阅读说明：本技术 基于细胞因子的可生物活化药物及其使用方法 (Cytokine-based bioactivatable agents and methods of use thereof ) 是由 李跃升 芮凌云 徐静 于 2019-06-20 设计创作，主要内容包括：本公开内容提供了一种基于细胞因子的可生物活化的药物构建体(“VitoKine”)平台,该平台旨在降低基于全身性机制的毒性,并使蛋白和细胞因子诸如IL-15和IL-2在治疗癌症、自身免疫性疾病、炎性疾病、病毒感染、移植和各种其他紊乱中具有更广泛的治疗用途。本发明的新的VitoKine构建体包含：1)组织或疾病部位靶向部分D1结构域(“D1”)、2)可生物活化部分D2结构域(“D2”)和遮蔽部分D3结构域(“D3”)。重要的是,因为VitoKine构建体的“活性部分”将保持惰性,直到被病变组织中上调的蛋白酶在局部活化,这将限制活性部分与非病变细胞和组织的外周中或细胞表面上的受体或靶的结合,以防止途径的过度活化并减少不期望的“组织外”“中靶”毒性。此外,在蛋白酶活化之前,VitoKine活性部分的惰性将显著降低潜在的抗原沉默或靶沉默,并从而延长体内半衰期且导致改进的生物分布、生物可利用度和治疗功效。(The present disclosure provides a cytokine-based bioactivatable pharmaceutical construct ("vitokinee") platform that aims to reduce systemic mechanism-based toxicity and to make proteins and cytokines such as IL-15 and IL-2 more broadly therapeutically useful in the treatment of cancer, autoimmune diseases, inflammatory diseases, viral infections, transplantation, and various other disorders. The novel vittokinene construct of the invention comprises: 1) a tissue or disease site targeting moiety D1 domain ("D1"), 2) a bioactivatable moiety D2 domain ("D2") and a masking moiety D3 domain ("D3"). Importantly, because the "active portion" of the vitokinene construct will remain inert until locally activated by proteases upregulated in diseased tissue, this will limit the binding of the active portion to receptors or targets in the periphery or on the cell surface of non-diseased cells and tissues to prevent over-activation of the pathway and reduce undesirable "out of tissue" or "in-target" toxicity. Furthermore, the inertness of the vitokinene active moiety prior to protease activation will significantly reduce potential antigen or target silencing and thereby prolong in vivo half-life and lead to improved biodistribution, bioavailability and therapeutic efficacy.)

1. A bioactivatable polypeptide drug construct comprising in the N-terminal to C-terminal direction (D1-D2-D3): 1) a tissue or disease site targeting moiety D1 domain ("D1"), 2) a bioactivatable moiety D2 domain ("D2") and 3) a shielding moiety D3 domain ("D3"); wherein D1 is functional in targeting the bioactivatable moiety to the intended treatment site; and wherein D3 is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site.

2. A bioactivatable polypeptide drug construct comprising in the N-terminal to C-terminal direction (D1-D2-D3): 1) a half-life extending moiety D1 domain ("D1"), 2) a bioactivatable moiety D2 domain ("D2") and 3) a shielding moiety D3 domain ("D3"); wherein D1 functions to target the bioactivatable moiety to the intended site of treatment and to extend the half-life of D2; and wherein D3 is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site.

3. A bioactivatable polypeptide drug construct comprising in the N-terminal to C-terminal direction (D1-D2-D3): 1) a dual function portion D1 domain ("D1"), 2) a bioactivatable portion D2 domain ("D2") and 3) a shielding portion D3 domain ("D3"); wherein D1 is functional to target and maintain the bioactivatable moiety at the intended treatment site; and wherein D3 is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site.

4. A bioactivatable polypeptide drug construct comprising in the N-terminal to C-terminal direction (D3-D2-D1): 1) a shielding moiety D3 domain ("D3"), 2) a bioactivatable moiety D2 domain ("D2") and 3) a tissue or disease site targeting moiety D1 domain ("D1"); wherein D1 is functional in targeting the bioactivatable moiety to the intended treatment site; and wherein D3 is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site.

5. A bioactivatable polypeptide drug construct comprising in the N-terminal to C-terminal direction (D3-D2-D1): 1) a shielding moiety D3 domain ("D3"), 2) a bioactivatable moiety D2 domain ("D2") and 3) a half-life extending moiety D1 domain ("D1"); wherein D1 functions to target the bioactivatable moiety to the intended site of treatment and to extend the half-life of D2; and wherein D3 is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site.

6. A bioactivatable polypeptide drug construct comprising in the N-terminal to C-terminal direction (D3-D2-D1): 1) a shielding moiety D3 domain ("D3"), 2) a bioactivatable moiety D2 domain ("D2") and 3) a dual-functional moiety D1 domain ("D1"); wherein D1 is functional to target and maintain the bioactivatable moiety at the intended treatment site; and wherein D3 is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site.

7. The construct of any one of claims 1 to 6, wherein the D1, D2, and D3 domains of the construct are each in a monomeric form, each in a dimeric form, or collectively in a combination of dimers and monomers.

8. The construct according to any one of claims 1 to 7, wherein the D1 domain is selected from the group consisting of: an antibody, or antibody fragment, or ligand or variant thereof, or receptor or variant thereof, capable of binding to a Tumor Associated Antigen (TAA) or tissue specific antigen or target; a cell surface molecule or extracellular matrix protein; proteases and any post-translationally modified residues.

9. The construct of claim 8, wherein the D1 domain is an antibody or antibody fragment directed against an immune checkpoint modulator.

10. The construct of any one of claims 1 to 7, wherein the D1 domain is an Fc domain selected from the group consisting of: a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain, an IgG Fc domain, and an IgM Fc domain.

11. The construct of claim 10, wherein the Fc domain is an Fc domain with silent effector function and/or with half-life extending function.

12. The construct according to any one of claims 10 to 11, wherein the Fc domain is an Fc domain having an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 156, and SEQ ID No. 166-168.

13. The construct according to any one of claims 1 to 12, wherein the D2 domain is a cytokine selected from the group consisting of: interleukin-2 (IL-2) (SEQ ID NO:8), interleukin-4 (IL-4) (SEQ ID NO:17), interleukin-7 (IL-7) (SEQ ID NO:18), interleukin-9 (IL-9) (SEQ ID NO:19), interleukin-10 (IL-10) (SEQ ID NO:20), interleukin-12 alpha (12 alpha) (SEQ ID NO:21), interleukin-12 beta (IL-12 beta) (SEQ ID NO:22), interleukin-15 (IL-15) (SEQ ID NO:2), interleukin-23 alpha (IL-23 alpha) (SEQ ID NO:23), and transforming growth factor beta (TGF beta) (SEQ ID NO:24), or variants thereof.

14. The construct of claim 13, wherein the D2 domain is IL-15(SEQ ID NO: 2).

15. The construct of claim 14, wherein the D2 domain is an IL-15 variant polypeptide comprising one or more amino acid substitutions or deletions at positions 30, 31, 32, 58, 62, 63, 67, 68 or 108 of SEQ ID No. 2.

16. The construct of claim 15, wherein the D2 domain is an IL-15 variant polypeptide having the amino acid sequence set forth in SEQ ID No. 3.

17. The construct of claim 13, wherein the D2 domain is IL-2(SEQ ID NO: 8).

18. The construct of claim 13, wherein the D2 domain is an IL-2 variant polypeptide comprising one or more amino acid substitutions or deletions at positions 19, 20, 38, 41, 42, 44, 88, 107, 125, or 126 of SEQ ID No. 8.

19. The construct according to any one of claims 1 to 18, wherein D2 is attached to D1 through a peptide linker ("L1") selected from the group consisting of a protease cleavable peptide linker and a non-cleavable peptide linker.

20. The construct according to claim 19, wherein the protease cleavable peptide linker is selected from the group of sequences listed in SEQ ID NO 71-96 and 157-161.

21. The construct according to claim 19, wherein the non-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NO 107-127.

22. The construct according to any one of claims 1 to 21, wherein the D3 domain is selected from the group consisting of: cognate receptor/binding partner (or variant thereof) and any binding partner identified for D2 and capable of masking the activity of D2.

23. The construct according to any one of claims 1 to 21, wherein the D3 domain is selected from the group consisting of: proteins, peptides, DNA fragments, RNA fragments, polymers, antibodies and antibody fragments capable of masking the activity of D2.

24. The construct of claim 22, wherein the D3 domain is a cognate receptor/binding partner for IL-15 (or a variant thereof) and comprises the amino acid sequence set forth in SEQ ID No. 4 or any functional fragment thereof.

25. The construct of claim 22, wherein the D3 domain is a cognate receptor/binding partner for IL-15 (or a variant thereof) and comprises the amino acid sequence set forth in SEQ ID No. 5.

26. The construct of claim 22, wherein the D3 domain is a cognate receptor/binding partner for IL-2 (or a variant thereof) and comprises the amino acid sequence set forth in SEQ ID No. 10.

27. The construct according to any one of claims 1 to 26, wherein D2 is attached to D3 through a peptide linker ("L2") selected from the group consisting of a protease cleavable peptide linker and a non-cleavable peptide linker.

28. The construct according to claim 27, wherein the protease cleavable peptide linker is selected from the group of sequences listed in SEQ ID NO 71-96 and 157-161.

29. The construct according to claim 27, wherein the non-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NO: 107-127.

30. The construct of any one of claims 1 to 29, wherein both L1 and L2 are protease cleavable peptide linkers.

31. The construct of any one of claims 1 to 29, wherein both L1 and L2 are non-cleavable peptide linkers.

32. The construct of any one of claims 1 to 29, wherein L1 is a protease cleavable peptide linker and L2 is a non-cleavable peptide linker.

33. The construct of any one of claims 1 to 29, wherein L1 is a non-cleavable peptide linker and L2 is a protease cleavable peptide linker.

34. A pharmaceutical composition comprising the construct of any one of claims 1 to 33 in admixture with a pharmaceutically acceptable carrier.

35. A method of treating cancer or cancer metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 34.

36. The method of claim 35, wherein the method further comprises a second therapeutic agent or therapy capable of treating cancer or cancer metastasis in the subject.

37. A method of treating an autoimmune disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 34.

38. The method of claim 37, wherein the method further comprises a second therapeutic agent or therapy capable of treating an autoimmune disease in a subject.

39. A method of treating an inflammatory disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 34.

40. The method of claim 39, wherein the method further comprises a second therapeutic agent or therapy capable of treating an inflammatory disease in a subject.

41. A method of treating a viral infection in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 34.

42. The method of claim 41, wherein the method further comprises a second therapeutic agent or therapy capable of treating a viral infection in a subject.

43. A nucleic acid molecule encoding the construct of any one of claims 1 to 33.

44. An expression vector comprising the nucleic acid molecule of claim 43.

45. A host cell comprising the expression vector of claim 44 or the expression vector of claim 43.

46. A method of producing a bioactivatable polypeptide drug construct according to any one of claims 1 to 33, comprising culturing a host cell according to claim 45 under conditions promoting expression of the bioactivatable polypeptide drug construct and recovering the bioactivatable polypeptide drug construct protein.

47. An isolated bioactivatable polypeptide drug construct protein produced by the method of claim 46.

Background

Many cytokines have been evaluated as potential therapeutic agents for the treatment of diseases. However, their systemic over-stimulation or over-suppression of the body's immune system severely hampers their development and clinical use.

Interleukin-2 (IL-2) and interleukin-15 (IL-15) share common receptor components (yc and IL-2R β) and signaling pathways, and have several similar functions. Both cytokines stimulate proliferation of T cells; inducing the production of Cytotoxic T Lymphocytes (CTLs); promoting proliferation of B cells and immunoglobulin synthesis; and inducing the generation and persistence of Natural Killer (NK) cells. Based on a number of preclinical studies and multiple clinical assessments, these two cytokines are considered potentially valuable therapeutic agents for cancer, autoimmune disorders, inflammatory disorders, transplantation, and a variety of other disorders. Recombinant IL-2 has been approved for use in patients with metastatic renal cell carcinoma and malignant melanoma. There are several ongoing clinical trials in oncology for IL-15, but the use has not been approved. In addition, both IL-2 and IL-15 have a third unique non-signaling receptor alpha-subunit: IL-2R α (also known as CD25) or IL-15R α, which may contribute to their different receptor specificities and biological functions.

Recombinant human IL-2 is an effective immunotherapy being used for metastatic melanoma and renal cancer, with a sustained response in about 10% of patients. However, the short half-life and severe toxicity limit the optimal administration of IL-2. In addition, IL-2 also binds with high affinity to its heterotrimeric receptor IL-2R α β γ, preferentially expanding immunosuppressive regulatory T cells (Tregs) that express high constitutive IL-2R α levels. Expansion of tregs may represent an undesirable effect of IL-2 on cancer immunotherapy. However, the ability of IL-2 to stimulate Treg cells even at low doses can be used to treat autoimmune and chronic inflammatory disorders. It has recently been discovered that IL-2 can be modified to selectively stimulate cytotoxic effector T cells or Treg cells. Various approaches have resulted in IL-2 variants with improved and selective immunomodulatory activity.

Both IL-2 and IL-15 are potent immune effector cell agonists, and it is critical that cytotoxic immune cells be fully activated only when at or very near the site of disease (e.g., cancer site) in order to specifically destroy only tumor cells; or the cytotoxic immune cells are fully activated only when at or very near the site of the inflammatory problem, so as to exert only an effect against autoimmune disorders and against chronic inflammatory disorders. For all cytokines, chemokines and growth factors, it is very important to improve specificity and selectivity for targets and to keep healthy cells and tissues intact and undamaged.

Disclosure of the invention

In one aspect, the present invention provides a cytokine-based bioactivatable drug ("VitoKine") platform that aims to reduce systemic mechanism-based toxicity and to allow for broader therapeutic use of cytokines, chemokines, hormones, and growth factors, such as IL-15 and IL-2, in the treatment of cancer, autoimmune disorders, inflammatory disorders, and various other disorders. The vitokinene platform is defined by the construct as depicted in fig. 1 and the proposed activation method as depicted in fig. 2. Referring to fig. 1, the novel vitokinene construct of the present invention comprises 3 domains: 1) a D1 domain ("D1") selected from the group consisting of: a tissue targeting domain, half-life extending domain, or dual function part domain, 2) a D2 domain ("D2"), which is the "active part domain," and 3) a D3 domain ("D3"), which is the "shielding part domain. Importantly, the D2 domain of the vitekine construct remains nearly inert or minimally active until locally activated by proteases upregulated in diseased tissue or hydrolysis of disease sites, which would limit binding of the active moiety to receptors in the periphery or on the cell surface of non-diseased cells or normal tissue to prevent over-activation of the pathway and reduce undesirable "out-of-tissue", "in-target" toxicity and undesirable target silencing (target sink).

In various embodiments, the vitokinene construct of the invention comprises D1, which D1 is a targeting moiety, such as an antibody or antibody fragment that binds to a Tumor Associated Antigen (TAA) or tissue specific antigen, a cell surface molecule or extracellular matrix protein or protease, or any post-translationally modified residue. In various embodiments, the vitokinene construct of the invention comprises D1, which D1 is a targeting moiety, such as a protein or peptide that exhibits binding affinity for diseased cells or tissues. In various embodiments, the vitokinene construct of the invention comprises D1, the D1 being a modified protein or peptide, such as glycan modified, that exhibits binding affinity for a particular receptor, such as the c-type lectin receptor, expressed on the diseased cell or tissue. In various embodiments, the vitokinene construct of the invention comprises a D1 domain, the D1 being an antibody directed to an immune checkpoint modulator. In various embodiments, the vitokinene construct of the invention comprises D1, the D1 acting to maintain the cytokine at the tissue site. In various embodiments, the vitokinene construct of the invention comprises D1, the D1 being dual functional, e.g., tissue targeting and maintenance. In various embodiments, the vitokinene constructs of the invention comprise a D1 domain, the D1 domain being a polymer. In various embodiments, the vitokinene constructs of the invention comprise a D1 domain, the D1 domain being a half-life extending moiety. In various embodiments, the vitokinene construct of the invention comprises a D1 domain, which D1 domain is an Fc domain (or functional fragment thereof).

"Fc domain" refers to a dimer of two Fc domain monomers, typically comprising all or part of a hinge region. In various embodiments, the Fc domain is selected from the group consisting of: a human IgG1Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, an IgA Fc domain, an IgD Fc domain, an IgE Fc domain, an IgG Fc domain, and an IgM Fc domain, or any combination thereof. In various embodiments, the Fc domain comprises amino acid changes that result in the Fc domain having altered complement binding properties or Fc receptor binding properties. Amino acid changes known to produce Fc domains with altered complement binding properties or Fc receptor binding properties are known in the art. In various embodiments, the Fc domain sequence used to prepare the VitroKine construct is the human IgG1-Fc domain sequence set forth in SEQ ID NO 13. In various embodiments, the Fc domain sequence used to prepare the VitroKine construct is that set forth in SEQ ID NO:14, and SEQ ID NO:14 comprises amino acid substitutions that abrogate (ablate) Fc γ R and C1q binding. In various embodiments, the Fc domain includes amino acid changes that result in further extended half-life in vivo. Amino acid changes known to produce Fc domains with further extended half-lives are known in the art. In various embodiments, the Fc domain sequence used to prepare the VitroKine construct is that set forth in SEQ ID NO:156 or 166, both of which contain amino acid substitutions that abrogate Fc γ R and C1q binding and extend half-life in vivo. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is derived from the Knob-Fc domain sequence set forth in SEQ ID NO. 15. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitroKine construct is derived from the Hole-Fc domain sequence set forth in SEQ ID NO 16. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is derived from the Knob-Fc domain with an extended in vivo half-life sequence set forth in SEQ ID NO: 167. In various embodiments, the heterodimeric Fc domain sequence used to prepare the VitoKine construct is derived from the Hole-Fc domain of the sequence set forth in SEQ ID NO:168 having an extended half-life in vivo.

In various embodiments, the vitokinene construct of the invention comprises a D2 domain, the D2 domain being a protein. In various embodiments, the VitoKine constructs of the invention comprise a D2 domain, the D2 domain being a cytokine (selected from the group including but not limited to IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-15, IL-23) and a ligand of the transforming growth factor beta (TGF β) superfamily, such as TGF β (SEQ ID NO: 24). In various embodiments, the VitoKine construct of the invention comprises a D2 domain, the D2 domain being IL-15. In various embodiments, the VitoKine constructs of the invention comprise a D2 domain, which D2 domain is an IL-15 variant (or mutant) comprising one or more amino acid substitutions, deletions, or insertions to the IL-15 polypeptide. In various embodiments, the VitoKine construct of the invention comprises a D2 domain, the D2 domain being IL-2. In various embodiments, the VitoKine constructs of the invention comprise a D2 domain, which D2 domain is an IL-2 variant (or mutant) comprising one or more amino acid substitutions, deletions, or insertions to the IL-2 polypeptide.

In various embodiments, the D2 domain of the VitroKine construct is an IL-15 domain comprising the sequence of a mature human IL-15 polypeptide (also referred to herein as huIL-15 or IL-15 wild-type (wt)) as set forth in SEQ ID NO: 2. In various embodiments, the IL-15 domain is an IL-15 variant (or mutant) comprising a sequence derived from a mature human IL-15 polypeptide sequence as set forth in SEQ ID NO: 2. In various embodiments, the IL-15 domain is an IL-15 variant (or mutant) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology to SEQ ID NO. 2. Natural amino acids, the position of natural amino acids in the mature sequence, and variant amino acids are used herein to refer to variants (or mutants) of IL-15. For example, huIL-15 "S58D" refers to a human IL-15 comprising an S to D substitution at position 58 of SEQ ID NO: 2. In various embodiments, the IL-15 variants function as IL-15 agonists, as demonstrated by, for example, increased binding activity to the IL-15R β γ c receptor as compared to the native IL-15 polypeptide. In various embodiments, the IL-15 variant functions as an IL-15 antagonist, as shown by: for example, binding activity to the IL-15R β γ c receptor is reduced, or binding activity to the IL-15R β γ c receptor is similar or increased, but signaling activity is reduced or eliminated, as compared to the native IL-15 polypeptide. In various embodiments, the IL-15 variant has increased binding affinity or decreased binding activity for the IL-15R β γ c receptor as compared to the native IL-15 polypeptide. In various embodiments, the sequence of the IL-15 variant has at least one (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid change as compared to the native IL-15 sequence. Amino acid changes may include one or more amino acid substitutions, deletions or insertions in the IL-15 polypeptide, such as in the domain of IL-15 that interacts with IL-15R β and/or IL-15R β γ c. In various embodiments, the amino acid change is one or more amino acid substitution or deletion at position 30, 31, 32, 58, 62, 63, 67, 68, or 108 of SEQ ID No. 2. In various embodiments, the amino acid change is a D to T substitution at position 30, a V to Y substitution at position 31, an H to E substitution at position 32, an S to D substitution at position 58, a T to D substitution at position 61, a V to F substitution at position 63, an I to V substitution at position 67, an I to F or H or D or K substitution at position 68, or a Q to a or M or S substitution at position 108 of the mature human IL-15 sequence, or any combination of these substitutions. In various embodiments, the amino acid change is an S to D substitution at position 58 of the mature human IL-15 sequence. In various embodiments, the IL-15 polypeptide comprises an IL-15 variant of SEQ ID NO 3. In various embodiments, the IL-15 domain has any combination of amino acid substitutions, deletions, and insertions.

In various embodiments, the D2 domain of the viteokine construct of the invention comprises an IL-2 polypeptide. In various embodiments, the vitokinene constructs of the invention comprise a D2 domain, which D2 domain is an IL-2 variant (or mutant) comprising one or more amino acid substitutions, deletions, or insertions. In various embodiments, the VitroKine construct comprises a D2 domain, wherein the IL-2 domain comprises the sequence of a mature human IL-2 polypeptide (also referred to herein as huIL-2 or IL-2 wild-type (wt)) as set forth in SEQ ID NO: 8. In various embodiments, the IL-2 domain is an IL-2 variant (or mutant) comprising a sequence derived from a mature human IL-2 polypeptide sequence as set forth in SEQ ID NO: 8. In various embodiments, the IL-2 domain is an IL-2 variant (or mutant) comprising a sequence having at least 80%, at least 85%, at least 90%, or at least 95% sequence homology to SEQ ID NO. 8. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the amino acid change is one or more amino acid substitutions at position 19, 20, 38, 41, 42, 44, 88, 107, 125, or 126 of SEQ ID No. 8. In various embodiments, the amino acid change is a L to D or H or N or P or Q or R or S or Y substitution at position 19, a D to E or I or N or Q or S or T or Y substitution at position 20, a R to E or a substitution at position 38, a T to a or G or V substitution at position 41, a F to a substitution at position 42, a F to G or V substitution at position 44, a N to D, E or G or I or M or Q or T or R substitution at position 88, a Y to G or H or L or V substitution at position 107, a S to E, H, K, I or W substitution at position 125, a Q to D or E or K or L or M or N substitution at position 126, or any combination of these substitutions in the mature human IL-2 sequence.

In various embodiments, the vitokinene constructs of the invention comprise a "masking moiety domain" (D3), which D3 domain is the cognate receptor/binding partner or any binding partner identified for the D2 protein or cytokine. In various embodiments, the D3 domain is a variant of the cognate receptor/binding partner of the D2 domain. In various embodiments, the D3 domain has enhanced binding to the D2 domain compared to the wild-type cognate receptor/binding partner. In various embodiments, the D3 domain has reduced or eliminated binding to the D2 domain compared to the wild-type cognate receptor/binding partner. In various embodiments, the D3 domain is a protein, or peptide, or antibody fragment capable of masking the activity of D2. In various embodiments, the D3 domain is a DNA, RNA fragment, or polymer, such as PEG. In various embodiments, the VitoKine construct of the invention comprises a D3 domain, the D3 domain being the extracellular domain of IL-15R α or a functional fragment thereof. In various embodiments, the VitoKine construct of the invention comprises a D3 domain, the D3 domain being an IL-15 Ra Sushi domain. In various embodiments, the VitoKine construct of the invention comprises a D3 domain, the D3 domain being an IL-2R α extracellular domain or a functional fragment thereof. In various embodiments, the VitoKine construct of the invention comprises a D3 domain, the D3 domain being an IL-2R α Sushi domain. In various embodiments, the D3 domain is capable of masking the functional activity of D2 until activated at the intended treatment site.

In various embodiments, the D1 domain, D2 domain, and D3 domain of the vitokinene construct are linked by a protease-cleavable polypeptide linker sequence. In various embodiments, the D1 domain, D2 domain, and D3 domain of the vitokinene construct are linked by a non-cleavable polypeptide linker sequence. In various embodiments, both L1 and L2 of the vitokinene constructs of the invention are protease cleavable peptide linkers. In various embodiments, L1 of the vitokinene construct of the invention is a protease cleavable peptide linker and L2 is a non-cleavable peptide linker. In various embodiments, L1 of the vitokinene construct of the invention is a non-cleavable peptide linker and L2 is a protease cleavable peptide linker. In various embodiments, both L1 and L2 of the vitokinene constructs of the invention are non-cleavable linkers. In various embodiments, the linker is rich in G/S content (e.g., at least about 60%, 70%, 80%, 90% or more of the amino acids in the linker are G or S). Each peptide linker sequence may be independently selected. In various embodiments, the protease cleavable linker is selected from the group of sequences set forth in SEQ ID NOS 71-96 and 157-161. In various embodiments, the protease cleavable linker may have an additional peptide spacer of variable length at the N-terminus of the cleavable linker or at the C-terminus of the cleavable linker or both termini of the cleavable linker. In various embodiments, the non-cleavable linker is selected from the group of sequences listed in SEQ ID NO: 107-127. In various embodiments, the joints are flexible or rigid and have various lengths.

In various embodiments, the D2 domain and the D3 domain of the vitekine construct are placed N-terminal to the D1 domain, as depicted in fig. 1. In various embodiments, the D2 domain and the D3 domain of the vitekine construct are placed C-terminal to the D1 domain, as depicted in fig. 1.

In various embodiments, the D1, D2, and D3 domains of the vitokinene construct may be a monomer or dimer or a combination of dimer and monomer, such as D1 being a dimer and D2 and D3 being a monomer.

In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma, or any cancer.

In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation, cell therapy including CAR-T, CAR-NK, iPS induced CAR-T or iPS induced CAR-NK, and vaccines such as bacillus Calmette-guerin (BCG). In various embodiments, combination therapy may comprise administering to a subject a therapeutically effective amount of immunotherapy, including, but not limited to, treatment with depleting antibodies against specific tumor antigens Treating; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic or blocking antibodies against costimulatory or cosuppressive molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec 7, Siglec 8, Siglec 9, Siglec 15 and VISTA, treatment with bispecific T cell binding antibodiesTreatments such as bornatemumab (blinatumomab); therapies involving administration of biological response modifiers (such as IL-12, IL-21, GM-CSF, IFN- α, IFN- β, and IFN- γ); treatment with a therapeutic vaccine such as sipuleucel-T; treatment with dendritic cell vaccines or tumor antigen peptide vaccines; treatment with Chimeric Antigen Receptor (CAR) -T cells; treatment with CAR-NK cells; treatment with Tumor Infiltrating Lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo expanded T cells and/or TCR transgenic T cells); treatment with TALL-104 cells; and treatment with immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment with vaccines such as BCG; wherein the combination therapy provides increased effector cell killing of the tumor cells, i.e., there is a synergy between the VitoKine construct and the immunotherapy when co-administered.

In another aspect, the present disclosure provides a method for treating a viral infection in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the virus is HIV.

In another aspect, the present disclosure provides methods for treating a viral infection in a subject comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention in combination with a second therapy including, but not limited to, acyclovir, echolucan (Epclusa), Mavyret, zidovudine, and enfuvirtide.

In another aspect, the present disclosure provides a method for treating an autoimmune disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the invention. In one embodiment, the subject is a human subject. In various embodiments, the autoimmune disease is selected from the group consisting of: systemic Lupus Erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenic purpura, Graves ' disease, Sjogren's syndrome, dermatomyositis, Hashimoto's disease, polymyositis, inflammatory bowel disease, Multiple Sclerosis (MS), diabetes, rheumatoid arthritis, and scleroderma.

In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the inflammatory disease is selected from the group consisting of: crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, metastatic colitis, Behcet's syndrome and indeterminate colitis.

In various embodiments, the inflammatory disease is selected from the group consisting of other autoimmune and inflammatory diseases such as: achalasia of cardia, adult still's disease, agammaglobulinemia, amyloidosis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune inner ear disease, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuronal neuropathies, barlow's disease, behcet's disease, benign mucosal pemphigoid, casteman's disease, chagas ' disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, allergic granulomatous vasculitis, cicatricial pemphigoid, kefir syndrome, coxsackie viral myocarditis, CREST syndrome, dermatitis herpetiformis, Devic/neuromyelitis optica, discoid lupus, lupus erythematosus, Deslerian syndrome, eosinophilic esophagitis, eosinophilic fasciitis, erythema nodosum, mixed cryoglobulinemia, fibropneumonitis, giant cell arteritis, giant cell myocarditis, allergic purpura, herpes gestationis or pemphigoid gestationis, IgA nephropathy, IgG 4-associated sclerosing disease, immune-related adverse events, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile myositis, Lambert-Eton syndrome, leukocyte-disrupting vasculitis, lichen planus, lichen sclerosis, lignoconjunctivitis, linear IgA disease, chronic Lyme disease, Meniere disease, microscopic polyangiitis, mixed connective tissue disease, silkworm's Ulcer, Mucha-Habermann disease, multifocal motor neuropathy, optic neuritis, recurrent rheumatism, PANDAS, paraneoplastic cerebellar degeneration, Parry Romber syndrome, Pars plana, Parsonage-Turner syndrome, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary sclerosing cholangitis, progesterone dermatitis, psoriatic arthritis, pure red blood cell regeneration disorder, pyoderma gangrenosum, raynaud's phenomenon, reflex sympathetic dystrophy, recurrent polychondritis, retroperitoneal fibrosis, scleritis, sperm and testis autoimmunity, stiff person syndrome, subacute bacterial endocarditis, Susac syndrome, sympathetic ophthalmia, takayatitis, thrombocytopenic purpura, painful ophthalmoplegia syndrome, transverse myelitis, undifferentiated connective tissue disease, Vogt-koyanagi-Harada disease.

In another aspect, the disclosure provides the use of a vitokinene construct for the manufacture of a medicament for the treatment of cancer.

In another aspect, the disclosure provides the use of a vitokinene construct for the manufacture of a medicament for the treatment of a viral infection.

In another aspect, the disclosure provides the use of a vitokinene construct for the manufacture of a medicament for the treatment of an autoimmune disease.

In another aspect, the disclosure provides the use of a vitokinene construct for the manufacture of a medicament for the treatment of inflammation.

In another aspect, the disclosure provides the use of the vitokinene constructs of the invention in combination with a second therapeutic agent or cell therapy capable of treating cancer, viral infection, or autoimmune disease or inflammation.

In another aspect, the disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding the vitokinene construct of the disclosure. In another aspect, the present disclosure provides a vector comprising a nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising a nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the present disclosure. In another aspect, the method of making the vitokinene construct is provided by culturing a host cell under conditions that promote expression of the protein or polypeptide.

In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated vittokine construct in admixture with a pharmaceutically acceptable carrier.