Cancer therapy using IL-2 conjugates and chimeric antigen receptor therapy

Abstract

Disclosed herein is a method for treating cancer in a subject in need thereof, the method comprising administering (a) an IL-2 conjugate and (b) chimeric antigen receptor (CAR) therapy. Also disclosed herein is a method for treating diffuse large B-cell lymphoma (DLBCL) in a subject in need thereof, the method comprising administering an IL-2 conjugate.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to International Application No. PCT / US2022 / 082151, filed December 21, 2022, the contents of which are incorporated herein by reference for all purposes. [Background technology]

[0002] Distinct populations of T cells regulate the immune system to maintain immune homeostasis and immune tolerance. For example, regulatory T (Treg) cells prevent inappropriate responses by the immune system by preventing pathological autoreactivity, while cytotoxic T cells target and destroy infected and / or cancer cells. In some cases, modulating different populations of T cells provides options for treating diseases or indications.

[0003] Cytokines include a family of cell signaling proteins, such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors that play a role in the homeostasis of innate and adaptive immune cells. Cytokines are produced by immune cells, such as macrophages, B lymphocytes, T lymphocytes, and mast cells, endothelial cells, fibroblasts, and different stromal cells. In some cases, cytokines regulate the balance between humoral and cell-based immune responses.

[0004] Interleukins are signaling proteins that regulate the development and differentiation of T and B lymphocytes, cells of the monocyte lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by helper CD4+ T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue-resident cells.

[0005] In some cases, interleukin 2 (IL-2) signaling is used to regulate T cell responses, which are subsequently used to treat cancer. Thus, in one aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate and (b) chimeric antigen receptor (CAR) therapy. In another aspect, provided herein is a method of treating diffuse large B-cell lymphoma (DLBCL) in a subject, comprising administering an IL-2 conjugate. Summary of the Invention [Problem to be solved by the invention]

[0006] Described herein are methods of treating cancer in a subject in need thereof, comprising administering to the subject (a) an IL-2 conjugate and (b) a CAR therapy, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, e.g., the amino acid sequence of SEQ ID NO: 2, having an unnatural amino acid residue at position 64, as described herein.

[0007] Also described herein is a method of treating diffuse large B-cell lymphoma (DLBCL) in a subject in need thereof, comprising administering to the subject an IL-2 conjugate, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, e.g., the amino acid sequence of SEQ ID NO: 2, having an unnatural amino acid residue at position 64, as described herein. [Means for solving the problem]

[0008] Exemplary embodiments include the following:

[0009] Embodiment 1 is a method of treating cancer in a subject in need thereof, comprising administering to the subject: (a) an IL-2 conjugate; and (b) a chimeric antigen receptor (CAR) therapy; The IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is of formula (I): [ka] (In the formula, Z is CH2 and Y is [ka] and Y is CH2 and Z is [ka] and Z is CH2 and Y is [ka] or Y is CH2 and Z is [ka] and W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa; q is 1, 2, or 3; X has the structure: [ka] is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue) is replaced by the structure of

[0010] Embodiment 2 is the method of embodiment 1, wherein the cancer is leukemia, myeloma, or lymphoma.

[0011] Embodiment 3 is the method of embodiment 2, wherein the leukemia is acute lymphoblastic leukemia or chronic lymphocytic leukemia.

[0012] Embodiment 4 is the method of embodiment 2, wherein the myeloma is multiple myeloma.

[0013] Embodiment 5 is the method of embodiment 2, wherein the lymphoma is non-Hodgkin's lymphoma, follicular lymphoma, transformed follicular lymphoma, mantle cell lymphoma, primary mediastinal B-cell lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma (DLBCL).

[0014] Embodiment 6 is the method of embodiment 2 or embodiment 5, wherein the lymphoma is Burkitt's lymphoma.

[0015] Embodiment 7 is the method of embodiment 2 or embodiment 5, wherein the lymphoma is diffuse large B-cell lymphoma (DLBCL).

[0016] Embodiment 8 is a method of treating diffuse large B-cell lymphoma (DLBCL) in a subject in need thereof, comprising administering to the subject an IL-2 conjugate; The IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is of formula (I): [ka] (In the formula, Z is CH2 and Y is [ka] and Y is CH2 and Z is [ka] and Z is CH2 and Y is [ka] or Y is CH2 and Z is [ka] and W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa; q is 1, 2, or 3; X has the structure: [ka] is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue) is replaced by the structure of

[0017] Embodiment 9 is the method of embodiment 7 or embodiment 8, wherein the DLBCL is relapsed or refractory DLBCL, or the DLBCL has relapsed after two or more prior lines of systemic therapy for DLBCL.

[0018] Embodiment 10 is the method of any one of embodiments 7 to 9, further comprising selecting a subject to be administered the IL-2 conjugate based at least in part on the subject having received two or more prior lines of systemic therapy for DLBCL.

[0019] Embodiment 11 is the method of embodiment 9 or embodiment 10, wherein the two or more prior lines of systemic therapy for DLBCL comprise an anthracycline, an anti-CD20 agent, or a combination of an anthracycline and an anti-CD20 agent.

[0020] Embodiment 12 is the method of embodiment 11, wherein the anti-CD20 agent comprises rituximab.

[0021] Embodiment 13 is the method of any one of embodiments 9 to 12, wherein two or more prior lines of systemic therapy for DLBCL include CAR therapy.

[0022] Embodiment 14 is the method of embodiment 13, wherein the CAR therapy is the last line of two or more prior lines of systemic therapy for DLBCL.

[0023] Embodiment 15 is the method of any one of embodiments 8 to 14, further comprising selecting a subject to be administered the IL-2 conjugate based at least in part on the subject having received CAR therapy.

[0024] Embodiment 16 is the method of any one of embodiments 8 to 15, further comprising administering CAR therapy to the subject.

[0025] Embodiment 17 is the method of any one of embodiments 1-7 and 13-16, wherein the CAR therapy comprises T cells expressing a CAR.

[0026] Embodiment 18 is the method of any one of embodiments 1-7 and 13-17, wherein the CAR therapy comprises gamma delta T cells expressing a CAR.

[0027] Embodiment 19 is the method of any one of embodiments 1-7 and 13-17, wherein the CAR therapy comprises natural killer (NK) T cells expressing a CAR.

[0028] Embodiment 20 is the method of any one of embodiments 1-7 and 13-16, wherein the CAR therapy comprises natural killer (NK) cells expressing a CAR.

[0029] Embodiment 21 is the method of any one of embodiments 17 to 20, wherein the CAR comprises an anti-CD19 domain comprising an anti-CD19 heavy chain variable domain (VH) and an anti-CD19 light chain variable domain (VL).

[0030] Embodiment 22 is the method of embodiment 21, wherein the anti-CD19 VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5.

[0031] Embodiment 23 is the method of embodiment 21 or 22, wherein the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:6.

[0032] Embodiment 24 is the method of any one of embodiments 21 to 23, wherein the anti-CD19 VH comprises the amino acid sequence of SEQ ID NO:6.

[0033] Embodiment 25 is the method described in any one of embodiments 21 to 24, wherein the anti-CD19 VL comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 7, CDR-L2 having the amino acid sequence of SEQ ID NO: 8, and CDR-L3 having the amino acid sequence of SEQ ID NO: 9.

[0034] Embodiment 26 is the method of any one of embodiments 21 to 25, wherein the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10.

[0035] Embodiment 27 is the method of any one of embodiments 21 to 26, wherein the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO:10.

[0036] Embodiment 28 is the method described in embodiment 21, wherein the anti-CD19 VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13.

[0037] Embodiment 29 is the method of embodiment 21 or 28, wherein the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0038] Embodiment 30 is the method of any one of embodiments 21, 28, and 29, wherein the anti-CD19 VH comprises the amino acid sequence of SEQ ID NO: 14.

[0039] Embodiment 31 is the method described in any one of embodiments 21 and 28 to 30, wherein the anti-CD19 VL comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 15, CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and CDR-L3 having the amino acid sequence of SEQ ID NO: 17.

[0040] Embodiment 32 is the method of any one of embodiments 21 and 28 to 31, wherein the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18.

[0041] Embodiment 33 is the method of any one of embodiments 21 and 28 to 32, wherein the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO:18.

[0042] Embodiment 34 is the method described in embodiment 21, wherein the anti-CD19 VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21.

[0043] Embodiment 35 is the method of embodiment 21 or 34, wherein the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22.

[0044] Embodiment 36 is the method of any one of embodiments 21, 34, and 35, wherein the anti-CD19 VH comprises the amino acid sequence of SEQ ID NO: 22.

[0045] Embodiment 37 is the method described in any one of embodiments 21 and 34 to 36, wherein the anti-CD19 VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 25.

[0046] Embodiment 38 is the method of any one of embodiments 21 and 34 to 37, wherein the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 26.

[0047] Embodiment 39 is the method of any one of embodiments 21 and 34 to 38, wherein the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO:26.

[0048] Embodiment 40 is the method of any one of embodiments 17 to 39, wherein the CAR comprises an anti-CD22 domain comprising an anti-CD22 heavy chain variable domain (VH) and an anti-CD22 light chain variable domain (VL).

[0049] Embodiment 41 is the method described in embodiment 40, wherein the anti-CD22 VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29.

[0050] Embodiment 42 is the method of embodiment 40 or 41, wherein the anti-CD22 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30.

[0051] Embodiment 43 is the method of any one of embodiments 40 to 42, wherein the anti-CD22 VH comprises the amino acid sequence of SEQ ID NO: 30.

[0052] Embodiment 44 is a method according to any one of embodiments 40 to 43, wherein the anti-CD22 VL comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 31, CDR-L2 having the amino acid sequence of SEQ ID NO: 32, and CDR-L3 having the amino acid sequence of SEQ ID NO: 33.

[0053] Embodiment 45 is the method of any one of embodiments 40 to 44, wherein the anti-CD22 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34.

[0054] Embodiment 46 is the method of any one of embodiments 40 to 45, wherein the anti-CD22 VL comprises the amino acid sequence of SEQ ID NO:34.

[0055] Embodiment 47 is the method of any one of embodiments 17 to 39, wherein the CAR is an anti-CD19 CAR.

[0056] Embodiment 48 is the method of embodiment 47, wherein the anti-CD19 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45.

[0057] Embodiment 49 is the method of embodiment 47 or embodiment 48, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 45.

[0058] Embodiment 50 is the method of embodiment 47, wherein the anti-CD19 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47.

[0059] Embodiment 51 is the method of embodiment 47 or 50, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 47.

[0060] Embodiment 52 is the method of any one of embodiments 17 to 46, wherein the CAR is an anti-CD19 / CD22 CAR.

[0061] Embodiment 53 is the method of embodiment 52, wherein the anti-CD19 / CD22 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43.

[0062] Embodiment 54 is the method of embodiment 52 or 53, wherein the anti-CD19 / CD22 CAR comprises the amino acid sequence of SEQ ID NO: 43.

[0063] Embodiment 55 is the method of any one of embodiments 17 to 20, wherein the CAR is an anti-BCMA CAR.

[0064] Embodiment 56 is the method of embodiment 55, wherein the anti-BCMA CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57.

[0065] Embodiment 57 is the method of embodiment 55 or embodiment 56, wherein the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 57.

[0066] Embodiment 58 is the method of embodiment 55, wherein the anti-BCMA CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 67.

[0067] Embodiment 59 is the method of embodiment 55 or embodiment 56, wherein the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 67.

[0068] Embodiment 60 is the method of any one of embodiments 17 to 20, wherein the CAR comprises an anti-BCMA domain comprising an anti-BCMA heavy chain variable domain (VH) and an anti-BCMA light chain variable domain (VL).

[0069] Embodiment 61 is the method of embodiment 60, wherein the anti-BCMA VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51.

[0070] Embodiment 62 is the method of embodiment 60 or 61, wherein the anti-BCMA VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52.

[0071] Embodiment 63 is the method of any one of embodiments 60 to 62, wherein the anti-BCMA VH comprises the amino acid sequence of SEQ ID NO: 52.

[0072] Embodiment 64 is the method of any one of embodiments 60 to 63, wherein the anti-BCMA VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 55.

[0073] Embodiment 65 is the method of any one of embodiments 60 to 64, wherein the anti-BCMA VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 56.

[0074] Embodiment 66 is the method of any one of embodiments 60 to 65, wherein the anti-BCMA VL comprises the amino acid sequence of SEQ ID NO: 56.

[0075] Embodiment 67 is a method for treating a CAR comprising administering to a subject a first anti-BCMA single domain antibody (V H H) and / or a second anti-BCMA V H 21. The method of any one of embodiments 17 to 20, comprising an anti-BCMA domain comprising H.

[0076] Embodiment 68 is directed to a first anti-BCMA V H 68. The method of embodiment 67, wherein H comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61.

[0077] Embodiment 69 is directed to a first anti-BCMA V H 69. The method of embodiment 67 or 68, wherein H comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 62.

[0078] Embodiment 70 is directed to a first anti-BCMA V H 70. The method of any one of embodiments 67-69, wherein H comprises the amino acid sequence of SEQ ID NO: 62.

[0079] Embodiment 71 is directed to a second anti-BCMA V H 71. The method of any one of embodiments 67 to 70, wherein H comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 63, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65.

[0080] Embodiment 72 is directed to a second anti-BCMA V H 72. The method of any one of embodiments 67 to 71, wherein H comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66.

[0081] Embodiment 73 is directed to a second anti-BCMA V H 73. The method of any one of embodiments 67-72, wherein H comprises the amino acid sequence of SEQ ID NO: 66.

[0082] In embodiment 74, CAR therapy is administered in a dose of 0.2 to 5.0 × 10 per kg of subject body weight. 6 or 0.5 to 1.0 x 10 6 74. The method of any one of embodiments 13 to 73, comprising cells.

[0083] Embodiment 75 is an embodiment in which the CAR therapy is administered in a dose of 1.0 x 10 per kg of subject body weight. 6 or 2.0 x 10 6 75. The method of any one of embodiments 13 to 74, comprising cells.

[0084] In embodiment 76, CAR therapy is 0.1 to 2.5 × 10 8 or 0.6 to 6.0 x 10 8 76. The method of any one of embodiments 13 to 75, comprising cells.

[0085] In embodiment 77, CAR therapy is 0.9 to 1.1 × 10 8 pieces, 0.5~1.1×10 8 pieces, or 3.0 to 4.6 x 10 8 77. The method of any one of embodiments 13 to 76, comprising cells.

[0086] Embodiment 78 is the method of any one of embodiments 16 to 77, wherein the CAR therapy is administered to the subject by intravenous administration.

[0087] Embodiment 79 is the method of any one of embodiments 1-7 and 16-78, wherein the CAR therapy is administered to the subject before administering any dose of the IL-2 conjugate to the subject.

[0088] Embodiment 80 is the method of any one of embodiments 1-7 and 16-78, wherein one dose of the IL-2 conjugate is administered to the subject before the CAR therapy is administered to the subject, and one or more additional doses of the IL-2 conjugate are administered to the subject after the CAR therapy is administered to the subject.

[0089] Embodiment 81 is the method of any one of embodiments 1-7 and 16-78, wherein one dose of the IL-2 conjugate is administered to the subject one day before the CAR therapy is administered to the subject, and one or more additional doses of the IL-2 conjugate are administered to the subject about once per week after the one dose of the IL-2 conjugate is administered to the subject.

[0090] Embodiment 82 is the method of any one of embodiments 1 to 81, comprising administering about 16 μg / kg of IL-2 as an IL-2 conjugate to the subject.

[0091] Embodiment 83 is the method of any one of embodiments 1 to 81, comprising administering about 24 μg / kg of IL-2 as an IL-2 conjugate to the subject.

[0092] Embodiment 84 is the method of any one of embodiments 1 to 81, comprising administering about 32 μg / kg of IL-2 as an IL-2 conjugate to the subject.

[0093] Embodiment 85 is the method of any one of embodiments 1 to 84, wherein the PEG group in the IL-2 conjugate has an average molecular weight of about 30 kDa.

[0094] Embodiment 86 is an IL-2 conjugate wherein Z is CH2 and Y is [ka] 86. The method of any one of embodiments 1 to 85, wherein:

[0095] Embodiment 87 is an IL-2 conjugate wherein Y is CH2 and Z is [ka] 86. The method of any one of embodiments 1 to 85, wherein:

[0096] Embodiment 88 is an IL-2 conjugate wherein Z is CH2 and Y is [ka] 86. The method of any one of embodiments 1 to 85, wherein:

[0097] Embodiment 89 is an IL-2 conjugate wherein Y is CH2 and Z is [ka] 86. The method of any one of embodiments 1 to 85, wherein:

[0098] Embodiment 90 is directed to a compound having the structure of formula (I) having the structure of formula (IV) or formula (V), or a mixture of formula (IV) and formula (V): [ka] (In the formula, q is 1, 2, or 3; X has the structure: [ka] is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue) 86. The method of any one of embodiments 1 to 85, wherein:

[0099] Embodiment 91 is an embodiment of the present invention, wherein the structure of formula (I) has the structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII): [ka] (In the formula, n is -(OCH2CH2) n is an integer such that -OCH3 has a molecular weight of about 30 kDa; q is 1, 2, or 3, and The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 1 that is not substituted. 86. The method of any one of embodiments 1 to 85, wherein:

[0100] Embodiment 92 is the method of any one of embodiments 1 to 91, wherein q is 1.

[0101] Embodiment 93 is the method of any one of embodiments 1 to 91, wherein q is 2.

[0102] Embodiment 94 is the method of any one of embodiments 1 to 91, wherein q is 3.

[0103] Embodiment 95 is the method of any one of embodiments 1 to 94, wherein the IL-2 conjugate is administered to the subject about once a week, about once every two weeks, about once every three weeks, or about once every four weeks.

[0104] Embodiment 96 is the method of embodiment 95, wherein the IL-2 conjugate is administered to the subject about once every three weeks.

[0105] Embodiment 97 is the method of any one of embodiments 1 to 96, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate or hydrate.

[0106] Embodiment 98 is the method of any one of embodiments 1 to 97, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

[0107] Embodiment 99 is the method of any one of embodiments 1 to 97, wherein the IL-2 conjugate is administered to the subject by subcutaneous administration.

[0108] Embodiment 100 is the method of any one of embodiments 1 to 99, further comprising administering acetaminophen to the subject.

[0109] Embodiment 101 is the method of any one of embodiments 1 to 100, further comprising administering diphenhydramine to the subject.

[0110] Embodiment 102 is the method of embodiment 100 or 101, wherein acetaminophen and / or diphenhydramine is administered to the subject prior to administering the IL-2 conjugate.

[0111] Embodiment 103 is an IL-2 conjugate for use in a method according to any one of embodiments 1 to 102.

[0112] Embodiment 104 is the use of an IL-2 conjugate for the manufacture of a medicament for a method according to any one of embodiments 1 to 102.

[0113] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0114] [Figure 1A]

[0023] Figure 1 shows the change in peripheral CD8+ Teff counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. Herein and elsewhere, designations such as "C1D1" refer to the treatment cycle and day (e.g., treatment cycle 1, day 1). "PRE" refers to the baseline measurement before administration, and 24HR refers to 24 hours after administration. [Figure 1B] Figure 1 shows peak peripheral CD8+ Teff cell proliferation after administration of a first dose of 24 μg / kg [Q3W] IL-2 conjugate. Data are normalized to pre-treatment (C1D1) CD8+ T cell counts. [Figure 1C] 1 shows peripheral CD8+ Teff cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. [Figure 2] Shown is the percentage of CD8+ Teffs expressing Ki67 in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 3A]1 shows the change in CD8+ memory cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 3B] 1 shows the number of CD8+ memory cells in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 4A] 1 shows the change in peripheral natural killer (NK) cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 4B] Figure 1 shows peak peripheral NK cell proliferation after administration of a first dose of 24 μg / kg [Q3W] IL-2 conjugate. Data are normalized to pre-treatment (C1D1) NK cell counts. [Figure 4C] 1 shows the change in peripheral natural killer (NK) cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 4D] 1 shows peripheral natural killer (NK) cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 5] Shown is the percentage of NK cells expressing ki67 in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 6A] 1 shows the change in peripheral CD4+ Treg counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. [Figure 6B] Figure 1 shows peak peripheral CD4+ Treg cell proliferation after administration of a first dose of 24 μg / kg [Q3W] IL-2 conjugate. Data are normalized to pre-treatment (C1D1) CD4+ T cell counts. [Figure 6C] 1 shows peripheral CD4+ Treg cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. [Figure 7] Shown is the percentage of CD4+ TJs expressing Ki67 in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 8A] 1 shows the change in eosinophil cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 8B] Figure 1 shows peak peripheral eosinophil cell proliferation after administration of a first dose of 24 μg / kg [Q3W] IL-2 conjugate. Data are normalized to pre-treatment (C1D1) eosinophil cell counts. [Figure 8C] 1 shows eosinophil cell counts in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 9A] Serum levels of IFN-γ, IL-5, and IL-6 are shown in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 9B] Figure 1 shows serum levels of IL-5 after administration of 24 μg / kg [Q3W] of IL-2 conjugate. BLQ = below limit of quantification. Data are plotted as mean (range BLQ - max). [Figure 9C] Figure 1 shows serum levels of IL-6 after administration of 24 μg / kg [Q3W] of IL-2 conjugate. BLQ = below limit of quantification. Data are plotted as mean (range BLQ - max). [Figure 10] Serum levels of the indicated cytokines in the indicated subjects treated with 8 μg / kg [Q3W] at the indicated times after administration of the IL-2 conjugate are shown. [Figure 11]Serum levels of the indicated cytokines in the indicated subjects treated with IL-2 conjugate at 16 μg / kg [Q3W] at the indicated times after administration are shown. [Figure 12A] Shown are eosinophil cell counts, as measured by cytometry or CBC (complete blood count), in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 13A] 1 shows lymphocyte counts, as measured by cytometry or CBC, in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 14A] Peripheral CD8+ Teff counts are shown in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated times after administration of the IL-2 conjugate. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 15A] Shown is the percentage of CD8+ Teff cells expressing Ki67 in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 15B] Shown is the percentage of CD8+ Teff cells expressing Ki67 in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 16A]Peripheral memory CD8+ counts are shown in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated times after administration of the IL-2 conjugate. [Figure 16B] Peripheral memory CD8+ counts are shown in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated times after administration of the IL-2 conjugate. [Figure 17A] Peripheral natural killer (NK) cell counts are shown in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 18A] Shown is the percentage of NK cells expressing Ki67 in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 18B] Shown is the percentage of NK cells expressing Ki67 in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 19A] Peripheral CD4+ Treg counts are shown in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated times after administration of the IL-2 conjugate. [Figure 19B] Peripheral CD4+ Treg counts are shown in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated times after administration of the IL-2 conjugate. [Figure 20A] Shown is the percentage of CD4+ Treg cells expressing Ki67 in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 20B]Shown is the percentage of CD4+ Treg cells expressing Ki67 in the indicated subjects treated with 8 μg / kg [Q3W] or 16 μg / kg [Q3W] at the indicated time points after administration of the IL-2 conjugate. [Figure 21A] 1 shows the change in peripheral CD8+ Teff counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. [Figure 21B] 1 shows peripheral CD8+ Teff cell counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. [Figure 22A] 1 shows the change in CD8+ memory cell counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 22B] 1 shows the number of CD8+ memory cells in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 23A] 1 shows the change in peripheral natural killer (NK) cell counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 23B] 1 shows peripheral natural killer (NK) cell counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 24A] 1 shows the change in peripheral CD4+ Treg counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. [Figure 24B] 1 shows peripheral CD4+ Treg cell counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at specified times after administration of the IL-2 conjugate. [Figure 25A] 1 shows the change in eosinophil cell counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 25B] 1 shows eosinophil cell counts in the indicated subjects treated with 32 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 26] Serum levels of IFN-γ, IL-5, and IL-6 are shown in the indicated subjects treated with 24 μg / kg [Q3W] of IL-2 conjugate at the indicated times after administration of the IL-2 conjugate. [Figure 27A] Figure 1 shows the cell count in a co-culture of CAR-T cells and large B-cell lymphoma cells. [Figure 27B] Flow cytometry data of cells in co-cultures are shown, which demonstrate an increased proportion of effector memory (EM) cells. [Figure 28] Shows the mean radiance in mice implanted with Raji-Luc cells and treated with CAR-T cells and IL-2 conjugates. [Figure 29] 1 shows radiance imaging in mice. [Figure 30] The changes in mouse body weight are shown. [Figure 31] Shown is the copy number of the CAR-T construct in mice as measured by droplet digital polymerase chain reaction (ddPCR). [Figure 32A] Shows the mean radiance in mice implanted with Raji-Luc cells and treated with CAR-T cells and IL-2 conjugates. [Figure 32B] The changes in mouse body weight are shown. [Figure 33] The amount of CD4+ CAR-T cells and CD8+ CAR-T cells in mice is shown. [Figure 34] The amounts of different cell types in the mice are shown. [Figure 34-1] Same as above. [Figure 34-2] Same as above. [Figure 35] The copy number of the CAR-T construct as determined by ddPCR is shown. [Figure 36] CAR expression in NK cells from two donors is shown. [Figure 37] 1 shows the killing of Nalm6 cancer cells by CAR-NK cells in the presence of IL-2 conjugates. [Figure 37-1] Same as above. [Figure 38] Shows IFN-γ production from co-cultures of CAR-NK cells and Nalm6 cells in the presence of IL-2 conjugates. [Figure 38-1] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0115] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of any claimed subject matter. To the extent that any material incorporated herein by reference is inconsistent with the language of the present disclosure, the language shall control. In this application, the use of the singular includes the plural unless expressly stated otherwise. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless the context otherwise requires. Furthermore, the use of the term "including" and other forms such as "include," "includes," and "included" is not limiting.

[0116] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is present in at least some embodiments, but not necessarily all embodiments of the present invention.

[0117] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About includes the exact amount. Thus, "about 5 μL" means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error, such as within 15%, 10%, or 5%.

[0118] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way.

[0119] As used herein, the terms "subject" and "patient" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. Neither term requires or is limited to a situation characterized by the supervision (e.g., constant or intermittent) of a healthcare professional (e.g., a physician, registered nurse, practice nurse, physician assistant, nursing assistant, or hospice worker).

[0120] As used herein, terms such as "unnatural amino acid" refer to an amino acid other than one of the 20 naturally occurring amino acids. Exemplary unnatural amino acids are described in Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry, 285(15):11039-11044 (2010), the disclosure of which is incorporated herein by reference.

[0121] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0122] As used herein, "nucleotide" refers to a compound comprising a nucleoside moiety and a phosphate moiety. Exemplary naturally occurring nucleotides include adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), and guanosine monophosphate (GMP), deoxyadenosine triphosphate (dATP), deoxythymidine Deoxyribonucleotides include, but are not limited to, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary naturally occurring deoxyribonucleotides containing deoxyribose as the sugar moiety include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary naturally occurring ribonucleotides that contain ribose as the sugar moiety include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.

[0123] As used herein, "base" and "nucleobase" refer to at least the nucleobase portion of a nucleoside or nucleotide (nucleosides and nucleotides include ribovariants or deoxyribovariants), which in some cases may contain further modifications to the sugar portion of the nucleoside or nucleotide. In some cases, "base" is also used to refer to the entire nucleoside or nucleotide (e.g., a "base" can be incorporated into DNA by a DNA polymerase or into RNA by an RNA polymerase). However, terms such as "base" should not be construed as necessarily referring to the entire nucleoside or nucleotide unless the context requires otherwise. In the chemical structures of a base or nucleobase provided herein, only the base of the nucleoside or nucleotide is shown, and for clarity, the sugar portion and, optionally, any phosphate residues are omitted. As used in the chemical structures of a base or nucleobase provided herein, a wavy line represents the bond to the nucleoside or nucleotide, and the sugar portion of the nucleoside or nucleotide may be further modified. In some embodiments, the wavy line represents the attachment of a base or nucleobase to a sugar moiety, such as a pentose, of a nucleoside or nucleotide. In some embodiments, the pentose is a ribose or deoxyribose.

[0124] In some embodiments, a nucleobase is generally the heterocyclic base portion of a nucleoside. A nucleobase may be naturally occurring, modified, or may bear no similarity to a natural base, and / or may be synthesized, for example, by organic synthesis. In certain embodiments, a nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide that can interact with a base of another nucleic acid, with or without the use of hydrogen bonds. In certain embodiments, a non-natural nucleobase is not derived from a natural nucleobase. Note that non-natural nucleobases are not necessarily basic, but are referred to as nucleobases for simplicity. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase can be attached to deoxyguanosine or ribose, while "d" without a parenthetical indicates that the nucleobase is attached to deoxyribose.

[0125] As used herein, a "nucleoside" is a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (found in DNA and RNA), basic nucleosides, modified nucleosides, and nucleosides with mimetic base and / or sugar groups. Nucleosides include nucleosides containing any of a variety of substituents. A nucleoside may be a glycosidic compound formed by a glycosidic bond between a nucleobase and a reducing group of a sugar.

[0126] As used herein, an "analog" of a chemical structure refers to a chemical structure that retains substantial similarity to the parent structure, even though it may not be readily synthetically derived from the parent structure. In some embodiments, a nucleotide analog is a non-naturally occurring nucleotide. In some embodiments, a nucleoside analog is a non-naturally occurring nucleotide. Related chemical structures that are readily synthetically derived from the parent chemical structure are referred to as "derivatives."

[0127] As used herein, a "dose-limiting toxicity" (DLT) is defined as an adverse event occurring within a defined time frame of a treatment cycle (e.g., within ±1 day of days 1 to 29, inclusive) that is not clearly related or related only to clearly unrelated causes and that meets one or more of the criteria set forth in Example 2 for a DLT.

[0128] As used herein, "severe cytokine release syndrome" refers to level 4 or 5 cytokine release syndrome as described in Teachey et al., Cancer Discov. 2016;6(6);664-79, the disclosure of which is incorporated herein by reference.

[0129] As used herein, "chimeric antigen receptor" or "CAR" describes a fusion protein that comprises an extracellular antigen-binding component, which may be a single-chain variable fragment (scFv or sFv) derived from a VH and a VL (e.g., a monoclonal antibody, Fab, etc.), fused to a transmembrane domain and an intracellular signaling domain that can activate or stimulate an immune cell.

[0130] As used herein, the assignment of amino acids in a complementarity-determining region (CDR) to a heavy chain variable domain (VH) or a light chain variable domain (VL) is determined by the amino acid sequence determined by the complementarity-determining region (CDR) as ... As defined in Molecular Biology 262:732-745.

[0131] As used herein, "percent identity" and related terms refer to a quantitative measure of similarity between two polypeptide or two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions shared by the two polypeptide sequences, taking into account the number of gaps and the length of each gap that may need to be introduced to optimize the alignment of the two polypeptide sequences. Similarly, the percent identity between two polynucleotide sequences is a function of the number of identical amino acids at aligned positions shared by the two polynucleotide sequences, taking into account the number of gaps and the length of each gap that may need to be introduced to optimize the alignment of the two polynucleotide sequences. Comparison of sequences and determination of percent identity between two polypeptide or two polynucleotide sequences can be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences can be determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. A phrase such as "comprises a sequence having at least X% identity to Y" with respect to a test sequence means that the test sequence, when aligned to sequence Y as described above, contains residues that are identical to at least X% of the residues in Y.

[0132] While various features of the invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein for clarity in the context of separate embodiments, the invention may also be practiced in a single embodiment.

[0133] IL-2 conjugates Interleukin-2 (IL-2) is a pleiotropic type 1 cytokine whose structure contains a four-α-helical bundle of 15.5 kDa. The precursor form of IL-2 is 153 amino acid residues in length, with the first 20 amino acids forming a signal peptide and residues 21–153 forming the mature form. IL-2 is produced primarily by CD4+ T cells after antigen stimulation, but to a lesser extent by CD8+ cells, natural killer (NK) and natural killer T (NKT) cells, activated dendritic cells (DCs), and mast cells. IL-2 signaling occurs through interaction with a specific combination of IL-2 receptor (IL-2R) subunits: IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132). The interaction of IL-2 with IL-2Rα is approximately 10 -8 K of M d The interaction of IL-2 with IL-2Rβ and IL-2Rγ forms a “low affinity” IL-2 receptor complex with approximately 10 -9 K of M d The interaction of IL-2 with all three subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, is approximately 10 -11 Super M K d The IL-2 receptor forms a "high affinity" IL-2 receptor having the following structure:

[0134] In some cases, IL-2 signaling through the "high affinity" IL-2Rαβγ complex regulates the activation and proliferation of regulatory T cells. Regulatory T cells, or CD4 + CD25 + Foxp3 + Regulatory T (Treg) cells are CD4 + T cells, CD8 +Treg cells mediate immune homeostasis by suppressing effector cells such as T cells, B cells, NK cells, and NKT cells. In some cases, Treg cells are generated from the thymus (tTreg cells) or derived from peripheral naive T cells (pTreg cells). In some cases, Treg cells are considered mediators of peripheral tolerance. In fact, one study showed that CD25-depleted peripheral CD4 + The transfer of T cells caused various autoimmune diseases in nude mice, but CD4 + CD25 + Co-transfer of T cells suppressed the development of autoimmunity (Sakaguchi, et al., "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)," J. Immunol. 155(3):1151-1164 (1995), the disclosure of which is incorporated herein by reference). Expanding the Treg cell population down-regulates the proliferation of effector T cells and suppresses autoimmunity and T cell anti-tumor responses.

[0135] IL-2 signaling through the "intermediate affinity" IL-2Rβγ complex is conserved among CD8 + Regulates the activation and proliferation of effector T (Teff) cells, NK cells, and NKT cells. CD8 + Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, Tcon or killer T cells) are T lymphocytes that recognize and kill damaged cells, cancer cells and pathogen-infected cells. NK cells and NKT cells are CD8 + Teff cells are a type of lymphocyte that target cancer cells and pathogen-infected cells.

[0136] In some cases, IL-2 signaling is used to regulate T cell responses and subsequently utilized for cancer treatment. For example, IL-2 is administered in high-dose forms to induce the expansion of Teff cell populations to treat cancer. However, high-dose IL-2 also results in costimulation of Treg cells, which reduces anti-tumor immune responses. High-dose IL-2 induces toxic adverse events mediated by the binding of IL-2Rα chain-expressing cells in the vasculature, including type 2 innate immune cells (ILC-2), eosinophils, and endothelial cells. This results in eosinophilia, capillary leak syndrome, and vascular leak syndrome (VLS).

[0137] Provided herein is a method of treating diffuse large B-cell lymphoma (DLBCL) in a subject in need of such treatment, comprising administering an IL-2 conjugate to the subject.

[0138] In some embodiments, the IL-2 sequence is SEQ ID NO:1: [ka] Including, wherein the amino acid at position P64 is of formula (I): [ka] (In the formula, Z is CH2 and Y is [ka] and Y is CH2 and Z is [ka] and Z is CH2 and Y is [ka] or Y is CH2 and Z is [ka] and W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa; q is 1, 2, or 3; X has the structure: [ka] is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue) is replaced by the structure

[0139] In any of the embodiments or variations of Formula (I) described herein, the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. In some embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt. In some embodiments, the IL-2 conjugate is a solvate. In some embodiments, the IL-2 conjugate is a hydrate.

[0140] In some embodiments of Formula (I), Z is CH2 and Y is [ka] is. In some embodiments of Formula (I), Y is CH2 and Z is [ka] In some embodiments of Formula (I), Z is CH2 and Y is [ka] In some embodiments of Formula (I), Y is CH2 and Z is [ka] is.

[0141] In some embodiments of Formula (I), q is 1. In some embodiments of Formula (I), q is 2. In some embodiments of Formula (I), q is 3.

[0142] In some embodiments of Formula (I), W is a PEG group having an average molecular weight of about 25 kDa. In some embodiments of Formula (I), W is a PEG group having an average molecular weight of about 30 kDa. In some embodiments of Formula (I), W is a PEG group having an average molecular weight of about 35 kDa.

[0143] In some embodiments of Formula (I), q is 1 and the structure of Formula (I) is Formula (Ia): [ka] (In the formula, Z is CH2 and Y is [ka] and Y is CH2 and Z is [ka] and Z is CH2 and Y is [ka] or Y is CH2 and Z is [ka] and W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa; X has the structure: [ka] is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue) The structure is as follows.

[0144] In some embodiments of Formula (Ia), Z is CH2 and Y is [ka] In some embodiments of Formula (Ia), Y is CH2 and Z is [ka] In another embodiment of Formula (Ia), Z is CH2 and Y is [ka] In some embodiments of Formula (Ia), Y is CH2 and Z is [ka] is.

[0145] In some embodiments of Formula (Ia), the PEG group has an average molecular weight of about 30 kDa.

[0146] In some embodiments, the IL-2 conjugate is SEQ ID NO:2: [ka] wherein [AzK_L1_PEG30kD] is N6-((2-azidoethoxy)-carbonyl)-L-lysine that is stably conjugated to PEG via DBCO-mediated click chemistry to form a compound comprising the structure of Formula (IV) or Formula (V), where q is 1 (e.g., Formula (IVa) or Formula (Va)), and the PEG group has an average molecular weight of about 25-35 kDa (e.g., about 30 kDa) capped with a methoxy group. The term "DBCO" refers to chemical moieties that contain a dibenzocyclooctyne group, including, for example, the mPEG-DBCO compounds shown in Schemes 1 and 2 of Example 1.

[0147] The ratio of regioisomers produced from the Click reaction is about 1:1 or greater than 1:1.

[0148] PEG typically comprises several (OCH2CH2) monomers (or CH2CHO monomers, depending on how PEG is defined). In some embodiments, the number of (OCH2CH2) monomers (or (CH2CHO) monomers) is such that the average molecular weight of the PEG group is about 30 kDa.

[0149] In some cases, PEG is an end-capped polymer, i.e., a polymer containing a relatively inert group, e.g., a lower C 1~6 A PEG group is a polymer having at least one terminus capped with an alkoxy or hydroxyl group. In some embodiments, the PEG group is a methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG in which one terminus of the polymer is a methoxy (-OCH3) group and the other terminus is a hydroxyl or other functional group that may be chemically modified.

[0150] In some embodiments, the PEG group is a linear or branched PEG group. In some embodiments, the PEG group is a linear PEG group. In some embodiments, the PEG group is a branched PEG group. In some embodiments, the PEG group is a methoxy PEG group. In some embodiments, the PEG group is a linear or branched methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG group. In some embodiments, the PEG group is a branched methoxy PEG group. For example, IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da ± 3,000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 5,000 Da are included within the scope of the present disclosure.

[0151] In some embodiments, the IL-2 conjugate has the structure of formula (IV) or formula (V), or a mixture of formulas (IV) and (V), at amino acid residue P64: [ka] (In the formula, W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa; q is 1, 2, or 3, and X has the structure: [ka] and X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue) The amino acid sequence of SEQ ID NO: 1 is substituted by:

[0152] In some embodiments of Formula (IV) or Formula (V), or mixtures of Formula (IV) or Formula (V), q is 1. In some embodiments of Formula (IV) or Formula (V), or mixtures of Formula (IV) or Formula (V), q is 2. In some embodiments of Formula (IV) or Formula (V), or mixtures of Formula (IV) or Formula (V), q is 3.

[0153] In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), W is a PEG group having an average molecular weight of about 25 kDa. In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), W is a PEG group having an average molecular weight of about 30 kDa. In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) or Formula (V), W is a PEG group having an average molecular weight of about 35 kDa.

[0154] In any embodiment described herein, the structure of Formula (I) has the structure of Formula (IV) or Formula (V), or is a mixture of Formula (IV) and Formula (V). In some embodiments, the structure of Formula (I) has the structure of Formula (IV). In some embodiments, the structure of Formula (I) has the structure of Formula (V). In some embodiments, the structure of Formula (I) is a mixture of Formula (IV) and Formula (V).

[0155] In some embodiments of Formula (IV) or Formula (V), or a mixture of Formula (IV) and Formula (V), q is 1, and the structure of Formula (IV) is the structure of Formula (IVa) and the structure of Formula (V) is the structure of Formula (Va): [ka] (In the formula, W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa, and X has the structure: [ka] and X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue) is.

[0156] In some embodiments of Formula (IVa) or Formula (Va), or a mixture of Formula (IVa) and Formula (Va), the PEG group has an average molecular weight of about 30 kDa.

[0157] In any embodiment described herein, the structure of Formula (I) has the structure of Formula (IVa) or Formula (Va), or is a mixture of Formula (IVa) and Formula (Va). In some embodiments, the structure of Formula (I) has the structure of Formula (IVa). In some embodiments, the structure of Formula (I) has the structure of Formula (Va). In some embodiments, the structure of Formula (I) is a mixture of Formula (IVa) and Formula (Va).

[0158] In some embodiments, the IL-2 conjugate has a structure in which amino acid residue P64 has the structure of formula (XII) or formula (XIII), or a mixture of formulas (XII) and (XIII): [ka] (In the formula, n is -(OCH2CH2) n -OCH3 is an integer such that the molecular weight is between about 25 kDa and 35 kDa; q is 1, 2, or 3, and The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 1 that is not substituted. The amino acid sequence of SEQ ID NO: 1 is substituted by:

[0159] In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), q is 1. In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), q is 2. In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), q is 3.

[0160] In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), n is —(OCH2CH2) n is an integer such that -OCH3 has a molecular weight of approximately 30 kDa.

[0161] In any embodiment described herein, the structure of Formula (I) has the structure of Formula (XII) or Formula (XIII), or is a mixture of Formula (XII) and Formula (XIII). In some embodiments, the structure of Formula (I) has the structure of Formula (XII). In some embodiments, the structure of Formula (I) has the structure of Formula (XIII). In some embodiments, the structure of Formula (I) is a mixture of Formula (XII) and Formula (XIII).

[0162] In some embodiments of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), q is 1, and the structure of Formula (XII) is the structure of Formula (XIIa), and the structure of Formula (XIII) is the structure of Formula (XIIIa): [ka] (In the formula, n is -(OCH2CH2) n -OCH3 is an integer such that the molecular weight is between about 25 kDa and 35 kDa; and The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 1 that is not substituted. is.

[0163] In some embodiments of Formula (XIIa) or Formula (XIIIa), or a mixture of Formula (XIIa) and Formula (XIIIa), n is —(OCH2CH2) n is an integer such that -OCH3 has a molecular weight of approximately 30 kDa.

[0164] In any embodiment described herein, the structure of Formula (I) has the structure of Formula (XIIa) or Formula (XIIIa), or is a mixture of Formula (XIIa) and Formula (XIIIa). In some embodiments, the structure of Formula (I) has the structure of Formula (XIIa). In some embodiments, the structure of Formula (I) has the structure of Formula (XIIIa). In some embodiments, the structure of Formula (I) is a mixture of Formula (XIIa) and Formula (XIIIa).

[0165] In some embodiments, the IL-2 conjugate has a structure in which amino acid residue P64 has the structure of formula (XIV) or formula (XV), or a mixture of formulas (XIV) and (XV): [ka] (In the formula, m is an integer from 0 to 20; p is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa to 35 kDa, and The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 1 that is not substituted. The amino acid sequence of SEQ ID NO: 1 is substituted by:

[0166] In some embodiments of Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV), n is an integer such that the PEG groups have an average molecular weight of about 30 kDa.

[0167] In some embodiments, m is an integer from 0 to 15. In some embodiments, m is an integer from 0 to 10. In some embodiments, m is an integer from 0 to 5. In some embodiments, m is an integer from 1 to 5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

[0168] In some embodiments, p is an integer from 0 to 15. In some embodiments, p is an integer from 0 to 10. In some embodiments, p is an integer from 0 to 5. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is 1. In some embodiments, p is 2. In some embodiments, p is 3. In some embodiments, p is 4. In some embodiments, p is 5.

[0169] In some embodiments, m and p are each 2.

[0170] In any embodiment described herein, the structure of Formula (I) has the structure of Formula (XIV) or Formula (XV), or is a mixture of Formula (XIV) and Formula (XV). In some embodiments, the structure of Formula (I) has the structure of Formula (XIV). In some embodiments, the structure of Formula (I) has the structure of Formula (XV). In some embodiments, the structure of Formula (I) is a mixture of Formula (XIV) and Formula (XV).

[0171] In some embodiments, the IL-2 conjugate has a structure in which amino acid residue P64 has the structure of formula (XVI) or formula (XVII), or a mixture of formulas (XVI) and (XVII): [ka] (In the formula, m is an integer from 0 to 20; n is an integer such that the PEG group has an average molecular weight of about 25 kDa to 35 kDa, and The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 1 that is not substituted. The amino acid sequence of SEQ ID NO: 1 is substituted by:

[0172] In some embodiments of Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), n is an integer such that the PEG groups have an average molecular weight of about 30 kDa.

[0173] In some embodiments, m is an integer from 0 to 15. In some embodiments, m is an integer from 0 to 10. In some embodiments, m is an integer from 0 to 5. In some embodiments, m is an integer from 1 to 5. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5.

[0174] In any embodiment described herein, the structure of Formula (I) has the structure of Formula (XVI) or Formula (XVII), or is a mixture of Formula (XVI) and Formula (XVII). In some embodiments, the structure of Formula (I) has the structure of Formula (XVI). In some embodiments, the structure of Formula (I) has the structure of Formula (XVII). In some embodiments, the structure of Formula (I) is a mixture of Formula (XVI) and Formula (XVII).

[0175] Conjugation Chemistry In some embodiments, the IL-2 conjugates described herein can be prepared by a conjugation reaction comprising a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises the reaction of an azide with an alkyne (a "click" reaction). In some embodiments, the conjugation reaction described herein comprises the reaction outlined in Scheme I, where X is an unnatural amino acid at position P64 of SEQ ID NO:1. Scheme I. [ka]

[0176] In some embodiments, the conjugate moiety comprises a PEG group as described herein, hi some embodiments, the reactive group comprises an alkyne or an azide.

[0177] In some embodiments, the conjugation reactions described herein include the reaction outlined in Scheme II, where X is an unnatural amino acid at position P64 of SEQ ID NO:1. Scheme II. [ka]

[0178] In some embodiments, the conjugation reactions described herein include the reaction outlined in Scheme III, where X is an unnatural amino acid at position P64 of SEQ ID NO:1. Scheme III. [ka]

[0179] In some embodiments, the conjugation reactions described herein include the reaction outlined in Scheme IV, where X is an unnatural amino acid at position P64 of SEQ ID NO:1. Scheme IV. [ka]

[0180] In some embodiments, the conjugation reactions described herein involve a cycloaddition reaction between an azide moiety, such as those found in proteins containing amino acid residues derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as those derived from DBCO, a chemical moiety containing a dibenzocyclooctyne group. PEG groups containing DBCO moieties are commercially available or can be prepared by methods known to those skilled in the art. Exemplary reactions are shown in Schemes V and VI. Scheme V. [ka] Scheme VI. [ka]

[0181] Conjugation reactions, such as the Click reactions described herein, can produce a single positional isomer or a mixture of positional isomers. In some cases, the ratio of positional isomers is about 1:1. In some cases, the ratio of positional isomers is about 2:1. In some cases, the ratio of positional isomers is about 1.5:1. In some cases, the ratio of positional isomers is about 1.2:1. In some cases, the ratio of positional isomers is about 1.1:1. In some cases, the ratio of positional isomers is greater than 1:1.

[0182] IL-2 polypeptide production In some cases, the IL-2 conjugates described herein containing either natural or non-natural amino acid mutations are recombinantly produced or chemically synthesized. In some cases, the IL-2 conjugates described herein are recombinantly produced, for example, by either a host cell system or a cell-free system.

[0183] In some cases, the IL-2 conjugate is recombinantly produced via a host cell system. In some cases, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell) or a prokaryotic cell (e.g., a gram-positive or gram-negative bacterium). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is a stable cell line, or a cell line that has integrated the genetic material of interest into its genome and is capable of expressing the product of the genetic material after many generations of cell division. In other cases, the mammalian host cell is a transient cell line, or a cell line that has not integrated the genetic material of interest into its genome and is not capable of expressing the product of the genetic material after many generations of cell division.

[0184] Exemplary mammalian host cells include 293T cell line, 293A cell line, 293FT cell line, 293F cells, 293H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F(TM) cells, Flp-In(TM) T-REx(TM) 293 cell line, Flp-In(TM)-293 cell line, Flp-In(TM)-3T3 cell line, Flp-In(TM)-BHK cells strain, Flp-In(TM)-CHO cell line, Flp-In(TM)-CV-1 cell line, Flp-In(TM)-Jurkat cell line, FreeStyle(TM) 293-F cells, FreeStyle(TM) CHO-S cells, GripTite(TM) 293 MSR cell line, GS-CHO cell line, HepaRG™ cell line, T-REx™ Jurkat cell line, Per.C6 cell line, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[0185] In some embodiments, the eukaryotic host cell is an insect host cell. Exemplary insect host cells include Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[0186] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris (K. phaffii) yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0187] In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, the plant cell comprises a cell derived from algae. Exemplary plant cell lines include strains derived from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[0188] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB survivor, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0189] In some cases, suitable polynucleic acid molecules or vectors for producing the IL-2 polypeptides described herein include any suitable vector derived from either eukaryotic or prokaryotic sources. Exemplary polynucleic acid molecules or vectors include those derived from bacterial (e.g., E. coli), insect, yeast (e.g., Pichia pastoris, K. phaffii), algae, or mammalian sources. Examples of bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, PRSET A, PRSET B, PRSET C, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, and pTAC-MAT-2.

[0190] Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[0191] Yeast vectors include, for example, Gateway® PDEST™ 14 vector, Gateway® PDEST™ 15 vector, Gateway® PDEST™ 17 vector, Gateway® PDEST™ 24 vector, Gateway® pYES-DEST52 vector, pBAD-DEST49 Gateway® destination vector, pAO815 Pichia vector, pFLD1 Pichia pastoris (K. phaffii) vector, pGAPZA, B, & C Pichia pastoris (K. phaffii) vector, pPIC3.5K Pichia vector, pPIC6 Examples include A, B, &C Pichia vectors, pPIC9K Pichia vectors, pTEF1 / Zeo, pYES2 yeast vectors, pYES2 / CT yeast vectors, pYES2 / NT A, B, &C yeast vectors, or pYES3 / CT yeast vectors.

[0192] Algal vectors include, for example, pChlamy-4 vectors or MCS vectors.

[0193] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, b, c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b, c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1 or pBICEP-CMV 2.

[0194] In some cases, cell-free systems are used for the production of the IL-2 polypeptides described herein. In some cases, the cell-free system comprises a mixture of cytoplasmic and / or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some cases, the cell-free system utilizes prokaryotic components. In other cases, the cell-free system utilizes eukaryotic components. Nucleic acid synthesis is obtained in cell-free systems based on, for example, Drosophila cells, Xenopus eggs, archaea, or HeLa cells. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or PURExPRess®, XPRessCF, and XPRessCF+.

[0195] Cell-free translation systems include a variety of components, such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and / or unnatural amino acids, and / or other components used in protein expression. Such components are optionally modified to improve yield, increase synthesis rate, increase fidelity of the protein product, or incorporate unnatural amino acids. In some embodiments, cytokines described herein are synthesized using the cell-free translation system described in U.S. Patent No. 8,778,631, U.S. Patent Application Publication No. 2017 / 0283469, U.S. Patent Application Publication No. 2018 / 0051065, U.S. Patent Application Publication No. 2014 / 0315245, or U.S. Patent No. 8,778,631 (the disclosures of each of which are incorporated herein by reference). In some embodiments, the cell-free translation system includes modified release factors or even the removal of one or more release factors from the system. In some embodiments, the cell-free translation system comprises a reduced protease concentration. In some embodiments, the cell-free translation system comprises a modified tRNA with a reassigned codon used to encode the unnatural amino acid. In some embodiments, a synthetic enzyme described herein for incorporating an unnatural amino acid is used in the cell-free translation system. In some embodiments, the tRNA is preloaded with the unnatural amino acid using enzymatic or chemical methods before being added to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from a modified organism, such as a modified bacterium, yeast, or other organism.

[0196] In some embodiments, the IL-2 polypeptide is produced in a circularly permuted form, either via an expression host system or a cell-free system.

[0197] Production of cytokine polypeptides containing unnatural amino acids The present disclosure allows for the use of an orthogonal or expanded genetic code in which one or more specific codons present in the nucleic acid sequence of an IL-2 polypeptide are assigned to encode an unnatural amino acid, which can then be genetically incorporated into IL-2 by using an orthogonal tRNA synthetase / tRNA pair that can add the unnatural amino acid to a tRNA, which can incorporate the unnatural amino acid into the polypeptide chain in response to a codon.

[0198] In some cases, the codon is an amber, ochre, opal, or quadruple codon. In some cases, the codon corresponds to an orthogonal tRNA used to deliver the unnatural amino acid. In some cases, the codon is an amber. In other cases, the codon is an orthogonal codon.

[0199] In some cases, the codon is a quadruple codon that can be decoded by the orthogonal ribosome riboQ1. In some cases, the quadruple codon is as shown in Neumann, et al., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature, 464(7287):441-444 (2010), the disclosure of which is incorporated herein by reference.

[0200] In some cases, the codons used in this disclosure are recoded codons, e.g., rare codons replaced with synonymous codons or alternative codons. In some cases, the recoded codons are as described in Napolitano, et al., "Emergent rules for codon checked by editing rare arginine codons in Escherichia coli," PNAS, 113(38):E5588-5597 (2016), the disclosure of which is incorporated herein by reference. In some cases, the recoded codons are as described in Ostrov et al., "Design, synthesis, and testing toward a 57-codon genome," Science 353(6301):819-822 (2016), the disclosure of which is incorporated herein by reference.

[0201] In some cases, unnatural nucleic acids are utilized, and one or more unnatural amino acids are incorporated into IL-2. Exemplary unnatural nucleic acids include uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine. cytosine, 6-azouracil, cytosine and thymine, 5-uracil (psouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, 5-methylcytosine, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine Cytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][l,4]benzoxazin-2(3H)-one), ... nothiazine cytidine (1H-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamp, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), in which the purine or pyrimidine base is replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio- Thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and the amino acids described in U.S. Pat. Nos. 3,687,808, 4,845,205, 4,910,300, 4,948,882, 5,093,232, 5,130,302, 5,134,066, 5,175,273, and 5,367,066.Nos. 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653, and 6,005,096, WO 99 / 62923, Kandimalla et al. al., (2001) Bioorg. Med. Chem. 9:807-813, The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Additional base modifications can be found, for example, in U.S. Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993, the disclosures of each of which are incorporated herein by reference.

[0202] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and nucleic acids sometimes contain one or several heterocyclic bases other than the five major base components of naturally occurring nucleic acids. For example, heterocyclic bases in some cases include uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, and 2-amino-4-oxopyrrolo[2.3-d]pyrimidin-3-yl groups, in which purines are linked to the sugar moiety of the nucleic acid through the 9-position, pyrimidines through the 1-position, pyrrolopyrimidines through the 7-position, and pyrazolopyrimidines through the 1-position.

[0203] In some embodiments, nucleotide analogs are also modified at the phosphate moiety. Modified phosphate moieties include those with modifications at the bond between two nucleotides, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and 3'-alkylene phosphonates and other alkyl phosphonates, including chiral phosphonates, phosphinates, 3'-amino phosphoramidates, and phosphoramidates, such as aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate or modified phosphate bonds between two nucleotides are understood to be via a 3'-5' or 2'-5' bond, and the bond may be reversed, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Numerous U.S. patents teach methods for making and using nucleotides containing modified phosphates, including U.S. Patents 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,196, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, and 5,405,939. Nos. 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, and 5,625,050, the disclosures of each of which are incorporated herein by reference.

[0204] In some embodiments, non-naturally occurring nucleic acids include 2',3'-dideoxy-2',3'-didehydro-nucleosides (PCT / US2002 / 006460), 5'-substituted DNA and RNA derivatives (PCT / US2011 / 033961, Saha et al., J. Org Chem., 1995, 60, 788-789, Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890, and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343, Leonid et al., 1995, 14(3-5), 901-905, and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020, PCT / JP2000 / 004720, PCT / JP2003 / 002342, PCT / JP2004 / 013216, PCT / JP2005 / 020435, PCT / JP2006 / 315479, PCT / JP2006 / 324484, PCT / JP2009 / 056718, PCT / JP2010 / 067560), or 5'-substituted monomers made as monophosphates with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23(1&2), 317-337), the disclosures of each of which are incorporated herein by reference.

[0205] In some embodiments, unnatural nucleic acids contain modifications at the 5' and 2' positions of the sugar ring (PCT / US94 / 02993), such as 5'-CH2-substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some cases, unnatural nucleic acids include amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, in which the 3'-linked nucleoside in the dimer (5' to 3') contains 2'-OCH3 and 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids can include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Non-natural nucleic acids can include 5'-methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids can include 5'-phosphonate monomers with 2' substitutions (U.S. Patent Application Publication No. 2006 / 0074035) and other modified 5'-phosphonate monomers (WO 1997 / 35869). The non-natural nucleic acids may include 5' modified methylene phosphonate monomers (EP 614907 and EP 629633).Non-naturally occurring nucleic acids can include 5'- or 6'-phosphonate ribonucleoside analogs containing hydroxyl groups at the 5' and / or 6' positions (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033). Non-naturally occurring nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers with a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids may include nucleosides having a 6'-phosphonate group that is unsubstituted or substituted at the 5' and / or 6' positions with a thio-tert-butyl group (SC(CH3)3) (and its analogs), a methyleneamino group (CH2NH2) (and its analogs), or a cyano group (CN) (and its analogs) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al. al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc., 1983, 105(26), 7638-7640; and Hampton et al., J. Am. Chem. Soc., 1973, 95(13), 4404-4414. The disclosure of each reference listed in this paragraph is incorporated herein by reference.

[0206] In some embodiments, non-natural nucleic acids also include modifications to the sugar moiety. In some cases, the nucleic acid includes one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer improved nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, the nucleic acid includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include substituents (including 5' and / or 2' substituents), bridging of two ring atoms to form bicyclic nucleic acids (BNAs), and substitution of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, C1-C2). 12 Examples of chemically modified sugars include, but are not limited to, alkyl, hydroxyl, hydroxypropyl ...

[0207] In some cases, modified nucleic acids contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be linked to the respective heterocyclic base in either the [α] or [β] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs, and the respective "nucleosides" in which such sugars or analogs are attached to a heterocyclic base (nucleobase), is known. Sugar modifications can be made or combined with other modifications.

[0208] Modifications to the sugar moiety include natural and non-natural modifications of ribose and deoxyribose. Sugar modifications include the following modifications at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl (where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6 alkyl groups). 10 , alkyl or C2-C 10 2' sugar modifications include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n ONH2 and -O(CH2) nExamples include, but are not limited to, ON[(CH2)nCH3)]2, where n and m are from 1 to about 10.

[0209] Other modifications at the 2' position include C1-C 10Modified sugars include, but are not limited to, lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties of an oligonucleotide, or groups that improve the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar of the 3'-terminal nucleotide or 2'-5'-linked oligonucleotides and the 5' position of the 5'-terminal nucleotide. Modified sugars also include those containing modifications to the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar.Numerous United States patents exist that teach the preparation of such modified sugar structures and detail and describe the range of base modifications, e.g., U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,5 Specification No. 14,785, Specification No. 5,519,134, Specification No. 5,567,811, Specification No. 5,576,427, Specification No. 5,591,722, Specification No. 5,597,9 Specification No. 09, Specification No. 5,610,300, Specification No. 5,627,053, Specification No. 5,639,873, Specification No. 5,646,265, Specification No. 5,658,873 5,670,633, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,175,273, Specification No. 5,367,066, Specification No. 5,432,272, Specification No. 5,457,187, Specification No. 5,459,255, Specification No. 5,484,908, Specification No. 5,502 ,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,681,941, and 5,700,920, the disclosures of each of which are incorporated herein by reference.

[0210] Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH, and 2'-O(CH)OCH substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-(C1-C2). 1O alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ) can also be selected from each Rm and R n are independently H or substituted or unsubstituted C1-C 10 It is alkyl.

[0211] In certain embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acids comprise a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids in which the bridge comprises a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include those of the following formula: 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2', and analogs thereof (see U.S. Pat. No. 7,399,845), 4'-C(CH3)(CH3)-O-2' and analogs thereof (see WO 2009 / 006478, WO 2008 / 150729, U.S. Pat. Appl. Publ. No. 2004 / 0171570, U.S. Pat. No. 7,427,672, Chattopadhyaya et al. al., J. Org. Chem., 209, 74, 118-134 and WO 2008 / 154401). Also, for example, Singh et al.,Chem.Commun.,1998,4,455-456, Koshkin et al.,Tetrahedron,1998,54,3607-3630, Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638, Kumar et al. al.,Bioorg.Med.Chem.Lett.,1998,8,2219-2222, Singh et al.,J.Org.Chem.,1998,63,10035-10039, Srivastava et al.,J.Am.Chem.Soc.,2007,129(26)8362-8379,Elayadi et al. al.,Curr.Opinion Invens.Drugs,2001,2,558-561, Braasch et al.,Chem.Biol,2001,8,1-7,Oram et al.,Curr.Opinion Mol.Ther.,2001,3,239-243, U.S. Patent Nos. 4,849,513, 5,015,733, 5,118,800, 5,118,802, 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845, WO 2004 / 106356, WO 1994 / 14226, WO 2005 / 02157 0, WO 2007 / 090071, and WO 2007 / 134181, U.S. Patent Application Publication Nos. 2004 / 0171570, 2007 / 0287831, and 2008 / 0039618, U.S. Provisional Patent Application Nos. 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, and 61 / 099,844, and International Application Nos. PCT / US2008 / 064591, PCT See US2008 / 066154, PCT US2008 / 068922, and PCT / DK98 / 00393. The disclosure of each reference listed in this paragraph is incorporated herein by reference.

[0212] In certain embodiments, nucleic acids include linked nucleic acids. Nucleic acids can be linked together using any internucleic acid linkage. Two major classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleic acid linkages having chiral atoms can be prepared as separate enantiomers, such as alkylphosphonates and phosphorothioates, or as racemic mixtures. Non-naturally occurring nucleic acids can contain a single modification, or they can contain multiple modifications within one moiety or between different moieties.

[0213] Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or unbridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate bridges may also be used.

[0214] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity to the modified nucleic acids and / or enhance their stability in vivo.

[0215] In some cases, the phosphorus derivative (or modified phosphate group) is attached to a sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like. Exemplary polynucleotides containing modified phosphate bridges or non-phosphate bridges are described in Peyrottes et al., 1996, Nucleic Acids Res. 24:1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, "Oligonucleotide Analogs: an Overview," Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.), John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates," Protocols for Oligonucleotides and Analogs, Synthesis and Procedures, Humana Press, pp. 165-190; Miller et al. al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Pat. No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem. 8:1157-1179, the disclosures of each of which are incorporated herein by reference.

[0216] In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include the following: anionic internucleoside linkages, N3'-P5' phosphoramidate modifications, boranophosphate DNA, prooligonucleotides, neutral internucleoside linkages such as methylphosphonates, amide-linked DNA, methylene (methylimino) linkages, formal and thioformal linkages, backbones containing sulfonyl groups, morpholino oligos, peptide nucleic acids (PNAs), and positively charged deoxyribonucleic guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8:1157-1179, the disclosure of which is incorporated herein by reference). Modified nucleic acids can also include chimeric or mixed backbones containing one or more modifications, such as combinations of phosphate bridges, e.g., combinations of phosphodiester and phosphorothioate bridges.

[0217] Phosphate substitutes include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonic acid and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties. Numerous U.S. patents disclose how to make and use these types of phosphate substitutes, including U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,6 77, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439. It is also understood that in nucleotide substitutes both the sugar and phosphate moieties of the nucleotide can be replaced, for example, by an amide type bond (aminoethylglycine) (PNA).U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262 teach how to make and use PNA molecules, and are each incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. Other types of molecules (conjugates) can also be linked to nucleotides or nucleotide analogs, for example, to enhance cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs.Such conjugates include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973, or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).Numerous United States patents teach the preparation of such conjugates, including U.S. Pat. Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138 ,045 specification, 5,414,077 specification, 5,486,603 specification, 5,512,439 specification, 5,5 Specification No. 78,718, Specification No. 5,608,046, Specification No. 4,587,044, Specification No. 4,605,735, Specification No. 4, Specification No. 667,025, Specification No. 4,762,779, Specification No. 4,789,737, Specification No. 4,824,941, Specification No. Specification No. 4,835,263, Specification No. 4,876,335, Specification No. 4,904,582, Specification No. 4,958,013, Specification No. Specification No. 5,082,830, Specification No. 5,112,963, Specification No. 5,214,136, Specification No. 5,082,830 , Specification No. 5,112,963, Specification No. 5,214,136, Specification No. 5,245,022, Specification No. 5,254,469 Specification, Specification No. 5,258,506, Specification No. 5,262,536, Specification No. 5,272,250, Specification No. 5,292,873 Specification No. 5,317,098, Specification No. 5,371,241, Specification No. 5,391,723, Specification No. 5,416,20 Nos. 3, 5,451,463, 5,510,475, 5,512,667, 5,514,785, 5,565,552, 5,567,810, 5,574,142, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, and 5,688,941.The disclosure of each reference listed in this paragraph is incorporated herein by reference.

[0218] In some cases, unnatural nucleic acids also form unnatural base pairs. Exemplary unnatural nucleotides that can form unnatural DNA or RNA base pairs (UBPs) under in vivo conditions include, but are not limited to, TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. In some embodiments, the unnatural nucleotides include: [ka] Exemplary unnatural base pairs include (D)TPT3-(d)NaM, (d)5SICS-(d)NaM, (d)CNMO-(d)TAT1, (d)NaM-(d)TAT1, (d)CNMO-(d)TPT3, (d)5FM-(d)TAT1.

[0219] Other examples of unnatural nucleotides capable of forming unnatural UBPs that can be used to prepare the IL-2 conjugates disclosed herein include those described in Dien et al., J Am Chem Soc., 2018, 140:16115-16123; Feldman et al., J Am Chem Soc., 2017, 139:11427-11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res. 2014, 42:10235-10244; Malyshev et al., Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc. Soc. 2013, 135:5408-5419 and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010, the disclosures of each of which are incorporated herein by reference. In some embodiments, the non-natural nucleotides include: [ka] Examples include:

[0220] In some embodiments, non-naturally occurring nucleotides that may be used to prepare the IL-2 conjugates disclosed herein have the formula: [ka] wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide; and The wavy line indicates a bond to a ribosyl or 2'-deoxyribosyl, where the 5'-hydroxy group of the ribosyl or 2'-deoxyribosyl moiety may be in the free form, attached to a monophosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or may be derived from RNA or DNA or an RNA or DNA analog.

[0221] In some embodiments, non-naturally occurring nucleotides that may be used to prepare the IL-2 conjugates disclosed herein have the formula: [ka] (In the formula, each X is independently carbon or nitrogen; R2 is absent when X is nitrogen and present when X is carbon and is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azido; Y is sulfur, oxygen, selenium, or a secondary amine; E is oxygen, sulfur, or selenium, and The wavy line indicates the point of attachment to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety or analog thereof, which may be in the free form, attached to a mono-phosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or derived from RNA or DNA or an RNA or DNA analog.

[0222] In some embodiments, each X is carbon. In some embodiments, at least one X is carbon. In some embodiments, one X is carbon. In some embodiments, at least two X are carbon. In some embodiments, two X are carbon. In some embodiments, at least one X is nitrogen. In some embodiments, one X is nitrogen. In some embodiments, at least two X are nitrogen. In some embodiments, two X are nitrogen.

[0223] In some embodiments, Y is sulfur. In some embodiments, Y is oxygen. In some embodiments, Y is selenium. In some embodiments, Y is a secondary amine.

[0224] In some embodiments, E is sulfur. In some embodiments, E is oxygen. In some embodiments, E is selenium.

[0225] In some embodiments, R2 is present when X is carbon. In some embodiments, when X is nitrogen, R 2 is absent. In some embodiments, each R2, if present, is hydrogen. In some embodiments, R2 is alkyl, e.g., methyl, ethyl, or propyl. In some embodiments, R2 is alkenyl, e.g., —CH2═CH2. In some embodiments, R2 is alkynyl, e.g., ethynyl. In some embodiments, R2 is methoxy. In some embodiments, R2 is methanethiol. In some embodiments, R2 is methaneseleno. In some embodiments, R2 is halogen, e.g., chloro, bromo, or fluoro. In some embodiments, R2 is cyano. In some embodiments, R2 is azide.

[0226] In some embodiments, E is sulfur, Y is sulfur, and each X is independently carbon or nitrogen. In some embodiments, E is sulfur, Y is sulfur, and each X is carbon.

[0227] In some embodiments, non-naturally occurring nucleotides that may be used to prepare the IL-2 conjugates disclosed herein include: [ka] In some embodiments, non-naturally occurring nucleotides that may be used to prepare the IL-2 conjugates disclosed herein include: [ka] or a salt thereof.

[0228] In some embodiments, the unnatural base pair generates an unnatural amino acid as described in Dumas et al., "Designing logical codon reassignment - Expanding the chemistry in biology," Chemical Science, 6:50-69 (2015), the disclosure of which is incorporated herein by reference.

[0229] In some embodiments, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) via synthetic codons comprising unnatural nucleic acids. In some cases, unnatural amino acids are incorporated into cytokines via orthogonal modified synthetase / tRNA pairs. Such orthogonal pairs include a non-natural synthetase that can charge a non-natural tRNA with an unnatural amino acid while minimizing a) charging of other endogenous amino acids onto the non-natural tRNA and b) charging of other endogenous tRNAs with the unnatural amino acid. Such orthogonal pairs include a tRNA that can be charged by the non-natural synthetase while avoiding charging of other endogenous amino acids by the endogenous synthetase. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, an orthogonal synthetase / tRNA pair includes components from a single organism. In some embodiments, an orthogonal synthetase / tRNA pair includes components from two different organisms. In some embodiments, an orthogonal synthetase / tRNA pair includes components that facilitate translation of two different amino acids prior to modification. In some embodiments, the orthogonal synthetase is a modified alanine synthetase. In some embodiments, the orthogonal synthetase is a modified arginine synthetase. In some embodiments, the orthogonal synthetase is a modified asparagine synthetase. In some embodiments, the orthogonal synthetase is a modified aspartate synthetase. In some embodiments, the orthogonal synthetase is a modified cysteine ​​synthetase. In some embodiments, the orthogonal synthetase is a modified glutamine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamate synthetase. In some embodiments, the orthogonal synthetase is a modified alanineglycine synthetase. In some embodiments, the orthogonal synthetase is a modified histidine synthetase. In some embodiments, the orthogonal synthetase is a modified leucine synthetase. In some embodiments, the orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, the orthogonal synthetase is a modified lysine synthetase. In some embodiments, the orthogonal synthetase is a modified methionine synthetase. In some embodiments, the orthogonal synthetase is a modified phenylalanine synthetase.In some embodiments, the orthogonal synthetase is a modified proline synthase. In some embodiments, the orthogonal synthetase is a modified serine synthase. In some embodiments, the orthogonal synthetase is a modified threonine synthase. In some embodiments, the orthogonal synthetase is a modified tryptophan synthase. In some embodiments, the orthogonal synthetase is a modified tyrosine synthase. In some embodiments, the orthogonal synthetase is a modified valine synthase. In some embodiments, the orthogonal synthetase is a modified phosphoserine synthase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartate tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine ​​tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine glycine. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

[0230] In some embodiments, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) by aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pairs. Exemplary aaRS-tRNA pairs include the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair and the E. coli TyrRS (Ec-Tyr) / B. stearothermophilus tRNA pair. CUA E. coli LeuRS (Ec-Leu) vs. B. stearothermophilus tRNA CUA Examples of UAAs that can be incorporated by Mj-TyrRS / tRNA pairs include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine, meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine, phenylselenocysteine, p-boronophenylalanine, and o-nitrobenzyltyrosine. In some cases, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) by Mj-TyrRS / tRNA pairs. Exemplary UAAs that can be incorporated by Mj-TyrRS / tRNA pairs include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine, meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine, phenylselenocysteine, p-boronophenylalanine, and o-nitrobenzyltyrosine.

[0231] In some cases, the unnatural amino acid is Ec-Tyr / tRNA CUA or Ec-Leu / tRNA CUA Ec-Tyr / tRNA pair is incorporated into cytokines (e.g., IL polypeptides). CUA or Ec-Leu / tRNA CUA Exemplary UAAs that can be incorporated by pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents, O-propargyl tyrosine, α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine, and 3-(naphthalen-2-ylamino)-2-amino-propanoic acid.

[0232] In some cases, unnatural amino acids are incorporated into cytokines (e.g., IL polypeptides) via pyrrolysine-tRNA pairs. In some cases, PylRS is obtained from archaea, e.g., methanogenic archaea. In some cases, PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated via pyrrolysine-tRNA pairs include amide and carbamate substituted lysines, e.g., 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N-ε- D -Prolyl- L -Lysine and N-ε-cyclopentyloxycarbonyl- L -Lysine, N-ε-acryloyl- L -Lysine, N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]- L N-(2-azidoethoxy)-carbonyl)-l-lysine and N-ε-(1-methylcyclopropyl-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein may be prepared by using M. mazei tRNA selectively charged with an unnatural amino acid, such as n6-((2-azidoethoxy)-carbonyl)-l-lysine (Azk), via M. barkeri pyrrolysyl-tRNA synthetase (Mb pylrs). Other methods, such as those disclosed in Zhang et al., Nature 2017, 551(7682):644-647, are known to those of skill in the art, the disclosure of which is incorporated herein by reference.

[0233] In some cases, unnatural amino acids are incorporated into the cytokines (e.g., IL polypeptides) described herein by synthetic enzymes as disclosed in U.S. Pat. No. 9,988,619 and U.S. Pat. No. 9,938,516, the disclosures of each of which are incorporated herein by reference.

[0234] Host cells into which a construct or vector disclosed herein is introduced are cultured or maintained in an appropriate medium to produce tRNA, tRNA synthetase, and the protein of interest. The medium also includes an unnatural amino acid so that the protein of interest incorporates the unnatural amino acid. In some embodiments, a nucleoside triphosphate transporter (NTT) from bacteria, plants, or algae is also present in the host cells. In some embodiments, the IL-2 conjugates disclosed herein are prepared using host cells that express NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter used in the host cell is TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulfuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis), trachomatis), PamNTT1, PamNTT2 (Protochlamia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some embodiments, an NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, an NTT is PtNTT1. In some embodiments, an NTT is PtNTT2. In some embodiments, an NTT is PtNTT3. In some embodiments, an NTT is PtNTT4. In some embodiments, an NTT is PtNTT5. In some embodiments, an NTT is PtNTT6.Other NTTs that can be used are disclosed in Zhang et al., Nature 2017, 551(7682):644-647, Malyshev et al. Nature 2014(509(7500), 385-388, and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322.

[0235] Orthogonal tRNA synthetase / tRNA pairs charge tRNAs with unnatural amino acids and incorporate the unnatural amino acids into polypeptide chains in response to codons. Exemplary aaRS-tRNA pairs include the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair and the Escherichia coli (E. coli) TyrRS (Ec-Tyr) / B. stearothermophilus tRNA pair. CUA E. coli LeuRS (Ec-Leu) vs. B. stearothermophilus tRNA CUA Other aaRS-tRNA pairs that can be used according to the present disclosure include, but are not limited to, those obtained from M. mazei, as described in Feldman et al., J Am Chem Soc., 2018 140:1447-1454, and Zhang et al., Proc Natl Acad Sci USA, 2017, 114:1317-1322, the disclosures of each of which are incorporated herein by reference.

[0236] In some embodiments, methods are provided for preparing the IL-2 conjugates disclosed herein in a cell line expressing an NTT and a tRNA synthetase. In some embodiments described herein, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr) / B. stearothermophilus, and M. mazei. In some embodiments, the NTT is PtNTT1 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT2 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei.In some embodiments, the NTT is PtNTT4 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT5 and the tRNA synthetase is from Methanococcus jannaschii, E. coli TyrRS(Ec-Tyr) / B. stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii, E. coli TyrRS (Ec-Tyr) / B. stearothermophilus, or M. mazei.

[0237] In some embodiments, the IL-2 conjugates disclosed herein can be prepared in cells such as E. coli containing (a) the nucleotide triphosphate transporter PtNTT2 (including a truncated mutant in which the first 65 amino acid residues of the full-length protein are deleted), (b) a plasmid comprising a double-stranded oligonucleotide encoding an IL-2 variant having a desired amino acid sequence and comprising an unnatural base pair comprising a first unnatural nucleotide and a second unnatural nucleotide to provide a codon at a desired position for incorporation of an unnatural amino acid, such as n6-((2-azidoethoxy)-carbonyl)-L-lysine (azk), (c) a plasmid encoding a tRNA derived from M. mazei and comprising an unnatural nucleotide to provide an anticodon (for the codon of the IL-2 variant) recognized in place of the natural sequence, and (D) a plasmid encoding a pyrrolysine-tRNA synthetase (mb pylrs) from M. barkeri, which may be the same plasmid as the tRNA or a different plasmid. In some embodiments, the cells are further supplemented with deoxyribotriphosphates comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with ribotriphosphates comprising one or more unnatural bases. In some embodiments, the cells are further supplemented with one or more unnatural amino acids, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the double-stranded oligonucleotide encoding the amino acid sequence of a desired IL-2 variant comprises the codon AXC at position 64 of the sequence encoding the protein having SEQ ID NO:1, where X is an unnatural nucleotide. In some embodiments, the cells further comprise a plasmid, which may be a protein expression plasmid or another plasmid, encoding an orthogonal tRNA gene from M. mazei that comprises the AXC-matching anticodon GYT in place of its native sequence, where Y is complementary and is an unnatural nucleotide that may be the same or different from the unnatural nucleotide in the codon.In some embodiments, the unnatural nucleotide in the codon is different from and complementary to the unnatural nucleotide in the anticodon. In some embodiments, the unnatural nucleotide in the codon is the same as the unnatural nucleotide in the anticodon. In some embodiments, the first and second unnatural nucleotides comprising an unnatural base pair in the double-stranded oligonucleotide are [ka] In some embodiments, the first and second unnatural nucleotides comprising the unnatural base pair in the double-stranded oligonucleotide can be derived from: [ka] In some embodiments, the triphosphates of the first and second non-natural nucleotides can be derived from: [ka] or a salt thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides include: [ka] or a salt thereof. In some embodiments, the mRNA derived from the double-stranded oligonucleotide comprising the first non-natural nucleotide and the second non-natural nucleotide is [ka] In some embodiments, the M. mazei tRNA may contain a codon containing a non-natural nucleotide obtained from an mRNA. In some embodiments, the M. mazei tRNA may contain an anticodon containing a non-natural nucleotide that recognizes a codon containing a non-natural nucleotide in the mRNA. The anticodon of the M. mazei tRNA may be [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] In some embodiments, the tRNA may include unnatural nucleotides obtained from [ka] In some embodiments, the tRNA may include unnatural nucleotides obtained from [ka] In some embodiments, the tRNA may include unnatural nucleotides obtained from [ka] In some embodiments, the tRNA may include unnatural nucleotides obtained from [ka] In some embodiments, the tRNA may include unnatural nucleotides obtained from [ka] In some embodiments, the tRNA may include unnatural nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] and the tRNA may comprise unnatural nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] and the tRNA may comprise unnatural nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] and the tRNA may comprise unnatural nucleotides obtained from [ka] In some embodiments, the mRNA may include non-naturally occurring nucleotides obtained from [ka] and the tRNA may comprise unnatural nucleotides obtained from [ka] The host cells may contain unnatural nucleotides obtained from (a) deoxyribonucleoside triphosphates containing one or more unnatural bases necessary for replication of the plasmid encoding the cytokine gene having codons, (b) ribonucleoside triphosphates containing one or more unnatural bases necessary for transcription of (i) mRNA corresponding to the cytokine coding sequence and containing codons containing one or more unnatural bases, and (ii) tRNA containing anticodons containing one or more unnatural bases, and (c) unnatural amino acids to be incorporated into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that allow for expression of the protein of interest.

[0238] The resulting expressed AzK-containing protein can be purified by methods known to those of skill in the art and then reacted with an alkyne, such as DBCO, containing a PEG chain having the desired average molecular weight as disclosed herein under conditions known to those of skill in the art to obtain the IL-2 conjugates disclosed herein. Other methods are known to those skilled in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682):644-647, WO 2015157555, WO 2015021432, WO 2016115168, WO 2017106767, WO 2017223528, WO 2019014262, WO 2019014267, WO 2019028419, and WO 2019 / 028425, the disclosures of each of which are incorporated herein by reference.

[0239] The resulting protein containing one or more expressed unnatural amino acids, e.g., Azk, can be purified by methods known to those of skill in the art and then reacted with an alkyne, e.g., a DBCO containing a PEG chain having a desired average molecular weight as disclosed herein, under conditions known to those of skill in the art to provide an IL-2 conjugate as disclosed herein. Other methods are known to those skilled in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682):644-647, WO 2015157555, WO 2015021432, WO 2016115168, WO 2017106767, WO 2017223528, WO 2019014262, WO 2019014267, WO 2019028419, and WO 2019 / 028425, the disclosures of each of which are incorporated herein by reference.

[0240] Alternatively, IL-2 polypeptides containing unnatural amino acids are prepared by introducing into host cells a nucleic acid construct described herein that includes a tRNA and an aminoacyl-tRNA synthetase and a nucleic acid sequence of interest with one or more in-frame orthogonal (stop) codons. The host cells are cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases necessary for replication of a plasmid encoding a cytokine gene containing a new codon and anticodon, (b) ribonucleoside triphosphates necessary for transcription of mRNA corresponding to (i) the cytokine sequence containing the codon and (ii) the orthogonal tRNA containing the anticodon, and (c) the unnatural amino acid. The host cells are then maintained under conditions that allow expression of the protein of interest. The unnatural amino acid is incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more unnatural amino acids are incorporated into an IL-2 polypeptide. Alternatively, two or more unnatural amino acids can be incorporated into an IL-2 polypeptide at two or more sites in the protein.

[0241] Once produced in a host cell, the IL-2 polypeptide comprising an unnatural amino acid can be extracted therefrom by a variety of techniques known in the art, including enzymatic, chemical, and / or osmotic lysis and physical disruption. The IL-2 polypeptide can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic chromatography, affinity chromatography, or any other suitable technique known to those of skill in the art.

[0242] Suitable host cells include bacterial cells (e.g., E. coli, BL21(DE3)), but most suitable host cells are eukaryotic cells, such as insect cells (e.g., Drosophila, such as Drosophila melanogaster), yeast cells, nematodes (e.g., C. elegans), mouse (e.g., Mus musculus) or mammalian cells (e.g., Chinese hamster ovary cells (CHO) or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells and murine erythroleukemia (MEL) cells) or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Suitably, the host cell is a mammalian cell, such as a human cell or an insect cell. In some embodiments, a suitable host cell comprises E. coli.

[0243] Other suitable host cells that can be commonly used in embodiments of the present invention are those mentioned in the Examples section. Vector DNA can be introduced into host cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various well-known techniques for introducing foreign nucleic acid molecules (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

[0244] When creating a cell line, it is generally preferable to prepare a stable cell line. For example, for stable transfection of mammalian cells, it is known that only a small proportion of cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a gene encoding a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, or methotrexate. A nucleic acid molecule encoding a selectable marker can be introduced into the host cells on the same vector or on a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g., cells that have integrated the selectable marker gene survive, while other cells are killed).

[0245] In one embodiment, the constructs described herein are integrated into the genome of a host cell. An advantage of stable integration is that uniformity between individual cells or clones is achieved. Another advantage is that selection of the best producers can be performed. Therefore, it is desirable to generate stable cell lines. In another embodiment, the constructs described herein are transfected into host cells. An advantage of transfecting the constructs into host cells is that protein yield can be maximized. In one aspect, cells comprising the nucleic acid constructs or vectors described herein are described.

[0246] Chimeric antigen receptor therapy In one embodiment, the chimeric antigen receptor (CAR) therapy described in any of the methods provided herein comprises T cells expressing a CAR. In one embodiment, the CAR therapy described in any of the methods provided herein comprises gamma delta T cells expressing a CAR. In one embodiment, the CAR therapy described in any of the methods provided herein comprises natural killer (NK) T cells expressing a CAR. In one embodiment, the CAR therapy described in any of the methods provided herein comprises natural killer (NK) cells expressing a CAR. In one embodiment, the CAR binds to CD19, CD22, CD19 and CD22, or B-cell maturation antigen (BCMA). In one embodiment, the CAR binds to CD20, claudin 6, or Her2. In some embodiments, the immune cells used in the CAR therapy described herein can be autologous. In some embodiments, the immune cells used in the CAR therapy described herein can be allogeneic. In some embodiments, the immune cells used in the CAR therapy described herein can be "off-the-shelf" immune cells.

[0247] In one embodiment, a CAR (e.g., expressed by a T cell, a gamma delta T cell, an NK T cell, or an NK cell) comprises an anti-CD19 domain comprising an anti-CD19 heavy chain variable domain (VH) and an anti-CD19 light chain variable domain (VL). In one embodiment, the anti-CD19 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5. In one embodiment, the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the anti-CD19 VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9. In one embodiment, the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10. In one embodiment, the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO: 10.

[0248] In one embodiment, a CAR (e.g., expressed by a T cell, a gamma delta T cell, an NK T cell, or an NK cell) comprises an anti-CD19 domain, including, for example, an anti-CD19 VH and an anti-CD19 VL. In one embodiment, the anti-CD19 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 13. In one embodiment, the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD19 VH comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD19 VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO: 18.

[0249] In one embodiment, a CAR (e.g., expressed by a T cell, a gamma delta T cell, a NK T cell, or a NK cell) comprises an anti-CD19 domain comprising an anti-CD19 VH and an anti-CD19 VL. In one embodiment, the anti-CD19 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD19 VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 25. In one embodiment, the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 26. In one embodiment, the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO:26.

[0250] In one embodiment, in addition to or instead of comprising an anti-CD19 domain, a CAR (e.g., expressed by a T cell, gamma delta T cell, NK T cell, or NK cell) comprises an anti-CD22 domain, including, for example, an anti-CD22 VH and an anti-CD22 VL. In one embodiment, the anti-CD22 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29. In one embodiment, the anti-CD22 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30. In one embodiment, the anti-CD22 VH comprises the amino acid sequence of SEQ ID NO: 30. In one embodiment, the anti-CD22 VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33. In one embodiment, the anti-CD22 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34. In one embodiment, the anti-CD22 VL comprises the amino acid sequence of SEQ ID NO: 34.

[0251] In one embodiment, instead of comprising an anti-CD19 domain or an anti-CD22 domain, the CAR (e.g., expressed by a T cell, gamma delta T cell, NK T cell, or NK cell) comprises an anti-B cell maturation antigen (BCMA) domain, e.g., an anti-BCMA VH and an anti-BCMA VL, or a first anti-bcma single domain antibody (V H H) and / or a second anti-BCMA V HIn one embodiment, the anti-BCMA VH comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51. In one embodiment, the anti-BCMA VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52. In one embodiment, the anti-BCMA VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 55. In one embodiment, the anti-BCMA VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 56. In one embodiment, the anti-BCMA VL comprises the amino acid sequence of SEQ ID NO: 56. In one embodiment, the CAR comprises a first anti-BCMA VL H H and / or a second anti-BCMA V H In one embodiment, the first anti-BCMA domain comprises an anti-BCMA V H In one embodiment, the first anti-BCMA V comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61. H H comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 62. In one embodiment, the first anti-BCMA V H H comprises the amino acid sequence of SEQ ID NO: 62. In one embodiment, the second anti-BCMA V H In one embodiment, the second anti-BCMA V comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 63, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65. H H comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 66. In one embodiment, the second anti-BCMA V HH comprises the amino acid sequence of SEQ ID NO:66.

[0252] In one embodiment, a CAR (e.g., expressed by a T cell, a gamma delta T cell, a NK T cell, or a NK cell) comprises a CD8 alpha chain comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, e.g., SEQ ID NO: 35, 36, 69, or 70. In one embodiment, a CAR comprises a CD8 alpha chain comprising the amino acid sequence of SEQ ID NO: 35, 36, 69, or 70. In one embodiment, a CAR comprises a 4-1BB domain comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37 or 38. In one embodiment, a CAR comprises a 4-1BB domain comprising the amino acid sequence of SEQ ID NO: 37 or 38. In one embodiment, the CAR comprises a CD28 domain comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39. In one embodiment, the CAR comprises a CD28 domain comprising the amino acid sequence of SEQ ID NO: 39. In one embodiment, the CAR comprises a CD137 domain comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 71. In one embodiment, the CAR comprises a CD137 domain comprising the amino acid sequence of SEQ ID NO: 71. In one embodiment, the CAR comprises a CD3 zeta domain comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 40, 41, 42, 72, or 73. In one embodiment, the CAR comprises a CD3 zeta domain comprising the amino acid sequence of SEQ ID NO: 40, 41, 42, 72, or 73.

[0253] In one embodiment, the CAR (e.g., expressed by a T cell, a gamma delta T cell, a NK T cell, or a NK cell) is an anti-CD19 CAR. In one embodiment, the anti-CD19 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45. In one embodiment, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 45. In one embodiment, the anti-CD19 CAR is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 46. In one embodiment, the anti-CD19 CAR is encoded by the nucleic acid sequence of SEQ ID NO: 46.

[0254] In one embodiment, the CAR (e.g., expressed by a T cell, a gamma delta T cell, a NK T cell, or a NK cell) is an anti-CD19 CAR. In one embodiment, the anti-CD19 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47. In one embodiment, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 47. In one embodiment, the anti-CD19 CAR is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 48. In one embodiment, the anti-CD19 CAR is encoded by the nucleic acid sequence of SEQ ID NO: 48.

[0255] In one embodiment, the CAR (e.g., expressed by a T cell, a gamma delta T cell, a NK T cell, or a NK cell) is an anti-CD19 / CD22 CAR. In one embodiment, the anti-CD19 / CD22 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 43. In one embodiment, the anti-CD19 / CD22 CAR comprises the amino acid sequence of SEQ ID NO: 43. In one embodiment, the anti-CD19 / CD22 CAR is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 44. In one embodiment, the anti-CD19 / CD22 CAR is encoded by the nucleic acid sequence of SEQ ID NO: 44.

[0256] In one embodiment, the CAR (e.g., expressed by a T cell, a gamma delta T cell, a NK T cell, or a NK cell) is an anti-BCMA CAR. In one embodiment, the anti-BCMA CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 57. In one embodiment, the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 57. In one embodiment, the anti-BCMA CAR is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 58. In one embodiment, the anti-BCMA CAR is encoded by the nucleic acid sequence of SEQ ID NO: 58.

[0257] In one embodiment, the CAR (e.g., expressed by a T cell, a gamma delta T cell, an NK T cell, or an NK cell) is an anti-BCMA CAR. In one embodiment, the anti-BCMA CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 67. In one embodiment, the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO: 67. In one embodiment, the anti-BCMA CAR is encoded by a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 68. In one embodiment, the anti-BCMA CAR is encoded by the nucleic acid sequence of SEQ ID NO: 68.

[0258] In one embodiment, CAR therapy is administered at a dose of 0.2-5.0 x 10 per kg of subject body weight, e.g., as a single dose. 6 or 0.5 to 1.0 x 10 6 In one embodiment, CAR therapy comprises 0.2-5.0 x 10 cells per kg of subject body weight, e.g., as a single dose. 6 In one embodiment, the CAR therapy comprises 0.5-1.0 x 10 cells per kg of subject body weight, e.g., as a single dose. 6 In one embodiment, the CAR therapy comprises 1.0 x 10 cells per kg of subject body weight, e.g., as a single dose. 6 or 2.0 x 10 6 In one embodiment, the CAR therapy comprises 1.0 x 10 cells per kg of subject body weight, e.g., as a single dose. 6 In one embodiment, the CAR therapy comprises 2.0 x 10 cells per kg of subject body weight, e.g., as a single dose. 6 In one embodiment, the CAR therapy comprises 0.1-2.5 x 10 cells, for example, as a single dose. 8 or 0.6-6.0 x 10 8 In one embodiment, the CAR therapy comprises 0.1-2.5 x 10 cells, for example, as a single dose. 8 In one embodiment, the CAR therapy comprises 0.6-6.0 x 10 cells, for example, as a single dose. 8In one embodiment, the CAR therapy comprises 0.9-1.1 x 10 cells, for example, as a single dose. 8 pieces, 0.5~1.1×10 8 pieces, or 3.0 to 4.6 x 10 8 In one embodiment, the CAR therapy comprises 0.9-1.1 x 10 cells. 8 In one embodiment, the CAR therapy comprises 0.5-1.1 x 10 cells, for example, as a single dose. 8 In one embodiment, the CAR therapy comprises 3.0-4.6 x 10 cells, for example, as a single dose. 8 Contains cells.

[0259] Treatment method In one aspect, provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject (a) an IL-2 conjugate described herein, and (b) a chimeric antigen receptor (CAR) therapy.

[0260] In one aspect, the cancer is leukemia, myeloma, or lymphoma. In one aspect, the cancer is leukemia. In one aspect, the leukemia is acute lymphoblastic leukemia or chronic lymphocytic leukemia. In one aspect, the cancer is myeloma. In one aspect, the myeloma is multiple myeloma. In one aspect, the lymphoma is non-Hodgkin's lymphoma, follicular lymphoma, transformed follicular lymphoma, mantle cell lymphoma, primary mediastinal B-cell lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma (DLBCL). In one aspect, the lymphoma is Burkitt's lymphoma. In one aspect, the lymphoma is DLBCL. In one aspect, the cancer is a solid tumor, melanoma, or glioblastoma.

[0261] In one aspect, provided herein are methods of treating diffuse large B-cell lymphoma (DLBCL) in a subject in need of such treatment, the method comprising administering to the subject an IL-2 conjugate described herein. In some embodiments, the method of treating DLBCL in a subject in need of such treatment comprises administering to the subject about 8 μg / kg of IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating DLBCL in a subject in need of such treatment comprises administering to the subject about 16 μg / kg of IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating DLBCL in a subject in need of such treatment comprises administering to the subject about 24 μg / kg of IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating DLBCL in a subject in need of such treatment comprises administering to the subject about 32 μg / kg of IL-2 as an IL-2 conjugate as described herein. In some embodiments, the method of treating DLBCL in a subject in need thereof further comprises administering a CAR therapy to the subject.

[0262] In a further aspect, provided herein is the use of an IL-2 conjugate for the manufacture of a medicament for the methods disclosed herein of treating cancer, including DLBCL, in a subject in need thereof.

[0263] The embodiments described in the following sections apply to any of the aforementioned aspects.

[0264] Administration In some embodiments, the IL-2 conjugate is administered as at least a third or subsequent line of therapy.

[0265] In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-articular, intradermal, intravitreal, intraosseous, intraperitoneal, or intrathecal administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous, subcutaneous, or intramuscular administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous administration. In some embodiments, the IL-2 conjugate is administered to the subject by subcutaneous administration. In some embodiments, the IL-2 conjugate is administered to the subject by intramuscular administration. In some embodiments, the IL-2 conjugate is administered to the subject by intravenous administration.

[0266] The IL-2 conjugate can be administered two or more times, e.g., two, three, four, five or more times. In some embodiments, the administration period is up to 24 months, such as 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, or 24 months. In some embodiments, the administration period is further extended for up to an additional 24 months.

[0267] In some embodiments, the IL-2 conjugate is administered to a subject in need thereof about once per week, about once per two weeks, about once per three weeks, or about once per four weeks. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once per week. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once per two weeks. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once per three weeks. In some embodiments, the IL-2 conjugate is administered to a subject in need thereof once every four weeks. In some embodiments, the IL-2 conjugate is administered about every 14, 15, 16, 17, 18, 19, 20, or 21 days.

[0268] In some embodiments, the CAR therapy is administered to the subject by intravenous administration. In some embodiments, the CAR therapy is administered to the subject before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 1 week before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 2 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 3 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 4 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 5 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 6 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 7 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 8 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 9 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 10 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 11 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 12 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 13 weeks before any dose of the IL-2 conjugate is administered to the subject.In some embodiments, the CAR therapy is administered to the subject at least about 14 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject at least about 100 days before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject about 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 days before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, the CAR therapy is administered to the subject about 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, or 14 to 15 weeks before any dose of the IL-2 conjugate is administered to the subject. In some embodiments, one dose of the IL-2 conjugate is administered to the subject before the CAR therapy is administered to the subject, and one or more additional doses of the IL-2 conjugate are administered to the subject after the CAR therapy is administered to the subject. In some embodiments, one dose of the IL-2 conjugate is administered to the subject one day before the CAR therapy is administered to the subject, and one or more additional doses of the IL-2 conjugate are administered to the subject about once per week after the dose of the IL-2 conjugate is administered to the subject. In any of the above embodiments, after the initial dose of the IL-2 conjugate is administered to the subject, the remaining doses of the IL-2 conjugate can be administered to the subject as described in any of the above embodiments, including, but not limited to, about once per week, about once per two weeks, about once per three weeks, or about once per four weeks, or about once every 14, 15, 16, 17, 18, 19, 20, or 21 days, and including, but not limited to, 1, 2, 3, 4, or 5 doses in addition to the initial dose, and / or a total duration of treatment with the IL-2 conjugate of 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, or 24 months (counting from the initial dose of the IL-2 conjugate).

[0269] In some cases, the desired dose may conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[0270] In some embodiments, the methods described herein further comprise administering one or more additional therapeutic agents. In some embodiments, the additional therapeutic agents comprise an antihistamine, such as diphenhydramine. In some embodiments, the additional therapeutic agents comprise a chemotherapeutic agent and an antihistamine, e.g., diphenhydramine. In some embodiments, the additional therapeutic agents comprise any one of the aforementioned chemotherapeutic agents and an antihistamine, such as diphenhydramine.

[0271] In some embodiments, the additional therapeutic agent comprises an analgesic, such as acetaminophen. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent and an analgesic, e.g., acetaminophen. In some embodiments, the additional therapeutic agent comprises any one of the aforementioned chemotherapeutic agents and an analgesic, such as acetaminophen.

[0272] In some embodiments, the additional therapeutic agents include one or more vitamins, such as folic acid and / or vitamin B 12. In some embodiments, the additional therapeutic agents include a chemotherapeutic agent and one or more vitamins, e.g., folic acid and / or vitamin B 12. In some embodiments, the additional therapeutic agents include any one of the aforementioned chemotherapeutic agents and one or more vitamins, such as folic acid and / or vitamin B 12.

[0273] In some embodiments, the additional therapeutic agents include an antihistamine and an analgesic, e.g., diphenhydramine and acetaminophen. In some embodiments, the additional therapeutic agents include an antihistamine and one or more vitamins, e.g., diphenhydramine and one or both of folic acid and vitamin B12. In some embodiments, the additional therapeutic agents include an analgesic and one or more vitamins, e.g., acetaminophen and one or both of folic acid and vitamin B12. In some embodiments, the additional therapeutic agents include an antihistamine, an analgesic, and one or more vitamins, e.g., diphenhydramine, acetaminophen, and one or both of folic acid and vitamin B12. In any of the foregoing embodiments, the additional therapeutic agents can further include a chemotherapeutic agent, such as any one of the chemotherapeutic agents described above.

[0274] subject In some embodiments, the IL-2 conjugate is administered to an adult subject. In some embodiments, the adult subject is male. In other embodiments, the adult subject is female. In some embodiments, the adult subject is at least 12, 15, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years old. In some embodiments, the adult subject has relapsed or refractory cancer, for example, relapsed or refractory DLBCL.

[0275] In some embodiments, the subject is 12 years of age or older. In some embodiments, the subject's disease location is suitable for tumor biopsy, for example, at baseline or before initiation of treatment. In some embodiments, the subject has measurable disease. In some embodiments, the subject, if female, is not pregnant or lactating, or is not a woman of childbearing potential (WOCBP), or is a WOCBP who agrees to: (1) use an approved method of contraception and follow regular pregnancy testing before treatment and for at least 180 days after discontinuation of study treatment, and (2) refrain from egg donation or cryopreservation for 180 days after discontinuation of study treatment. In some embodiments, the subject, if male, agrees to refrain from sperm donation or cryopreservation and abstain from heterosexual intercourse or use approved contraception during study treatment and for at least 210 days after discontinuation of study treatment. In some embodiments, the subject is capable of providing signed informed consent. In some embodiments, the subject has a histologically or cytologically confirmed diagnosis of DLBCL. In some embodiments, the subject has adequate cardiovascular, hematological, hepatic, and renal function, as determined by a physician. In some embodiments, the subject has been determined (e.g., by a physician) to have a life expectancy of 12 weeks or greater. In some embodiments, the subject has received at least two prior lines of systemic therapy for DLBCL before administering the initial therapeutic dose of the IL-2 conjugate. In some embodiments, the at least two prior lines of systemic therapy for DLBCL comprise an anthracycline, an anti-CD20 agent, or a combination of an anthracycline and an anti-CD20 agent. In some embodiments, the anti-CD20 agent comprises rituximab. In some embodiments, the at least two prior lines of systemic therapy for DLBCL include CAR therapy, including any one of the CAR therapies disclosed herein. In some embodiments, the CAR therapy is the last line of the at least two prior lines of systemic therapy for DLBCL. In some embodiments, the subject meets each of the aforementioned criteria.

[0276] In some embodiments, the subject does not have an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or greater (for subjects 16 years of age or older). In some embodiments, the subject does not have a Lansky scale of less than 50% (for subjects under 16 years of age). In some embodiments, the subject does not have inadequate bone marrow reserve. In some embodiments, the subject does not have inadequate organ function. In some embodiments, the subject does not have a baseline SpO2 of 92% or less. In some embodiments, the subject does not have lymphomatous involvement of the central nervous system. In some embodiments, the subject does not have a history of allogeneic organ transplant or solid organ transplant. In some embodiments, the subject has not received the last dose of a previous anti-tumor therapy or any investigational treatment within 21 days or less than five half-lives of receiving the IL-2 conjugate, whichever is shorter. In some embodiments, the subject has not undergone major surgery or local intervention within 21 days of receiving the IL-2 conjugate. In some embodiments, the subject has not received prior IL-2-based anti-cancer therapy. In some embodiments, the subject does not have comorbidities requiring corticosteroid therapy. In some embodiments, the subject has not used antibiotics (other than topical antibiotics) within 14 days prior to the first dose of the IL-2 conjugate. In some embodiments, the subject has not had a severe or unstable cardiac condition within 6 months prior to starting study treatment. In some embodiments, the subject has not had an active, known, or suspected autoimmune disease that required systemic treatment within the past 2 years. In some embodiments, the subject has not had a known second malignancy that has progressed or required active treatment within the past 3 years. In some embodiments, the subject has not received a live or live-attenuated virus vaccination (excluding seasonal influenza or SARS-CoV-2 vaccines that do not contain live virus) within 28 days of the planned start of treatment. In some embodiments, the subject does not have any of the characteristics listed in this paragraph.In some embodiments, the subject does not have any of the characteristics listed in this paragraph and meets each of the criteria in the previous paragraph.

[0277] In some embodiments, the subject has no known hypersensitivity or contraindication to the administered IL-2 conjugate or PEG. In some embodiments, the subject has no known hypersensitivity or contraindication to the administered IL-2 conjugate, PEG, or PEGylated drug.

[0278] In some embodiments, the subject does not have any serious medical conditions (including pre-existing autoimmune or inflammatory disorders), laboratory abnormalities, psychiatric conditions, or other significant or unstable concurrent medical illnesses that would prevent treatment or make treatment inappropriate.

[0279] In some embodiments, the subject is not pregnant or lactating, hi some embodiments, the subject is not expecting to conceive or give birth during the course of treatment and up to 1, 2, 3, 4, 5, 6, or 7 months after administration of the final therapeutic dose.

[0280] In some embodiments, the subject has not received concomitant therapy with any investigational drug, vaccine, or device during the course of treatment, hi some embodiments, the subject has received concomitant therapy with an investigational drug, vaccine, or device during the course of treatment after physician approval.

[0281] Effect of administration In some embodiments, administration of the IL-2 conjugate provides a complete response, a partial response, or stable disease.

[0282] In some embodiments, after administration of the IL-2 conjugate, the subject experiences a response as measured by the Lugano Response Criteria 2014. In some embodiments, after administration of the IL-2 conjugate, the subject experiences an objective response rate (ORR) according to the Lugano Response Criteria 2014. In some embodiments, after administration of the IL-2 conjugate, the subject experiences a duration of response (DoR) according to the Lugano Response Criteria 2014. In some embodiments, after administration of the IL-2 conjugate, the subject experiences progression-free survival (PFS) according to the Lugano Response Criteria 2014. In some embodiments, after administration of the IL-2 conjugate, the subject experiences overall survival according to the Lugano Response Criteria 2014. In some embodiments, after administration of the IL-2 conjugate, the subject experiences a time to response (TTR) according to the Lugano Response Criteria 2014. In some embodiments, after administration of the IL-2 conjugate, the subject experiences a clinical benefit rate (CBR) according to the Lugano response criteria 2014.

[0283] In some embodiments, administration of the IL-2 conjugate to a subject does not cause vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 2, grade 3, or grade 4 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 2 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 3 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 4 vascular leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause loss of vascular tone in the subject.

[0284] In some embodiments, administration of the IL-2 conjugate to a subject does not cause extravasation of plasma proteins and fluids into the extravascular space of the subject.

[0285] In some embodiments, administration of the IL-2 conjugate to a subject does not cause hypotension and decreased organ perfusion in the subject.

[0286] In some embodiments, administration of the IL-2 conjugate to a subject does not cause neutrophil dysfunction in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause a decrease in chemotaxis in the subject.

[0287] In some embodiments, administration of an IL-2 conjugate to a subject is not associated with an increased risk of disseminated infection in the subject. In some embodiments, the disseminated infection is sepsis or bacterial endocarditis. In some embodiments, the disseminated infection is sepsis. In some embodiments, the disseminated infection is bacterial endocarditis. In some embodiments, the subject is treated for an existing bacterial infection prior to administration of the IL-2 conjugate. In some embodiments, the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate.

[0288] In some embodiments, administration of an IL-2 conjugate to a subject does not exacerbate an existing or early manifestation of an autoimmune disease or inflammatory disorder in the subject. In some embodiments, administration of an IL-2 conjugate to a subject does not exacerbate an existing or early manifestation of an autoimmune disease in the subject. In some embodiments, administration of an IL-2 conjugate to a subject does not exacerbate an existing or early manifestation of an inflammatory disorder in the subject. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, ophthalmic myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is Crohn's disease. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is scleroderma. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is thyroiditis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is inflammatory arthritis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is diabetes mellitus. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is ocular myasthenia gravis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is crescentic IgA glomerulonephritis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cholecystitis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is cerebral vasculitis. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is Stevens-Johnson syndrome. In some embodiments, the autoimmune disease or inflammatory disorder in the subject is bullous pemphigoid.

[0289] In some embodiments, administration of the IL-2 conjugate to a subject does not cause changes in the subject's mental status, speech impairment, cortical blindness, limb or gait ataxia, hallucinations, agitation, slowness, or coma. In some embodiments, administration of the IL-2 conjugate to a subject does not cause seizures in the subject. In some embodiments, administration of the IL-2 conjugate to a subject is not contraindicated in subjects with known seizure disorders.

[0290] In some embodiments, administration of the IL-2 conjugate to a subject does not cause capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 2, grade 3, or grade 4 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 2 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 3 capillary leak syndrome in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause grade 4 capillary leak syndrome in the subject.

[0291] In some embodiments, administration of an IL-2 conjugate to a subject does not cause a decrease in the subject's mean arterial pressure after administration. In some embodiments, administration of an IL-2 conjugate to a subject causes hypotension in the subject. In some embodiments, administration of an IL-2 conjugate to a subject does not cause the subject to experience a systolic blood pressure of less than 90 mmHg or a decrease of 20 mmHg from baseline systolic blood pressure.

[0292] In some embodiments, administration of the IL-2 conjugate to a subject does not cause edema or impairment of kidney or liver function in the subject.

[0293] In some embodiments, administration of the IL-2 conjugate to a subject does not cause eosinophilia in the subject. In some embodiments, administration of the IL-2 conjugate to a subject does not cause the subject's peripheral blood eosinophil count to exceed 500 / μL. In some embodiments, administration of the IL-2 conjugate to a subject does not cause the subject's peripheral blood eosinophil count to exceed 500 / μL to 1500 / μL. In some embodiments, administration of the IL-2 conjugate to a subject does not cause the subject's peripheral blood eosinophil count to exceed 1,500 / μL to 5,000 / μL. In some embodiments, administration of the IL-2 conjugate to a subject does not cause the subject's peripheral blood eosinophil count to exceed 5,000 / μL. In some embodiments, administration of the IL-2 conjugate to a subject is not contraindicated in the subject by an existing regimen of psychotropic medication.

[0294] In some embodiments, administration of an IL-2 conjugate to a subject is not contraindicated in a subject receiving a pre-existing regimen of nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs. In some embodiments, administration of an IL-2 conjugate to a subject is not contraindicated in a subject receiving a pre-existing regimen of aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase. In some embodiments, administration of an IL-2 conjugate to a subject is not contraindicated in a subject receiving a concomitant regimen containing an anti-neoplastic agent. In some embodiments, the anti-neoplastic agent is selected from dacarbazine, cis-platinum, tamoxifen, and interferon-alpha.

[0295] In some embodiments, administration of the IL-2 conjugate to a subject does not result in one or more grade 4 adverse events in the subject following administration. In some embodiments, the Grade 4 adverse event is selected from hypothermia, shock, bradycardia, premature ventricular contractions, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, second-degree AV block, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disease, stomatitis, nausea and vomiting, abnormal liver function tests, gastrointestinal bleeding, hematemesis, bloody diarrhea, gastrointestinal disorders, intestinal perforation, pancreatitis, anemia, leukopenia, leukocytosis, hypocalcemia, increased alkaline phosphatase, increased blood urea nitrogen (BUN), hyperuricemia, increased non-protein nitrogen (NPN), respiratory acidosis, somnolence, restlessness, neuropathy, paranoid reaction, convulsions, grand mal seizures, delirium, asthma, pulmonary edema, hyperventilation, hypoxia, hemoptysis, hypoventilation, pneumothorax, mydriasis, pupillary disturbances, abnormal renal function, renal failure, and acute tubular necrosis. In some embodiments, administration of the IL-2 conjugate to a group of subjects does not result in one or more grade 4 adverse events in more than 1% of the subjects following administration. In some embodiments, the Grade 4 adverse event is selected from hypothermia, shock, bradycardia, premature ventricular contractions, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, second-degree AV block, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disease, stomatitis, nausea and vomiting, abnormal liver function tests, gastrointestinal bleeding, hematemesis, bloody diarrhea, gastrointestinal disorders, intestinal perforation, pancreatitis, anemia, leukopenia, leukocytosis, hypocalcemia, increased alkaline phosphatase, increased blood urea nitrogen (BUN), hyperuricemia, increased non-protein nitrogen (NPN), respiratory acidosis, somnolence, restlessness, neuropathy, paranoid reaction, convulsions, grand mal seizures, delirium, asthma, pulmonary edema, hyperventilation, hypoxia, hemoptysis, hypoventilation, pneumothorax, mydriasis, pupillary disturbances, abnormal renal function, renal failure, and acute tubular necrosis.

[0296] In some embodiments, administration of the IL-2 conjugate to a group of subjects does not cause one or more adverse events in more than 1% of the subjects following administration, wherein the one or more adverse events are selected from duodenal ulcer formation, intestinal necrosis, myocarditis, supraventricular tachycardia, permanent or transient blindness secondary to optic neuritis, transient ischemic attack, meningitis, cerebral edema, pericarditis, allergic interstitial nephritis, and tracheoesophageal fistula.

[0297] In some embodiments, administration of the IL-2 conjugate to a group of subjects does not result in one or more adverse events in more than 1% of the subjects following administration, wherein the one or more adverse events are selected from malignant hyperthermia, cardiac arrest, myocardial infarction, pulmonary embolism, stroke, intestinal perforation, liver or kidney failure, severe depression leading to suicide, pulmonary edema, respiratory arrest, and respiratory failure.

[0298] In some embodiments, administration of an IL-2 conjugate to a subject stimulates CD8+ cells in the subject. In some embodiments, administration of an IL-2 conjugate to a subject stimulates NK cells in the subject. Stimulation can include, for example, an increase in the number of CD8+ cells in the subject about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. In some embodiments, the CD8+ cells comprise memory CD8+ cells. In some embodiments, the CD8+ cells comprise effector CD8+ cells. Stimulation can include, for example, an increase in the proportion of CD8+ cells that are Ki67 positive in the subject about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration. Stimulation can include, for example, an increase in the number of NK cells in the subject about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration.

[0299] In some embodiments, CD8+ cells are expanded by at least 1.5-fold in a subject after administration of the IL-2 conjugate, e.g., at least 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, or 2.5-fold. In some embodiments, NK cells are expanded by at least 3.5-fold in a subject after administration of the IL-2 conjugate, e.g., at least 4.0-fold, 4.5-fold, 5.5-fold, 6-fold, 6.5-fold, 7.0-fold, or 7.5-fold. In some embodiments, eosinophils are expanded by about 2-fold or less in a subject after administration of the IL-2 conjugate, e.g., about 1.5-fold, 1.4-fold, or 1.3-fold or less. In some embodiments, CD4+ cells are expanded by about 2-fold or less in a subject after administration of the IL-2 conjugate, e.g., about 1.8-fold, 1.7-fold, or 1.6-fold or less. In some embodiments, the proliferation of CD8+ cells and / or NK cells in a subject after administration of the IL-2 conjugate is greater than the proliferation of CD4+ cells and / or eosinophils. In some embodiments, the proliferation of CD8+ cells is greater than the proliferation of CD4+ cells. In some embodiments, the proliferation of NK cells is greater than the proliferation of CD4+ cells. In some embodiments, the proliferation of CD8+ cells is greater than the proliferation of eosinophils. In some embodiments, the proliferation of NK cells is greater than the proliferation of eosinophils. The fold proliferation is determined relative to a baseline value measured before administration of the IL-2 conjugate. In some embodiments, the fold proliferation is determined at any time after administration, for example, about 4, 5, 6, or 7 days after administration, or about 1, 2, 3, or 4 weeks after administration.

[0300] In some embodiments, administering an IL-2 conjugate to a subject increases the number of peripheral CD8+ T cells and NK cells in the subject without increasing the number of peripheral CD4+ regulatory T cells in the subject. In some embodiments, administering an IL-2 conjugate to a subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject. In some embodiments, administering an IL-2 conjugate to a subject increases the number of peripheral CD8+ T cells and NK cells in the subject without increasing the number of intratumoral CD8+ T cells and NK cells in the subject, and without increasing the number of intratumoral CD4+ regulatory T cells in the subject.

[0301] In some embodiments, administration of the IL-2 conjugate to a subject does not require the availability of an intensive care facility or a specialist skilled in cardiopulmonary or critical care medicine. In some embodiments, administration of the IL-2 conjugate to a subject does not require the availability of an intensive care facility or a specialist skilled in cardiopulmonary or critical care medicine. In some embodiments, administration of the IL-2 conjugate to a subject does not require the availability of an intensive care facility. In some embodiments, administration of the IL-2 conjugate to a subject does not require the availability of a specialist skilled in cardiopulmonary or critical care medicine.

[0302] In some embodiments, administration of the IL-2 conjugate does not cause dose-limiting toxicity. In some embodiments, administration of the IL-2 conjugate does not cause severe cytokine release syndrome. In some embodiments, the IL-2 conjugate does not induce anti-drug antibodies (ADA), i.e., antibodies to the IL-2 conjugate. In some embodiments, the lack of ADA induction is determined by direct immunoassay of antibodies to PEG and / or ELISA of antibodies to the IL-2 conjugate. An IL-2 conjugate is considered not to induce ADA if the measured ADA levels are statistically indistinguishable from baseline (pre-treatment) levels or levels of untreated controls.

[0303] Kit / manufactured product Disclosed herein are kits and articles of manufacture for use with one or more of the methods and compositions described in certain embodiments. Such kits include carriers, packages, or containers that are compartmentalized to receive one or more containers, such as vials, tubes, etc., each of which contains one of the individual components used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials, such as glass or plastic.

[0304] The kit typically includes a label and / or instructions describing the contents, as well as a package insert containing the instructions. A set of instructions is also typically included.

[0305] In one embodiment, the label is on or associated with the container. In one embodiment, the label is present on the container when letters, numbers, or other characters forming the label are affixed, molded, or etched into the container itself, or is associated with the container when present in a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, the label is used to indicate that the contents should be used for a particular therapeutic application. The label also provides instructions for using the contents, such as in the methods described herein.

[0306] In certain embodiments, the pharmaceutical compositions are supplied in a pack or dispenser device containing one or more unit dosage forms containing a compound provided herein. For example, the pack contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser device is also accompanied by a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals. The notice reflects approval by the agency of the drug form for administration to humans or veterinary animals. Such notice may be, for example, a label approved by the U.S. Food and Drug Administration or an approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. [Example]

[0307] These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

[0308] Example 1. Preparation of PEGylated IL-2 conjugates Exemplary methods with details for preparing the IL-2 conjugates described herein are provided in the Examples.

[0309] IL-2 for bioconjugation was expressed as inclusion bodies in E. coli using the methods disclosed herein using (a) an expression plasmid encoding a tRNA from M. mazei Pyl containing (i) a protein having a desired amino acid sequence, including a first unnatural base pair to provide a codon at the desired position incorporating the unnatural amino acid N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and (ii) a second unnatural nucleotide to provide a compatible anticodon in place of the natural sequence; (b) a plasmid encoding pyrrolysine-tRNA synthetase (Mb PylRS) from M. barkeri; (c) N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK); and (d) a truncated variant of the nucleotide triphosphate transporter PtNTT2 lacking the first 65 amino acid residues of the full-length protein. The double-stranded oligonucleotide encoding the amino acid sequence of the desired IL-2 variant contained the codon AXC as codon 64 of the protein-encoding sequence of SEQ ID NO: 1, in which P64 was replaced with an unnatural amino acid described herein. A plasmid encoding an orthogonal tRNA gene from M. mazei contained the AXC-matching anticodon GYT in place of its native sequence, where Y is an unnatural nucleotide as disclosed herein. X and Y were selected from the unnatural nucleotides dTPT3 and DNAM, as disclosed herein. After the expressed protein was extracted from inclusion bodies and refolded using standard procedures, the AzK-containing IL-2 product was site-specifically PEGylated using DBCO-mediated copper-free click chemistry to attach a stable, covalent mPEG moiety to AzK. Exemplary reactions are shown in Schemes 1 and 2 (where n indicates the number of repeating PEG units). The reaction of the AzK moiety with the DBCO alkynyl moiety can yield a single regioisomeric product or a mixture of regioisomeric products. Scheme 1. [ka] Scheme 2. [ka]

[0310] Example 2. Clinical trial of biomarker effects after administration of IL-2 conjugates (8 μg / kg, 16 μg / kg, and 24 μg / kg [Q3w]) Cohort using 24 μg / kg dose [Q3W] Studies were conducted to characterize the immunological effects of in vivo administration of the IL-2 conjugate described herein. The IL-2 conjugate comprises SEQ ID NO: 2, where position 64 is AzK_L1_PEG30kD, where AzK_L1_PEG30kD is defined as the structure of formula (IV) or formula (V), or a mixture of formulas (IV) and (V), and a 30 kDa linear mPEG chain. This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, where position 64 is replaced by the structure of formula (IV) or formula (V), or a mixture of formulas (IV) and (V), and a 30 kDa linear mPEG chain. This IL-2 conjugate can also be described as an IL-2 conjugate comprising SEQ ID NO: 1, where position 64 is replaced by the structure of Formula (XII) or Formula (XIII), or a mixture of Formulas (XII) and (XIII), and a 30 kDa linear mPEG chain. This IL-2 conjugate has a proposed International Nonproprietary Name (pINN) of pegendiloquin. The compound was prepared using a method that first prepares a protein having SEQ ID NO: 1 in which the proline at position 64 is replaced with N6-((2-azidoethoxy)-carbonyl)-L-lysine AzK. The AzK-containing protein was then reacted with DBCO containing a methoxy linear PEG group having an average molecular weight of 30 kDa under click chemistry conditions, followed by purification and formulation using standard procedures. Herein and throughout the Examples, the drug mass per kg of subject (e.g., 24 μg / kg) refers to the IL-2 mass excluding the PEG and linker mass.

[0311] The IL-2 conjugate was administered by IV infusion over 30 minutes at a dose of 24 μg / kg every 3 weeks [Q3W]. The effects on the following biomarkers were analyzed as surrogate predictors of safety and / or efficacy: Eosinophilia (increased peripheral eosinophil count): A cellular surrogate marker for IL-2-induced proliferation of cells (eosinophils) associated with vascular leak syndrome (VLS). Interleukin 5 (IL-5): A cytokine surrogate marker for IL-2-induced activation of type 2 innate lymphoid cells and release of this chemoattractant, which leads to eosinophilia and potentially VLS. Interleukin 6 (IL-6): A cytokine surrogate marker for IL-2-induced cytokine release syndrome (CRS), and Interferon-gamma (IFN-γ): A cytokine surrogate marker for IL-2-induced activation of CD8+ cytotoxic T lymphocytes.

[0312] The effects on the following biomarkers were analyzed as surrogate predictors of antitumor immune activity: Peripheral CD8+ effector cells: a marker of IL-2-induced proliferation of these target cells in the periphery, which, upon infiltration, serves as a surrogate marker for inducing a potential therapeutic response. Peripheral CD8+ memory cells: a marker of IL-2-induced proliferation of these target cells in the periphery, which, upon infiltration, becomes a surrogate marker for inducing healing and maintenance of a potentially permanent latent memory population. Peripheral NK cells: a marker of IL-2-induced proliferation of these target cells in the periphery, potentially serving as a surrogate marker for rapid therapeutic responses upon infiltration; and Peripheral CD4+ regulatory cells: a marker of IL-2-induced proliferation of these target cells in the periphery that induce an immunosuppressive TME upon infiltration and provide a surrogate marker for counteracting the effects of effector-based therapies.

[0313] Subjects were human males or females aged 18 years or older at the time of screening. All subjects had been previously treated with anticancer therapy and had at least one of the following: treatment-related toxicity (excluding alopecia) resolved to Grade 0 or 1 according to NCI CTCAE v5.0, or treatment-related toxicity resolved to at least Grade 2 according to NCI CTCAE v5.0 with prior approval from a Medical Monitor. The most common tumors were colorectal or melanoma.

[0314] Subjects also met the following criteria: provided informed consent; Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; life expectancy of 12 weeks or greater as determined by the investigator; histologically or cytologically confirmed diagnosis of advanced and / or metastatic solid tumor; subjects with advanced or metastatic solid tumors who refused standard treatment, or for whom no reasonable standard of care exists that would confer clinical benefit, or for whom standard treatment is unacceptable, ineffective, or unavailable; measurable disease per RECIST v1.1; appropriate laboratory parameters, e.g., absolute lymphocyte count >0.5 times the lower limit of normal, platelet count ≥100 x 10 9 / L, hemoglobin ≥ 9.0 g / dL (absence of growth factors or transfection within 2 weeks; a 1-week washout for ESA and CSF administration is sufficient), absolute neutrophil count ≥ 1.5 × 10 9 / L (absence of growth factors within 2 weeks), prothrombin time (PT) and partial thromboplastin time (PTT) ≤ 1.5 times the upper limit of normal (ULN), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 times the ULN and may be ≤ 5 times the ULN except in the presence of liver metastases, total bilirubin ≤ 1.5 x ULN. Premenopausal women and women who were postmenopausal for less than 12 months had a negative serum pregnancy test within 7 days before starting study treatment.

[0315] Q3W Administration. Eleven individuals (4 [36.4%] male, 7 [63.6%] female, [9 [81.8%] Caucasian]) with advanced or metastatic solid tumors and a median age of 67 years (range, 37-78 years) received up to 9 cycles (1 dose per cycle) of the IL-2 conjugate at a 24 μg / kg dose Q3W. Note that throughout the discussion of the first cohort in Example 2, the drug mass per kg of subject (e.g., 24 μg / kg) refers to the IL-2 mass excluding PEG and linker mass.

[0316] One subject had a partial response at the first scan, confirmed by second and third scans (prior PD-1 exposure) that continued for 6+ months. Five subjects had initial disease stabilization (at the 6-week assessment), three subjects had progressive disease at the first assessment, and one subject discontinued treatment due to an adverse event. All subjects had peak post-dose CD8+Ki67 expression levels greater than 50% (range 50%-85%).

[0317] A 73-year-old male subject with squamous cell carcinoma of unknown etiology who received seven cycles of treatment (24 μg / kg Q3W) and also received two lines of systemic therapy, including anti-PD-1 (best response to anti-PD-1: SD), showed a 31% tumor reduction after two cycles. The greatest tumor responses in other patients with immune-sensitive tumors were found in renal cell carcinoma (RCC) (16% growth) and melanoma (10% growth, 2% reduction, and 20% reduction observed in two subjects).

[0318] Peripheral proliferation of CD8+ T effector cells averaged 4.47-fold greater than baseline. All subjects had high post-treatment NK cell Ki67 expression levels. Subjects had a peak post-treatment peripheral proliferation of NK cells averaged 7.67-fold greater than baseline.

[0319] Efficacy biomarkers. Efficacy biomarker data were based on available data for up to 10 subjects receiving 24 μg / kg of IL-2 conjugate. Peripheral CD8+ T effCell counts were measured (Figure 1A-C). Prolonged CD8+ proliferation (e.g., 2-fold or greater change) above baseline was observed in some subjects 3 weeks after the previous dose. Ki67-expressing CD8+ T cells were also observed. eff The percentage of peripheral CD8+ memory cells was also measured (Figure 2). The number of peripheral CD8+ memory cells is shown in Figures 3A-3B.

[0320] Peripheral NK cell counts are shown in Figures 4A-4D. Increased NK cell counts were observed in each subject. The percentage of NK cells expressing ki67 was also measured (Figure 5).

[0321] peripheral CD4+T reg The numbers are shown in Figures 6A to 6C. reg The percentage of cells was also determined (Fig. 7).

[0322] Eosinophil counts were measured (Figures 8A-8C). Measurements did not exceed a fourfold increase and were consistently below the range of 2,328 to 15,958 eosinophils / μL reported in patients with IL-2-induced eosinophilia (Pisani et al., Blood 1991 Sep 15, 78(6):1538-44). IFN-γ, IL-5, and IL-6 levels were also measured (Figures 9A-9C). Measurements indicated that IFN-γ was induced, but low amounts of IL-5 and IL-6 (cytokines associated with VLS and CRS, respectively), except in one subject whose IL-6 levels increased to approximately 1,100 pg / mL 24 hours after treatment (after receiving tocilizumab) but subsequently decreased.

[0323] Anti-drug antibodies (ADA). Samples from treated subjects were assayed for anti-drug antibodies (ADA) after each dosing cycle. Anti-polyethylene glycol autoantibodies were detected by direct immunoassay (detection limit: 36 ng / mL). A bridging MesoScale Discovery ELISA was performed using a labeled form of the IL-2 conjugate with a detection limit of 4.66 ng / mL. Additionally, a cell-based assay for neutralizing antibodies to the IL-2 conjugate was performed using the CTLL-2 cell line with STAT5 phosphorylation as the readout (detection limit: 6.3 μg / mL).

[0324] Samples were collected and analyzed after each dosing cycle from two subjects who received five dosing cycles and one subject who received four dosing cycles. During assay qualification, assay-specific cutpoints were determined as signal-to-negative ratios of 1.09 or greater for the IL-2 conjugate ADA assay and 2.08 or greater for the PEG ADA assay. Samples that gave positive or indeterminate results in the IL-2 conjugate assay were subjected to a confirmatory test, in which the sample and control were assayed in the presence and absence of a confirmation buffer (10 μg / mL IL-2 conjugate in blocking solution). Samples that gave positive or indeterminate results in the PEG assay were subjected to a confirmatory test, in which the sample and control were assayed in the presence and absence of a confirmation buffer (10 μg / mL IL-2 conjugate in 6% horse serum). During the detection step, a sample was considered "confirmed" if the absorbance signal was inhibited at or above the assay-specific cutpoint (14.5% for the IL-2 conjugate or 42.4% for PEG) determined during assay qualification. No confirmed ADAs were detected against the IL-2 conjugate or PEG (data not shown).

[0325] Summary and Discussion of Results: All subjects tested had post-administration CD8+ Ki67 expression levels greater than 50% (50%-85%) and peripheral proliferation of CD8+ T effector (Teff) cells at one or more time points. All subjects tested also had post-administration NK cell Ki67 expression levels greater than 50% (50%-100%) at one or more time points accompanied by peripheral proliferation of NK cells. There was no significant increase in IL-5 levels, and subjects with elevated IL-6 levels on day 3 showed a decrease the following day. ADA was not induced in any of the subjects tested.

[0326] An AE was any untoward medical occurrence in a clinical investigational subject administered a medicinal product, regardless of attribution of cause. Dose-limiting toxicity was defined as an AE occurring within ±1 day of days 1 to 29 (inclusive) of a treatment cycle that was not related solely to a clearly or apparently unrelated cause and met at least one of the following criteria: Grade 3 neutropenia (absolute neutrophil count <1,000 / mm) lasting for 7 days or more 3 >500 / mm 3 ), or grade 4 neutropenia of any duration Grade 3+ febrile neutropenia Grade 4+ thrombocytopenia (platelet count <25,000 / mm 3 ) Grade 3+ thrombocytopenia (platelet count <50,000-25,000 / mm3) lasting for ≥5 days or associated with clinically significant bleeding or requiring platelet transfusion 3 ) Absolute neutrophil count of 1,000 / mm within 10 days 3 Over, platelet count 75,000 / mm 3 Not meeting the above recovery criteria Any other grade 4+ hematologic toxicity lasting 5 days or more Grade 3+ ALT or AST combined with bilirubin greater than 2x ULN without evidence of cholestasis or other causes such as viral infection or other drugs (i.e., Hy's law) Grade 3 infusion-related reactions and Grade 4 infusion-related reactions occurring with premedication Grade 3 vascular leak syndrome, defined as hypotension associated with fluid retention and pulmonary edema Grade 3+ anaphylaxis Grade 3+ hypotension Grade 3+ acceptable care AEs that do not resolve to less than Grade 2 within 7 days after initiation of medical management Grade 3+ cytokine release syndrome The following exceptions applied to non-hematologic AEs: Grade 3: Fatigue, nausea, vomiting, or diarrhea that resolves to Grade 2 or less with optimal medical management within 3 days Grade 3 fever (defined as >40°C for ≤24 hours) Grade 3 infusion-related reaction occurring without premedication, subsequent doses should be administered with premedication, and if the reaction recurs, it becomes a DLT. Grade 3 arthralgia or rash resolves to Grade 2 or less within 7 days of initiating accepted standard treatment (e.g., systemic corticosteroid therapy) If the subject had a grade 1 or 2 elevation of ALT or AST at baseline considered secondary to liver metastases, a grade 3 elevation must also be ≥3x baseline and must persist for >7 days.

[0327] A serious AE was defined as any AE that resulted in any of the following outcomes: death, a life-threatening AE, inpatient hospitalization or prolongation of an existing hospitalization, persistent or significant incapacity or substantial disruption of the ability to perform normal life functions, or a congenital anomaly / birth defect. Significant medical events that do not result in death, life-threatening, or require hospitalization may be considered serious if, based on appropriate medical judgment, they have the potential to endanger the patient and require medical or surgical intervention to prevent one of the outcomes listed above. Examples of such medical events include allergic bronchospasm requiring intensive care in the emergency room or at home, a blood disorder or seizure that does not result in inpatient hospitalization, or the development of drug dependence or abuse.

[0328] No dose-limiting toxicities were reported. There was no cumulative toxicity. There were two treatment-related SAEs (one G3 acute kidney injury and one G4 cytokine release syndrome), which resolved with accepted standard of care. Overall, the IL-2 conjugates were considered well tolerated.

[0329] All subjects had at least one treatment-emergent AE (TEAE). TEAEs are detailed in Table 1. There were no grade 5 TEAEs. Two subjects had grade 3 events and three subjects had grade 4 events. Grade 3 events included: one ALT / AST elevation, one neutrophil count decrease, and one acute kidney injury. Grade 4 events included: one CRS, one lymphocyte count increase, and two lymphocyte count decreases.

[0330] [Table 1]

[0331] TEAEs consisted primarily of flu-like symptoms, nausea, or vomiting. TEAEs resolved with accepted standard therapy. TEAEs were transient. The fever, hypotension, and hypoxia AEs did not correlate with elevated IL-5 / IL-6 cytokines. One subject had elevated IL-6 (after tocilizumab treatment) to 1,000 pg / mL at 24 hours, which decreased to less than 100 pg / mL by 72 hours. There was no significant effect on vital signs, QTc prolongation, or other cardiac toxicity.

[0332] Thus, the IL-2 conjugate showed promising PD data and was generally well tolerated. The in vivo half-life of the IL-2 conjugate was determined to be approximately 10 hours. Overall, the results appear to support the non-alpha-preferential activity of the IL-2 conjugate, have a tolerable safety profile, and provide preliminary evidence of activity in patients with PD and immune-sensitive tumors.

[0333] Cohorts using 8 μg / kg and 16 μg / kg doses [Q3W] The IL-2 conjugate was administered by IV infusion over 30 minutes every 3 weeks at a dose of 8 μg / kg or 16 μg / kg [Q3W]. Effects on the same biomarkers listed above for studies using the 24 μg / kg dose were analyzed as surrogate predictors of safety and / or efficacy. Subjects in these studies met the same criteria as those receiving the 24 μg / kg dose.

[0334] Cohort 1: 8μg / kg [Q3W] Cohort 1 (individuals with malignant solid tumors) received IL-2 conjugate at an 8 μg / kg dose Q3W for 5 treatment cycles.

[0335] Four individuals with early disease stabilization (one patient had approximately 12% tumor regression at the 6-week evaluation) were treated with IL-2 conjugates. These four subjects had post-treatment CD8+Ki67 expression levels greater than 60% (65%-80%).

[0336] Biomarkers were determined for four subjects in Cohort 1 as follows: CD8+ T effector cell peripheral proliferation averaged >1.53-fold above baseline, with one subject at 2.1-fold above baseline; all four subjects had near 100% post-dose NK cell Ki67 expression levels; all four subjects had post-dose NK cell peripheral proliferation averaged >3.9-fold above baseline on Day 3, with one subject at 5.0-fold above baseline on Day 3; there were no changes in PK parameters from Cycle 1 to Cycle 2; no anti-drug antibodies were detected in the first three subjects, and these were measured in two subjects until Cycle 5 and in one subject until Cycle 4.

[0337] Serum IFNγ, IL-6, and IL-5 levels were measured on days 1, 2, and 3 post-dose during cycles 1 and 2. The means and ranges are shown in Table 2. The highest values ​​in the range were observed on day 1 post-dose for all subjects.

[0338] [Table 2]

[0339] The measured cytokine levels are shown graphically in FIG.

[0340] Teachey et al., Cancer Discov. 2016;6(6);664-79, reported that in patients with acute lymphoblastic leukemia treated with CAR-T cells, severe cytokine release syndrome (CRS levels 4 or 5) was associated with higher values ​​of each of the three cytokines measured in this study than non-severe cytokine release syndrome (CRS levels 0-3). The data for IFNγ, IL-6, and IL-5 (expressed as median (range)) from Teachey et al. are reproduced in Table 3.

[0341] [Table 3]

[0342] Therefore, the results in Table 2 and Figure 10 are consistent with the absence of severe CRS.

[0343] IL-2 conjugates showed encouraging PD data and were generally well tolerated. Overall, the results appear to support the non-alpha-preferred activity of IL-2 conjugates, have a tolerable safety profile, and provide preliminary evidence of activity in patients with PD and immune-sensitive tumors.

[0344] Cohort 2: 16 μg / kg [Q3W] This example reports the results for up to six individuals with malignant solid tumors who received at least two cycles of IL-2 conjugate at a 16 μg / kg dose Q3W. After the first dose, one subject had a 4.1-fold post-administration peripheral proliferation of CD8+ T effector cells, with an average of 2.2-fold proliferation across three patients. All three subjects had a post-administration peripheral proliferation of NK cells greater than four times baseline on day 3, with one subject experiencing an 11.4-fold increase over baseline, with an average of 7.2-fold.

[0345] Serum IFNγ, IL-6, and IL-5 levels were measured 1, 2, and 3 days after dosing during cycles 1 and 2. The means and ranges are shown in Table 4. The highest values ​​in the range were observed 1 day after dosing for the three subjects shown.

[0346] [Table 4]

[0347] The cytokine levels measured for the four subjects are shown graphically in Figure 11. These results are also consistent with the absence of severe CRS.

[0348] Eosinophil counts were measured by FACS and CBC for cohorts 1 and 2 (Figures 12A-12D). Measurements consistently fell below the range of 2328 to 15958 eosinophils / μl in patients with IL-2-induced eosinophilia, as reported by Pisani et al., Blood 1991 Sep 15;78(6):1538-44. Peripheral lymphocyte counts were also measured for cohorts 1 and 2 (Figures 13A-13D).

[0349] Efficacy biomarkers for cohorts 1 and 2. Peripheral CD8+ T eff The number of Ki67-expressing CD8+ T cells was measured for cohorts 1 and 2 (Figures 14A-14D). Prolonged CD8+ proliferation (e.g., 2-fold or greater change) above baseline was observed in some subjects 3 weeks after the previous dose. effThe percentage of cells was also determined for cohorts 1 and 2 (FIGS. 15A-15B).

[0350] Peripheral memory CD8+ counts are shown in Figures 16A-16B. Peripheral NK cell counts are shown in Figures 17A-17D. Prolonged NK cell proliferation (e.g., 5-fold or greater change) above baseline was observed in some subjects 3 weeks after the previous dose. The percentage of NK cells expressing ki67 was also measured for Cohorts 1 and 2 (Figures 18A-18B).

[0351] Peripheral CD4+T in Cohorts 1 and 2 reg The numbers are shown in Figures 19A-19B. reg The percentage of cells was also determined for cohorts 1 and 2 (FIGS. 20A-B).

[0352] Summary and Discussion of Results: The subjects receiving the 8 μg / kg dose had post-administration CD8+ Ki67 expression levels greater than 60% (65%-80%), and peripheral proliferation of CD8+ T effector (Teff) cells was an average of 1.53-fold greater than baseline. All four subjects also had post-administration NK cell Ki67 expression levels near 100%, and peripheral proliferation of NK cells was an average of 3.9-fold greater than baseline on Day 3. Of the three subjects receiving the 16 μg / kg dose, one had post-administration peripheral proliferation of CD8+ Teff cells 4.1-fold greater than baseline on Day 7, with an average proliferation of 2.2-fold across the three subjects. No anti-drug antibodies (IL-2 or PEG) were observed, and there was no significant increase in IL-5 or IL-6 levels. PK data also showed no decrease in AUC from Cycle 1 to Cycle 2 (data not shown).

[0353] No dose-limiting toxicities were reported at any dose, and there were no treatment-related adverse events (TRAEs) leading to discontinuation or treatment-related serious AEs reported.

[0354] TEAEs in the 10 subjects who received the Q3W 8 or 16 μg / kg dose are detailed in Table 5. There were no Grade 5 TEAEs. Two subjects had Grade 4 events (one AST elevation and one lymphocyte count decrease).

[0355] [Table 5]

[0356] TEAEs consisted primarily of flu-like symptoms, nausea, or vomiting. TEAEs resolved with accepted standard therapy. TEAEs were transient. Fever, hypotension, and hypoxia were not correlated with elevated IL-5 / IL-6 cytokines. There was no significant effect on vital signs, QTc prolongation, or other cardiac toxicity. Thus, the IL-2 conjugate demonstrated encouraging PD data and was generally well tolerated. The in vivo half-life of the IL-2 conjugate was determined to be approximately 10 hours. Overall, the results appear to support the non-alpha-preferential activity of the IL-2 conjugate, have a tolerable safety profile, and provide preliminary evidence of activity in patients with PD and immune-sensitive tumors.

[0357] Selected individual results: One subject with prostate adenocarcinoma received 10 cycles of the 16 μg / kg dose Q3W and experienced stable disease (24% reduction after 2 cycles). This subject discontinued treatment after the 10th cycle due to a rising PSA.

[0358] One subject with non-small cell lung cancer received at least six cycles of the Q3W 16 μg / kg dose and showed stable disease (17.9% reduction after five cycles).

[0359] Anti-drug antibodies (ADA). Samples from treated subjects were assayed for anti-drug antibodies (ADA) after each dosing cycle. Anti-polyethylene glycol autoantibodies were detected by direct immunoassay (detection limit: 36 ng / mL). A bridging MesoScale Discovery ELISA was performed using a labeled form of the IL-2 conjugate with a detection limit of 4.66 ng / mL. Additionally, a cell-based assay for neutralizing antibodies to the IL-2 conjugate was performed using the CTLL-2 cell line with STAT5 phosphorylation as the readout (detection limit: 6.3 μg / mL).

[0360] Samples were collected and analyzed after each dosing cycle from two subjects who received five dosing cycles and one subject who received four dosing cycles. During assay qualification, assay-specific cutpoints were determined as signal-to-negative ratios of 1.09 or greater for the IL-2 conjugate ADA assay and 2.08 or greater for the PEG ADA assay. Samples that gave positive or indeterminate results in the IL-2 conjugate assay were subjected to a confirmatory test, in which the sample and control were assayed in the presence and absence of a confirmation buffer (10 μg / mL IL-2 conjugate in blocking solution). Samples that gave positive or indeterminate results in the PEG assay were subjected to a confirmatory test, in which the sample and control were assayed in the presence and absence of a confirmation buffer (10 μg / mL IL-2 conjugate in 6% horse serum). During the detection step, a sample was considered "confirmed" if the absorbance signal was inhibited at or above the assay-specific cutpoint (14.5% for the IL-2 conjugate or 42.4% for PEG) determined during assay qualification. No confirmed ADAs were detected against the IL-2 conjugate or PEG (data not shown).

[0361] Example 3. Clinical trial of biomarker effects after administration of IL-2 conjugate (32 μg / kg [Q3W]) An extension of the above study for the 24 μg / kg dose was conducted to characterize the immunological effects of in vivo administration of the IL-2 conjugate administered by IV infusion at a dose of 32 μg / kg over 30 minutes every 3 weeks [Q3W]. The effects on the same biomarkers described in Example 2 were analyzed as surrogate predictors of safety and / or efficacy.

[0362] Subjects in this second cohort met the same criteria as those in Example 2. Tumor types included cervical, colorectal, pancreatic, and sarcoma.

[0363] Q3W Administration. Six individuals (5 [83.3%] male, 4 [66.7%] Caucasian) with advanced or metastatic solid tumors were administered the IL-2 conjugate at a 32 μg / kg dose Q3W (one dose per cycle). Here and throughout the Examples, the drug mass per kg of subject (e.g., 32 μg / kg) refers to the IL-2 mass excluding PEG and linker mass.

[0364] Efficacy biomarker: Peripheral CD8+T eff Cell counts were measured (Figures 21A-21B). Prolonged CD8+ expansion above baseline (e.g., 4-fold or greater change) was observed in some subjects at 3 weeks. Peripheral CD8+ memory cell counts are shown in Figures 22A-22B.

[0365] Peripheral NK cell counts are shown in Figures 23A-23B. An increase in NK cell counts was observed in each subject.

[0366] peripheral CD4+T reg The numbers are shown in Figures 24A-24B.

[0367] Eosinophil counts were measured (Figures 25A-25B). Measured values ​​did not exceed a four-fold increase and were consistently below the range of 2,328 to 15,958 eosinophils / μl reported in patients with IL-2-induced eosinophilia (Pisani et al., Blood 1991 Sep 15;78(6):1538-44).

[0368] Levels of IFN-γ, IL-5, and IL-6 were also measured ( FIG. 26 ). Measurements showed that IFN-γ was induced, but low amounts of IL-5 and IL-6 (cytokines associated with VLS and CRS, respectively), except in one subject whose IL-6 levels increased to approximately 700 pg / mL 4 hours after treatment but then decreased.

[0369] Summary and discussion of results. All subjects tested had CD8+ T effector (Teff) cells, CD8+ memory cells, NK cells, and CD4+ T cells. reg There was peripheral proliferation after cell administration. There was no significant increase in IL-5 levels, and subjects who had elevated IL-6 levels on day 3 showed a decrease the following day.

[0370] One subject experienced fever at 16 hours on Day 1 of the first cycle and at 9 hours on Day 1 of the second cycle. A second subject had elevated blood pressure (162 / 9 mmHg) 16 hours after dosing in the first cycle. A third subject experienced two infusion reactions. The first was a Grade 1 reaction 2.5 hours after dosing on Day 1 of the first cycle. The second was a Grade 3 reaction 4 hours after dosing on Day 1 of the second cycle. On Day 1 of the first cycle, a fourth subject experienced Grade 1 CRS, including fever, rigors, and hypotension (135 / 63 to 106 / 61 mmHg). A fifth patient experienced G2 CRS, consisting of fever and hypotension, which was managed with hydration and dexamethasone. He subsequently developed G3 transaminitis, which was treated with dexamethasone. At C2D1, the subject experienced a second episode of G2 CRS, which was managed with steroids and hydration.

[0371] In summary, all six subjects had at least one treatment-emergent AE (TEAE). The TEAEs are detailed in Table 6. There were no grade 4 or 5 TEAEs.

[0372] [Table 6]

[0373] Two patients had SAEs: one patient had grade 1 CRS (fever, chills, and a drop in BP from 135 / 62 mmHg to 106 / 61 mmHg) requiring hospitalization and was managed with hydration and electrolyte replacement; another patient had grade 3 meningitis in the first cycle and grade 2 CRS (fever and hypotension) in both the first and second cycles and was managed with dexamethasone.

[0374] One DLT occurred, an infusion-related reaction requiring a dose reduction. There were no drug discontinuations due to TEAEs, but one patient's dose reduction occurred as a result of a TEAE (G3 infusion reaction). No subjects experienced anaphylaxis.

[0375] TEAEs resolved with accepted standard therapy. There was no significant elevation of IL-5, no cumulative toxicity, no end-organ toxicity, no QTc prolongation associated with G3 hypertension and G4 lymphopenia, or other cardiac toxicities. Thus, the IL-2 conjugate demonstrated encouraging PD data and was generally well tolerated. PK data (Table 7) were consistent with an in vivo half-life of the IL-2 conjugate of approximately 10 hours. Overall, the results appear to support the non-alpha-preferential activity of the IL-2 conjugate, have a tolerable safety profile, and provide preliminary evidence of activity in patients with PD and immune-sensitive tumors.

[0376] For PK evaluation, samples were taken from subjects to determine drug concentrations in the blood over time. Table 7 reports the mean and standard deviation of the blood concentration levels measured in cycles 1 and 2.

[0377] [Table 7]

[0378] Example 4. Clinical trial of diffuse large B-cell lymphoma treatment using IL-2 conjugates A Phase 2, non-randomized, open-label, multicenter study will be conducted to evaluate the clinical benefit of the IL-2 conjugate described in Example 1 as at least a third-line therapy for the treatment of participants with diffuse large B-cell lymphoma (DLBCL). Participants must be 12 years of age or older with DLBCL, have received at least two lines of systemic therapy, and be post-chimeric antigen receptor T-cell (CAR-T) therapy.

[0379] Participants will receive the IL-2 conjugate every 3 weeks (21 days per cycle) on day 1 of each cycle for up to 35 cycles.

[0380] The following inclusion criteria apply: Participants must be 12 years of age or older at the time of signing the informed consent. Participants' disease location must be suitable for tumor biopsy at baseline. Participants must have measurable disease. If participants are female, they are eligible to participate if they are not pregnant or breastfeeding and are not women of childbearing potential (WOCBP) or are WOCBP who agree to the following: (1) use an approved method of contraception and follow routine pregnancy testing before treatment and for at least 180 days after discontinuation of study treatment, and (2) refrain from egg donation or cryopreservation for 180 days after discontinuation of study treatment. If participants are male, they are eligible to participate if they agree to refrain from sperm donation or cryopreservation and abstain from heterosexual intercourse or use an approved method of contraception during study treatment and for at least 210 days after discontinuation of study treatment. Participants must be able to give signed informed consent. Participants had to have a histologically or cytologically confirmed diagnosis of DLBCL and had to have received at least two prior lines of systemic therapy for DLBCL, including at least one containing a combination of an anthracycline and rituximab (or another anti-CD20 agent), with the last line of treatment being a commercially available anti-CD19 CAR-T therapy.

[0381] Participants will be excluded from the study if any of the following criteria apply: Eastern Cooperative Oncology Group (ECOG) performance status ≥ 2 (≥ 16 years old) - Lansky scale (<16 years) <50% - Poor bone marrow reserve - Organ dysfunction - Participants with baseline SpO2 ≤ 92% - Lymphomatous involvement of the central nervous system - History of allogeneic or solid organ transplant - The last dose of prior antineoplastic therapy or any investigational treatment was within 21 days or less than 5 half-lives, whichever is shorter, or major surgery or local intervention within 21 days - Previous IL-2-based anti-cancer therapy - Comorbidities requiring corticosteroid treatment - Use of antibiotics (excluding topical antibiotics) within 14 days prior to the first dose of IL-2 conjugate - Severe or unstable cardiac condition within 6 months prior to starting study treatment - Active, known, or suspected autoimmune disease requiring systemic treatment within the past two years - Known second malignancy that is progressing or requiring active treatment within the past 3 years - Receipt of live or live-attenuated virus vaccination within 28 days of planned treatment initiation (seasonal influenza vaccine or SARS-CoV-2 vaccine that does not contain live virus are acceptable).

[0382] Disease progression can be monitored in patients according to various criteria.

[0383] Complete response rate (CRR), defined as the proportion of participants with a determined complete response (CR), as determined according to the Lugano response criteria 2014, can be assessed. CRR can be assessed until the date of first documented progression or initiation of subsequent anticancer therapy, or approximately 8 months after the last participant received their first dose.

[0384] Objective response rate (ORR) can be assessed as the proportion of participants with a CR or partial response (PR) as determined according to the Lugano response criteria 2014. ORR can be assessed until the date of first documented progression or initiation of subsequent anticancer therapy, or approximately 8 months after the last participant received their first dose.

[0385] Time to response can be assessed, defined as the time from the first dose of IL-2 conjugate to the first documented evidence of PR or CR, determined according to the Lugano response criteria 2014. Time to response can be assessed from the date of the first dose to the date of first documented progression or death from any cause, whichever occurs first, for up to 36 months.

[0386] Duration of response (DoR), defined as the time from first documented evidence of CR or PR to progressive disease (PD) or death from any cause, whichever occurs first, can be assessed, as determined according to the Lugano response criteria 2014. DoR can be assessed from the date of first dose to the date of first documented progression or death from any cause, whichever occurs first, up to 36 months.

[0387] Clinical benefit rate (CBR), including CR or PR or at least 6 months of stable disease (SD) at any time from the first dose of IL-2 conjugate to PD (per Lugano response criteria 2014), or death from any cause, whichever occurs first, can be assessed. CBR can be assessed until the date of first documented progression or initiation of subsequent anticancer therapy, or approximately 8 months after the last participant received their first dose.

[0388] Progression-free survival (PFS), defined as the time from the first date of IL-2 conjugate administration to the date of first documented disease progression by Lugano Response Criteria 2014, or death from any cause, whichever occurs first, can be assessed. PFS can be assessed from the date of first dose to the date of first documented progression or death from any cause, whichever occurs first, for up to 36 months.

[0389] CAR-T cell activation and proliferation, along with suppression of circulating B cells, can be assessed until the date of first documented progression or initiation of subsequent anticancer therapy, or approximately 8 months after the last participant received their first dose.

[0390] Pharmacokinetic parameters, such as the concentration of the IL-2 conjugate and the incidence of any anti-drug antibodies (ADAs) to the IL-2 conjugate, can also be assessed in patients at various time points throughout the study. For example, the plasma concentration of the IL-2 conjugate can be assessed on days 1 and 15 of cycle 1, on day 1 of cycles 2-4-7-10, and every 5 cycles (each cycle is 21 days) for up to about 24 months. The incidence of any ADAs to the IL-2 conjugate can also be assessed on days 1 and 15 of cycle 1, on day 1 of cycles 2-4-7-10, and every 5 cycles (each cycle is 21 days) and 30 days after the last administration of the IL-2 conjugate for up to about 24 months.

[0391] To confirm the dose of IL-2 conjugate with or without other anticancer therapy, the incidence of any dose-limiting toxicity (DLT) can be assessed over one cycle (21 days). DLTs include, for example, grade 3 neutropenic fever (absolute neutrophil count (ANC) <1,000 / mm3). 3 , a single temperature >38.3°C (101°F) or a sustained temperature >38°C (100.4°F) for more than 1 hour).

[0392] To assess the safety profile of IL-2 conjugates in combination with or without other anticancer therapies, the incidence of any treatment-emergent adverse events (TEAEs) and laboratory abnormalities can be assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) V5.0 and the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading. All such TEAEs and laboratory abnormalities can be assessed from the first dose of IL-2 conjugate to 30 days after the last dose of IL-2 conjugate.

[0393] To assess the safety profile of IL-2 conjugates in combination with or without other anticancer therapies, the incidence of serious adverse events (SAEs) and laboratory abnormalities can be assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) V5.0 and the American Society for Transplantation and Cellular Therapy (ASTCT) consensus grading. Both such SAEs and laboratory abnormalities can be assessed from the first dose of IL-2 conjugate to 90 days after the last dose of IL-2 conjugate.

[0394] The duration of the study period per participant is a maximum of 6 years (screening period [28 days], treatment period [maximum 35 cycles = 735 days], and follow-up period [approximately 3 years]).

[0395] Example 5. Use of IL-2 conjugates to stimulate proliferation and persistence of CAR-T cells in co-culture with large B-cell lymphoma (LBCL) cells A study was conducted to evaluate the addition of the IL-2 conjugate described in Example 1 to CAR-T cells cocultured with two wild-type LBCL cell lines (HT and RL) using three patient peripheral blood mononuclear cell samples to generate CAR-T cells. The CAR-T cells expressed a construct equivalent to the CD3 / CD28 design used in the anti-CD19 CAR-T cell axicabtagene ciloleucel. Cocultures were generated at a 1:1 effector-to-target ratio, and fresh cancer cells were added every two days with the IL-2 conjugate or vehicle control until day 6. Flow cytometry analysis was performed on days 4 and 8 to determine T cell counts and immunophenotypes.

[0396] The addition of IL-2 conjugates significantly increased the number of expanded CAR-T cells, increased the expression of activation markers, and decreased the expression of exhaustion markers. As shown in Figure 27A, IL-2 conjugates led to sustained proliferation of CAR-T cells for at least 8 days. This effect was more robust when compared with the control condition (Figure 27A, right) and the condition with IL-2 addition (Figure 27A, center). Furthermore, the addition of IL-2 conjugates led to an increase in the proportion of effector memory (EM) cells on day 8. See Figure 27B, Q4 in each section. Thus, IL-2 conjugates can inhibit the terminal differentiation of CAR-T cells, as evidenced by the sustained proliferation of CAR-T cells and the increased generation of effector memory cells, contributing to the sustained killing of LBCL cells.

[0397] Example 6. Evaluation of concentrations of IL-2 conjugates in combination with anti-CD19 / CD22 CAR-T cells in lymphoma mouse models Materials and Methods MHC I / II double knockout NOD.Cg-Prkdc scid H2-K1 tm1Bpe H2-Ab1 em1Mvw H2-D1 tm1Bpe Il2rg tm1WjlA study was conducted to evaluate the concentration of the IL-2 conjugate of Example 1 for use with anti-CD19 / CD22 CAR-T cells against Raji-Luc Burkitt lymphoma cells in / SzJ (NSG-MHC I / II DKO) mice (The Jackson Laboratory, Bar Harbor, Maine).

[0398] Mice were treated and evaluated as described in Table 8. On day 0, mice received 0.5 x 10 6 10 Raji-Luc cells were transplanted. Mice were enrolled into four groups as shown in Table 8. On days 9, 16, and 23, IL-2 conjugates were injected intravenously according to Table 8. On day 10, mice were injected with 2 x 10 6 Anti-CD19 / CD22 CAR-T cells (ProMab Biotechnologies, Richmond, California) were administered intravenously. The anti-CD19 / CD22 CAR of the CAR-T cells contained the sequence of SEQ ID NO: 43.

[0399] Mice were imaged on days 7, 11, 15, 22, 29, and 36. Prior to imaging, mice were intraperitoneally injected with 150 mg / kg D-luciferin at a dose volume of 10 mL / kg. Mice were weighed on selected days, as shown in Figure 30. For droplet digital polymerase chain reaction (ddPCR) analysis on selected days, 50 μL of whole blood was collected from the submandibular vein of the mice into K2EDTA tubes on wet ice and then stored at -80°C, as shown in Figure 31.

[0400] [Table 8]

[0401] result Figure 28 shows the average radiance reflecting the growth of engrafted Raji-Luc cells in the four groups of mice. Figure 29 shows radiance imaging in the four groups of mice. Addition of CAR-T cells in group 2 resulted in suppression of Raji-Luc cell growth, and addition of both CAR-T cells and IL-2 conjugates in groups 3 and 4 resulted in even greater suppression of Raji-Luc cell growth. Figure 30 shows the weight change in the four groups of mice.

[0402] Figure 31 shows the copy number of anti-CD19 / CD22 CAR-T constructs as determined by ddPCR. Group 1 (control) has numbers around 0 and is not apparent in this figure (because mice in Group 1 did not receive anti-CD19 / CD22 CAR-T cells).

[0403] Example 7. Evaluation of the antitumor activity of IL-2 conjugate and anti-CD19 / CD22 CAR-T cells as single agents and in combination in lymphoma mouse models Materials and Methods MHC I / II double knockout NOD.Cg-Prkdc scid H2-K1 tm1Bpe H2-Ab1 em1Mvw H2-D1 tm1Bpe Il2rg tm1Wjl A study was conducted to evaluate the antitumor activity of the IL-2 conjugate of Example 1 and anti-CD19 / CD22 CAR-T cells, as single agents and in combination, against Raji-Luc Burkitt's lymphoma cells in / SzJ (NSG-MHC I / II DKO) mice (The Jackson Laboratory, Bar Harbor, Maine).

[0404] Mice were treated and evaluated as described in Table 9. On day 0, 40 mice were weighed and then 2.5 x 10 6 0.5 x 10 cells / mL in a 200 µL volume with a concentration of 610 Raji-Luc cells were transplanted. On day 7, the mice were weighed. On day 8, the mice were enrolled into five groups as shown in Table 9. On days 9, 16, and 23, IL-2 conjugates were intravenously injected according to Table 9, and the mice were monitored for the first 1 and 6 hours after administration, and the mice were also weighed. On day 10, 2 x 10 6 Mice were weighed before intravenous administration of non-transduced T (mock-T) cells or anti-CD19 / CD22 CAR-T cells (PRoMab Biotechnologies, Richmond, California). The anti-CD19 / CD22 CAR of the CAR-T cells contained the sequence of SEQ ID NO: 43. Mice were monitored for the first 1 and 6 hours after administration.

[0405] Mice were imaged on days 7, 11, 18, 23, 25, and 29. Prior to imaging, mice were intraperitoneally injected with 150 mg / kg D-luciferin at a dose volume of 10 mL / kg. From day 21 onward, mice were monitored daily, and the date of death and phenotype were recorded.

[0406] On day 14, 50 μL of whole blood was collected from the mandibular vein of mice into K2EDTA tubes on wet ice for ddPCR analysis and then stored at -80°C. On day 21, 100 μL of whole blood was collected from the mandibular vein of mice into K2EDTA tubes, aliquoted into 50 μL portions, and then stored at -80°C for flow cytometry and ddPCR analysis. On days 22 and 25, 50 μL of whole blood was collected from the mandibular vein of mice into K2EDTA tubes on wet ice for ddPCR analysis of select groups and then stored at -80°C. On day 28, 100 μL of whole blood was collected from the mandibular vein of mice into K2EDTA tubes, aliquoted into 50 μL portions, and then stored at -80°C for flow cytometry and ddPCR analysis.

[0407] Mice underwent body weight (BW) measurements at least twice weekly and were clinically observed. Mice were euthanized if they lost more than 20% BW or developed hind limb paralysis. By day 21, one mouse was euthanized in each of groups 1, 2, and 4. By day 22, one mouse was euthanized in each of groups 1 and 2, and two mice were euthanized in group 4. By day 25, one mouse was euthanized in each of groups 1, 2, 3, and 5, and two mice were euthanized in group 4. By day 28, all mice were euthanized in each of groups 1, 2, and 4. Four mice were euthanized in group 3, and three mice were euthanized in group 5. By day 31, all mice were euthanized in each of groups 1, 2, and 4. In group 3, 4 mice were euthanized, and in group 5, 3 mice were euthanized.

[0408] If mice were euthanized early or terminally, on day 31, 100 μL of whole blood was collected for ddPCR analysis and stored at -80°C, and the remaining whole blood (>200 μL) was collected and processed for cytokine analysis. Mouse livers and spleens were weighed and then fixed for immunohistochemical analysis.

[0409] [Table 9]

[0410] result Figure 32A shows the mean radiance reflecting the growth of engrafted Raji-Luc cells in the five groups of mice. Addition of CAR-T cells in group 3 resulted in suppression of Raji-Luc cell proliferation, and addition of both CAR-T cells and IL-2 conjugates in group 5 resulted in even greater suppression of Raji-Luc cell proliferation. Figure 32B shows the weight change in the five groups of mice.

[0411] Figure 33 shows the CD4+ quantification by flow cytometry in Groups 3 and 5 on days 21 and 28. + CAR-T cells and CD8 +The amount of CAR-T cells is shown. The addition of IL-2 conjugates increased CD4 + CAR-T cells and CD8 + Both CAR-Ts significantly increased the number of patients.

[0412] Figure 34 shows the amount of different types of cells at day 21, as quantified by flow cytometry. The addition of both CAR-T cells and IL-2 conjugates in Group 5 significantly increased the number of hCD45 cells compared to any one of Groups 1-4. + cells, hCD4 + cells, hCD8 + cells, scFvCD9 + hCD8 + Cells and scFvCD9 + hCD4 + It significantly increased the number of Raji_Luc cells and significantly decreased the number of Raji_Luc cells.

[0413] Figure 35 shows the copy number of the anti-CD19 / CD22 CAR-T construct as determined by ddPCR. The addition of IL-2 conjugate in group 5 significantly increased the copy number of the CAR-T construct compared to group 3. Groups 1, 2, and 4 had a copy number of approximately 0 and are not apparent in this figure (because mice in groups 1, 2, and 4 did not receive anti-CD19 / CD22 CAR-T cells).

[0414] Example 8. Use of IL-2 conjugates to improve killing of BL-ALL cells by anti-CD19 CAR-NK cells Materials and Methods Preparation of lentivirus: One day before transfection, 5 x 10 6 HEK293T cells were seeded in 10 cm dishes in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS) and incubated overnight so that the cells were approximately 80% confluent the next day.

[0415] The next day, a plasmid DNA mixture was prepared by adding 1 μg of pUC.BaEV-Rless plasmid, 6.54 μg of Gagpol plasmid, 3.27 μg of REV plasmid, and 18 μg of CAR19 transfection plasmid encoding the CAR of axicabagin-ciloleucel to 2 mL of serum-free medium in a 50 mL tube and mixed by vortexing.

[0416] 30 μL of Lipofectamine 2000 was added to 2 mL of serum-free medium and mixed by vortexing. The diluted Lipofectamine 2000 mixture was then added to the DNA mixture, vortexed, and incubated at room temperature for 20 minutes. 5 mL of DMEM and 10% FBS was then added and gently mixed by inverting the tube several times.

[0417] The dish containing HEK293T cells was removed from the incubator and examined under a microscope. The medium was carefully aspirated, and the transfection mixture was then carefully added without disrupting the cell monolayer. The plate was then incubated overnight at 37°C. The transfection medium was then replaced with fresh DMEM and 10% FBS, and after 48 hours of incubation, the virus-containing supernatant was collected. The supernatant was then filtered through a 0.45 μm polyvinylidene fluoride (PVDF) filter. One dose of Lenti-X™ Concentrator (Takara, catalog number 631231) was mixed with three volumes of medium containing the virus and left in the refrigerator overnight. The Lenti-X and virus mixture was then centrifuged at 600 g for 45 minutes, the supernatant was discarded, and the pellet was resuspended in 0.5 mL of phosphate-buffered saline (PBS). The presence of virus was confirmed using Lenti-X™ GoStix™ (Takara, catalog number 631280). Virus aliquots were stored at -80°C.

[0418] NK cell isolation and transduction: NK cells were isolated from two peripheral blood mononuclear cells (PBMCs) using Stemcell isolation reagent (Cat. No. 17955, 20144) at a concentration of 0.5 × 106 The cells were resuspended at 1.25 x 10 / mL. 1.5 mL / well was plated in a 12-well plate. IL-2 and PM21, particles made from K562 derivative cells expressing IL-21 and 4-1BB, were added and incubated for 7 days. The cells were then washed, counted, and collected at 1.25 x 10 6 Cells / ml were resuspended in CTS Xpander medium (gibco, catalogue no. A49161-01) containing 10% human ab serum and IL-2 (20 ng / ml).

[0419] 250,000 cells / well were seeded into a 96-well flat-bottom plate containing 10 μg / mL Vectofusin (Miltenyi, Cat. No. 130-111-163) and thawed virus. An untransduced control was included. The plate was centrifuged at 600 g in a Beckman Coulter benchtop centrifuge for 45 minutes at room temperature. The plate was then incubated for 72 hours. Cells were then harvested, washed, and aliquots were taken to verify CAR expression using flow cytometry. Briefly, cells were treated for 20 minutes at 4°C in staining buffer containing TruStain FcXFc block (BioLegend, Cat. No. 422302), anti-CD56 BV421 (BioLegend, Cat. No. 318328), and CD19 Fc chimera Atto647 (R&D Biosystems, Cat. No. DMUL0522101). The cells were then washed twice with staining buffer before being analyzed on a BD Fortessa flow cytometer. The percentage of cells expressing CAR was determined as CD56+ and CAR+ (Figure 36). FlowJo was used to analyze the flow cytometry data.

[0420] Labeling of Nalm6 target cells: The CD19-expressing B-lineage acute lymphoblastic leukemia (BL-ALL) cell line, Nalm6, was grown in the presence of PM21 and selected as the target cell line for cytotoxicity experiments using NK cells expressing CAR (CAR-NK cells). Sensitivity to puromycin in Nalm6 cells was determined by incubating them in RPMI 1640 and 10% FBS culture medium with several concentrations of puromycin (ranging from 10 μg / mL to 0.5 μg / mL) and observing cell death using a microscope. 1 μg / mL was determined to be the appropriate concentration for selection. Nalm6 cells were transfected into 48-well plates in the presence of NucLight Red lentivirus (Sartorius, catalog no. 4625). The plates were centrifuged at 600 g for 45 minutes in a benchtop centrifuge and then incubated overnight at 37°C. The medium was replaced, and the cells were incubated for an additional day before undergoing puromycin selection. After puromycin addition, the cells were incubated until only NucLight Red-positive cells survived. Non-transduced Nalm6 cells were included as a control for selection. For culture maintenance, cells were cultured in the presence of 0.5 μg / mL puromycin.

[0421] CAR-NK killing assay: Duplicate wells were set up. A 96-well flat-bottom plate (black with a clear bottom) was coated with 50 μL of poly-L-ornithine and placed in a hood for 30 minutes. After 30 minutes, the solution was aspirated and the plate was allowed to dry in the hood. Target Nalm6 cells expressing NucLight Red were seeded into the plate at 15,000 cells / well in 100 μL of RPMI and 10% FBS medium.

[0422] The IL-2 conjugate of Example 1 was prepared in a 5-fold dilution series to final concentrations of 2 μg / mL, 0.4 μg / mL, 0.08 μg / mL, 0.016 μg / mL, and 0 μg / mL in PBS. (5 μL of the 2 mg / mL IL-2 conjugate stock was added to 500 μL of medium, from which 100 μL was transferred to 400 μL for a 5-fold dilution, repeated two more times, including 0 μg / mL.) 22 μL of the IL-2 conjugate was added to target Nalm6 cells.

[0423] CAR-NK cells were added in 100 μL at 45,000 cells / well at a 3:1 effector-to-target ratio, and the plate was centrifuged at 90 g for 10 minutes with a deceleration setting of 3. The plate was then placed in an Incucyte® S3 Live-Cell Imaging and Analysis System (Sartorius) and set to scan every 1-2 hours using a 4X objective, capturing one picture in the selected red fluorescent channel. Red object counts were used to determine the number of viable cells. Counts obtained from wells containing only target cells (no CAR-NK cells) were defined as 100% viable cells or 0% killing and used to calculate the % killing in experimental wells (Figure 37).

[0424] Every 2–4 days, 100 μL of supernatant was carefully removed without touching the bottom of the well and transferred to a new 96-well plate. The supernatant was then frozen for subsequent IFN-γ analysis. The removed supernatant was replaced with 100 μL of fresh medium containing 15,000 Nalm6 NucLight Red cells in the wells, and the plate was centrifuged as before and returned to the Incucyte® System. (The first time point (T=1) was 2 days, the second time point (T=2) was 4 days, the third time point (T=3) was 8 days, and the fourth time point (T=4) was 10 days.)

[0425] IFN-γ levels in the supernatants were determined using an IFN-γ detection kit (Mesoscale Discovery, catalog number K151QOD-4) and a Sector Imager 6000 (Mesoscale Discovery) according to the manufacturer's protocol (FIG. 38).

[0426] result Figure 36 shows CAR expression in NK cells from two donors. As shown in the figure, CAR-NK cells generated from NK cells from the two donors expressed CAR at levels of 12% and 11%, respectively, as shown above, compared to non-transduced control NK cells.

[0427] Figure 37 shows the killing of Nalm6 cancer cells by CAR-NK cells measured at different time points (T=1, T=2, T=3, =4) in the presence of various concentrations of IL-2 conjugates. Specifically, the figure shows the rate of killing of Nalm6 target cells by CAR-NK cells generated from NK cells from two different donors at each indicated time point, with or without IL-2 conjugates, after each addition of Nalm6 cells. Nalm6 cell killing continues in an IL-2 conjugate dose-dependent manner.

[0428] Figure 38 shows IFN-γ production from co-cultures of CAR-NK cells and Nalm6 cells in the presence of IL-2 conjugates. Specifically, the figure shows the IFN-γ response from CAR-NK cells at each indicated time point after each addition of Nalm6 cancer cells. Over time, IFN-γ is detected in the supernatant in a dose-response manner to IL-2 conjugate concentration for both donors.

[0429] As demonstrated, the presence of the IL-2 conjugate enhanced the ability and / or persistence of CAR-NK cells to kill Nalm6 target cells. This killing effect persisted for more than 10 days, particularly when higher concentrations of the IL-2 conjugate were used. IFN-γ continued to be produced over time in a dose-dependent manner and correlated with the IL-2 conjugate concentration.

[0430] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

[0431] Sequence Listing The following sequence listing provides the sequences referenced in the claims and specification.

[0432] [Table 10]

[0433] [Table 11]

[0434] [Table 12]

[0435] [Table 13]

[0436] [Table 14]

[0437] [Table 15]

[0438] Table 16

[0439] Table 17

[0440] Table 18

[0441] Table 19

[0442] Table 20

[0443] Table 21

Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising administering to said subject (a) an IL-2 conjugate, and (b) a chimeric antigen receptor (CAR) therapy; The IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is of formula (I): 【Chemistry 1】 (In the formula, Z is CH ２ and Y is 【Chemistry 2】 and Y is CH ２ and Z is 【Transformation 3】 and Z is CH ２ and Y is 【Chemistry 4】 or Y is CH ２ and Z is 【Transformation 5】 and W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa; q is 1, 2, or 3; X has the structure: 【Transformation 6】 is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue. The method is replaced by the structure of

2. 10. The method of claim 1, wherein the cancer is leukemia, myeloma, or lymphoma.

3. 3. The method of claim 2, wherein the leukemia is acute lymphoblastic leukemia or chronic lymphocytic leukemia.

4. 3. The method of claim 2, wherein the myeloma is multiple myeloma.

5. 3. The method of claim 2, wherein the lymphoma is non-Hodgkin's lymphoma, follicular lymphoma, transformed follicular lymphoma, mantle cell lymphoma, primary mediastinal B-cell lymphoma, Burkitt's lymphoma, or diffuse large B-cell lymphoma (DLBCL).

6. 10. The method of claim 2 or claim 5, wherein the lymphoma is Burkitt's lymphoma.

7. 10. The method of claim 2 or claim 5, wherein the lymphoma is diffuse large B-cell lymphoma (DLBCL).

8. 1. A method of treating diffuse large B-cell lymphoma (DLBCL) in a subject in need thereof, comprising administering to the subject an IL-2 conjugate; The IL-2 conjugate comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at position P64 is of formula (I): 【Transformation 7】 (In the formula, Z is CH ２ and Y is 【Transformation 8】 and Y is CH ２ and Z is 【Chemistry 9】 and Z is CH ２ and Y is 【Chemistry 10】 or Y is CH ２ and Z is 【Chemistry 11】 and W is a PEG group with an average molecular weight of about 25 kDa to 35 kDa; q is 1, 2, or 3; X has the structure: 【Chemistry 12】 is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue. The method is replaced by the structure of

9. 9. The method of claim 7 or claim 8, wherein the DLBCL is relapsed or refractory DLBCL, or the DLBCL has relapsed after two or more prior lines of systemic therapy for DLBCL.

10. 10. The method of any one of claims 7-9, further comprising selecting a subject to be administered the IL-2 conjugate based at least in part on the subject having received two or more prior lines of systemic therapy for DLBCL.

11. 11. The method of claim 9 or claim 10, wherein the two or more prior lines of systemic therapy for DLBCL comprise an anthracycline, an anti-CD20 agent, or a combination of an anthracycline and an anti-CD20 agent.

12. 12. The method of claim 11, wherein the anti-CD20 agent comprises rituximab.

13. The method of any one of claims 9 to 12, wherein two or more prior lines of systemic therapy for DLBCL include CAR therapy.

14. 14. The method of claim 13, wherein the CAR therapy is the last line of two or more prior lines of systemic therapy for DLBCL.

15. 15. The method of any one of claims 8-14, further comprising selecting a subject to be administered the IL-2 conjugate based at least in part on the subject having undergone the CAR therapy.

16. 16. The method of any one of claims 8-15, further comprising administering a CAR therapy to the subject.

17. 17. The method of any one of claims 1-7 and 13-16, wherein the CAR therapy comprises T cells expressing a CAR.

18. 18. The method of any one of claims 1-7 and 13-17, wherein the CAR therapy comprises gamma delta T cells expressing a CAR.

19. 18. The method of any one of claims 1-7 and 13-17, wherein the CAR therapy comprises natural killer (NK) T cells expressing a CAR.

20. 17. The method of any one of claims 1-7 and 13-16, wherein the CAR therapy comprises natural killer (NK) cells expressing a CAR.

21. The method of any one of claims 17 to 20, wherein the CAR comprises an anti-CD19 domain comprising an anti-CD19 heavy chain variable domain (VH) and an anti-CD19 light chain variable domain (VL).

22. 22. The method of claim 21, wherein the anti-CD19 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

5.

23. 23. The method of claim 21 or claim 22, wherein the anti-CD19 VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

6.

24. The method of any one of claims 21 to 23, wherein the anti-CD19 VH comprises the amino acid sequence of SEQ ID NO:

6.

25. The method of any one of claims 21 to 24, wherein the CD19 VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

9.

26. 26. The method of any one of claims 21 to 25, wherein the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

10.

27. The method of any one of claims 21 to 26, wherein the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO:

10.

28. 22. The method of claim 21, wherein the anti-CD19 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 11, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

13.

29. 29. The method of claim 21 or claim 28, wherein the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

14.

30. 30. The method of any one of claims 21, 28 and 29, wherein the anti-CD19 VH comprises the amino acid sequence of SEQ ID NO:

14.

31. The method of any one of claims 21 and 28 to 30, wherein the CD19 VL comprises CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

17.

32. 32. The method of any one of claims 21 and 28-31, wherein the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

8.

33. 33. The method of any one of claims 21 and 28 to 32, wherein the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO:

18.

34. 22. The method of claim 21, wherein the anti-CD19 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

21.

35. 35. The method of claim 21 or claim 34, wherein the anti-CD19 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

22.

36. 36. The method of any one of claims 21, 34 and 35, wherein the anti-CD19 VH comprises the amino acid sequence of SEQ ID NO:

22.

37. 37. The method of any one of claims 21 and 34 to 36, wherein the CD19 VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

25.

38. 38. The method of any one of claims 21 and 34-37, wherein the anti-CD19 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

26.

39. 39. The method of any one of claims 21 or 34-38, wherein the anti-CD19 VL comprises the amino acid sequence of SEQ ID NO:

26.

40. The method of any one of claims 17 to 39, wherein the CAR comprises an anti-CD22 domain comprising an anti-CD22 heavy chain variable domain (VH) and an anti-CD22 light chain variable domain (VL).

41. 41. The method of claim 40, wherein the anti-CD22 VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

29.

42. 42. The method of claim 40 or claim 41, wherein the anti-CD22 VH comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

30.

43. The method of any one of claims 40 to 42, wherein the anti-CD22 VH comprises the amino acid sequence of SEQ ID NO:

30.

44. 44. The method of any one of claims 40 to 43, wherein the CD22 VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

33.

45. 45. The method of any one of claims 40-44, wherein the anti-CD22 VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

34.

46. The method of any one of claims 40 to 45, wherein the anti-CD22 VL comprises the amino acid sequence of SEQ ID NO:

34.

47. The method of any one of claims 17 to 39, wherein the CAR is an anti-CD19 CAR.

48. 48. The method of claim 47, wherein the anti-CD19 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

45.

49. 49. The method of claim 47 or claim 48, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO:

45.

50. 48. The method of claim 47, wherein the anti-CD19 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

47.

51. 51. The method of claim 47 or claim 50, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO:

47.

52. The method of any one of claims 17 to 46, wherein the CAR is an anti-CD19 / CD22 CAR.

53. 53. The method of claim 52, wherein the anti-CD19 / CD22 CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

43.

54. 54. The method of claim 52 or claim 53, wherein the anti-CD19 / CD22 CAR comprises the amino acid sequence of SEQ ID NO:

43.

55. The method of any one of claims 17 to 20, wherein the CAR is an anti-BCMA CAR.

56. 56. The method of claim 55, wherein the anti-BCMA CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

57.

57. 57. The method of claim 55 or claim 56, wherein the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO:

57.

58. 56. The method of claim 55, wherein the anti-BCMA CAR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

67.

59. 57. The method of claim 55 or claim 56, wherein the anti-BCMA CAR comprises the amino acid sequence of SEQ ID NO:

67.

60. The method of any one of claims 17 to 20, wherein the CAR comprises an anti-BCMA domain comprising an anti-BCMA heavy chain variable domain (VH) and an anti-BCMA light chain variable domain (VL).

61. 61. The method of claim 60, wherein the anti-BCMA VH comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO:

51.

62. 62. The method of claim 60 or claim 61, wherein the anti-BCMA VH comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

52.

63. The method of any one of claims 60 to 62, wherein the anti-BCMA VH comprises the amino acid sequence of SEQ ID NO:

52.

64. 64. The method of any one of claims 60 to 63, wherein the BCMA VL comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 54, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

55.

65. 65. The method of any one of claims 60-64, wherein the anti-BCMA VL comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

56.

66. 66. The method of any one of claims 60 to 65, wherein the anti-BCMA VL comprises the amino acid sequence of SEQ ID NO:

56.

67. The CAR comprises a first anti-BCMA single domain antibody (V Ｈ H) and / or a second anti-BCMA V Ｈ The method of any one of claims 17 to 20, comprising an anti-BCMA domain comprising H.

68. The first anti-BCMA V Ｈ 68. The method of claim 67, wherein H comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:

61.

69. The first anti-BCMA V Ｈ 69. The method of claim 67 or claim 68, wherein H comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

62.

70. The first anti-BCMA V Ｈ 70. The method of any one of claims 67 to 69, wherein H comprises the amino acid sequence of SEQ ID NO:

62.

71. The second anti-BCMA V Ｈ 71. The method of any one of claims 67 to 70, wherein H comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 63, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:

65.

72. The second anti-BCMA V Ｈ 72. The method of any one of claims 67-71, wherein H comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

66.

73. The second anti-BCMA V Ｈ 73. The method of any one of claims 67 to 72, wherein H comprises the amino acid sequence of SEQ ID NO:

66.

74. The CAR therapy is administered at a dose of 1.0.2 to 5.0 x 10 per kg of the subject's body weight. ６ or 0.5 to 1.0 x 10 ６ 74. The method of any one of claims 13 to 73, comprising cells.

75. The CAR therapy is administered at a dose of 1.0 x 10 per kg of the subject's body weight. ６ or 2.0 x 10 ６ 75. The method of any one of claims 13 to 74, comprising cells.

76. The CAR therapy is 0.1 to 2.5 × 10 ８ or 0.6 to 6.0 × 10 ８ 76. The method of any one of claims 13 to 75, comprising cells.

77. The CAR therapy is 0.9 to 1.1 × 10 ８ pieces, 0.5 to 1.1×10 ８ or 3.0 to 4.6 x 10 ８ 77. The method of any one of claims 13 to 76, comprising cells.

78. 78. The method of any one of claims 16-77, wherein the CAR therapy is administered to the subject by intravenous administration.

79. 79. The method of any one of claims 1-7 and 16-78, wherein the CAR therapy is administered to the subject prior to administering to the subject any dose of an IL-2 conjugate.

80. 79. The method of any one of claims 1-7 and 16-78, wherein one dose of an IL-2 conjugate is administered to the subject before the CAR therapy is administered to the subject, and one or more additional doses of an IL-2 conjugate are administered to the subject after the CAR therapy is administered to the subject.

81. 79. The method of any one of claims 1-7 and 16-78, wherein one dose of an IL-2 conjugate is administered to the subject one day before the CAR therapy is administered to the subject, and one or more additional doses of an IL-2 conjugate are administered to the subject about once per week after the one dose of the IL-2 conjugate is administered to the subject.

82. 82. The method of any one of claims 1 to 81, comprising administering to the subject about 16 μg / kg of IL-2 as an IL-2 conjugate.

83. 82. The method of any one of claims 1 to 81, comprising administering to the subject about 24 μg / kg of IL-2 as an IL-2 conjugate.

84. 82. The method of any one of claims 1 to 81, comprising administering to the subject about 32 μg / kg of IL-2 as an IL-2 conjugate.

85. 85. The method of any one of claims 1 to 84, wherein the PEG groups in the IL-2 conjugate have an average molecular weight of about 30 kDa.

86. The IL-2 conjugates wherein Z is CH ２ and Y is 【Chemistry 13】 The method according to any one of claims 1 to 85, wherein

87. The IL-2 conjugate wherein Y is CH ２ and Z is 【Chemistry 14】 The method according to any one of claims 1 to 85, wherein

88. The IL-2 conjugate wherein z is CH ２ and Y is 【Chemistry 15】 The method according to any one of claims 1 to 85, wherein

89. The IL-2 conjugate wherein Y is CH ２ and Z is 【Chemistry 16】 The method according to any one of claims 1 to 85, wherein

90. The structure of formula (I) has the structure of formula (IV) or formula (V), or a mixture of formula (IV) and formula (V). 【Chemistry 17】 (In the formula, q is 1, 2, or 3; X has the structure: [Chemistry 18] is an L-amino acid having the formula X-1 indicates the point of attachment to the preceding amino acid residue, and X+1 indicates the point of attachment to the subsequent amino acid residue. The method according to any one of claims 1 to 85, wherein

91. The structure of formula (I) has the structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII): 【Chemistry 19】 (In the formula, n is -(OCH ２ CH ２ ) ｎ -OCH ３ is an integer such that it has a molecular weight of about 30 kDa, q is 1, 2, or 3, and The wavy line indicates a covalent bond to an amino acid residue in SEQ ID NO: 1 that is not substituted. The method according to any one of claims 1 to 85, wherein

92. 92. The method of any one of claims 1 to 91, wherein q is 1.

93. 92. The method of any one of claims 1 to 91, wherein q is 2.

94. 92. The method of any one of claims 1 to 91, wherein q is 3.

95. 95. The method of any one of claims 1-94, wherein the IL-2 conjugate is administered to the subject about once per week, about once per two weeks, about once per three weeks, or about once per four weeks.

96. 96. The method of claim 95, wherein the IL-2 conjugate is administered to the subject about once every three weeks.

97. 97. The method of any one of claims 1 to 96, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate or hydrate.

98. 98. The method of any one of claims 1 to 97, wherein the IL-2 conjugate is administered to the subject by intravenous administration.

99. 98. The method of any one of claims 1 to 97, wherein the IL-2 conjugate is administered to the subject by subcutaneous administration.

100. 100. The method of any one of claims 1-99, further comprising administering acetaminophen to the subject.

101. 101. The method of any one of claims 1 to 100, further comprising administering diphenhydramine to the subject.

102. 102. The method of claim 100 or claim 101, wherein the acetaminophen and / or diphenhydramine is administered to the subject prior to administering the IL-2 conjugate.

103. 103. An IL-2 conjugate for use in a method according to any one of claims 1 to 102.

104. Use of an IL-2 conjugate for the manufacture of a medicament for the method according to any one of claims 1 to 102.

Images