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CN-115151279-B - Cytokine-based bioactive agents and methods of use thereof

CN115151279BCN 115151279 BCN115151279 BCN 115151279BCN-115151279-B

Abstract

The present disclosure provides a bioactivated pharmaceutical construct "VitoKine" platform that aims to reduce toxicity based on systemic mechanisms and to make proteins and cytokines more widely therapeutically useful in the treatment of cancer, autoimmune diseases, inflammatory diseases, viral infections, transplantation, and various other disorders. The novel VitoKine constructs of the present invention comprise 1) a tissue or disease site targeting moiety D1 domain ("D1"), 2) a bioactivator moiety D2 domain ("D2") and a mask moiety D3 domain ("D3"). Because the "active portion" of VitoKine construct will remain inert until locally activated by proteases upregulated in diseased tissue, this will prevent excessive activation of the pathway and reduce undesirable "off-tissue" in-target "toxicity. Furthermore, the inertness of the VitoKine active moiety prior to protease activation will significantly reduce potential antigen or target absorption, thereby extending the in vivo half-life and resulting in improved biodistribution, bioavailability, and therapeutic efficacy.

Inventors

  • LI YUESHENG
  • RUI LINGYUN
  • XU JING

Assignees

  • 科优基因公司

Dates

Publication Date
20260505
Application Date
20201211
Priority Date
20191213

Claims (20)

  1. 1. A biologically activatable polypeptide pharmaceutical construct D1-D2-D3, said construct D1-D2-D3 consisting of, in the N-terminal to C-terminal direction, 1) a dual function moiety D1 domain, 2) a biologically activatable moiety D2 domain, and 3) a masking moiety D3 domain, wherein D1 functions to target the biologically activatable moiety to an intended treatment site, wherein D3 is capable of masking the functional activity of D2 until D2 is activated at the intended treatment site, and wherein D2 domain is an IL-2 variant polypeptide having the amino acid sequence set forth in SEQ ID NO: 240, wherein D1 is an antagonistic humanized PD-1 antibody having the sequences of heavy and light chains set forth in SEQ ID NO: 195 and 196, heavy and light chain amino acid sequences set forth in SEQ ID NO: 197 and 198, heavy and light chain amino acid sequences set forth in SEQ ID NO: 275 and 276, or heavy and light chain amino acid sequences set forth in SEQ ID NO: 277 and 278, and the amino acid sequences set forth in SEQ ID NO: 268, Wherein D2 is attached to D1 by a peptide linker L1, and D2 is attached to D3 by a peptide linker L2, The L1 and L2 are protease cleavable peptide linkers or non-cleavable peptide linkers, respectively.
  2. 2. The construct of claim 1, wherein the protease-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs 71-96 and 157-161.
  3. 3. The construct of claim 1, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs 107-127.
  4. 4. A construct according to any one of claims 1 to 3, wherein both L1 and L2 are protease cleavable peptide linkers.
  5. 5. A construct according to any one of claims 1 to 3, wherein both L1 and L2 are non-cleavable peptide linkers.
  6. 6. A construct according to any one of claims 1 to 3, wherein L1 is a protease cleavable peptide linker and L2 is a non-cleavable peptide linker.
  7. 7. A construct according to any one of claims 1 to 3, wherein L1 is a non-cleavable peptide linker and L2 is a protease cleavable peptide linker.
  8. 8. The construct of any one of claims 1-3, wherein the D1, D2, and D3 domains of the construct are each in monomeric form, are each in dimeric form, or are collectively in a combination of dimers and monomers.
  9. 9. The construct of claim 4, wherein the D1, D2, and D3 domains of the construct are each in monomeric form, are each in dimeric form, or are collectively in a combination of dimers and monomers.
  10. 10. The construct of claim 5, wherein the D1, D2, and D3 domains of the construct are each in monomeric form, are each in dimeric form, or are collectively in a combination of dimers and monomers.
  11. 11. The construct of claim 6, wherein the D1, D2, and D3 domains of the construct are each in monomeric form, are each in dimeric form, or are collectively in a combination of dimers and monomers.
  12. 12. The construct of claim 7, wherein the D1, D2, and D3 domains of the construct are each in monomeric form, are each in dimeric form, or are collectively in a combination of dimers and monomers.
  13. 13. A pharmaceutical composition comprising a construct according to any one of claims 1 to 12 in admixture with a pharmaceutically acceptable carrier.
  14. 14. Use of a construct according to any one of claims 1 to 12 in the manufacture of a pharmaceutical composition for the treatment of colon cancer.
  15. 15. The use of claim 14, wherein the treatment further comprises a second therapeutic agent or therapy capable of treating cancer or cancer metastasis in a subject.
  16. 16. A nucleic acid molecule encoding the biologically activatable polypeptide pharmaceutical construct of any one of claims 1 to 12.
  17. 17. An expression vector comprising the nucleic acid molecule of claim 16.
  18. 18. A host cell comprising the expression vector of claim 17.
  19. 19. A method of producing a biologically activatable polypeptide pharmaceutical construct according to any one of claims 1 to 12, the method comprising culturing the host cell of claim 18 under conditions that promote expression of the biologically activatable polypeptide pharmaceutical construct and recovering the biologically activatable polypeptide pharmaceutical construct protein.
  20. 20. An isolated biologically activatable polypeptide pharmaceutical construct protein, the isolated biologically activatable polypeptide pharmaceutical construct protein produced by the method of claim 19.

Description

Cytokine-based bioactive agents and methods of use thereof Background Many cytokines have been evaluated as potential therapeutic agents for the treatment of diseases. However, their systemic overstimulation or oversuppression 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-2Rβ) and signaling pathways and have several similar functions. Both cytokines stimulate proliferation of T cells, induce production of Cytotoxic T Lymphocytes (CTLs), promote proliferation of B cells and synthesis of immunoglobulins, and induce production and persistence of Natural Killer (NK) cells. Based on a number of preclinical studies and multiple clinical evaluations, these two cytokines are considered potentially valuable therapeutic agents for cancer, autoimmune disorders, inflammatory disorders, transplantation, and various 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 for oncology for IL-15, but no use has been approved. In addition, both IL-2 and IL-15 have a third unique non-signaling receptor alpha-subunit, IL-2Ralpha (also known as CD 25) or IL-15Ralpha, respectively, 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 optimal dosing of IL-2. In addition, IL-2 binds with higher affinity to its heterotrimeric receptor IL-2Rαβγ, preferentially expanding immunosuppressive regulatory T cells (Tregs) expressing high constitutive IL-2Rα 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. Recently it has been discovered that IL-2 can be modified to selectively stimulate cytotoxic effector T cells or Treg cells. Various approaches have led to the generation of 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 at or very near the site of disease (e.g. cancer site) in order to specifically destroy tumor cells only, or that cytotoxic immune cells be fully activated only at or very near the site of inflammatory problems in order to exert an anti-autoimmune and anti-chronic inflammatory disorder effect locally. For all cytokines, chemokines and growth factors, it is important to improve the specificity and selectivity for targets and to keep healthy cells and tissues intact and intact. Disclosure of the invention In one aspect, the present invention provides a cytokine-based bioactive drug ("VitoKine") platform that aims to reduce toxicity based on systemic mechanisms and to make cytokines, chemokines, hormones, and growth factors, such as IL-15 and IL-2, more widely therapeutically useful in the treatment of cancer, autoimmune disorders, inflammatory disorders, and various other disorders. The VitoKine 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 VitoKine construct of the present invention comprises 3 domains, 1) a D1 domain ("D1") selected from the group consisting of a tissue targeting domain, a half-life extending domain, or a dual-function portion domain, 2) a D2 domain ("D2"), which is an "active portion domain", and 3) a D3 domain ("D3"), which is a "masking portion domain". Importantly, the D2 domain of the VitoKine construct remains nearly inert or minimally active until locally activated by hydrolysis of the up-regulated protease or disease site in diseased tissue, which will limit binding of the active moiety to receptors in or on the periphery of non-diseased or normal tissue to prevent over-activation of the pathway and reduce unwanted "off-target" toxicity and unwanted target uptake (TARGET SINK). In various embodiments, the VitoKine constructs of the invention comprise D1, which D1 is a targeting moiety, such as an antibody or antibody fragment that binds to a tumor-associated antigen (TAA), or an immune checkpoint modulator, or a tissue-specific antigen, a cell surface molecule, or an extracellular matrix protein or protease, or any post-translational modification residue. In various embodiments, vitoKine constructs of the invention comprise D1, which D1 is a targeting moiety, such as a protein or peptide that exhibits binding affinity to diseased cells or tissue. Exemplary antibodies contemplated for use as D1 in VitoKine constructs of the invention include various PD-1 antagonist antibodies, PD