Human interleukin 2 variant or derivative thereof
阅读说明：本技术 一种人白细胞介素2变体或其衍生物 (Human interleukin 2 variant or derivative thereof ) 是由 陈磊 胡齐悦 葛虎 林�源 王宏伟 欧阳超 孔祥林 廖成 张连山 于 2020-03-03 设计创作，主要内容包括：本公开涉及一种人白细胞介素2变体或其衍生物。具体而言,本公开涉及具有一个或多个氨基酸突变的IL-2或其衍生物,具有消除或降低的对高亲和力受体(IL-2Rα/β/γ)的亲和力,并保留对中等亲和力受体(IL-2Rβ/γ)的亲和力。所述IL-2变体或其衍生物为蛋白成药提供了良好的基础。(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.)
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.
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
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
In some embodiments, the IL-2 variant or derivative comprises a mutation at position 26 to Gln (q) and/or a mutation at
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
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
In some embodiments, the IL-2 variant or derivative thereof comprises a mutation of Asn 26 to Gln (N26Q) and/or a mutation of
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
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
(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
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
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 "
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.
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