CN111647068A - Human interleukin 2 variant or derivative thereof - Google Patents

Human interleukin 2 variant or derivative thereof Download PDF

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CN111647068A
CN111647068A CN202010138036.1A CN202010138036A CN111647068A CN 111647068 A CN111647068 A CN 111647068A CN 202010138036 A CN202010138036 A CN 202010138036A CN 111647068 A CN111647068 A CN 111647068A
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陈磊
胡齐悦
葛虎
林�源
王宏伟
欧阳超
孔祥林
廖成
张连山
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Shanghai Hengrui Pharmaceutical Co Ltd
Shanghai Shengdi Pharmaceutical Co Ltd
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Abstract

The present disclosure relates to a human interleukin 2 variant or derivative thereof. In particular, the disclosure relates to IL-2 or derivatives thereof having one or more amino acid mutations, having an eliminated or reduced affinity for a high affinity receptor (IL-2R α/β/γ) and retaining affinity for a medium affinity receptor (IL-2R β/γ). The IL-2 variant or derivative thereof provides a good basis for protein pharmacy.

Description

Human interleukin 2 variant or derivative thereof
Technical Field
The present disclosure relates to human interleukin-2 (IL-2) variants or derivatives thereof having one or more amino acid mutations. The IL-2 variants or derivatives have an eliminated or reduced affinity for the high affinity receptor (IL-2R α/β/γ) and retain affinity for the medium affinity receptor (IL-2R β/γ). The disclosure also relates to immunoconjugates, encoding polynucleotides, vectors, host cells, pharmaceutical compositions, methods of preparation and methods of treatment and uses comprising the human IL-2 variants or derivatives thereof.
Background
Human interleukin-2 (interleukin-2, IL-2), also known as T Cell Growth Factor (TCGF), is located on chromosome 4 (4q27) and comprises a total 7kb sequence consisting of 133 amino acids and a molecular weight of about 15 kD. In 1976 and 1977, culture broth of activated T cells was found to promote T cell proliferation by Doris Morgan, Francis Rusceti, Robert Gallo and Steven Gillis, Kendal Smith, respectively. The stimulatory factor in the culture broth is then purified and identified as a single protein, i.e., IL-2.
Initial in vitro cell experiments showed that T cells, after activation by TCR and CD28, can secrete IL-2 and express the IL-2 receptor (IL-2R) on the cell surface. The binding of IL-2 to its receptor can cause the proliferation and effect of T cells. This model makes IL-2 a molecule that plays a central role in T cell immune responses. However, subsequent in vivo experiments have shown that following IL-2 knock-out or its receptor, the animals develop autoimmunity. Subsequent experiments have shown that IL-2 can activate not only effector cells such as T cells and NK cells, but also regulatory T cells, thereby suppressing excessive immunity against itself.
IL-2 acts through IL-2R. IL-2R includes three subunits, IL-2R α (i.e., CD25), IL-2R β (i.e., CD122), and IL-2R γ (i.e., CD 132). Three subunits can form three receptor forms: the high binding force receptor comprises all three subunits IL-2R α/β/γ, and the binding force receptor comprises IL-2R β/γ and the low binding force receptor IL-2R α. Wherein, IL-2R beta and IL-2R gamma are necessary for IL-2 to activate a downstream signal path, when IL-2 is combined with IL-2R beta and IL-2R gamma, two receptor subunits form heterodimer, phosphorylate STAT5 in cells, enter cell nucleus and cause corresponding gene transcription and expression; IL-2R α is not required for signaling, but can promote the binding of IL-2 to IL-2R β and IL-2R γ. IL-2R gamma is expressed in all immune cells; IL-2R beta is expressed in CD8+ T cells, NK cells and regulatory T cells, and the expression level is also increased after the T cells are activated; IL-2R alpha is continuously highly expressed in regulatory T cells, and is transiently expressed in activated CD8+ T cells, and then expression level is down-regulated.
IL-2 is synthesized predominantly by activated T cells, especially CD4+ helper T cells. It stimulates proliferation and differentiation of T cells, induces production of Cytotoxic T Lymphocytes (CTLs) and differentiation of peripheral blood lymphocytes into cytotoxic and Lymphokine Activated Killer (LAK) cells, promotes expression of cytokines and cytolytic molecules by T cells, promotes proliferation and differentiation of B cells and immunoglobulin synthesis by B cells, and stimulates production, proliferation and activation of Natural Killer (NK) cells.
The ability of IL-2 to expand lymphocyte populations and enhance effector functions of these cells in vivo makes it an anti-tumor effect, IL-2 immunotherapy is the treatment of choice in certain patients with metastatic cancer, and high doses of IL-2 are currently approved for the treatment of metastatic renal cell carcinoma and malignant melanoma.
Previous studies of IL-2 variants have shown that IL-2 variants with mutations in at least four of the positions 38, 42, 45, 62, 68 have reduced stimulatory effects on regulatory T cells (WO 2012062228); IL-2 variants containing mutations at positions 72, 42, 45, have reduced or eliminated binding to high binding IL-2 receptors, but retain binding to intermediate binding IL-2 receptors (CN 201280017730.1); variants with mutations at positions 91, 126, which mutations allow IL-2 to bind to CD25(IL2R α) but do not activate IL-2R on regulatory T cells (US 8906356); IL-2R comprising at least one E15, H16, Q22, D84, N88 or E95 mutation for use in the treatment of graft-versus-host disease in a subject (US 9732134); IL-2 variants comprising at least R38W have the ability to decrease vascular permeability and treat solid tumors (US 7371371; US 7514073; US 8124066; US 7803361); fusion protein of IL-2 variant and Fc for treating diseases, wherein the IL-2 has N88R mutation (WO 2016014428); a chimeric polypeptide comprising a cytokine linked to an immune cell surface protein targeting ligand, wherein the cytokine may be a variant of IL-2 (WO2017136818) or the like.
However, there is still a lack in the prior art of IL-2 variants with higher stability, reduced level of activation of regulatory T cells, and no influence on the activation of immune effector cells. Providing such IL-2 variants and derivatives thereof to enhance the effectiveness of IL-2 therapy is a problem that needs to be addressed in the art.
Disclosure of Invention
The present disclosure relates to human interleukin 2 variants or derivatives thereof having one or more amino acid mutations.
In a first aspect, the present disclosure provides an IL-2 variant or derivative thereof comprising one or more amino acid mutations in the region that binds to the IL-2 receptor alpha subunit (IL-2 ra), said mutation(s) being a substitution of one or more amino acids of the region on human interleukin 15(IL-15) that binds to IL-2 ra to the region on IL-2 that binds to IL-2 ra.
In some embodiments, the IL-2 variant or derivative thereof has reduced affinity for IL-2R α and unchanged or increased affinity for IL-2R β and/or IL-2R γ.
In some embodiments, the IL-2 variant or derivative thereof has reduced affinity for a high affinity receptor (IL-2R α/β/γ), but retains affinity for a medium affinity receptor (IL-2R β/γ).
In some embodiments, the IL-2 variant or derivative thereof described above has reduced activation of regulatory T cells (tregs), and/or immune effector cells (e.g., T cells, NK cells) are not affected or increased.
In some embodiments, the mutation of the IL-2 variant or derivative thereof occurs at one or more of amino acid residues 29-44, or 41-44, or 35-44 of IL-2.
In some embodiments, the variants are asparagine (N) at position 26 and/or asparagine (N) at position 30 and/or glutamine (Q) at position 11 and/or leucine (L) at position 132 and/or leucine (L) at position 70 and/or proline (P) at position 82 and/or glycine (G) at position 27 and/or phenylalanine (F) at position 78 and/or asparagine (N) at position 29 and/or asparagine (N) at position 30 and/or tyrosine (Y) at position 31 and/or lysine (K) at position 32 and/or asparagine (N) at position 33 and/or proline (P) at position 34 and/or lysine (K) at position 35 and/or leucine (L) at position 36 and/or threonine (T) at position 37 and/or arginine (R) at position 38 and/or methionine (M) at position 39 and/or methionine (M) at position 35 of the wild-type human interleukin 2(SEQ ID NO:2) sequence Or leucine (L) at position 40 and/or threonine (T) at position 41 and/or phenylalanine (F) at position 42 and/or lysine (K) at position 43 and/or phenylalanine (F) at position 44 and/or tyrosine (Y) at position 45 and/or asparagine (N) at position 71 to other amino acids. The numbering of the sites is from position 2 of the mature human IL-2 protein.
In some embodiments, the IL-2 variant or derivative comprises a mutation at position 26 to Gln (q) and/or a mutation at position 30 to Ser(s) and/or a mutation at position 11 to cys (c) and/or a mutation at position 132 to cys (c) and/or a mutation at position 70 to cys (c) and/or a mutation at position 82 to cys (c) and/or a mutation at position 27 to cys (c) and/or a mutation at position 78 to cys (c) and/or a mutation at positions 29 to 44 to Gln-Ser-Met-His-Ile-Asp-Ala-Thr-leu qsmhidatl and/or a mutation at position 45 to alanine (a) and/or a mutation at position 72 to glycine (G) and/or a mutation at position 71 to Gln (q).
In some embodiments, the IL-2 variant or derivative thereof comprises a mutation at position 26 to gln (q) and/or a mutation at position 29 to ser(s) and/or a mutation at position 30 to ser(s) and/or a mutation at position 11 to cys (c) and/or a mutation at position 132 to cys (c) and/or a mutation at position 70 to cys (c) and/or a mutation at position 82 to cys (c) and/or a mutation at position 27 to cys (c) and/or a mutation at position 78 to cys (c) and/or a mutation at positions 41 to 44 Asp-Ala-Thr-leu (datl) and/or a mutation at position 45 to alanine (a) and/or a mutation at position 72 to glycine (G) and/or a mutation at position 71 to gln (q).
In some embodiments, the IL-2 variant or derivative thereof comprises a mutation at position 26 to gln (q) and/or a mutation at position 29 to ser(s) and/or a mutation at position 30 to ser(s) and/or a mutation at position 11 to cys (c) and/or a mutation at position 132 to cys (c) and/or a mutation at position 70 to cys (c) and/or a mutation at position 82 to cys (c) and/or a mutation at position 27 to cys (c) and/or a mutation at position 78 to cys (c) and/or a mutation at positions 35 to 44 to His-Ile-Asp-Ala-Thr-leu (mhidatl) and/or a mutation at position 42 to alanine (a) and/or a mutation at position 45 to alanine (a) and/or a mutation at position 72 to glycine (G) and/or a mutation at position 71 to gln q.
In some embodiments, the IL-2 variant or derivative thereof comprises a mutation of Asn 26 to Gln (N26Q) and/or a mutation of Asn 30 to Ser (N30S) and/or a mutation of Gln 11 to Cys (Q11C) and/or a mutation of Leu 132 to Cys (L132C) and/or a mutation of Leu 70 to Cys (L70C) and/or a mutation of Pro 82 to Cys (P48382) and/or a mutation of Gly 27 to Cys (G27C) and/or a mutation of Phe 78 to Cys (F78C) and/or a mutation of Gln-Gln-Tyr-Lys-Gln-Pro-Lys-Leu-Thr-Arg-Met-Lys-Thr-Phe-NNNNPKMLTFT) to Gln-Ser-His-Asp-Thr-Ser-His-Asp-Ser The leucine mutation glycine (L72G) and/or the 71 Asn mutation Gln (N71Q).
In some embodiments, the above-mentioned IL-2 variant or derivative thereof comprises a mutation of Asn 26 to gin (N26Q) and/or Asn 29 to Ser (N29S) and/or Asn 30 to Ser (N30S) and/or Gln 11 to Cys (Q11C) and/or Leu 132 to Cys (L132C) and/or Leu 70 to Cys (L70C) and/or Pro 82 to Cys (P82C) and/or Gly 27 to Cys (G27C) and/or Phe 78 to Cys (F78C) and/or Lys-Leu-Thr-Arg-Met-Lys-Thr-Phe-kltrmltff 35-44 (G27G C)) and/or a mutation of Met-His-Asp-Ala-Thr-Leu-Phe) and/or a mutation of Asn 45 to Gly (e) and/or Lys 72 to Leu 72 and/or Leu 72 (e) and/or a mutation of Asn 45 to Cys (e and/or Cys 72) and/or Leu 72 to Cys 72 (G72) and/or a mutation of Phe 72 to Cys The Asn at position 71 was mutated to Gln (N71Q).
In some embodiments, the variant IL-2 or derivative thereof comprises mutation of Asn 26 to gin (N26Q) and/or Asn 29 to Ser (N29S) and/or Asn 30 to Ser (N30S) and/or Asn 11 to Cys (Q11C) and/or Leu 132 to Cys (L132C) and/or Leu 70 to Cys (L70C) and/or Pro 82 to Cys (P82C) and/or Gly 27 to Cys (G27C) and/or Phe 78 to Cys (F78C) and/or Thr-Phe-Lys-Phe (tfkf) 41-44 to Asp-Ala-Thr-Leu-dall and/or tyr 45 to alanine (Y A) and/or Leu 72 to Gly (72) and/48371 and/or Gln 71).
In some embodiments, the IL-2 variant or derivative thereof described above comprises Q11C/L132C and/or L70C/P82C and/or G27C/F78C.
In some embodiments, in the IL-2 variant or derivative thereof described above, when Q11C/L132C is comprised, a disulfide bond is formed between the C at position 11 and the C at position 132; when L70C/P82C is contained, a disulfide bond is formed between C at position 70 and C at position 82; when G27C/F78C is contained, a disulfide bond is formed between C at position 27 and C at position 78.
In some embodiments, IL-2 has one or more amino acid mutations selected from any one of the following (I) - (VII), or any combination thereof:
(I) NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL, or TFKF mutation at position 41-44 is DATL, or KLTRMLTFKF mutation at position 35-44 is MHIDATL;
(II)N26Q；
(III)N29S；
(IV)N30S；
(V) Q11C/L132C, or L70C/P82C, or G27C/F78C;
(VI) F42A/Y45A, or F42A/L72G, or Y45A/L72G, or F42A, or Y45A, or L72G, or F42A/Y45A/L72G;
(VII)N71Q。
the above combinations of mutations include, but are not limited to, any of the following groups (1) to (540):
(1) NNYKNPKLTRMLTFKF mutation at position N26Q/29-44 to QSMHIDATL;
(2) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/L132C;
(3) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/L70C/P82C;
(4) NNYKNPKLTRMLTFKF mutation at G27C/29-44 to QSMHIDATL/F78C;
(5) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/Y45A;
(6) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/N71Q;
(7) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/L72G;
(8) NNYKNPKLTRMLTFKF mutations at positions 26Q/29-44 of Q11C/N26 are QSMHIDATL/L132C;
(9) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/L70C/P82C;
(10) the NNYKNPKLTRMLTFKF mutation at the 27C/29-44 position of N26Q/G27 is QSMHIDATL/F78C;
(11) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A;
(12) NNYKNPKLTRMLTFKF mutation at position N26Q/29-44 to QSMHIDATL/N71Q;
(13) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/L72G;
(14) NNYKNPKLTRMLTFKF mutations at positions Q11C/N26Q/29-44 are QSMHIDATL/Y45A/L132C;
(15) NNYKNPKLTRMLTFKF mutations at positions from Q11C/N26Q/29-44 are QSMHIDATL/N71Q/L132C;
(16) NNYKNPKLTRMLTFKF mutations at positions 26Q/29-44 of Q11C/N26 are QSMHIDATL/L72G/L132C;
(17) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/L70C/P82C;
(18) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/L70C/N71Q/P82C;
(19) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/L70C/L72G/P82C;
(20) NNYKNPKLTRMLTFKF mutation at the 27C/29-44 position of N26Q/G27 is QSMHIDATL/Y45A/F78C;
(21) the NNYKNPKLTRMLTFKF mutation at the 27C/29-44 position of N26Q/G27 is QSMHIDATL/N71Q/F78C;
(22) the NNYKNPKLTRMLTFKF mutation at the 27C/29-44 position of N26Q/G27 is QSMHIDATL/L72G/F78C;
(23) NNYKNPKLTRMLTFKF mutations at positions from Q11C/N26Q/29-44 are QSMHIDATL/Y45A/N71Q/L132C;
(24) NNYKNPKLTRMLTFKF mutations at positions 26Q/29-44 of Q11C/N26 are QSMHIDATL/Y45A/L72G/L132C;
(25) NNYKNPKLTRMLTFKF mutations at positions from Q11C/N26Q/29-44 are QSMHIDATL/N71Q/L72G/L132C;
(26) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/L70C/P82C;
(27) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/L70C/N71Q/P82C;
(28) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/L70C/L72G/P82C;
(29) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/L70C/N71Q/P82C;
(30) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/L70C/L72G/P82C;
(31) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/L70C/N71Q/L72G/P82C;
(32) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/Y45A/L132C;
(33) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/N71Q/L132C;
(34) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/L72G/L132C;
(35) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/Y45A/N71Q/L132C;
(36) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/Y45A/L72G/L132C;
(37) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/N71Q/L72G/L132C;
(38) NNYKNPKLTRMLTFKF mutation of Q11C/29-44 position is QSMHIDATL/Y45A/N71Q/L72G/L132C;
(39) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/Y45A/L70C/P82C;
(40) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/L70C/N71Q/P82C;
(41) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/L70C/L72G/P82C;
(42) the NNYKNPKLTRMLTFKF mutation at the 29-44 position is QSMHIDATL/Y45A/L70C/N71Q/P82C;
(43) the NNYKNPKLTRMLTFKF mutation at the 29-44 position is QSMHIDATL/Y45A/L70C/L72G/P82C;
(44) the NNYKNPKLTRMLTFKF mutation at the 29-44 position is QSMHIDATL/L70C/N71Q/L72G/P82C;
(45) the NNYKNPKLTRMLTFKF mutation at the 29-44 position is QSMHIDATL/Y45A/L70C/N71Q/L72G/P82C;
(46) NNYKNPKLTRMLTFKF mutation at the G27C/29-44 position is QSMHIDATL/Y45A/F78C;
(47) NNYKNPKLTRMLTFKF mutation at the G27C/29-44 position is QSMHIDATL/N71Q/F78C;
(48) NNYKNPKLTRMLTFKF mutation at the G27C/29-44 position is QSMHIDATL/L72G/F78C;
(49) NNYKNPKLTRMLTFKF mutation at G27C/29-44 is QSMHIDATL/N71Q/L72G/F78C;
(50) NNYKNPKLTRMLTFKF mutation at the G27C/29-44 position is QSMHIDATL/Y45A/N71Q/L72G/F78C;
(51) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/Y45A/N71Q;
(52) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/Y45A/L72G;
(53) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/N71Q/L72G;
(54) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL/Y45A/N71Q/L72G;
(55) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/N71Q;
(56) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/L72G;
(57) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/N71Q/L72G;
(58) the NNYKNPKLTRMLTFKF mutation at the Q11C/N26Q/29-44 position is QSMHIDATL/Y45A/N71Q/L72G/L132C;
(59) NNYKNPKLTRMLTFKF mutation at the N26Q/29-44 position is QSMHIDATL/Y45A/L70C/N71Q/L72G/P82C;
(60) the NNYKNPKLTRMLTFKF mutation at the 27C/29-44 position of N26Q/G27 is QSMHIDATL/Y45A/N71Q/L72G/F78C;
groups (61) - (120), corresponding to the substitution of "NNYKNPKLTRMLTFKF mutation at positions 29-44 to QSMHIDATL" to "TFKF mutation at positions 41-44 to DATL" in groups (1) - (60);
groups (121) - (180) corresponding to groups (61) - (120) further comprising a mutation of N29S;
groups (181) - (240) corresponding to groups (61) - (120) further comprising a mutation of N30S;
groups (241) - (300) corresponding to groups (61) - (120) further comprising mutations of N29S/N30S;
groups (301) - (360), corresponding to the substitution of "NNYKNPKLTRMLTFKF mutation at positions 29-44 to QSMHIDATL" to "KLTRMLTFKF mutation at positions 35-44 to MHIDATL" in groups (1) - (60);
groups (361) - (420) corresponding to groups (301) - (360) further comprising the mutation of N29S;
groups (421) to (480) corresponding to groups (301) to (360) further comprising a mutation of N30S;
groups (481) to (540), which correspond to groups (301) to (360), further comprise mutations N29S/N30S.
In some embodiments, the amino acid sequence of the IL-2 variant or derivative thereof comprises SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40, or a fragment thereof. The amino acid sequence numbers are shown in the following table:
TABLE 1 human IL-2 wild-type and variant sequences
Figure BDA0002398016630000081
Figure BDA0002398016630000091
Figure BDA0002398016630000101
Figure BDA0002398016630000111
(Note 1: the above-mentioned mutation site numbering is calculated as the numbering of the mature human IL-2 protein, which does not contain amino acid M at position 1, so the numbering is counted starting with amino acid A at position 2. wherein, "/" indicates that the mutations are present simultaneously in the same IL-2 variant. Note 2: all mutants contain C125A, in order not to form dimers.)
In some embodiments, derivatives of the IL-2 variants include muteins, functional derivatives, functional fragments, bioactive peptides, fusion proteins, isoforms, or salts thereof that are related to the full-length, partial proteins of the IL-2 variants of the disclosure or that are obtained by further mutation based on the IL-2 variants of the disclosure. For example, fusion proteins comprising IL-2 variants, monomers or dimers or trimers or multimers of said IL-2 variants, various modified forms of said IL-2 variants (e.g., PEGylation, glycosylation, albumin conjugation or fusion, Fc fusion or conjugation, hydroxyethylation, removal of O-glycosylation, etc.), and homologs of said IL-2 variants in various species. The modification of IL-2 does not result in adverse effects on the immunogenicity associated with the treatment.
In some embodiments, the IL-2 variant or derivative is PEGylated (may be referred to as PEG-IL-2), e.g., is a mono-or di-PEGylated IL-2 variant or derivative. PEG-IL-2 variants or derivatives include SC-PEG linkers. In other embodiments, a PEG-IL-2 variant or derivative includes a methoxy-PEG-aldehyde (mPEG-ALD) linker. In certain embodiments, the PEG moiety has an average molecular weight of about 5KD to about 50KD, specifically 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 KD; or about 5KD to about 40KD, or about 10KD to about 30KD, or about 15KD to about 20 KD. In certain embodiments, the mPEG-ALD linker comprises a PEG molecule having an average molecular weight selected from the group consisting of: about 5KDa, about 12KDa, or about 20 KDa. In certain embodiments, the aldehyde group of mPEG-ALD can be acetaldehyde, propionaldehyde, butyraldehyde, or the like. In one embodiment, the IL-2 variant or derivative thereof has an extended serum half-life compared to wild-type IL-2 or derivative thereof.
In some embodiments, the IL-2 variant or derivative thereof is capable of eliciting one or more cellular responses selected from the group consisting of: proliferation in activated T lymphocytes, differentiation in activated T lymphocytes, cytotoxic T Cell (CTL) activity, proliferation in activated B cells, differentiation in activated B cells, proliferation in Natural Killer (NK) cells, differentiation in NK cells, cytokine secretion by activated T cells or NK cells, and NK/lymphocyte-activated killer (LAK) anti-tumor cytotoxicity. In some embodiments, the IL-2 variant or derivative thereof has a reduced ability to induce IL-2 signaling in regulatory T cells as compared to the wild-type IL-2 polypeptide. In one embodiment, the IL-2 variant or derivative thereof induces less activation-induced cell death (AICD) in the T cells compared to wild-type IL-2 or derivative thereof. In some embodiments, the IL-2 variant or derivative thereof has reduced in vivo toxicity compared to wild-type IL-2 or derivative thereof.
In some embodiments, IL-2 variants are provided that comprise mutations at positions 29-44 (mutations at positions 29-44 to QSMHIDATL, or mutations at positions 41-44 to DATL, or mutations at positions 35-44 to MHIDATL) that have reduced binding to IL-R α and substantially unchanged binding to IL-R β/γ.
In some embodiments, IL-2 variants are provided that comprise mutations at positions 29-44 (including mutations at positions 29-44 to QSMHIDATL, or mutations at positions 41-44 to DATL, or mutations at positions 35-44 to MHIDATL), while comprising one or more of N26Q, N29S, N30S, Q11C/L132C, L70C/P82C, G27C/F78C, that have reduced binding to IL-R α and substantially unchanged binding to IL-R β/γ with increased stability.
When the IL-2 variant has reduced binding force with IL-2R alpha and basically unchanged binding force with IL-2R beta/gamma, the IL-2 variant has reduced activation level on regulatory T cells and basically unchanged activation on immune effector cells relative to wild type IL-2, thereby increasing the curative effect.
In a second aspect, the present disclosure provides a linker or conjugate to which an IL-2 variant or derivative thereof is directly or indirectly attached via a linker to a non-IL-2 module. In some embodiments, it is an immunoconjugate, wherein the non-IL-2 moiety is an antigen binding moiety. In some embodiments, the antigen binding module targets an antigen presented on a tumor cell or in the environment of a tumor cell.
In some embodiments, the IL-2 variant is linked to at least one non-IL-2 module. In some embodiments, the IL-2 variant and the non-IL-2 module form a fusion protein, i.e., the IL-2 variant shares a peptide bond with the non-IL-2 module. In some embodiments, the IL-2 variant is linked to at least one non-IL-2 module, such as a first and second non-IL-2 module. In some embodiments, the non-IL-2 module is an antigen binding module. In some embodiments, the IL-2 variant shares an amino-or carboxy-terminal peptide bond with the first antigen-binding moiety, and the second antigen-binding moiety shares an amino-or carboxy-terminal peptide bond with: i) an IL-2 variant or ii) a first antigen binding moiety. In some particular embodiments, the IL-2 variant shares a carboxy-terminal peptide bond with the first non-IL-2 moiety and an amino-terminal peptide bond with the second non-IL-2 moiety. In some embodiments, the non-IL-2 moiety is an antigen-binding moiety. The antigen binding moiety may be an antibody or antigen binding fragment, including but not limited to an immunoglobulin molecule (e.g., an IgG (e.g., IgG1) -class immunoglobulin molecule), an antibody, or an antigen binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of polypeptide complexes comprising an antibody heavy chain variable region and an antibody light chain variable region, Fab, Fv, sFv, F (ab') 2, linear antibodies, single chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, peptidibodies, domain antibodies, and multispecific antibodies (bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scfvs, tandem tri-scfvs). Where the IL-2 variant is linked to more than one antigen binding moiety, e.g., first and second antigen binding moieties, each antigen binding moiety may be independently selected from various forms of antibodies and antigen binding fragments, e.g., the first antigen binding moiety may be a Fab molecule and the second antigen binding moiety may be an scFv molecule, or each of the first and second antigen binding moieties may be a Fab molecule. In some embodiments, where an IL-2 variant is linked to more than one antigen binding moiety, e.g., a first and a second antigen binding moiety, the antigen to which each antigen binding moiety is directed can be independently selected, e.g., the first and the second antigen binding moiety are directed to different antigens or to the same antigen.
In some embodiments, the antigen bound by the antigen binding moiety may be selected from the group consisting of: the a1 Domain of tenascin-C (TNC a1), the a2 Domain of tenascin-C (TNC a2), the external Domain of fibronectin (Extra Domain B) (EDB), carcinoembryonic antigen (CEA), and melanoma-associated chondroitin sulfate proteoglycan (MCSP). In some embodiments, tumor antigens include, but are not limited to, MAGE, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin (cyclophilin) b, colorectal-associated antigen (CRC) -C017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitope PSA-1, PSA-2 and PSA-3, Prostate Specific Membrane Antigen (PSMA), T cell receptor/CD 3-zeta chain, MAGE family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A5982, MAGE-A10, and the like, MAGE-A11, MAGE-A12, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the GAGE family of tumor antigens (e.g. GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, the MUC family, HER2/neu, p21, AS1, alpha-fetoprotein, E-calpain, alpha-catenin, beta-catenin (beta-catenin), prge-catenin, beta-catenin, PGN-P120, PmCOL-C-P-7, PmOCP-C-E, PmOCP-C-E, and Pm, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig idiotype, P15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA) -1, cerebroglycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2. In some embodiments, non-limiting examples of viral antigens include influenza hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, and HIV gp 120. In some embodiments, non-limiting examples of ECM antigens include syndecan (syndecan), heparanase (heparanase), integrin, osteopontin (osteopontin), link, cadherin, laminin, EGF-type laminin, lectin, fibronectin, notch, tenascin, and matrixin.
In a third aspect, the present disclosure provides a pharmaceutical composition comprising an IL-2 variant or derivative or immunoconjugate thereof as described above, optionally comprising a pharmaceutically acceptable diluent, carrier or adjuvant. The pharmaceutical composition may be a lyophilized formulation or an injectable solution.
In some embodiments, the pharmaceutical composition may contain from 0.01 to 99% by weight of the IL-2 variant or derivative or immunoconjugate thereof in a unit dose, or the amount of the IL-2 variant or derivative or immunoconjugate thereof in a unit dose of the pharmaceutical composition is from 0.1 to 2000mg (e.g., 1 to 1000 mg).
In a fourth aspect, the present disclosure provides nucleic acid sequences and amino acid sequences encoding the above IL-2 variants or derivatives thereof. The nucleic acid sequence may comprise SEQ ID NO: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17. SEQ ID NO: 19. SEQ ID NO: 21. SEQ ID NO: 23. SEQ ID NO: 25. SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO: 31. SEQ ID NO: 33. SEQ ID NO: 35. SEQ ID NO: 37. SEQ ID NO: 39, or a variant thereof. The amino acid sequence may comprise SEQ id no: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18. SEQ ID NO: 20. SEQ ID NO: 22. SEQ ID NO: 24. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO: 30. SEQ ID NO: 32. SEQ ID NO: 34. SEQ ID NO: 36. SEQ ID NO: 38. SEQ ID NO: 40, or a pharmaceutically acceptable salt thereof.
In a fifth aspect, the present disclosure provides an expression vector comprising a nucleic acid sequence of the above IL-2 variant or derivative thereof. The vector can be a eukaryotic expression vector, a prokaryotic expression vector and a viral vector.
In a sixth aspect, the present disclosure provides a host cell expressing the above vector. The host cell may be a prokaryotic or eukaryotic cell. In some embodiments, the host cell comprises a prokaryotic microorganism (e.g., e.coli) or various eukaryotic cells (e.g., Chinese Hamster Ovary (CHO), Human Embryonic Kidney (HEK) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells), insect cells, etc.). In some embodiments, host cells expressing glycosylated polypeptides may be used, derived from multicellular organisms (including, for example, invertebrates and vertebrates), such as plant and insect cells. Vertebrate cells can also be used as host cells, for example, suspension grown mammalian cell lines, monkey kidney CV1 line (COS-7), human embryonic kidney line (293 or 293T cells), young alendron kidney cells (BHK), mouse Sertoli (sertoli) cells (TM4 cells), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse breast tumor cells (MMT060562), TRI cells (as described, for example, in Mather et al, Annals N.Y.Acad Sci383, 44-68(1982), MR5639 cells and FS 367 cells, Chinese Hamster Ovary (CHO) cells, myeloma cell lines such as YO, NS0, P3X63, and Sp 2/360, and cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissues.
In a seventh aspect, the present disclosure provides the use of an IL-2 variant or a derivative, conjugate or pharmaceutical composition thereof for the manufacture of a medicament for the treatment of a proliferative disease, an immunological disease, for modulating a T cell mediated immune response, for stimulating the immune system of an individual. The proliferative disease may be a tumor or cancer (e.g., metastatic tumor or cancer), and may be a solid tumor (e.g., metastatic renal cell carcinoma and malignant melanoma).
In some embodiments, the IL-2 variants, or derivatives, immunoconjugates thereof, of the disclosure are useful in treating disease conditions that stimulate the host's immune system to benefit from, particularly conditions in which it is desirable to enhance a cellular immune response, which may include disease conditions in which the host's immune response is inadequate or deficient. In some embodiments, disease conditions for administration of IL-2 variants or derivatives thereof, immunoconjugates include tumors or infections where the cellular immune response is a key mechanism for specific immunity, such as cancer (e.g., renal cell carcinoma or melanoma), immunodeficiency (e.g., in HIV-positive patients, immunosuppressed patients), chronic infections, and the like. In some embodiments, enhancing the cellular immune response may include any one or more of: general elevation of immune function, elevation of T cell function, elevation of B cell function, restoration of lymphocyte function, elevation of IL-2 receptor expression, elevation of T cell responsiveness, elevation of natural killer cell activity or lymphokine-activated killer (LAK) cell activity, and the like.
In some embodiments, the disease for which the IL-2 variants, or derivatives, immunoconjugates of the disclosure are used to treat is a proliferative disorder, such as cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, hematologic cancer, skin cancer, squamous cell carcinoma, bone cancer, and renal cancer. Other cell proliferative disorders that can be treated using the IL-2 variants or derivatives thereof of the present disclosure include, but are not limited to, neoplasms located at: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Also included are precancerous conditions or lesions and cancer metastases. In certain embodiments, the cancer is selected from the group consisting of: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. Similarly, other cell proliferative disorders can also be treated with IL-2 variants of the disclosure or derivatives thereof, including but not limited to: hypergammaglobulinemia (hypergamma), lymphoproliferative disorders, pathoproteinemia (paraproteemias), purpura (purpura), sarcoidosis, Sezary Syndrome (Sezary Syn), Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis (histiocytosis) and any other cell proliferative disorder outside neoplasia (neoplasma) in the organ systems listed above. In other embodiments, the disease involves autoimmunity, transplant rejection, post-traumatic immune response, and infectious disease (e.g., HIV).
In some embodiments, methods are provided wherein IL-2 is administered to a subject at least 2 times daily, at least 1 time every 48 hours, at least once every 72 hours, at least once weekly, at least once every 2 weeks, at least once every month, at least once every 2 months, or at least once every 3 months. IL-2 may be administered by any effective route. In some embodiments, IL-2 is administered by parenteral injection, including subcutaneous injection. Particular embodiments relate to pharmaceutical compositions comprising a pharmaceutically acceptable amount of IL-2 (e.g., a therapeutically effective amount), including those agents described above, in combination with one or more pharmaceutically acceptable diluents, carriers or excipients (e.g., isotonic injection solutions). The pharmaceutical composition is typically a pharmaceutical composition suitable for human administration. Furthermore, in some embodiments, the pharmaceutical composition comprises at least one additional prophylactic or therapeutic agent. Some embodiments contain a sterile container of one of the above-described pharmaceutical compositions and optionally one or more additional components.
In an eighth aspect, the present disclosure provides a method of making an IL-2 variant or derivative, comprising introducing into wild-type human IL-2 a mutation of the aforementioned IL-2 variant or derivative, or using the aforementioned nucleic acid sequence, or using the aforementioned expression vector, or using the aforementioned host cell.
Drawings
FIG. 1: and (3) measuring the binding force of the wild type IL-2 and the variants IL-2-01, IL-2-02, IL-2-03, IL-2-04, IL-2-05, IL-2-06, IL-2-07, IL-2-08, IL-2-09, IL-2-13 and IL-2R alpha thereof detected by an ELISA experiment.
FIG. 2: the effect of wild type IL-2 and its variant IL-2-01, IL-2-02, IL-2-07, IL-2-08, IL-2-09 on STAT5 phosphorylation activity of mouse T lymphocyte CTLL2 was determined.
Fig. 3A-3F: the results of thermostability assays for wild-type IL-2 and its variants, A-F are the results of thermostability assays for IL-2(WT) (FIG. 3A), IL-2-01 (FIG. 3B), IL-2-02 (FIG. 3C), IL-2-03 (FIG. 3D), IL-2-04 (FIG. 3E), and IL-2-05 (FIG. 3F), respectively.
FIG. 4: determination that variant IL2-06 does not bind IL-15 Ra.
Detailed Description
In order that the disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The three letter codes and the one letter codes for amino acids used herein are as described in j. diol. chem,243, p3558 (1968).
Term(s) for
"Interleukin-2" or "IL-2" refers to any native IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 as well as any form of IL-2 that results from processing in a cell. The term also encompasses naturally occurring IL-2 variants, such as splice variants or allelic variants. Exemplary wild-type human IL-2 amino acid sequences are set forth in SEQ ID NO:2, respectively. Unprocessed human IL-2 additionally comprises a signal peptide of 20 amino acids N-terminal (as shown in SEQ ID NO: 272 in WO 2012107417), which is absent in the mature IL-2 molecule.
"amino acid mutation" includes amino acid substitutions, deletions, insertions, modifications, and any combination thereof, to achieve the final construct such that the final construct possesses the desired property, e.g., enhanced stability. Amino acid sequence deletions and insertions include amino and/or carboxy-terminal deletions and amino acid insertions. An example of a terminal deletion is the deletion of an alanine residue at position 1 of full-length human IL-2. Preferred amino acid mutations are amino acid substitutions. To alter the binding properties of, for example, an IL-2 polypeptide, non-conservative amino acid substitutions may be made, i.e., one amino acid is replaced with another amino acid having a different structural and/or chemical property. Preferred amino acid substitutions include the substitution of a hydrophilic amino acid for a hydrophobic amino acid. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art, including methods of site-directed mutagenesis, PCR, gene synthesis, chemical modification, and the like.
A "wild-type IL-2" is a form of IL-2 that is otherwise identical to a variant IL-2 polypeptide, except that it has a wild-type amino acid at each amino acid position in the variant IL-2 polypeptide. For example, if the IL-2 variant is full-length IL-2 (i.e., IL-2 that is not fused or conjugated to any other molecule), then the wild-type form of this variant is the full-length native IL-2. If the IL-2 variant is a fusion between IL-2 and another polypeptide encoded downstream of IL-2 (e.g., an antibody chain), then the wild-type form of this IL-2 variant is IL-2 having a wild-type amino acid sequence fused to the same downstream polypeptide. Furthermore, if the IL-2 variant is a truncated form of IL-2 (a mutated or modified sequence within the non-truncated portion of IL-2), then the wild-type form of the IL-2 variant is a similarly truncated IL-2 having a wild-type sequence. In order to compare the IL-2 receptor binding affinity or biological activity for various forms of IL-2 variants with the corresponding IL-2 wild-type form, the term "wild-type" encompasses forms of IL-2 that comprise one or more amino acid mutations that do not affect IL-2 receptor binding, e.g., a cysteine substitution to alanine C125A at a position corresponding to residue 125 of human IL-2, as compared to naturally-occurring, native IL-2. In some embodiments, the wild-type IL-2 comprises SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.
"derivatives" are intended to be interpreted broadly, including any IL-2 related product. Including but not limited to human and non-human IL-2 homologs, fragments or truncations, fusion proteins (e.g., fused to a signal peptide or other active, inactive component, such as an antibody or antigen-binding fragment thereof), modified forms (e.g., PEGylation, glycosylation, albumin conjugation/fusion, Fc conjugation and/or fusion, hydroxyethylation, etc.), conservatively modified proteins, and the like.
"CD 25" or "alpha subunit of the IL-2 receptor" refers to any native CD25 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), including "full-length" unprocessed CD25 as well as any form of CD25 that results from processing in cells, and also includes naturally occurring CD25 variants, such as splice variants or allelic variants. In certain embodiments, CD25 is human CD25, with exemplary sequences as set forth in SEQ ID NO: shown at 37.
"high affinity IL-2 receptor" refers to heterotrimeric forms of the IL-2 receptor that consist of a receptor gamma subunit (also known as the universal cytokine receptor gamma subunit, yc, or CD132), a receptor beta subunit (also known as CD122 or p70), and a receptor alpha subunit (also known as CD25 or p 55). In contrast, a "medium affinity IL-2 receptor" refers to an IL-2 receptor that contains only gamma and beta subunits and no alpha subunits (see, e.g., Olejniczak and Kasprzak, MedSci Monit14, RA179-189 (2008)).
"affinity ofForce "refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, "binding affinity" herein refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., a receptor and a ligand). The affinity of a molecule X for its partner Y can generally be determined by the dissociation constant (K)D) Expressed as dissociation and association rate constants (K, respectively)DissociationAnd KBonding of) The ratio of (a) to (b). As such, equal affinities may comprise different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by methods conventional in the art, including the methods described herein.
"regulatory T cells" or "TRegulatingBy cell "is meant a specialized CD4+ T cell type that can suppress the response of other T cells T regulatory cells are characterized by expression of the α subunit of the IL-2 receptor (CD25) and the transcription factor forkhead box P3(FOXP3) (Sakaguchi, annurev immunol22,531-62(2004)) and play a key role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors.
"Effector cells" refers to a population of lymphocytes that mediate the cytotoxic effects of IL-2. Effector cells include effector T cells such as CD8+ cytotoxic T cells, NK cells, lymphokine-activated killer (LAK) cells, and macrophages/monocytes.
An "antigen binding moiety" refers to a polypeptide molecule that specifically binds an antigenic determinant. In some embodiments, the antigen binding moiety is capable of directing the entity (e.g., cytokine or second antigen binding moiety) to which it is attached to a target site, e.g., to a specific type of tumor cell or to an antigenic determinant-bearing tumor stroma. Antigen binding moieties include antibodies and fragments thereof as further defined herein. Preferably the antigen binding moiety comprises an antigen binding domain of an antibody comprising an antibody heavy chain variable region and an antibody light chain variable region. In certain embodiments, the antigen binding moiety may comprise an antibody constant region, as further defined herein and known in the art. Useful heavy chain constant regions include any of the following 5 isoforms: α, γ, or μ. Useful light chain constant regions include any of the following 2 isoforms: κ and λ.
An "immunoconjugate" refers to a polypeptide molecule comprising at least one IL-2 moiety and at least one antigen binding moiety. In certain embodiments, the immunoconjugate comprises at least one IL-2 moiety and at least two antigen binding moieties. Specific immunoconjugates according to the disclosure consist essentially of one IL-2 moiety and two antigen binding moieties linked by one or more linker sequences. The antigen binding module can be linked to the IL-2 module by a variety of interactions and in a variety of configurations.
By "specific binding" is meant that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding module to bind specific antigenic determinants can be measured via enzyme-linked immunosorbent assays (ELISAs) or other techniques well known to those skilled in the art, such as surface plasmon resonance techniques (analyzed on BIAcore instruments) (Liljebelad et al, Glyco J17, 323-.
"antibody" is used herein 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 antigen-binding fragments, so long as they exhibit the desired antigen-binding activity. Antibodies may include murine, human, humanized, chimeric, and camelid antibodies. Illustratively, the antibody may be an immunoglobulin, a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, γ, α, and chain, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. In the five classes of igs, the second class of igs can have either kappa chains or lambda chains.
"antigen binding fragment" refers to a Fab fragment, Fab 'fragment, F (ab') 2 fragment, single chain Fv (i.e., sFv), nanobody (i.e., VHH), VH/VL domain that has antigen binding activity. The Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but lacks the constant region, and has the smallest antigen binding fragment of the total antigen binding site. Generally, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Two antibody variable regions can also be joined together with different linkers into a single polypeptide chain, known as single chain antibodies (scFv) or single chain fv (sFv).
"conservative modifications" apply to amino acid and nucleotide sequences. For a particular nucleotide sequence, conservative modifications refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or, in the case of nucleotides that do not encode amino acid sequences, to substantially identical nucleotide sequences. With respect to amino acid sequences, "conservative modifications" refer to the replacement of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) molecular μ lar Biology of The Gene, The Benjamin/Cummings Pub. Co., p. 224, (4 th edition)).
By "pegylated" is meant that at least one PEG molecule is linked to another molecule (e.g., a therapeutic protein). For example, Adagen (PEGylated formulation of adenosine deaminase) is approved for the treatment of severe combined immunodeficiency disorders. It has been shown that the attachment of polyethylene glycol can prevent proteolysis (see, e.g., Sada et al (1991) J. fermentation Bioengineering 71: 137-139). In the most common form, PEG is at one endA linear or branched polyether linked to a hydroxyl group and having the following general structure: HO- (CH)2CH2O)n-CH2CH2PEG conjugation of proteins can be activated by preparing derivatives of PEG with functional groups at some or both ends, which are suitable for reaction with lysine and N-terminal amino acid groups.a common approach to PEG conjugation of proteins is to activate PEG with functional groups, especially α or amino groups participating in conjugation.
"vector", "expression vector" is synonymous with "expression construct" and refers to a DNA molecule for introducing a specific gene in operable association therewith and directing its expression in a target cell, including vectors that are autonomously replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. The expression vectors herein comprise an expression cassette, allowing transcription of a large amount of stable mRNA. Once the expression vector is in the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and/or translation machinery.
"host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived therefrom (regardless of the number of passages). Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as the function or biological activity screened or selected in the originally transformed cell.
"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "administration", "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
By "treating" is meant administering an internal or external therapeutic agent, such as a composition comprising any of the IL-2 variants and derivatives thereof of the present disclosure, or comprising the variants or derivatives, to a subject diagnosed as having, suspected of having, or susceptible to one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in the subject or population being treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically unmeasurable degree.
The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce a desired therapeutic effect in the subject. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in each patient, they should alleviate the symptoms of the target disease in a statistically significant number of subjects, as determined according to any statistical test method known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpsra test, and Wilcoxon test.
An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular subject or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the subject, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
In this disclosure, "IL-2" may be used interchangeably with "IL 2".
Detailed Description
The present disclosure is further described below with reference to examples, but these examples do not limit the scope of the present disclosure.
Example 1 construction and expression of wild-type IL-2 and variants
1. Gene synthesis and recombinant expression vector construction
The wild-type IL-2 nucleic acid sequence was synthesized by Nanjing Kingsrei Biotech, Inc.
The synthetic wild-type IL-2 nucleic acid sequence is set forth in SEQ ID NO: 1, an Nde I cleavage site is added to the 5 'end, and a BamH I cleavage site is added to the 3' end. The nucleotide sequence is as follows:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTTGTCAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：1
the italic parts are the NdeI and BamHI cleavage sites, respectively.
The corresponding protein sequences are shown below:
MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
SEQ ID NO：2
the expression vector used in the present disclosure is E.coli expression vector pET-9a (Novagen, Cat.69431-3) available from Novagen. After the IL-2 nucleic acid sequence is synthesized, the DNA is connected to Nde I and BamH I enzyme cutting sites of pET-9a to obtain an expression vector of the wild type IL-2.
2. The amino acid mutations described in this disclosure are introduced in the wild-type IL-2 amino acid sequence.
In one variant, asparagine at position 26 is replaced with glutamine and the codons AAC at positions 76-78 of its corresponding nucleotide sequence are changed to CAG. The mutated nucleic acid sequence is as follows:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGCAGGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：3
the corresponding amino acid sequence is shown below, namely the IL-2-01 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILQGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：4
in one variant, asparagine at position 30 is replaced with serine and the codons AAC at positions 88-90 of its corresponding nucleotide sequence are changed to AGC. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAGCTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：5
the corresponding amino acid sequence is shown below, namely the IL-2-02 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNGINSYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：6
in one variant, the glutamine at position 11 is replaced with cysteine, and the codons CAG at positions 31-33 of its corresponding nucleotide sequence are changed to TGT; the leucine amino acid at position 132 was replaced with cysteine, and the codon CTG at the position 394-396 of the corresponding nucleotide sequence was changed to TGT. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCTGTCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCTGTACCTAATGAGGATCC
SEQ ID NO：7
the corresponding amino acid sequence is shown as follows, namely the IL-2-03 variant sequence:
MAPTSSSTKKTCLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTCT
SEQ ID NO：8
in one variant, the leucine at position 70 is replaced by a cysteine and the codon CTG at position 208 and 210 of its corresponding nucleotide sequence is changed to TGT; the proline at position 82 was replaced by cysteine, and the codon CCG at position 244-246 of the corresponding nucleotide sequence was changed to TGT. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTTGTAATCTGGCACAGAGCAAAAACTTTCATCTGCGTTGTCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：9
the corresponding amino acid sequence is shown as follows, namely the IL-2-04 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVCNLAQSKNFHLRCRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：10
in one variant, the glycine at position 27 is replaced by a cysteine and the codons GGC at positions 79-81 of its corresponding nucleotide sequence are changed to TGT; the phenylalanine at position 78 was replaced with cysteine, and the codon TTT at position 232-234 of the corresponding nucleotide sequence was changed to TGT. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACTGTATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTGTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：11
the corresponding amino acid sequence is shown below, namely the IL-2-05 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNCINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNCHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：12
in one variant, the peptide stretch at positions 29-44 is replaced with QSMHIDATL, and the codon AACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTC at the corresponding nucleotide sequence at positions 85-132 is changed to CAGAGCATGCATATTGATGCAACCCTG. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCCAGAGCATGCATATTGATGCAACCCTGTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：13
the corresponding amino acid sequence is shown below, namely the IL-2-06 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNGIQSMHIDATLYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：14
in one variant, the phenylalanine at position 42 is replaced by alanine, and the corresponding codon TTC at position 124 and 126 is changed to GCA; substitution of tyrosine at position 45 with alanine; the codon TAC at position 133-135 of the corresponding nucleotide sequence was changed to GCA. The mutated nucleic acid sequence is shown below: CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCGCAATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：15
The corresponding amino acid sequence is shown below, namely the IL-2-07 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：16
in one variant, the phenylalanine at position 42 is replaced by alanine, and the corresponding codon TTC at position 124 and 126 is changed to GCA; a substitution of the 72 th leucine to glycine; the CTG codon at the position of 214-216 of the corresponding nucleotide sequence is changed into GGC. The mutated nucleic acid sequence is shown below: CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATGGCGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：17
The corresponding amino acid sequence is shown below, namely the IL-2-08 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：18
in one variant, the tyrosine at position 45 is replaced by alanine, and the codon TAC at position 133 and 135 of the corresponding nucleotide sequence is changed to GCA; the leucine at position 72 was replaced by glycine, and the codon CTG at position 214-216 of the corresponding nucleotide sequence was changed to GGC. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTCGCAATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATGGCGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：19
the corresponding amino acid sequence is shown below, namely the IL-2-09 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFAMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：20
in one variant, asparagine at position 26 is replaced with glutamine, and the codons AAC at positions 76-78 of its corresponding nucleotide sequence are changed to CAG; replacing asparagine at position 30 with serine, and changing codon AAC at position 88-90 of the corresponding nucleotide sequence into AGC; the phenylalanine at position 42 is replaced by alanine, and the codon TTC at the position 124 and 126 of the corresponding nucleotide sequence is changed into GCA; the leucine at position 72 was replaced by glycine, and the codon CTG at position 214-216 of the corresponding nucleotide sequence was changed to GGC. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGCAGGGCATCAACAGCTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATGGCGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：21
the corresponding amino acid sequence is shown below, namely the IL-2-10 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILQGINSYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：22
in one variant, the glutamine at position 11 is replaced with cysteine, and the codons CAG at positions 31-33 of its corresponding nucleotide sequence are changed to TGT; replacing asparagine at position 26 with glutamine to change the codon AAC at positions 76-78 of the corresponding nucleotide sequence to CAG; replacing asparagine at position 30 with serine, and changing codon AAC at position 88-90 of the corresponding nucleotide sequence into AGC; the phenylalanine at position 42 is replaced by alanine, and the codon TTC at the position 124 and 126 of the corresponding nucleotide sequence is changed into GCA; replacing the leucine at the 72 th position with glycine, and changing the codon CTG at the 214-216 th position of the corresponding nucleotide sequence into GGC; the leucine amino acid at position 132 was replaced with cysteine, and the codon CTG at the position 394-396 of the corresponding nucleotide sequence was changed to TGT. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCTGTCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGCAGGGCATCAACAGCTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATGGCGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCTGTACCTAATGAGGATCC
SEQ ID NO：23
the corresponding amino acid sequence is shown below, namely the IL-2-11 variant sequence:
MAPTSSSTKKTCLQLEHLLLDLQMILQGINSYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTCT
SEQ ID NO：24
in one variant, asparagine at position 26 is replaced with glutamine, and the codons AAC at positions 76-78 of its corresponding nucleotide sequence are changed to CAG; replacing asparagine at position 30 with serine, and changing codon AAC at position 88-90 of the corresponding nucleotide sequence into AGC; the phenylalanine at position 42 is replaced by alanine, and the codon TTC at the position 124 and 126 of the corresponding nucleotide sequence is changed into GCA; the leucine at the position 70 is replaced by cysteine, and the codon CTG at the position 208-210 of the corresponding nucleotide sequence is changed into TGT; replacing the leucine at the 72 th position with glycine, and changing the codon CTG at the 214-216 th position of the corresponding nucleotide sequence into GGC; the proline at position 82 was replaced by cysteine, and the codon CCG at position 244-246 of the corresponding nucleotide sequence was changed to TGT. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGCAGGGCATCAACAGCTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTTGTAATGGCGCACAGAGCAAAAACTTTCATCTGCGTTGTCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：25
the corresponding amino acid sequence is shown below, namely the IL-2-12 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILQGINSYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVCNGAQSKNFHLRCRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：26
in one variant, asparagine at position 26 is replaced with glutamine, and the codons AAC at positions 76-78 of its corresponding nucleotide sequence are changed to CAG; replacing glycine at position 27 with cysteine, and changing codons GGC at positions 79-81 of the corresponding nucleotide sequence into TGT; replacing asparagine at position 30 with serine, and changing codon AAC at position 88-90 of the corresponding nucleotide sequence into AGC; the phenylalanine at position 42 is replaced by alanine, and the codon TTC at the position 124 and 126 of the corresponding nucleotide sequence is changed into GCA; replacing the leucine at the 72 th position with glycine, and changing the codon CTG at the 214-216 th position of the corresponding nucleotide sequence into GGC; the phenylalanine at position 78 was replaced with cysteine, and the codon TTT at position 232-234 of the corresponding nucleotide sequence was changed to TGT. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGCAGTGTATCAACAGCTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATGGCGCACAGAGCAAAAACTGTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：27
the corresponding amino acid sequence is shown below, namely the IL-2-13 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILQCINSYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNGAQSKNCHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：28
in one variant, asparagine at position 29 is replaced with serine, changing the codon AAC at position 85-87 of its corresponding nucleotide sequence to AGC; the phenylalanine at position 42 is replaced by alanine, and the codon TTC at the position 124 and 126 of the corresponding nucleotide sequence is changed into GCA; the leucine at position 72 was replaced by glycine, and the codon CTG at position 214-216 of the corresponding nucleotide sequence was changed to GGC. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAGCAACTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATGGCGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：29
the corresponding amino acid sequence is shown below, namely the IL-2-14 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILNGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：30
in one variant, asparagine at position 26 is replaced with glutamine, and the codons AAC at positions 76-78 of its corresponding nucleotide sequence are changed to CAG; replacing asparagine at position 29 with serine to change the codon AAC at positions 85-87 of the corresponding nucleotide sequence to AGC; the phenylalanine at position 42 is replaced by alanine, and the codon TTC at the position 124 and 126 of the corresponding nucleotide sequence is changed into GCA; the leucine at position 72 was replaced by glycine, and the codon CTG at position 214-216 of the corresponding nucleotide sequence was changed to GGC. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGCAGGGCATCAGCAACTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATGGCGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：31
the corresponding amino acid sequence is shown below, namely the IL-2-21 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：32
in one variant, asparagine at position 26 is replaced with glutamine, and the codons AAC at positions 76-78 of its corresponding nucleotide sequence are changed to CAG; replacing asparagine at position 29 with serine to change the codon AAC at positions 85-87 of the corresponding nucleotide sequence to AGC; the phenylalanine at position 42 is replaced by alanine, and the codon TTC at the position 124 and 126 of the corresponding nucleotide sequence is changed into GCA; the asparagine at position 71 is replaced by glutamine, and the corresponding codon AAT at position 211-213 of the nucleotide sequence is changed into CAG. The leucine at position 72 was replaced by glycine, and the codon CTG at position 214-216 of the corresponding nucleotide sequence was changed to GGC. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGCAGGGCATCAGCAACTACAAAAATCCGAAACTGACCCGTATGCTGACCGCAAAATTCTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGCAGGGCGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：33
the corresponding amino acid sequence is shown below, namely the IL-2-22 variant sequence:
MAPTSSSTKKTQLQLEHLLLDLQMILQGISNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLQGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：34
in one variant, the glutamine at position 11 is replaced with cysteine to change the codon CAG at positions 31-33 of its corresponding nucleotide sequence to TGT; the peptide fragment at positions 29 to 44 was replaced with QSMHIDATL, and the codon AACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTC at the position 85 to 132 of the corresponding nucleotide sequence was changed to CAGAGCATGCATATTGATGCAACCCTG. A substitution of the leucine at position 132 with cysteine; the codon CTG at position 394-396 of the corresponding nucleotide sequence was changed to TGT. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCTGTCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCCAGAGCATGCATATTGATGCAACCCTGTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCTGTACCTAATGAGGATCC
SEQ ID NO：35
the corresponding amino acid sequence is shown below, namely the IL-2-23 variant sequence:
MAPTSSSTKKTCLQLEHLLLDLQMILNGIQSMHIDATLYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTCT
SEQ ID NO：36
in one variant, the peptide stretch at positions 41-44 is replaced with DATL; the codon AACAACTACAAAAATCCGAAACTGACCCGTATGCTGACCTTCAAATTC at position 120-132 of its corresponding nucleotide sequence was changed to GATGCAACCCTG. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCAACAACTACAAAAATCCGAAACTGACCCGTATGCTGGATGCAACCCTGTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：37
the corresponding amino acid sequence is shown as follows, namely the IL2-G1 variant sequence:
MAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLDATLYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：38
in one variant, the substitution of the peptidyl fragment at positions 35-44 to MHIDATL; the codon AAACTGACCCGTATGCTGACCTTCAAATTC at position 85-132 of its corresponding nucleotide sequence was changed to ATGCATATTGATGCAACCCTG. The mutated nucleic acid sequence is shown below:
CATATGGCACCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAACTGGAACATCTGCTGTTAGATCTGCAAATGATTCTGAACGGCATCATGCATATTGATGCAACCCTGTACATGCCGAAAAAAGCAACCGAGCTGAAACATCTGCAGTGTCTGGAAGAAGAACTGAAACCGCTGGAAGAGGTTCTGAATCTGGCACAGAGCAAAAACTTTCATCTGCGTCCGCGTGATCTGATTAGCAATATTAACGTTATTGTGCTGGAACTGAAAGGTAGCGAAACCACCTTTATGTGTGAATATGCCGATGAAACCGCAACCATTGTGGAATTTCTGAATCGTTGGATTACCTTTGCACAGAGCATTATTAGCACCCTGACCTAATGAGGATCC
SEQ ID NO：39
the corresponding amino acid sequence is shown as follows, namely the IL2-G2 variant sequence:
MAPASSSTKKTQLQLEHLLLDLQMILNGINNYKNPMHIDATLYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT
SEQ ID NO：40
the gene sequence of the variant is synthesized and connected to Nde I and BamH I enzyme cutting sites of pET-9a to obtain the recombinant expression vector of each variant of IL-2.
3. Recombinant expression and production of wild-type IL-2 and variants
The recombinant expression vector was transformed into BL21(DE3) strain (Novagen, Cat.69450-3) purchased from Novagen to obtain a recombinant strain of wild type IL-2 and its variant, and the recombinant strain was spread on a LB plate containing Kan resistance and screened.
Selecting single colony after Kan screening, culturing in 10mL LB culture medium at 37 deg.C and 220rpm to OD6001.2 + -0.2, adding 50% glycerol to a final concentration of 10%, packaging into a freezing tube (1 mL/tube), and storing at-80 deg.C.
Collecting 1 frozen glycerol tube, recovering in 37 deg.C water bath, inoculating into 1L LB culture medium, culturing at 37 deg.C and 220rpm to OD6000.6-1.0, 1M IPTG was added to a final concentration of 1mM, and induced at 37 ℃ and 220rpm for 4 hours. After the induction, the cells were collected.
The thallus is crushed, and the inclusion body is denatured, renatured and purified to obtain the target protein.
Example 2 determination of the binding of wild-type IL-2 and variants to Interleukin 2 receptor alpha (IL-2R α)
The binding properties of IL-2 and its mutants prepared in example 1 to IL-2R α were tested using an ELISA assay. Coating the IL-2R alpha recombinant protein with his labels, adding IL-2, and detecting the activity of the antibody combined with the antigen by adding an HRP-coupled anti-IL-2 polyclonal antibody and an HRP substrate TMB.
96-well microtiter plates were coated with 2. mu.g/mL his-tagged recombinant IL-2R α protein (Sinobiological, Cat #10165-H08H) and incubated overnight at 4 ℃. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Add 200. mu.l/well blocking solution and incubate at room temperature for 2 hours. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Add 100. mu.l of diluted IL-2 and its mutants to each well. Incubate at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Mu.l of HRP-labeled anti-IL-2 polyclonal antibody (SinoBiological, Cat #11848-T16) diluted at 0.1. mu.g/mL was added to each well. Incubate at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Mu.l of TMB was added to each well and the reaction was protected from light for 15 minutes. 50 μ l of 0.16M sulfuric acid per well was added. Reading the OD value of 450nm by a Thermo MultiSkanFc plate reader, and calculating the binding EC50 value of the IL-2 and the mutant thereof and the IL-2R alpha.
The ELISA binding data for the mutants of example 1 to IL-2R α are shown in FIG. 1 and Table 2. The results show that the first class of mutations, namely N26Q, N30S, Q11C/L132C, L70C/P82C, G27C/F78C, N29S mutations do not affect the binding of IL-2 to IL-2R alpha. And the second class of mutations, namely F42A/Y45A, F42A/L72G, Y45A/L72G and 29-44 mutations are QSMHIDATL, TFKF at 41-44 is mutated into DATL, and KLTRMLTFKF at 35-44 is mutated into MHIDATL, so that the combination of IL-2 and IL-2R alpha is greatly reduced, and the combination of the two can not be observed under experimental conditions. The combination of the two classes of mutations also reduced IL-2 binding to IL-2R α due to the inclusion of the second class of mutations.
TABLE 2 ELISA binding of IL-2 wild type and its variants to the low affinity receptor IL-2 Ra EC50
Figure BDA0002398016630000301
Figure BDA0002398016630000311
(Note: N.A., not available, because these mutants have low binding to IL-2R α, EC50 could not be obtained by fitting the data in the concentration range used in the experiment.)
Example 3 binding of wild-type IL-2 and variants to IL-2 receptor beta/gamma (IL-2R β/γ) A Biacore experiment was used to examine the binding of IL-2 and its mutants to IL-2R β/γ in example 1.
First, IL-2R β and IL-2R γ subunits (SEQ ID NOS: 41 and 42) were cloned and fused to Fc hole and Fc knob, respectively, for the preparation of a tool molecule, IL-2R β/γ -Fc heterodimer. IL-2R β -Fc-hole and IL-2R γ -Fc-knob were transfected into HEK293 cells simultaneously. The heterodimer was purified using Protein a followed by molecular sieve Superdex 200.
MDMRVPAQLLGLLLLWFPGARCAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGAQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：41
MLKPSLPFTSLLFLQLPLLGVGLNTTILTPNGNEDTTADFFLTTMPTDSLSVSTLPLPEVQCFVFNVEYMNCTWNSSSEPQPTNLTLHYWYKNSDNDKVQKCSHYLFSEEITSGCQLQKKEIHLYQTFVVQLQDPREPRRQATQMLKLQNLVIPWAPENLTLHKLSESQLELNWNNRFLNHCLEHLVQYRTDWDHSWTEQSVDYRHKFSLPSVDGQKRYTFRVRSRFNPLCGSAQHWSEWSHPIHWGSNTSKENPFLFALEAGAQDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：42
IL-2R β/γ -Fc was captured with a Protein A sensor chip (GE, Cat #29127556) of a Biacore instrument (Biacore T200, GE), in which IL-2R β/γ -Fc was diluted to 1 μ g/mL with 1 × HBS-EP for 30 seconds at a flow rate of 10 μ L/min. Then IL-2 and its mutant under a series of concentration gradients were flowed on the surface of the chip at a flow rate of 30. mu.L/min for binding for 120 seconds and dissociation for 360 seconds, and reaction signals were detected in real time using a Biacore instrument (Biacore X100, GE) to obtain binding and dissociation curves. After each cycle of dissociation completion, the chip was washed and regenerated with 10mM Gly-HCl pH 1.5. The data obtained from the experiments were fitted to a 1:1binding model to give the values for binding of the mutants of example 1 to the intermediate affinity receptor IL-2R β/γ, see Table 3.
The results show that N26Q, N30S, Q11C/L132C, L70C/P82C, G27C/F78C, F42A/Y45A, F42A/L72G, Y45A/L72G, N26Q/N30S/F42A/L72G and 29-44 mutation are QSMHIDATL, and the combination of mutations is not large in the combination of IL-2 and IL-2R beta/gamma. The mutation of TFKF at positions 41-44 to DATL, the mutation of KLTRMLTFKF at positions 35-44 to MHIDATL, like the mutation at positions 29-44 to QSMHIDATL, had little effect on the binding of IL-2 and IL-2R beta/gamma, and the results are not shown here.
TABLE 3 affinity of IL-2 wild type and its variants for the intermediate affinity receptor IL-2 Rbeta/gamma
Name (R) Kon(M-1s-1) Koff(s-1) Kd(nM)
IL-2(WT) 9.36×105 2.40×10-4 0.257
IL-2-01 9.02×105 2.20×10-4 0.244
IL-2-02 1.03×106 2.12×10-4 0.206
IL-2-03 5.33×105 1.35×10-4 0.253
IL-2-04 4.98×105 1.50×10-4 0.302
IL-2-05 3.36×105 9.68×10-5 0.288
IL-2-06 8.56×105 4.36×10-4 0.509
IL-2-07 8.22×105 2.15×10-4 0.261
IL-2-08 9.20×105 2.06×10-4 0.224
IL-2-09 6.46×105 2.22×10-4 0.344
IL-2-10 1.62×106 4.30×10-4 0.266
Example 4 assay of the cell proliferation Activity of wild-type IL-2 and variants CTLL2
The biological activity of IL-2 was examined on the basis of the proliferation rate of the cell-dependent strain CTLL-2 at different concentrations of interleukin-2 (IL-2).
Complete culture solution: RPMI 1640+2mM L-glutamine +1mM sodium pyruvate + 10% fetal bovine serum + 10% T-STIM culture supplement with containment-A (containing IL-2); basic culture medium: RPMI 1640+2mM L-glutamine +1mM sodium pyruvate + 10% fetal bovine serum.
CTLL-2 cell proliferation assay CTLL-2 cells were cultured in complete medium at 37 ℃ and 5% carbon dioxide to 2.0 × 10/ml5Cell density, passage of cells, 3-4 days later centrifugation to collect CTLL-2 cells, PBS washing 3 times, heavy suspension in basic culture medium to make each ml containing 2.0 × 105Placing cell suspension of each cell in a 96-well plate, adding 90 microliter of cell per well, adding 10 microliter of 10 × corresponding concentration IL-2 concentrated solution prepared by using basic culture solution, placing the cell under the condition of 37 deg.C and 5% carbon dioxide, culturing for 24 hr, adding 100 microliter of lysate CELLTITER-Glo reagent (Promega) into each well, uniformly mixing the liquid in the cell plate, placing the cell plate into an enzyme-labeling instrument, measuring absorbance at the position of 570nm of wavelength by using 630nm as reference wavelength, recording the measurement result, fitting the data by using a computer program or a four-parameter regression calculation method, and calculating the result according to the following formula, namely the relative biological activity (%). of the sample is the reference substance EC50Test sample EC50(EC50: half maximal effect concentration).
The results show that the first type of mutation, namely N26Q, N30S, Q11C/L132C, L70C/P82C, G27C/F78C and N29S do not influence or even slightly enhance the CTLL2 cell proliferation activity of IL-2; and the second kind of mutation, F42A/Y45A, F42A/L72G, Y45A/L72G, TFKF with position QSMHIDATL, position 41-44 to DATL, and KLTRMLTFKF with position 35-44 to MHIDATL, reduces the combination of IL-2 and IL-2R alpha, and these mutants also reduce the CTLL2 cell proliferation activity of IL-2.
The activity data of the mutants in example 1 are shown in Table 4.
TABLE 4 CTLL 2-cell proliferation Activity of IL-2 wild type and its variants
Figure BDA0002398016630000331
Example 5 STAT5 phosphorylation Activity assay of wild-type IL-2 and variants on CTLL2 cells
The biological activity of IL-2 was determined based on the phosphorylation level of STAT5 in the cell-dependent strain CTLL-2 at different IL-2 concentrations.
Complete culture solution: RPMI 1640+2mM L-glutamine +1mM sodium pyruvate + 10% fetal bovine serum + 10% T-STIM culture supplement with containment-A (containing IL-2); basic culture medium: RPMI 1640+2mM L-glutamine +1mM sodium pyruvate + 10% fetal bovine serum.
STAT5 phosphorylation experiment on CTLL-2 cells, in which CTLL-2 cells were cultured in a complete medium at 37 ℃ and 5% carbon dioxide to 2.0 × 10/ml5The density of the individual cells was adjusted to 1.0 × 10/ml by washing 1 time with PBSA (PBS, pH7.2, 1% BSA)6And (3) subpackaging each cell into a flow tube according to the volume of 500 mu L per tube, adding IL-2 concentrated solution with corresponding concentration and prepared by using basic culture solution, incubating at room temperature for 20 minutes, immediately adding paraformaldehyde to the final concentration of 1.5%, uniformly mixing by vortex, and incubating at room temperature for 10 minutes. Add 1ml PBS, 4 ℃, 1400rpm centrifugal 5 minutes, remove paraformaldehyde. Resuspend cells, add 1ml of pre-cooled 100% methanol at 4 ℃, vortex and mix well, incubate for 20 min at 4 ℃. 3ml of PBSA buffer was added, centrifuged at 1400rpm for 5 minutes at 4 ℃ and the cells were washed 2 times. anti-STAT 5-pY694 antibody (BD, Cat #612599) coupled to Alexa Fluor 647 was added and incubated for 30 min at room temperature in the dark. Wash twice with 3mL PBSA and detect with flow cytometer. The data is processed by computer program or four-parameter regression calculation methodFitting, the calculation results are made as follows: relative biological activity (%) of the test sample as a control EC50Test sample EC50(EC50: half maximal effect concentration)
The results show that the mutations N26Q and N30S do not affect or even slightly enhance the STAT5 phosphorylation activity of CTLL2 cells of IL-2, while F42A/Y45A, F42A/L72G and Y45A/L72G, and the mutants also reduce the STAT5 phosphorylation activity of CTLL2 cells of IL-2 due to the reduction of the binding of IL-2 to IL-2R alpha.
The CTLL2 cell STAT5 phosphorylation activity data for the mutants in example 1 are shown in fig. 2 and table 5.
TABLE 5 CTLL 2-cell STAT5 phosphorylation activity of IL-2 wild type and variants thereof
Figure BDA0002398016630000341
Example 6 stability Studies of wild-type IL-2 and variants
Stability studies were performed on approximately 1mg/mL IL-2 samples (10 mM acetate-sodium acetate buffer, pH4.5, 10% trehalose) using a Uncle (Uncariamed labs) instrument. The temperature of the sample was raised from 25 ℃ to 95 ℃ at a rate of 0.3 ℃/min while exciting tryptophan in the sample with 266nm light, and the emission light (emission) of the sample at 300-400nm was observed. The melting temperature (T) was calculated according to the following formulam) Where λ is the wavelength (300-.
Figure BDA0002398016630000342
Figure BDA0002398016630000343
As the temperature increases, the BCM parameters of the samples change, reflecting the change in protein conformation. The presence of two melting temperatures for wild-type IL-2 suggests that IL-2 undergoes two stages before complete unfolding, with the first unfolding stage requiring a lower energy relative to the second stage. The mutations N26Q and N30S, while not substantially affecting the first melting temperature, caused a significant increase in the second melting temperature; and IL-2-03, IL-2-04 and IL-2-05 have no first melting temperature, and the second melting temperature is obviously increased compared with the wild type. The experimental results show that the mutations N26Q, N30S, Q11C/L132C, L70C/P82C and G27C/F78C improve the thermal stability of IL-2.
The results of the thermostability experiments for IL-2 mutants are shown in FIGS. 3A-3F and Table 6.
TABLE 6 solubilization temperatures of IL-2 wild type and its variants
Name (R) Tm1(℃) Tm2(℃)
IL-2 61.5 77.5
IL-2-01 60.8 90.5
IL-2-02 61.8 89.1
IL-2-03 Is absent from 92.1
IL-2-04 Is absent from 91.8
IL-2-05 Is absent from 92.5
IL-2-06 Not determined Not determined
Example 6 determination of the binding of mutant IL2-06 to IL-15 receptor alpha
ELISA experiments were used to test the binding properties of the interleukin 2 mutant IL2-6 of example 1 to IL-15 Ra.
First, a His tag (SEQ ID NO: 31) was added to IL 2-06. Transient expression was performed in HEK293 cells and purified by Ni-NTA affinity column and Superdex 200 molecular sieve column.
The 96-well plate was coated with 2. mu.g/mL of Fc-tagged recombinant IL-2R α protein (SinoBiological, Cat #183666-H02H) and incubated overnight at 4 ℃. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Add 200. mu.l/well blocking solution and incubate at room temperature for 2 hours. The wash solution was washed three times, 250. mu.l per well. Each wash was shaken for 10 seconds to ensure adequate cleaning. Mu.l of wild type IL15(SinoBiological, Cat #10360-H07E) or IL2-06 diluted in diluent was added to each well. Incubate at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Mu.l of anti-6 × His antibody conjugated with HRP at 0.5. mu.g/mL in diluent was added to each well. Incubate at room temperature for 1 hour. The wash solution was washed three times, 250. mu.l per well. Mu.l of TMB was added to each well and the reaction was protected from light for 15 minutes. 50 μ l of 0.16M sulfuric acid per well was added. The Thermo MultiSkanFc microplate reader reads the OD value of 450nm and calculates the binding EC50 value of IL2 and the mutant thereof with the low-affinity IL-2R alpha.
The ELISA binding data for wild-type IL15 or IL2-06 and IL-15 Ra are shown in FIG. 4. The results indicated that IL2-06 failed to bind IL-15R α.
Sequence listing
<110> Hengrui pharmaceutical Co., Ltd of Jiangsu
SHANGHAI HENGRUI PHARMACEUTICAL Co.,Ltd.
SHANGHAI SHENGDI PHARMACEUTICAL Co.,Ltd.
<120> a human interleukin 2 variant or derivative thereof
<160>42
<170>SIPOSequenceListing 1.0
<210>1
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> wild-type IL-2 nucleic acid sequence
<400>1
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctgacct tcaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatc tggcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttaccttttg tcagagcatt attagcaccc tgacctaatg aggatcc 417
<210>2
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> wild-type IL-2 amino acid sequence
<400>2
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>3
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-01
<400>3
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gcagggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctgacct tcaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatc tggcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>4
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-01
<400>4
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>5
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-02
<400>5
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacagctaca aaaatccgaa actgacccgt 120
atgctgacct tcaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatc tggcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>6
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-02
<400>6
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Ser Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>7
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-03
<400>7
catatggcac cgaccagcag cagcaccaaa aaaacctgtc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctgacct tcaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatc tggcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcacct gtacctaatg aggatcc 417
<210>8
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-03
<400>8
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Cys Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Cys Thr
130
<210>9
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>allele
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-04
<400>9
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctgacct tcaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gtttgtaatc tggcacagag caaaaacttt 240
catctgcgtt gtcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>10
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-04
<400>10
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Cys Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Cys Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>11
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-05
<400>11
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaactgtatc aacaactaca aaaatccgaa actgacccgt 120
atgctgacct tcaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatc tggcacagag caaaaactgt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>12
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-05
<400>12
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Cys Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Cys His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>13
<211>396
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(396)
<223> nucleic acid sequence of IL-2-06
<400>13
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc cagagcatgc atattgatgc aaccctgtac 120
atgccgaaaa aagcaaccga gctgaaacat ctgcagtgtc tggaagaaga actgaaaccg 180
ctggaagagg ttctgaatct ggcacagagc aaaaactttc atctgcgtcc gcgtgatctg 240
attagcaata ttaacgttat tgtgctggaa ctgaaaggta gcgaaaccac ctttatgtgt 300
gaatatgccg atgaaaccgc aaccattgtg gaatttctga atcgttggat tacctttgca 360
cagagcatta ttagcaccct gacctaatga ggatcc 396
<210>14
<211>127
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(124)
<223> amino acid sequence of IL-2-06
<400>14
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Gln Ser Met
20 25 30
His Ile Asp Ala Thr Leu Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys
35 40 45
His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu
50 55 60
Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile
65 70 75 80
Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr
85 90 95
Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu
100 105 110
Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile Ile Ser Thr Leu Thr
115 120 125
<210>15
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-07
<400>15
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcgc aatgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatc tggcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>16
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-07
<400>16
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>17
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-08
<400>17
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatg gcgcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>18
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-08
<400>18
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
3540 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>19
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-09
<400>19
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctgacct tcaaattcgc aatgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatg gcgcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>20
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-09
<400>20
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Ala Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>21
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-10
<400>21
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gcagggcatc aacagctaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatg gcgcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>22
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-10
<400>22
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Asn Ser Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>23
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-11
<400>23
catatggcac cgaccagcag cagcaccaaa aaaacctgtc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gcagggcatc aacagctaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatg gcgcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcacct gtacctaatg aggatcc 417
<210>24
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-11
<400>24
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Cys Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Asn Ser Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr CysThr
130
<210>25
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-12
<400>25
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gcagggcatc aacagctaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gtttgtaatg gcgcacagag caaaaacttt 240
catctgcgtt gtcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>26
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-12
<400>26
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Asn Ser Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Cys Asn Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Cys Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>27
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-13
<400>27
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gcagtgtatc aacagctaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatg gcgcacagag caaaaactgt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>28
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-13
<400>28
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Cys Ile Asn Ser Tyr
2025 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Cys His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>29
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-14
<400>29
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc agcaactaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatg gcgcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>30
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-14
<400>30
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Ser Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>31
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-21
<400>31
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gcagggcatc agcaactaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatg gcgcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>32
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-21
<400>32
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Ser Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val LeuGlu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>33
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> nucleic acid sequence of IL-2-22
<400>33
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gcagggcatc agcaactaca aaaatccgaa actgacccgt 120
atgctgaccg caaaattcta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgcagg gcgcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>34
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> amino acid sequence of IL-2-22
<400>34
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Gln Gly Ile Ser Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Gln Gly Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>35
<211>396
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>allele
<222>(1)..(396)
<223> nucleic acid sequence of IL-2-23
<400>35
catatggcac cgaccagcag cagcaccaaa aaaacctgtc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc cagagcatgc atattgatgc aaccctgtac 120
atgccgaaaa aagcaaccga gctgaaacat ctgcagtgtc tggaagaaga actgaaaccg 180
ctggaagagg ttctgaatct ggcacagagc aaaaactttc atctgcgtcc gcgtgatctg 240
attagcaata ttaacgttat tgtgctggaa ctgaaaggta gcgaaaccac ctttatgtgt 300
gaatatgccg atgaaaccgc aaccattgtg gaatttctga atcgttggat tacctttgca 360
cagagcatta ttagcacctg tacctaatga ggatcc 396
<210>36
<211>127
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(127)
<223> amino acid sequence of IL-2-23
<400>36
Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Cys Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Gln Ser Met
20 25 30
His Ile Asp Ala Thr Leu Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys
35 40 45
His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu
50 55 60
Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile
65 70 75 80
Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr
85 90 95
Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu
100 105 110
Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile Ile Ser Thr Cys Thr
115 120 125
<210>37
<211>417
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(417)
<223> IL2-G1 nucleic acid sequence
<400>37
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc aacaactaca aaaatccgaa actgacccgt 120
atgctggatg caaccctgta catgccgaaa aaagcaaccg agctgaaaca tctgcagtgt 180
ctggaagaag aactgaaacc gctggaagag gttctgaatc tggcacagag caaaaacttt 240
catctgcgtc cgcgtgatct gattagcaat attaacgtta ttgtgctgga actgaaaggt 300
agcgaaacca cctttatgtg tgaatatgcc gatgaaaccg caaccattgt ggaatttctg 360
aatcgttgga ttacctttgc acagagcatt attagcaccc tgacctaatg aggatcc 417
<210>38
<211>134
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(134)
<223> IL2-G1 amino acid sequence
<400>38
Met Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Lys Leu Thr Arg Met Leu Asp Ala Thr Leu Tyr Met Pro
35 40 45
Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu
50 55 60
Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
65 70 75 80
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu
85 90 95
Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr
100 105 110
Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser
115 120 125
Ile Ile Ser Thr Leu Thr
130
<210>39
<211>390
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>gene
<222>(1)..(390)
<223> IL2-G2 nucleic acid sequence
<400>39
catatggcac cgaccagcag cagcaccaaa aaaacccagc tgcaactgga acatctgctg 60
ttagatctgc aaatgattct gaacggcatc atgcatattg atgcaaccct gtacatgccg 120
aaaaaagcaa ccgagctgaa acatctgcag tgtctggaag aagaactgaa accgctggaa 180
gaggttctga atctggcaca gagcaaaaac tttcatctgc gtccgcgtga tctgattagc 240
aatattaacg ttattgtgct ggaactgaaa ggtagcgaaa ccacctttat gtgtgaatat 300
gccgatgaaa ccgcaaccat tgtggaattt ctgaatcgtt ggattacctt tgcacagagc 360
attattagca ccctgaccta atgaggatcc 390
<210>40
<211>131
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>PEPTIDE
<222>(1)..(131)
<223> IL2-G2 amino acid sequence
<400>40
Met Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
1 5 10 15
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr
20 25 30
Lys Asn Pro Met His Ile Asp Ala Thr Leu Tyr Met Pro Lys Lys Ala
35 40 45
Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu
50 55 60
Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro
65 70 75 80
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly
85 90 95
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile
100 105 110
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile Ile Ser
115 120 125
Thr Leu Thr
130
<210>41
<211>466
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>DOMAIN
<222>(1)..(466)
<223>IL-2Rβ-Fc-hole
<400>41
Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Phe Pro Gly Ala Arg Cys Ala Val Asn Gly Thr Ser Gln Phe Thr Cys
20 25 30
Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly
35 40 45
Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg
50 55 60
Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp
65 70 75 80
Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr
85 90 95
Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp
100 105 110
Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu
115 120 125
Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His Arg Cys
130 135 140
Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His
145 150 155 160
Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu
165 170 175
Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu
180 185 190
Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro
195 200 205
Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala
210 215 220
Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr Gly Ala Gln Asp
225 230 235 240
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
245 250 255
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
260 265 270
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
275 280 285
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
290 295 300
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
305 310 315 320
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
325 330 335
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
340 345 350
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
355 360 365
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
370 375 380
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
385 390 395 400
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
405 410 415
Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp
420 425 430
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
435 440 445
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
450 455 460
Gly Lys
465
<210>42
<211>492
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>DOMAIN
<222>(1)..(492)
<223>IL-2Rγ-Fc-knob
<400>42
Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu
1 5 10 15
Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly
20 25 30
Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp
35 40 45
Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val
50 55 60
Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro
65 70 75 80
Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn
85 90 95
Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr
100 105 110
Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe
115 120 125
Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln
130 135 140
Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu
145 150 155 160
Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn
165 170 175
Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp
180 185 190
Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe
195 200 205
Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg
210 215 220
Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp
225 230 235 240
Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe
245 250 255
Leu Phe Ala Leu Glu Ala Gly Ala Gln Asp Lys Thr His Thr Cys Pro
260 265 270
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
275 280 285
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
290 295 300
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
305 310 315 320
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
325 330 335
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
340 345 350
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
355 360 365
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
370 375 380
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
385 390 395 400
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
405 410 415
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
420 425 430
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
435 440 445
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
450 455 460
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
465 470 475 480
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
485 490

Claims (19)

1. A human interleukin 2(IL-2) variant or derivative thereof comprising one or more amino acid mutations in the region that binds to the IL-2 receptor alpha subunit (IL-2 ra), said mutation being a substitution of one or more amino acids of the region that binds human interleukin 15(IL-15) to IL-2 ra to the region that binds IL-2 to IL-2 ra.
2. The IL-2 variant or derivative thereof according to claim 1, having reduced affinity for IL-2 ra, unchanged or increased affinity for IL-2 rbeta and/or IL-2 rcgamma; or reduced affinity to IL-2R α/β/γ and retained affinity for IL-2R β/γ.
3. The IL-2 variant or derivative thereof of claims 1-2, which has reduced activation of regulatory T cells and/or has no effect or increased activation of immune effector cells.
4. The IL-2 variant or derivative thereof according to any of the preceding claims, wherein the mutation is at one or more of the amino acid residues in positions 29-44, or positions 41-44, or positions 35-44 of IL-2.
5. The IL-2 variant or derivative thereof of claim 4, wherein:
(1) one or more amino acid mutations at positions 11, 26, 27, 45, 70, 72, 78, 82, 132 in addition to one or more amino acid residue mutations at positions 29-44;
(2) one or more amino acid mutations at positions 11, 26, 27, 29, 30, 45, 70, 72, 78, 82, 132 in addition to one or more amino acid residue mutations at positions 41-44; or
(3) One or more amino acid mutations at positions 11, 26, 27, 29, 30, 45, 70, 72, 78, 82, 132 in addition to one or more amino acid residue mutations at positions 35-44;
preferably, the mutation types of the amino acids are: the 11 th mutation is C, the 26 th mutation is Q, the 27 th mutation is C, the 29 th mutation is S, the 30 th mutation is S, the 45 th mutation is A, the 70 th mutation is C, the 72 th mutation is G, the 78 th mutation is C, the 82 th mutation is C, and the 132 th mutation is C.
6. The IL-2 variant or derivative thereof of any preceding claim, wherein:
(1) the mutation at position 29-44 is QSMHIDATL;
(2) mutation at positions 41-44 to DATL; or
(3) Mutation of 35-44 bits to MHIDATL;
preferably, the first and second electrodes are formed of a metal,
(1) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL;
(2) the TFKF at the 41-44 position is mutated into DATL; or
(3) The KLTRMLTFKF mutation at positions 35-44 was MHIDATL.
7. The IL-2 variant or derivative thereof of claim 6, wherein:
(1) the NNYKNPKLTRMLTFKF mutation at position 29-44 is QSMHIDATL, and further comprises one or more mutations in N26Q, Q11C/L132C, L70C/P82C, G27C/F78C, Y45A, N71Q and L72G;
(2) the TFKF mutation at the 41-44 position is DATL, and the TFKF mutation also comprises one or more mutations of N26Q, N29S, N30S, Q11C/L132C, L70C/P82C, G27C/F78C, Y45A, N71Q and L72G;
(3) the KLTRMLTFKF mutation at position 35-44 is MHIDATL, and also comprises one or more mutations of N26Q, N29S, N30S, Q11C/L132C, L70C/P82C, G27C/F78C, Y45A, N71Q and L72G.
8. The IL-2 variant or derivative thereof of claim 7, comprising an amino acid sequence selected from SEQ ID NO: 14. SEQ ID NO: 38. SEQ ID NO: 40, or a pharmaceutically acceptable salt thereof.
9. The IL-2 variant or derivative according to any one of claims 1-8, which each comprises the C125A mutation.
10. The IL-2 variant or derivative thereof according to any one of claims 1-9, which is monomeric, and/or pegylated, and/or glycosylated, and/or albumin conjugated or fused, and/or Fc fused, and/or hydroxyethylated, and/or de-O-glycosylated;
preferably, the PEG is attached to the N-terminus of the IL-2 variant;
more preferably, the PEG has a molecular weight of 20 KD.
11. A conjugate comprising an IL-2 variant or derivative thereof as defined in any preceding claim, which IL-2 variant or derivative thereof is linked directly or indirectly via a linker to a non-IL-2 moiety;
preferably, the non-IL-2 moiety is an antigen-binding moiety;
more preferably, the antigen binding moiety is an antibody or antigen binding fragment thereof;
most preferably, the antibody or antigen-binding fragment thereof targets an antigen present on a tumor cell or in the environment of a tumor cell.
12. A pharmaceutical composition comprising an IL-2 variant or derivative thereof according to any one of claims 1 to 10, together with a pharmaceutically acceptable diluent, carrier or adjuvant.
13. A polynucleotide encoding the IL-2 variant of any one of claims 1-10 or a derivative thereof.
14. The polynucleotide of claim 13, comprising a sequence selected from SEQ ID NOs: 13. SEQ ID NO: 37. SEQ ID NO: 39, or a variant thereof.
15. A vector comprising the polynucleotide of claim 12 or 13.
16. A host cell comprising the expression vector of claim 15, or expressing the IL-2 variant or derivative thereof of any one of claims 1-10, the conjugate of claim 11,
preferably, the host cell is a prokaryotic or eukaryotic cell;
more preferably, the host cell is a bacterial or yeast or mammalian cell;
most preferably, the host cell is Saccharomyces cerevisiae or Escherichia coli.
17. Use of an IL-2 variant or derivative thereof according to any one of claims 1 to 10, a conjugate according to claim 11 or a pharmaceutical composition according to claim 12 for the preparation of a medicament for the treatment of a proliferative disease, an immunological disease, a regulatory T cell mediated immune response, stimulating the immune system of an individual.
18. The use according to claim 17, wherein the proliferative disease is a tumor or cancer,
preferably, the tumor or cancer is selected from the group consisting of epithelial cell carcinoma, endothelial cell carcinoma, squamous cell carcinoma, papilloma virus-induced carcinoma, adenocarcinoma, carcinoma, melanoma, sarcoma, teratocarcinoma, lung tumor, metastatic lung cancer, lymphoma, and metastatic renal cell carcinoma.
19. A method for producing a variant or derivative of IL-2, comprising introducing into wild-type human IL-2 a mutation as described in any one of claims 1 to 10, or using a nucleic acid sequence of claims 13 to 14, or using an expression vector of claim 15, or using a host cell of claim 16 for recombinant expression.
CN202010138036.1A 2019-03-04 2020-03-03 Human interleukin 2 variant or derivative thereof Pending CN111647068A (en)

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CN113831402A (en) * 2021-07-30 2021-12-24 西安龙腾景云生物科技有限公司 Human interleukin 2 variant and application thereof
CN114349843A (en) * 2022-01-18 2022-04-15 浙江博锐生物制药有限公司 Interleukin-2 derivative and preparation method and application thereof
WO2022078518A1 (en) * 2020-10-18 2022-04-21 北京志道生物科技有限公司 Modified il-2 molecule and use thereof
WO2022100686A1 (en) * 2020-11-13 2022-05-19 江苏恒瑞医药股份有限公司 A pharmaceutical composition comprising human il-2 variant or derivative and use thereof
WO2023005680A1 (en) * 2021-07-30 2023-02-02 西安龙腾景云生物科技有限公司 Human interleukin-2 variant and use thereof
WO2023046156A1 (en) * 2021-09-26 2023-03-30 Wuxi Biologics (Shanghai) Co. Ltd. Il-2 variants and fusion proteins thereof
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US11692020B2 (en) 2019-11-20 2023-07-04 Anwita Biosciences, Inc. Cytokine fusion proteins, and their pharmaceutical compositions and therapeutic applications
US11897930B2 (en) 2020-04-28 2024-02-13 Anwita Biosciences, Inc. Interleukin-2 polypeptides and fusion proteins thereof, and their pharmaceutical compositions and therapeutic applications
GB2613483A (en) * 2020-10-18 2023-06-07 Leto Laboratories Co Ltd Modified IL-2 molecule and use thereof
CN114380919B (en) * 2020-10-18 2024-06-25 北京志道生物科技有限公司 Modified IL-2 molecules and uses thereof
WO2022078518A1 (en) * 2020-10-18 2022-04-21 北京志道生物科技有限公司 Modified il-2 molecule and use thereof
WO2022100686A1 (en) * 2020-11-13 2022-05-19 江苏恒瑞医药股份有限公司 A pharmaceutical composition comprising human il-2 variant or derivative and use thereof
WO2023005680A1 (en) * 2021-07-30 2023-02-02 西安龙腾景云生物科技有限公司 Human interleukin-2 variant and use thereof
CN113831402A (en) * 2021-07-30 2021-12-24 西安龙腾景云生物科技有限公司 Human interleukin 2 variant and application thereof
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WO2023133595A2 (en) 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Methods of ex vivo dosing and administration of lipid particles or viral vectors and related systems and uses
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WO2023151661A1 (en) * 2022-02-11 2023-08-17 江苏恒瑞医药股份有限公司 Immunoconjugate and use thereof
WO2023193015A1 (en) 2022-04-01 2023-10-05 Sana Biotechnology, Inc. Cytokine receptor agonist and viral vector combination therapies

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