 6del / S267K A second IL-15 / R containing the human mature IL-15 Q108E variant linked to In some embodiments, the bivalent scIL-15 / Rα -Fc fusion protein contains the amino acid substitution C220S, the truncation substitutions E233P / L234V / L235A / G236del / S267K, and FcRn substitution M428L / N434S Human IL-15Rα (sushi) domain linked to an Fc monomer containing The first IL-15 / Rα(sushi)-Fc monoclonal antibody containing the mature IL-15 Q108E variant with amino acid substitution C220S, truncation substitutions E233P / L234V / L235A / G2 Fc monomer containing 36del / S267K and FcRn substitutions M428L / N434S Human mature IL-15 Q linked to the human IL-15Rα (sushi) domain linked to and a second IL-15 / Rα(sushi)-Fc monomer containing the 108E variant.

[0340] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 wild-type variants are utilized.

[0341] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is IL- 15 wild-type variant and scubariant vs. S364K / E357Q:L368D In the bivalent scIL-15 / Rα-Fc format, A new embodiment is an IL-15 wild type variant, S364K / E3 57Q:L368D / K370S, and 428L / 434S in each Fc monomer In the bivalent scIL-15 / Rα-Fc format, a variant is preferably used. A preferred embodiment is an IL-15 wild type variant, S364K / E35 variant. 7Q:L368D / K370S, and M428L / N434 in each Fc monomer Use the S variant.

[0342] In some embodiments, the bivalent ncIL-15 / Rα-Fc fusion protein comprises two Human mature IL-15 wild-type variant, two IL-15Rα (sushi) domains, and In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in Figure 6D. The bivalent scIL-15 / Rα-Fc fusion protein contains the amino acid substitution C220S and the truncation Fc motif containing the substitutions E233P / L234V / L235A / G236del / S267K Human mature IL-1 linked to the human IL-15Rα (sushi) domain linked to a symmetric α domain 5. First IL-15 / Rα(sushi)-Fc monomer containing the N1D / N65D variant ; amino acid substitution C220S and truncation substitutions E233P / L234V / L235A / G23 Human IL-15Rα (sushi) domain linked to Fc monomer containing 6del / S267K A second IL-1 containing the human mature IL-15 N1D / N65D variant linked to a cysteine. In some embodiments, the bivalent scIL-15 The / Rα-Fc fusion protein contains the amino acid substitution C220S, the truncation substitution E233P / L23 4V / L235A / G236del / S267K, and FcRn substitution M428L / N4 linked to the human IL-15Rα (sushi) domain linked to an Fc monomer containing 34S The first IL-15 / Rα (SEQ ID NO: 1) containing the mature human IL-15 N1D / N65D variant C)-Fc monomer; amino acid substitution C220S, truncation substitution E233P / L234V / L2 35A / G236del / S267K, and FcRn substitutions M428L / N434S Human mature IL-15Rα (sushi) domain linked to a Fc monomer containing A second IL-15 / Rα(sushi)-Fc monoclonal antibody containing the IL-15 N1D / N65D variant and dimers.

[0343] In the bivalent scIL-15 / Rα-Fc format, a preferred embodiment is shown in FIG. As shown in.

[0344] F.Fc-ncIL-15 / Rα In this embodiment, as shown in Figure 9F, the heterodimeric fusion protein comprises three The first monomer contains (from the N-terminus to the C-terminus) a CH2-CH3- domain. It contains the in-linker-IL-15 / Rα (sushi), and the Fc contains all of part of the hinge. The second monomer contains an "empty" Fc, including all or part of the hinge-CH2-CH3. The third monomer is IL-15. This is called "ncIL-15 / Rα-Fc" and "nc" stands for "non-covalent bond."

[0345] In the ncIL-15 / Rα-Fc (or Fc-ncIL-15 / Rα) format In this case, the preferred embodiment is the Scuba Variant pair S364K / E357Q:L368D / Use K370S.

[0346] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Utilizes the D30N / N65D variant.

[0347] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 D30N / E64Q / N65D variant and Scuba variant vs. S364K / E 357Q:L368D / K370S is used. Fc-ncIL-15 / Rα format In this regard, a preferred embodiment is a combination of the IL-15 D30N / N65D variant and Cubariant vs. S364K / E357Q:L368D / K370S, as well as both The monomer utilizes the 428L / 434S variant. Fc-ncIL-15 / Rα In the format, a preferred embodiment is the IL-15 D30N / N65D variant. and Scuba liant vs. S364K / E357Q:L368D / K370S, It utilizes the M428L / N434S variant in both monomers.

[0348] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The L-15 D30N / N65D variant, IL-15Rα (sushi) domain, and Fig. In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in Figure 6E. The ncIL-15 / Rα-Fc fusion protein binds to the human IL-15 D30N / N65D antibody. riant, amino acid substitution C220S, heterodimeric pI variant L368D / K370 S, and truncation substitutions E233P / L234V / L235A / G236del / S267K Fc containing a human IL-15Rα (sushi) domain linked to the C-terminus of an Fc monomer containing -IL-15 / Rα(sushi) monomer, and heterodimer with amino acid substitution C220S I variant S364K / E357Q, isosteric pI substitutions P217R / P228R / N2 76K, and truncation substitutions E233P / L234V / L235A / G236del / S26 In some embodiments, the ncIL-15 / Rα- The Fc fusion protein contains the human IL-15 D30N / N65D variant, an amino acid substitution C220S, heterodimeric pI variant L368D / K370S, truncation substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitution M428 Human IL-15Rα (sushi) domain linked to the C-terminus of Fc monomer containing L / N343S Fc-IL-15 / Rα(sushi) monomer containing sucrine, and the amino acid substitution C220S, Heterodimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P 228R / N276K, cut and replace E233P / L234V / L235A / G236del / S267K, and an empty Fc monomer containing the FcRn substitutions M428L / N343S. nothing.

[0349] In the ncIL-15 / Rα-Fc format, a preferred embodiment is IL-15 Utilizes the D30N / E64Q / N65D variants.

[0350] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 D30N / E64Q / N65D variant and Scuba variant vs. S364K / E 357Q:L368D / K370S is used. Fc-ncIL-15 / Rα format In this case, a preferred embodiment is the IL-15 D30N / E64Q / N65D variant. Scuba liant vs. S364K / E357Q:L368D / K370S, and each Utilizes the 428L / 434S variant in each Fc monomer. Fc-ncIL-1 In the IL-15 / Rα format, a preferred embodiment is IL-15 D30N / E64Q / N65D variant, Scuba liant vs. S364K / E357Q:L368D / K 370S, and utilizes the M428L / N434S variant in each Fc monomer. do.

[0351] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I L-15 D30N / E64Q / N65D variant, IL-15Rα (sushi) domain , and two Fc monomers containing the amino acid substitutions shown in Figure 6E. In this form, the ncIL-15 / Rα-Fc fusion protein is human IL-15 D30N / E64Q / N65D variant, amino acid substitution C220S, heterodimeric pI variant L368D / K370S, and truncated substitutions E233P / L234V / L235A / G23 Human IL-15Rα (Success) linked to the C-terminus of an Fc monomer containing 6del / S267K ) domain containing Fc-IL-15 / Rα(sushi) monomer, and the amino acid substitution C22 0S, heterodimeric pI variant S364K / E357Q, isosteric pI substitution P217 R / P228R / N276K, and truncated replacements E233P / L234V / L235A / G In some embodiments, the n The cIL-15 / Rα-Fc fusion protein is human IL-15 D30N / E64Q / N 65D variant, amino acid substitution C220S, heterodimeric pI variant L368D / K370S, cut and replace E233P / L234V / L235A / G236del / S267 K, and linked to the C-terminus of an Fc monomer containing the FcRn substitution M428L / N343S Fc-IL-15 / Rα(sushi) monomer containing the human IL-15Rα(sushi) domain, and the amino acid substitution C220S, heterodimeric pI variant S364K / E357Q, Isosteric pI substitution P217R / P228R / N276K, cleavage substitution E233P / L234 V / L235A / G236del / S267K, and FcRn substitution M428L / N34 Contains empty Fc monomers containing 3S.

[0352] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes the N4D variant.

[0353] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 N4D variant and scubariant vs. S364K / E357Q:L368D / In the Fc-ncIL-15 / Rα format, the preferred Embodiments include IL-15 N4D variant, scubariant vs. S364K / E357 Q: L368D / K370S and 428L / 434S variants in each Fc monomer In the Fc-ncIL-15 / Rα format, a preferred embodiment The IL-15 N4D variant, scuba variant vs. S364K / E357Q: L368D / K370S, and M428L / N434S variants in each Fc monomer Use Ant.

[0354] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The IL-15 N4D variant, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6E In some embodiments, the ncIL-1 The 5 / Rα-Fc fusion protein contains the human IL-15 N4D variant, amino acid substitution C 220S, heterodimeric pI variant L368D / K370S, and truncated substitution E23 C-terminus of Fc monomer containing 3P / L234V / L235A / G236del / S267K Fc-IL-15 / Rα(sushi) containing the human IL-15Rα(sushi) domain linked to ) monomer, as well as the amino acid substitution C220S, heterodimeric pI variant S364K / E357Q, isosteric pI substitutions P217R / P228R / N276K, and truncation substitution E Empty Fc monomer containing 233P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises IL-15 N4D variant, amino acid substitution C220S, heterodimeric pI variant L368D / K370S, cutting replacement E233P / L234V / L235A / G236d e1 / S267K, and the C-terminus of the Fc monomer containing the FcRn substitutions M428L / N343S Fc-IL-15 / Rα(sushi) containing the human IL-15Rα(sushi) domain linked to the termini C) Monomer, as well as the amino acid substitution C220S and heterodimer pI variant S364K / E357Q, isosteric pI substitution P217R / P228R / N276K, cleavage substitution E23 3P / L234V / L235A / G236del / S267K, and FcRn substitution M4 Contains empty Fc monomer containing 28L / N343S.

[0355] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Take advantage of the N65D variant.

[0356] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 N65D variant and scuba variant vs. S364K / E357Q:L368D In the Fc-ncIL-15 / Rα format, the preferred Embodiments include IL-15 N65D variant, scubariant vs. S364K / E3 57Q:L368D / K370S, and 428L / 434S in each Fc monomer In the ncIL-15 / Rα-Fc format, a preferred embodiment is The embodiment is directed to the N65D variant of IL-15, Scubariant vs. S364K / E35 7Q:L368D / K370S, and M428L / N434 in each Fc monomer Use the S variant.

[0357] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The IL-15 N65D variant, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6E In some embodiments, the ncIL- The 15 / Rα-Fc fusion protein is a human IL-15 N65D variant, recombinant C220S, heterodimeric pI variant L368D / K370S, and truncated substitution E C of Fc monomer containing 233P / L234V / L235A / G236del / S267K Fc-IL-15 / Rα ( Sushi) monomer, as well as the amino acid substitution C220S and heterodimeric pI variant S364 K / E357Q, isosteric pI substitutions P217R / P228R / N276K, and truncation substitutions Empty Fc molecules containing E233P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises , human IL-15 N65D variant, amino acid substitution C220S, heterodimeric pI variant Rianto L368D / K370S, Cut-off Replacement E233P / L234V / L235A / G2 Fc monomer containing 36del / S267K and FcRn substitutions M428L / N343S Fc-IL-15 / R containing the human IL-15Rα (sushi) domain linked to the C-terminus of α(sushi) monomer, and the amino acid substitution C220S, heterodimeric pI variant S3 64K / E357Q, isosteric pI substitution P217R / P228R / N276K, cleavage substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitutions Contains an empty Fc monomer containing the recombinant M428L / N343S.

[0358] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes the N4D / N65D variant.

[0359] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 N4D / N65D variant and scuba variant vs. S364K / E357Q:L 368D / K370S. In the Fc-ncIL-15 / Rα format, A preferred embodiment is the IL-15 N4D / N65D variant, scubariant vs. S364K / E357Q:L368D / K370S, and 4 in each Fc monomer Utilizes the 28L / 434S variant. In this regard, a preferred embodiment is the IL-15 N4D / N65D variant, skew barrier S364K / E357Q:L368D / K370S vs. S364K / E357Q:L368D / K370S, and in each Fc monomer It utilizes the M428L / N434S variant.

[0360] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The L-15 N4D / N65D variant, IL-15Rα (sushi) domain, and Fig. 6 In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in E. The cIL-15 / Rα-Fc fusion protein inhibits the human IL-15 N4D / N65D barrier. nt, amino acid substitution C220S, heterodimeric pI variant L368D / K370S, and truncation substitutions E233P / L234V / L235A / G236del / S267K Fc-I containing the human IL-15Rα (sushi) domain linked to the C-terminus of the Fc monomer L-15 / Rα (sushi) monomer and the heterodimer with the amino acid substitution C220S Reant S364K / E357Q, isosteric pI substitution P217R / P228R / N276 K, and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15 / Rα-Fc The fusion protein contains the human IL-15 N4D / N65D variant, with the amino acid substitution C22 0S, heterodimeric pI variant L368D / K370S, truncation substitution E233P / L2 34V / L235A / G236del / S267K, and FcRn substitution M428L / N The human IL-15Rα (sushi) domain linked to the C-terminus of the Fc monomer containing 343S Fc-IL-15 / Rα(sushi) monomer containing the amino acid substitution C220S, heterodimer Dimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P228 R / N276K, cut and replace E233P / L234V / L235A / G236del / S2 67K, and an empty Fc monomer containing the FcRn substitutions M428L / N343S.

[0361] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes the N1D / N65D variant.

[0362] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 N1D / N65D variant and scuba variant vs. S364K / E357Q:L Use 368D / K370S.

[0363] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 N1D / N65D variant, Scuba liant vs. S364K / E357Q:L36 8D / K370S and 428L / 434S variants in each Fc monomer. In the Fc-ncIL-15 / Rα format, a preferred embodiment is -15 N1D / N65D variant, Scuba liant vs S364K / E357Q: L368D / K370S, and M428L / N434S variants in each Fc monomer Use Ant.

[0364] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The L-15 N1D / N65D variant, IL-15Rα (sushi) domain, and Fig. 6 In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in E. The cIL-15 / Rα-Fc fusion protein inhibits the human IL-15 N1D / N65D barrier. nt, amino acid substitution C220S, heterodimeric pI variant L368D / K370S, and truncation substitutions E233P / L234V / L235A / G236del / S267K Fc-I containing the human IL-15Rα (sushi) domain linked to the C-terminus of the Fc monomer L-15 / Rα (sushi) monomer and the heterodimer with the amino acid substitution C220S Reant S364K / E357Q, isosteric pI substitution P217R / P228R / N276 K, and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15 / Rα-Fc The fusion protein contains the human IL-15 N1D / N65D variant, with the amino acid substitution C22 0S, heterodimeric pI variant L368D / K370S, truncation substitution E233P / L2 34V / L235A / G236del / S267K, and FcRn substitution M428L / N The human IL-15Rα (sushi) domain linked to the C-terminus of the Fc monomer containing 343S Fc-IL-15 / Rα(sushi) monomer containing the amino acid substitution C220S, heterodimer Dimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P228 R / N276K, cut and replace E233P / L234V / L235A / G236del / S2 67K, and an empty Fc monomer containing the FcRn substitutions M428L / N343S.

[0365] In the ncIL-15 / Rα-Fc format, a preferred embodiment is scuba Use the Rianto vs. S364K / E357Q:L368D / K370S.

[0366] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes the Q108E variant.

[0367] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Q108E variant and scuba variant vs. S364K / E357Q:L368 In the Fc-ncIL-15 / Rα format, A preferred embodiment is the IL-15 Q108E variant and the scuba variant vs. S364 K / E357Q:L368D / K370S, as well as 428L / 4 in both monomers. In the Fc-ncIL-15 / Rα format, the 34S variant is used. A new embodiment is the IL-15 Q108E variant and the scubariant vs. S36 4K / E357Q:L368D / K370S and M428L in both monomers / Utilizing the N434S variant.

[0368] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The L-15 Q108E variant, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6E In some embodiments, the ncIL-12 comprises two Fc monomers containing the amino acid substitutions. The IL-15 / Rα-Fc fusion protein is a human IL-15 Q108E variant, amino The acid substitution C220S, the heterodimeric pI variant L368D / K370S, and the truncation substitution Fc monomer containing the recombinant E233P / L234V / L235A / G236del / S267K Fc-IL-15 / R containing the human IL-15Rα (sushi) domain linked to the C-terminus of α(sushi) monomer, and the amino acid substitution C220S, heterodimeric pI variant S3 64K / E357Q, isosteric pI substitutions P217R / P228R / N276K, and Empty F containing E233P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises a ncIL-15 / Rα-Fc monomer. The quality is human IL-15 Q108E variant, amino acid substitution C220S, heterodimer pI variant L368D / K370S, truncation substitutions E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N343S Fc-IL-1 containing the human IL-15Rα (sushi) domain linked to the C-terminus of the monomer 5 / Rα (sushi) monomer and the amino acid substitution C220S, heterodimeric pI variant isosteric pI substitutions P217R / P228R / N276K, Substitutions E233P / L234V / L235A / G236del / S267K and Fc Contains an empty Fc monomer containing the Rn substitutions M428L / N343S.

[0369] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Use a wild-type variant.

[0370] In the Fc-ncIL-15 / Rα format, a preferred embodiment is IL-15 Wild-type variant and scubariant vs. S364K / E357Q:L368D / K In the Fc-ncIL-15 / Rα format, a preferred embodiment is 370S. Morphology: IL-15 wild-type variant, scubariant vs. S364K / E357Q: L368D / K370S, and 428L / 434S variants in each Fc monomer In the Fc-ncIL-15 / Rα format, a preferred embodiment is , IL-15 wild-type variant, scubariant vs. S364K / E357Q:L36 8D / K370S, and M428L / N434S variants in each Fc monomer Use.

[0371] In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human mature I The IL-15 wild-type variant, IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6E In some embodiments, the ncIL-15 comprises two Fc monomers containing amino acid substitutions. The / Rα-Fc fusion protein contains the wild-type variant of human IL-15, with the amino acid substitution C22 0S, heterodimeric pI variant L368D / K370S, and truncation substitution E233P / L234V / L235A / G236del / S267K linked to the C-terminus of the Fc monomer Fc-IL-15 / Rα(sushi) monoclonal antibody containing the fused human IL-15Rα(sushi) domain heterodimer, as well as the amino acid substitution C220S and the heterodimeric pI variant S364K / E3 57Q, isosteric pI substitutions P217R / P228R / N276K, and truncation substitution E23 Empty Fc monomer containing 3P / L234V / L235A / G236del / S267K In some embodiments, the ncIL-15 / Rα-Fc fusion protein comprises human I L-15 wild-type variant, amino acid substitution C220S, heterodimeric pI variant L3 68D / K370S, cutting replacement E233P / L234V / L235A / G236del / S267K, and FcRn substitutions M428L / N343S linked to the C-terminus of the Fc monomer. Fc-IL-15 / Rα(sushi) monoclonal antibody containing the fused human IL-15Rα(sushi) domain heterodimer, as well as the amino acid substitution C220S and the heterodimeric pI variant S364K / E3 57Q, isosteric pI substitution P217R / P228R / N276K, cleavage substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitution M428L / containing empty Fc monomers containing N343S.

[0372] In the Fc-ncIL-15 / Rα format, preferred A preferred embodiment is described in WO2 104T of JP 018 / 071919 and shown as SEQ ID NOs: 668-672 in the sequence listing. do.

[0373] In the Fc-ncIL-15 / Rα format, a preferred embodiment is 603) and XENP22637 as strand 2 (8927) and strand 3 (16484). Included, as shown in FIG.

[0374] G.Fc-scIL-15 / Rα In this embodiment, as shown in Figure 9G, the heterodimeric fusion protein comprises two The first monomer comprises (from the N-terminus to the C-terminus) CH2-CH3-any domain linker-IL-15 / Rα(sushi)-domain linker-IL-15 The second monomer contains the entire hinge, CH2-CH3, and the Fc monomer contains the entire hinge. or a portion thereof, including an "empty" Fc. This may be referred to as "Fc-scIL-15 / Rα" or It is called "scIL-15 / Rα-Fc," and the "sc" stands for "single chain" (e.g., IL-15 / Sushi complex)

[0375] In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises human mature IL-15. The IL-15 variant, IL-15Rα (sushi) domain, and amino acid sequence shown in Figure 6E In some embodiments, the Fc-scIL-15 comprises two Fc monomers containing acid substitutions. The / Rα fusion protein is a human Fc domain linked to a human mature IL-15 variant. The amino acid substitution C220S, heterodimer, and The dimeric pI variant L368D / K370S and the truncation substitution E233P / L234 Fc-IL-15 containing an Fc monomer containing V / L235A / G236del / S267K / Rα (sushi) monomer; and the amino acid substitution C220S, heterodimeric pI variant S364K / E357Q, isosteric pI substitutions P217R / P228R / N276K, and and truncation substitutions E233P / L234V / L235A / G236del / S267K In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises an Fc monomer. The protein is human IL-15Rα, in which the Fc domain is linked to a human mature IL-15 variant. (Sushi) domain, the amino acid substitution C220S, heterodimer pI balance Ant L368D / K370S, cutting replacement E233P / L234V / L235A / G23 6del / S267K, and FcRn substitutions M428L / N343S. Fc-IL-15 / Rα(sushi) monomer containing the amino acid substitution C220S, heterodimer Dimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P228 R / N276K, cut and replace E233P / L234V / L235A / G236del / S2 67K, and an empty Fc monomer containing the FcRn substitutions M428L / N343S.

[0376] In the Fc-scIL-15 / Rα format, a preferred embodiment is scuba Use the Rianto vs. S364K / E357Q:L368D / K370S.

[0377] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes the D30N / N65D variant.

[0378] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 D30N / E64Q / N65D variant and Scuba variant vs. S364K / E 357Q:L368D / K370S is used. Fc-scIL-15 / Rα format In this regard, a preferred embodiment is a combination of the IL-15 D30N / N65D variant and Cubariant vs. S364K / E357Q:L368D / K370S, as well as both Utilizes the 428L / 434S variant in the monomer. Fc-scIL-15 / Rα In the format, a preferred embodiment is the IL-15 D30N / N65D variant. and Scuba liant vs. S364K / E357Q:L368D / K370S, It utilizes the M428L / N434S variant in both monomers.

[0379] In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises human mature IL-15. The L-15 D30N / N65D variant, IL-15Rα (sushi) domain, and Fig. In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in Figure 6E. The Fc-scIL-15 / Rα fusion protein contains the Fc domain of human mature IL-15 D Linked to the human IL-15Rα (sushi) domain linked to the 30N / N65D variant As shown, the amino acid substitution C220S, heterodimeric pI variant L368D / K37 0S, and truncation substitutions E233P / L234V / L235A / G236del / S267 Fc-IL-15 / Rα (sushi) monomers, including Fc monomers containing K; and amino acid substitutions Substituted C220S, heterodimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P228R / N276K, and truncated substitutions E233P / L234V / L23 In some embodiments, the empty Fc monomer comprises 5A / G236del / S267K. The Fc-scIL-15 / Rα fusion protein contains the Fc domain of human mature IL-15. Human IL-15Rα (sushi) domain linked to the D30N / N65D variant As shown, the amino acid substitution C220S, the heterodimeric pI variant L368D / K 370S, cut and replace E233P / L234V / L235A / G236del / S267K , and Fc-IL-15, which contains an Fc monomer containing the FcRn substitution M428L / N343S. / Rα (sushi) monomer; and the amino acid substitution C220S, heterodimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P228R / N276K, cleavage Substitutions E233P / L234V / L235A / G236del / S267K, and FcR Includes an empty Fc monomer containing the n substitution M428L / N343S.

[0380] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes D30N / E64Q / N65D variants.

[0381] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 D30N / E64Q / N65D variant and Scuba variant vs. S364K / E 357Q:L368D / K370S is used. Fc-scIL-15 / Rα format In this case, a preferred embodiment is the IL-15 D30N / E64Q / N65D variant. Scuba liant vs. S364K / E357Q:L368D / K370S, and each Utilizes the 428L / 434S variant in each Fc monomer. Fc-scIL-1 In the IL-15 / Rα format, a preferred embodiment is IL-15 D30N / E64Q / N65D variant, Scuba liant vs. S364K / E357Q:L368D / K 370S, and utilizes the M428L / N434S variant in each Fc monomer. do.

[0382] In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises human mature IL-15. L-15 D30N / E64Q / N65D variant, IL-15Rα (sushi) domain , and two Fc monomers containing the amino acid substitutions shown in Figure 6E. In this form, the Fc-scIL-15 / Rα fusion protein contains the Fc domain of the human mature IL-15. Human IL-15Rα (SEQ ID NO: 1) linked to the -15 D30N / E64Q / N65D variant (c) Amino acid substitution C220S, heterodimeric pI variant, linked to the domain L368D / K370S, and truncated replacements E233P / L234V / L235A / G2 Fc-IL-15 / Rα(sushi) monomers, including Fc monomers containing 36del / S267K ; as well as the amino acid substitution C220S, heterodimeric pI variant S364K / E357 Q, isosteric pI substitutions P217R / P228R / N276K, and truncation substitution E233P / L234V / L235A / G236del / S267K containing empty Fc monomer. In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises an Fc domain. linked to the human mature IL-15 D30N / E64Q / N65D variant The amino acid substitution C220S allows for heterodimerization of the α-15Rα (sushi) domain. pI variant L368D / K370S, truncation substitutions E233P / L234V / L235 A / G236del / S267K, and FcRn substitutions M428L / N343S Fc-IL-15 / Rα(sushi) monomers containing the c monomer; and the amino acid substitution C220 S, heterodimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P228R / N276K, cut and replace E233P / L234V / L235A / G236d e1 / S267K, and an empty Fc monomer containing the FcRn substitutions M428L / N343S. include.

[0383] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 Take advantage of the N65D variant.

[0384] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 N65D variant and scuba variant vs. S364K / E357Q:L368D In the Fc-scIL-15 / Rα format, Embodiments include the N65D variant of IL-15, scubariant vs. S364K / E3 57Q:L368D / K370S, and 428L / 434S in each Fc monomer In the Fc-scIL-15 / Rα format, a preferred embodiment is The embodiment is directed to the N65D variant of IL-15, Scubariant vs. S364K / E35 7Q:L368D / K370S, and M428L / N434 in each Fc monomer Use the S variant.

[0385] In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises human mature IL-15. The IL-15 N65D variant, the IL-15Rα (sushi) domain, and the IL-15Rα (sushi) domain shown in Figure 6E In some embodiments, the Fc-sc comprises two Fc monomers containing the amino acid substitutions. The IL-15 / Rα fusion protein contains the human mature IL-15 N65D variant in the Fc domain. The amino acid sequence is linked to the human IL-15Rα (sushi) domain linked to the ant. substitution C220S, heterodimeric pI variant L368D / K370S, and truncation substitution Fc monomer containing E233P / L234V / L235A / G236del / S267K Fc-IL-15 / Rα(sushi) monomer containing the amino acid substitution C220S, heterodimer Dimeric pI variant S364K / E357Q, isosteric pI substitution P217R / P228 R / N276K, and truncation substitutions E233P / L234V / L235A / G236del In some embodiments, the Fc-scIL- The IL-15 / Rα fusion protein contains the human mature IL-15 N65D variant in the Fc domain. The amino acid substitution C was linked to the human IL-15Rα (sushi) domain linked to 220S, heterodimeric pI variant L368D / K370S, truncation substitution E233P / L234V / L235A / G236del / S267K, and FcRn substitution M428L / N343S-containing Fc-IL-15 / Rα(sushi) monomer; and Amino acid substitution C220S, heterodimeric pI variant S364K / E357Q, isoelectric child pI substitution P217R / P228R / N276K, truncation substitution E233P / L234V / L 235A / G236del / S267K, and FcRn substitutions M428L / N343S Contains empty Fc monomers.

[0386] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes the N4D / N65D variant.

[0387] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 N4D / N65D variant and scuba variant vs. S364K / E357Q:L 368D / K370S. In the Fc-scIL-15 / Rα format, A preferred embodiment is the IL-15 N4D / N65D variant, scubariant vs. S364K / E357Q:L368D / K370S, and 4 in each Fc monomer Utilizing the 28L / 434S variant, the ncIL-15 / Rα-Fc format In this regard, a preferred embodiment is the IL-15 N4D / N65D variant, skew barrier S364K / E357Q:L368D / K370S vs. S364K / E357Q:L368D / K370S, and in each Fc monomer It utilizes the M428L / N434S variant.

[0388] In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises human mature IL-15. The L-15 N4D / N65D variant, IL-15Rα (sushi) domain, and Fig. 6 In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in F. The c-scIL-15 / Rα fusion protein contains the Fc domain of human mature IL-15 N4 Linked to the human IL-15Rα (sushi) domain linked to the D / N65D variant As such, the amino acid substitution C220S, heterodimeric pI variant L368D / K370S , and truncation substitutions E233P / L234V / L235A / G236del / S267K Fc-IL-15 / Rα (sushi) monomers containing Fc monomers containing the amino acid substitution C 220S, heterodimeric pI variant S364K / E357Q, isosteric pI substitution P2 17R / P228R / N276K, and truncated replacements E233P / L234V / L235A / G236del / S267K. In some embodiments, the empty Fc monomer comprises: The Fc-scIL-15 / Rα fusion protein contains the Fc domain of human mature IL-15 N Linked to the human IL-15Rα (sushi) domain linked to the 4D / N65D variant As shown in Fig. 1, the amino acid substitution C220S and the heterodimeric pI variant L368D / K370 S, truncated substitutions E233P / L234V / L235A / G236del / S267K, and Fc-IL-15 / Rα containing Fc monomers containing FcRn substitutions M428L / N343S (Sushi) monomer; and amino acid substitution C220S, heterodimeric pI variant S36 4K / E357Q, isosteric pI substitution P217R / P228R / N276K, cleavage substitution E 233P / L234V / L235A / G236del / S267K and FcRn substitutions Contains an empty Fc monomer containing M428L / N343S.

[0389] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 Utilizes the N1D / N65D variant.

[0390] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 N1D / N65D variant and scuba variant vs. S364K / E357Q:L 368D / K370S. In the Fc-scIL-15 / Rα format, A preferred embodiment is the IL-15 N1D / N65D variant, scubariant vs. S364K / E357Q:L368D / K370S, and 4 in each Fc monomer Utilizes the 28L / 434S variant. In this regard, a preferred embodiment is the IL-15 N1D / N65D variant, skew barrier S364K / E357Q:L368D / K370S vs. S364K / E357Q:L368D / K370S, and in each Fc monomer It utilizes the M428L / N434S variant.

[0391] In some embodiments, the Fc-scIL-15 / Rα fusion protein comprises human mature IL-15. The L-15 N1D / N65D variant, IL-15Rα (sushi) domain, and Fig. 6 In some embodiments, the Fc monomer comprises two Fc monomers containing the amino acid substitutions shown in F. The c-scIL-15 / Rα fusion protein contains the Fc domain of human mature IL-15 N1 Linked to the human IL-15Rα (sushi) domain linked to the D / N65D variant As such, the amino acid substitution C220S, heterodimeric pI variant L368D / K370S , and truncation substitutions E233P / L234V / L235A / G236del / S267K Fc-IL-15 / Rα (sushi) monomers containing Fc monomers containing the amino acid substitution C 220S, heterodimeric pI variant S364K / E357Q, isosteric pI substitution P2 17R / P228R / N276K, and truncated replacements E233P / L234V / L235A / G236del / S267K. In some embodiments, the empty Fc monomer comprises: The Fc-scIL-15 / Rα fusion protein contains the Fc domain of human mature IL-15 N Linked to the human IL-15Rα (sushi) domain linked to the 1D / N65D variant As shown in Fig. 1, the amino acid substitution C220S and the heterodimeric pI variant L368D / K370 S, truncated substitutions E233P / L234V / L235A / G236del / S267K, and Fc-IL-15 / Rα containing Fc monomers containing FcRn substitutions M428L / N343S (Sushi) monomer; and amino acid substitution C220S, heterodimeric pI variant S36 4K / E357Q, isosteric pI substitution P217R / P228R / N276K, cleavage substitution E 233P / L234V / L235A / G236del / S267K and FcRn substitutions Contains an empty Fc monomer containing M428L / N343S.

[0392] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 Uses variant Q108E.

[0393] In the Fc-scIL-15 / Rα format, a preferred embodiment is IL-15 Variant Q108E and scubariant vs. S364K / E357Q:L368D In the Fc-scIL-15 / Rα format, Embodiments include IL-15 variants Q108E and S364K / E357Q:L368D / K370S, and 428L / 43 in both monomers ...

Claims

1. A pharmaceutical composition for use in a method for treating cancer in a patient in need thereof, comprising: A therapeutically effective amount of an IL-15 / IL-15Rα heterodimeric Fc fusion protein, a) from the N-terminus to the C-terminus: i) IL-15 receptor alpha (IL-15Rα) sushi domain; ii) a first domain linker, and iii) a first monomer comprising a first variant Fc domain comprising CH2-CH3; and b) from the N-terminus to the C-terminus: i) a variant IL-15 domain comprising the amino acid sequence of SEQ ID NO:2 and any one of the amino acid substitutions selected from the group consisting of N4D / N65D, D30N / N65D, and D30N / E64Q / N65D; ii) a second domain linker, and iii) a second monomer comprising a second variant Fc domain comprising CH2-CH3; the first and second variant Fc domains have a set of amino acid substitutions selected from the group consisting of: S267K / L368D / K370S:S267K / S364K / E357Q; S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L, and K370S:S364K / E357Q, according to EU numbering; The method comprises simultaneously administering a therapeutically effective amount of an anti-PD-L1 antibody to a pharmaceutical composition.

2. A pharmaceutical composition for use in a method of inducing T cell expansion in a patient, comprising: A therapeutically effective amount of an IL-15 / IL-15Rα heterodimeric Fc fusion protein, A therapeutically effective amount of an IL-15 / IL-15Rα heterodimeric Fc fusion protein, a) from the N-terminus to the C-terminus: i) IL-15 receptor alpha (IL-15Rα) sushi domain; ii) a first domain linker, and iii) a first monomer comprising a first variant Fc domain comprising CH2-CH3; and b) from the N-terminus to the C-terminus: i) a variant IL-15 domain comprising the amino acid sequence of SEQ ID NO:2 and any one of the amino acid substitutions selected from the group consisting of N4D / N65D, D30N / N65D, and D30N / E64Q / N65D; ii) a second domain linker, and iii) a second monomer comprising a second variant Fc domain comprising CH2-CH3; the first and second variant Fc domains have a set of amino acid substitutions selected from the group consisting of: S267K / L368D / K370S:S267K / S364K / E357Q; S364K / E357Q:L368D / K370S; L368D / K370S:S364K; L368E / K370S:S364K; T411E / K360E / Q362E:D401K; L368D / K370S:S364K / E357L, and K370S:S364K / E357Q, according to EU numbering; The method further comprises administering in combination with a therapeutically effective amount of an anti-PD-L1 antibody.

3. The pharmaceutical composition of claim 1, wherein the variant IL-15 domain comprises the amino acid sequence of SEQ ID NO:2 and amino acid substitutions D30N / E64Q / N65D.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein the IL-15Rα sushi domain has the amino acid sequence of SEQ ID NO:

4.

5. The pharmaceutical composition of claim 1, wherein the first and second variant FC domains have S364K / E357Q:L368D / K370S substitutions.

6. The pharmaceutical composition of claim 1, wherein the first variant Fc domain has an S364K / E357Q substitution and the second variant Fc domain has an L368D / K370S substitution.

7. The pharmaceutical composition of claim 1, wherein the first and second variant Fc domains each comprise an M428L / N434S substitution.

8. The pharmaceutical composition of claim 1, wherein the first and second variant FC domains each include E233P / L234V / L235A / G236del / S267K substitutions.

9. The pharmaceutical composition of claim 1, wherein the IL-15 / IL-15Rα heterodimeric Fc fusion protein and checkpoint blocking antibody are administered simultaneously or sequentially.

10. The pharmaceutical composition of claim 1, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, or darvalumab.

11. The pharmaceutical composition described in claim 10, wherein the IL-15 / IL-15Rα heterodimer Fc fusion protein has the amino acid sequences of SEQ ID NOs: 253 and 254.

12. The pharmaceutical composition described in claim 10, wherein the IL-15 / IL-15Rα heterodimeric Fc fusion protein comprises the amino acid sequences of SEQ ID NOs: 204 and 205.

13. The pharmaceutical composition of any one of claims 1 or 3 to 12, wherein the cancer is a metastatic cancer.

14. The pharmaceutical composition of any one of claims 1 or 3 to 12, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, kidney cancer, renal cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, and myeloma.

15. The pharmaceutical composition of any of claims 1 to 14, wherein the method results in a minimal level of vascular leakage in a patient.

16. The pharmaceutical composition of claim 15, wherein the level of vascular leakage is in the range of less than or equal to a 20% reduction in serum albumin in a patient following administration.

17. The pharmaceutical composition of any one of claims 2 to 12, 15, or 16, wherein the T cell expansion is at least a two-fold increase in T cells.

18. The pharmaceutical composition of any of claims 2-12, 15, or 16, wherein the T cell expansion is in the range of a 2-fold to 15-fold increase in T cells.

19. A pharmaceutical composition described in any of claims 1 to 18, wherein the method does not increase the likelihood of inducing hypoalbuminemia.

20. The pharmaceutical composition of any one of claims 2 to 12, 15, 16, 17, 18, or 19, wherein the T cells comprise tumor-infiltrating lymphocytes.

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