Brand new masked cytokines and uses thereof

Abstract

The invention discloses a brand new masked cytokine and application thereof, and further provides a prodrug, a preparation method thereof and application of the prodrug in stimulation of an immune system and treatment of cancers, autoimmune diseases or infectious diseases.

Description

[0001] This application is a divisional application of the Chinese invention patent with an application date of January 11, 2021, application number 202180007592.8, and the invention name is "New Masked Cytokines and Their Applications".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application also claims priority to U.S. Provisional Application No. 62 / 959,973 filed on January 11, 2020, U.S. Provisional Application No. 63 / 027,138 filed on May 19, 2020, U.S. Provisional Application No. 63 / 029,473 filed on May 23, 2020, and U.S. Provisional Application No. 63 / 126,393 filed on December 16, 2020, the contents of the above U.S. Provisional Applications are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy created on January 11, 2021 is named 025471_WO008_SL.txt and is 240,322 bytes in size. Background Art

[0006] Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21 have important effects on the proliferation, differentiation and survival of immune cells. The receptors of the above cytokines share the γ chain (γC), that is, CD132, so these cytokines are also called γC family cytokines.

[0007] IL-2 plays an important role in the generation, survival and in vivo stability of lymphocytes. The IL-2 has 133 amino acids and is composed of four antiparallel, amphipathic α-helices. The tetrameric structure formed by it is a necessary condition for the function of IL-2 (Smith, Science (1988) 240: 1169-76; Bazan, Science (1992) 257: 410-13). IL-2 achieves its activity by binding to the IL-2 receptor (IL-2R), which is composed of up to three independent subunits: a trimer composed of α (CD25 or Tac antigen), β (CD122), and γC subunits, forming a high affinity (K) for IL-2. D ~0.01nM) receptor. The IL-2 dimer receptor composed of β and γ subunits is called the intermediate affinity IL-2 receptor (K D ~1nM). The α subunit monomer constitutes a low affinity IL-2 receptor (KD ~10nM) (See, eg, Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66). Although the affinity of the dimeric intermediate affinity IL-2 receptor binding to IL-2 is reduced to about 1 / 100 compared to the trimeric high affinity receptor, both the dimeric and trimeric IL-2 receptors can transmit signals when bound to IL-2 (Minami et al., Annu Rev Immunol. (1993) 11:245-68).

[0008] IL-15 is a cytokine with a similar structure to IL-2. IL-15 is secreted by phagocytes and other immune cells after viral infection. IL-15 can induce the proliferation of natural killer cells and other cells of the immune system, and participate in killing virus-infected cells and tumor cells. Like IL-2, IL-15 binds to the IL-2 receptor (IL-2R) β / γ complex, also known as the intermediate affinity receptor, with K D IL-15 binds with an affinity of about 1 nM (Giri et al., EMBO J. (1994) 13: 2822-30). D ~0.05nM) and IL-15 receptor (IL-15R) α, IL-15Rα can form a specific, high-affinity (αβγ) functional receptor for IL-15 together with the IL-2Rβ / γ complex (Minami et al., Annu. Rev. Immunol. (1993) 11:245-67; ​​Giri et al., J Leukoc Biol. (1995) 5745:763-6; and Lehours et al., Eur Cytokine Netw. (2000) 11:207-15).

[0009] IL-21 is produced by activated CD4+ T cells, T-follicular helper cells and natural killer cells (NKT) (Spolski and Leonard, Ann Rev Immunol. (2008) 26:57008). IL-21 has been shown to exert pleiotropic effects on the proliferation, differentiation and cytotoxicity of various types of lymphocytes. Recently, IL-21 has been further shown to play a role in CD4 + T cells differentiate into helper T cells 17 (TH 17), which is a subtype of T cells associated with inflammation and autoimmune responses (Korn et al., Nature (2007) 448 (7152): 484-87; Nurieva et al., Nature (2007) 448 (7152): 480-83). The IL-21 receptor composition consists of a private chain IL-21Rα and a common chain γC (or Rγ). Human IL-21 binds to the IL-21 receptor with a very high affinity (K D ~70pM; Zhang et al., Biochem Biophys Res Commun. (2003) 300(2):291-6) and IL-21Rα, but with relatively low affinity (K D ~160μM) binds to IL-21Rγ.

[0010] The therapeutic potential of the above cytokines, their mutants and their fusions is being studied and developed, among which recombinant IL-2 is the first cytokine approved for cancer treatment. However, cytokine drugs and candidate drugs have obvious side effects. In addition, these drugs usually have a short half-life in the body, and even exist in the form of antibody-cytokine fusion proteins. The reason may be the "PK sedimentation" formed by cytokine receptors on immune cells.

[0011] Therefore, further research and development of cytokine-based tumor treatments with higher tumor site selectivity, improved PK, and efficacy to reduce side effects is necessary. SUMMARY OF THE INVENTION

[0013] The present invention provides a prodrug, comprising a cytokine portion, a masking portion, and a carrier portion, wherein the masking portion binds to the cytokine portion and inhibits the biological activity of the cytokine; the carrier portion comprises an antigen binding portion that can bind to an antigen expressed on the surface of a target cell; and the masking portion is not connected to the carrier portion via a cleavable linker (such as indirectly connected to the carrier portion via a non-cleavable peptide linker, or directly connected to the carrier portion via a peptide linker).

[0014] In some embodiments, the prodrug can be activated by a cell, the cell surface expressing antigens and cytokine receptors at the same time. In some embodiments, the biological activity of the prodrug at the lesion containing the above-mentioned cells is increased by at least 2 times, 5 times or 10 times compared to the biological activity at the site without the above-mentioned cells. In some specific embodiments, the receptor of the cytokine part includes two or more subunits.

[0015] In some embodiments, the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD30, CD38, BCMA, and signal regulatory protein alpha (SIRPα, named CD172a).

[0016] In some embodiments, the cytokine portion comprises an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, an IL-21 agonist polypeptide, an IL-1α agonist polypeptide, an IL-1β agonist polypeptide, an IL-4 agonist polypeptide, an IL-5 agonist polypeptide, an IL-6 agonist polypeptide, an IL-8 agonist polypeptide, an IL-10 agonist polypeptide, an IL-12 agonist polypeptide, an IL-17 agonist polypeptide, an IL-18 agonist polypeptide, an IL-22 agonist polypeptide, an IL-23 agonist polypeptide, an IL-31 agonist polypeptide, an IL-33 agonist polypeptide, an IL-36 agonist polypeptide, an interferon-α agonist polypeptide, an interferon gamma agonist polypeptide, a 4-1BB ligand, an OX-40 ligand, or a CD-40 ligand.

[0017] In some embodiments, the cytokine portion comprises an IL-2 agonist polypeptide or an IL-15 agonist polypeptide, and the masking portion comprises an IL-2 receptor β extracellular domain (IL-2RβECD) or a functional fragment thereof. In other embodiments, the cytokine portion comprises an IL-21 agonist polypeptide, and the masking portion comprises an anti-IL-21 antibody Fab, a single-chain Fv (scFv), or a single-domain antibody thereof. In other embodiments, the cytokine portion comprises an IL-2 agonist polypeptide, and the masking portion comprises an anti-IL-2 antibody Fab, scFv, or a single-domain antibody thereof. In other embodiments, the cytokine portion comprises an IL-15 agonist polypeptide, and the masking portion comprises an anti-IL-15 antibody Fab, scFv, or a single-domain antibody thereof. In other embodiments, the cytokine portion comprises an IL-15 agonist polypeptide, and the masking portion comprises an IL-15 receptor α Sushi domain (IL-15RαSushi domain).

[0018] In some embodiments, the cytokine portion comprises an IL-21 agonist polypeptide having a sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90% homology thereto. In other embodiments, the cytokine portion comprises an IL-2 agonist polypeptide having a sequence of SEQ ID NO: 6 or 62, or an amino acid sequence having at least 95% homology thereto. In other embodiments, the cytokine portion comprises an IL-15 agonist polypeptide having a sequence of SEQ ID NO: 7, or an amino acid sequence having at least 95% homology thereto.

[0019] In some embodiments, the masking portion includes the extracellular domain (ECD) of the cytokine receptor. In specific embodiments, the cytokine includes an IL-7 agonist polypeptide, and the masking portion includes the extracellular domain of IL-7 receptor α (IL-7RαECD) or a functional analog thereof. In specific embodiments, the cytokine portion is an IL-21 agonist polypeptide, and the masking portion includes the extracellular domain of IL-21 receptor α (IL-21RαECD) or a functional analog thereof. In specific embodiments, the masking portion includes IL-21RαECD or a functional analog thereof, and its amino acid sequence is selected from SEQ ID NO: 12, 13 and 63-73 or an amino acid sequence having at least 90% (such as at least 95%) homology thereto. In some embodiments, the masking portion includes IL-2RβECD or a functional analog thereof, including SEQ ID NO: 11 or an amino acid sequence having at least 95% homology thereto.

[0020] In some embodiments, the masking portion includes an scFv that can bind to a cytokine portion and inhibit or interfere with the interaction of the cytokine with its receptor. In a specific embodiment, the masking portion is an scFv that can bind to human IL-21, which includes the amino acid sequence SEQ ID NO: 20 or 21. In a specific embodiment, the masking portion includes an scFv that can bind to IL-2, and inhibits the interaction of IL-2 with IL-2Rα, the interaction of IL-2 with IL-2Rβ, and / or the interaction of IL-2 with IL-2Rγ by binding to IL-2, wherein the scFv optionally includes the complementary determining region (CDR) of the light chain and heavy chain of the antibody 4E12B2D10. In some embodiments, the scFv includes the light chain and heavy chain of the antibody 4E12B2D10. In a specific embodiment, the antibody 4E12B2D10, the amino acid sequences of its heavy chain and light chain are SEQ ID NO: 60 and 61, respectively. In some embodiments, the masking moiety is a scFv that can bind to IL-2, which includes SEQ ID NO: 22 or 23, or an amino acid sequence having at least 95% homology thereto. In a specific embodiment, the masking moiety includes a scFv that can bind to IL-15, which inhibits or interferes with the interaction of IL-15 with IL-2Rβ, and / or the interaction of IL-15 with IL-2Rγ by binding to the scFv, wherein the scFv optionally includes the CDRs of the light and heavy chains of antibodies 146B7, 146H5, or 404E4, or the scFv optionally includes the light and heavy chains of anti-IL-15 antibodies 146B7, 146H5, or 404E4, or the scFv optionally includes SEQ ID NO: 18 or 19. In some embodiments, the IL-15 agonist scFv or Fab comprises CDR1-3 of the heavy chain and CDR1-3 of the light chain of an anti-IL-15 antibody selected from 146B7, 146H5, or 404E4, all of which are described in WO2003 / 017935A2.

[0021] In a specific embodiment, the masking portion includes a scFv that can bind to IL-21, which inhibits or interferes with the interaction between IL-21 and IL-21Rα, and / or the interaction between IL-21 and IL-2Rγ by binding to IL-21. In a specific embodiment, the masking portion includes a scFv that can bind to a cytokine selected from IL-2, IL-7, IL-9, IL-15 or IL-21, and inhibits or interferes with the interaction between the cytokine and IL-2Rγ.

[0022] In some embodiments, the prodrug has a half-life increased by at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, or at least 200-fold compared to the corresponding wild-type cytokine in a non-human primate or human.

[0023] In certain embodiments, the prodrug comprises two light chains having an amino acid sequence of SEQ ID NO:44, and two heavy polypeptide chains having amino acid sequences comprising SEQ ID NOs:24 and 25; SEQ ID NOs:35 and 36; SEQ ID NOs:37 and 36; SEQ ID NOs:37 and 38; SEQ ID NOs:39 and 41, or SEQ ID NOs:42 and 43, respectively.

[0024] In certain embodiments, the prodrug comprises two light chains having an amino acid sequence of SEQ ID NO:50, and two heavy polypeptide chains having amino acid sequences comprising SEQ ID NO:51 and 54; SEQ ID NO:51 and 55; SEQ ID NO:51 and 56; SEQ ID NO:52 and 54; SEQ ID NO:53 and 58; SEQ ID NO:53 and 59; or SEQ ID NO:52 and 57, respectively.

[0025] In some embodiments, the present invention provides a pharmaceutical composition comprising a prodrug disclosed herein and a pharmaceutically acceptable excipient.

[0026] On the other hand, the present invention provides a polynucleotide encoding the prodrug, an expression vector comprising the polynucleotide, and a host cell (such as a mammalian host cell, such as a CHO cell, a NS0 cell, and a 293T cell) comprising the expression vector. The present invention also provides a method for preparing the prodrug, comprising culturing the mammalian host cell under conditions that allow the expression of the prodrug, and isolating (purifying) the prodrug.

[0027] Under the premise of ensuring that the cytokine part is masked, the Fc-based IL-21 prodrug cannot interact with the expression of IL-21 receptor ( Figure 2 The study further showed that the masked molecules with the antibody PD-1 as carrier had little activity against NK92 cells that did not express PD-1 ( Figure 3 The inventors surprisingly found that the IL-21 prodrug molecule based on PD-1 antibody has obvious activity against Mino cells before activation ( Figure 4A and Figure 4B IL-2 prodrug ( Fig. 6A and Figure 6B ) and IL-15 prodrug ( Figure 5A-C) has a similar phenomenon. Although not wishing to be bound by theory, it can be assumed that when cells express antigens and cytokine receptors at the same time, the cis binding of PD-1 antibodies to antigens and cytokines to cytokine receptors relieves the masking effect of the masking part. Therefore, the prodrug containing the targeting functional part has obvious selectivity for cells expressing specific antigens compared to cells without the corresponding antigen, wherein the antigen has targeting to the carrier molecule of the prodrug. Based on this, the prodrug can be constructed without the need for protease cleavage or removal of the masking part.

[0028] The present invention provides a prodrug that is selectively activated without the need for protease to cleave or remove the masking portion at the lesion. On the one hand, the present invention provides a completely new prodrug, which includes a cytokine portion (cytokine agonist polypeptide), a masking portion, and a carrier portion, wherein the masking portion inhibits the biological activity of the cytokine by binding to the cytokine agonist polypeptide, the carrier portion includes an antigen binding portion, and the masking portion is directly or indirectly connected to the carrier portion through a non-cleavable peptide connector or without a peptide connector, wherein the prodrug has a higher activity in activated cells that simultaneously express a cytokine receptor and a targeting antigen to the carrier portion than in activated cells that only express a cytokine receptor but do not express a targeting antigen to the carrier portion.

[0029] On the other hand, the prodrug provided by the present invention includes a cytokine portion (cytokine agonist polypeptide), a masking portion and a carrier portion, wherein the masking portion inhibits the biological activity of the cytokine by binding to the cytokine agonist polypeptide, and the carrier portion includes an antigen binding portion, and the masking portion is directly or indirectly connected to the carrier portion through a non-cleavable peptide connector or without a peptide connector, wherein the prodrug is activated at the lesion site (the cells at this site simultaneously express cytokine receptors and antigens that are targeted to the carrier portion), or the prodrug is activated at the lesion site (the cells at this site simultaneously express cytokine receptors and antigens that are targeted to the carrier portion), and its biological activity at the lesion site is increased by at least 2 times, at least 5 times, or at least 10 times.

[0030] In some embodiments, the prodrug does not contain any cleavable peptide linker.

[0031] In some embodiments, the carrier portion includes a binding portion for an antigen expressed by an immune cell. In some embodiments, the carrier portion includes a binding portion for an antigen selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, and CD16b.

[0032] In some embodiments, the cytokine portion includes a cytokine selected from an IL-2 agonist polypeptide, an IL-17 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, and an IL-21 agonist polypeptide. In some embodiments, the cytokine is selected from an IL-1α agonist polypeptide, an IL-1β agonist polypeptide, an IL-4 agonist polypeptide, an IL-5 agonist polypeptide, an IL-6 agonist polypeptide, an IL-8 agonist polypeptide, an IL-10 agonist polypeptide, an IL-12 agonist polypeptide, an IL-15 agonist polypeptide, an IL-17 agonist polypeptide, an IL-18 agonist polypeptide, an IL-22 agonist polypeptide, an IL-23 agonist polypeptide, an IL-31 agonist polypeptide, an IL-33 agonist polypeptide, an IL-36 agonist polypeptide, an interferon α agonist polypeptide, an interferon γ, a 4-1BB ligand, an OX-40 ligand, and a CD-40 ligand.

[0033] In some embodiments, the masking moiety is an extracellular domain (ECD) of a cytokine receptor and the masking moiety comprises an anti-cytokine antibody or a binding fragment of an antibody.

[0034] In some embodiments, the cytokine is an IL-21 agonist polypeptide, wherein the masking portion is an IL-21 receptor extracellular domain (IL-21RαECD) or a functional analog thereof. In some embodiments, the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide, wherein the masking portion is an IL-2 receptor extracellular domain (IL-2RβECD). In some embodiments, the cytokine is an IL-21 agonist polypeptide, wherein the masking portion is an anti-IL-21 Fab, a single-chain antibody (scFv) or a single-domain antibody.

[0035] In some embodiments, the cytokine is an IL-2 agonist polypeptide, wherein the masking moiety is an anti-IL-2 Fab, a single chain antibody (scFv), or a single domain antibody.

[0036] In some embodiments, the cytokine is an IL-15 agonist polypeptide, wherein the masking moiety is an anti-IL-15 Fab, a single chain antibody (scFv) or a single domain antibody. In some embodiments, the cytokine comprises an IL-15 agonist polypeptide, wherein the chimeric molecule further comprises the sushi domain of the α of the IL-15 receptor (IL-15Rαsushi domain).

[0037] In some embodiments, the IL-21 agonist polypeptide comprises SEQ ID NO: 1 or an amino acid sequence having at least 90% homology thereto. In some embodiments, the masking moiety is IL-21RαECD or a functional analog thereof, comprising an amino acid sequence selected from SEQ ID NO: 12, 63-72 and 73, or an amino acid sequence having at least 90% homology thereto with SEQ ID NO: 12. In some embodiments, the masking moiety is a scFv that binds to human IL-21, comprising the amino acid sequence SEQ ID NO: 20 or 21.

[0038] In some embodiments, the IL-2 agonist polypeptide comprises SEQ ID NO: 6 or 61, or an amino acid sequence having at least 95% homology to SEQ ID NO: 6 or 61. In some embodiments, the masking moiety comprises an IL-2RβECD or a functional analog thereof, wherein the IL-2RβECD comprises SEQ ID NO: 11 or an amino acid sequence having at least 95% homology thereto. In some embodiments, the masking moiety is an scFv that binds to IL-2, and the scFv affects the interaction between IL-2 and IL-2Rα. In some embodiments, the masking moiety is an scFv that binds to IL-2, and the scFv affects the interaction between IL-2 and IL-2Rβ. In some embodiments, the masking moiety is an scFv that binds to IL-2, and the scFv affects the interaction between IL-2 and IL-2Rγ. In some embodiments, the masking moiety is an scFv that binds to IL-2, and the scFv has the same light chain and heavy chain as the hybridoma 4E12B2D10. In some embodiments, the masking moiety is a scFv that binds IL-2, the scFv comprising SEQ ID NO: 22, 23, or an amino acid sequence having at least 95% homology thereto.

[0039] In some embodiments, the IL-15 agonist polypeptide comprises SEQ ID NO: 7, or an amino acid sequence having at least 95% homology thereto. In some embodiments, the IL-15 prodrug further comprises a sushi domain, the amino acid sequence of which is SEQ ID NO: 8, or an amino acid sequence having at least 95% homology thereto. In some embodiments, the masking moiety comprises an IL-2RβECD or a functional analog thereof, wherein the IL-2RβECD comprises SEQ ID NO: 11 or an amino acid sequence having at least 95% homology thereto. In some embodiments, the masking moiety is an scFv that binds to IL-15, and the scFv affects the interaction between IL-15 and IL-2Rβ. In some embodiments, the masking moiety is an scFv that binds to IL-15, and the scFv affects the interaction between IL-15 and IL-2Rγ. In some embodiments, the masking moiety is an scFv that binds to IL-15, and the scFv has the same light chain and heavy chain as the IL-15 antibody 146B7, 146H5 or 404E4. In some embodiments, the masking moiety is a scFv that binds IL-15, wherein the scFv comprises the amino acid sequence of SEQ ID NO: 18 or 19.

[0040] In some embodiments, the human IL-21 agonist polypeptide comprises one or more mutations selected from D18, Q19, E109 and K117 (sequence numbers are consistent with SEQ ID NO: 1). In a specific embodiment, the human IL-21 agonist polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 4, and 5.

[0041] In some embodiments of the prodrug, the cytokine portion and the masking portion are fused to the carrier portion via a non-cleavable peptide linker, for example, the non-cleavable peptide linker is selected from SEQ ID NOs: 27-34.

[0042] In some embodiments of the prodrug, the carrier portion is an IgG1 antibody comprising L234A and L235A ("LALA") (EU numbering) mutations or an IgG4 antibody comprising S228P / L234A / L235A (PAA) mutations. When the carrier portion is an Fc domain or an antibody Fc, other mutations that can reduce Fc functionality may also be included, such as the mutations described in Tam et al., Antibodies (2017) 6 (12): 1-34.

[0043] In certain embodiments, the carrier portion is an antibody comprising knobs-into-holes mutations, and the cytokine portion and the masking portion are fused to different heavy chains of the antibody, respectively. In certain embodiments, the knobs-into-holes mutation comprises a T366Y "knob" mutation on one polypeptide of the antibody heavy chain or Fc domain, and a Y407T "hole" mutation on another polypeptide of the antibody heavy chain or Fc domain (EU numbering). In certain embodiments, the knobs-into-holes mutation comprises a Y349C and / or T366W mutation in the CH3 domain of the "knob" chain, and an E356C, T366S, L368A, and / or Y407V mutation in the CH3 domain of the "hole" chain (EU numbering).

[0044] In some embodiments, the carrier portion is an antibody or antigen-binding fragment that specifically binds to a protein selected from amidinocyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), carcinoembryonic antigen CEA, insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), proteoglycan-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Li v-1, glycoprotein NMB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), six transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein (SIRPα), PD1, Claudin 18.2, Claudin 6, 5T4, BCMA, PD-L1, PD-1, fibroblast activation protein alpha (FAPα), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM) or one or more antigens. In a specific embodiment, the carrier portion is an antibody or a fragment thereof that binds to FAPα or 5T4.

[0045] On the other hand, the present invention provides a polynucleotide encoding the prodrug or fusion molecule, an expression vector comprising the polynucleotide, and a host cell (such as a mammalian host cell CHO, NS0 cell and 293T cell) comprising the expression vector. The present invention also provides a method for preparing the prodrug or fusion molecule, comprising culturing the host cell under conditions allowing the expression of the prodrug or fusion molecule and isolating the prodrug or fusion molecule.

[0046] The present invention also provides a method for treating cancer, infectious diseases, or stimulating the immune system for a patient (such as a patient) in need thereof, comprising administering to the patient a therapeutically effective amount of a cytokine prodrug or a pharmaceutical composition provided by the present invention. The patient may suffer from, for example, a viral infection (such as HIV, HBV, HCV or HPV infection) or any cancer selected from breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer and gastric cancer. Also provided are the use of cytokine prodrugs in treating cancer, infectious diseases, or stimulating the immune system, the use of cytokine prodrugs in preparing drugs for treating cancer, infectious diseases, or stimulating the immune system, and products (such as kits) comprising one or more dosage units of the cytokine prodrug.

[0047] The present invention also provides the use of prodrugs in treating cancer, infectious diseases, or stimulating the immune system, the use of prodrugs in preparing drugs for treating cancer, infectious diseases, or stimulating the immune system, and preparations (such as kits) comprising one or more dosage units of the prodrug.

[0048] Other features, purposes and beneficial effects of the present invention may be apparent from the following detailed description, and it should be understood that the detailed descriptions describing the embodiments and aspects of the present invention are intended to be illustrative only and not limiting. Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1A -E lists heterodimeric cytokine prodrugs using antibodies as carriers. Figure 1A It is a prodrug with a typical antibody having two light chains and two heavy chains as its carrier; Figure 1B It is a prodrug with a bispecific antibody as its carrier, each antigen-binding portion is a monomer, one antigen-binding portion includes a Fab domain, and the other antigen-binding portion includes a scFv or a single-domain antibody; Figure 1C It is a prodrug with a bispecific antibody as its carrier, in which each antigen-binding part is a dimer; Figure 1D A prodrug in which one polypeptide chain of a dimeric Fc domain is fused to a Fab domain, and the other polypeptide chain is fused to a cytokine polypeptide, wherein the cytokine is fused to a masking portion at the N-terminus; Figure 1E It is a prodrug in which one polypeptide chain of the dimer Fc domain is fused to the Fab domain, and the other polypeptide chain is fused to the cytokine polypeptide, wherein the N-terminus of the masking part is fused to the cytokine.

[0050] Figure 2 The binding results of PD-1 antibody, Fc-IL-21 fusion molecule and control molecule to Mino cells were analyzed by FACS.

[0051] Figure 3 The results show the effects of IL-21 prodrug and control molecules on the biological activity of NK-92 cells before and after MMP2 protease activation. PW04-38αPD1-IL21-a* and PW09-16αPD1-IL21-a* are the same molecule from different batches, which are PD-1 antibody-IL-21 prodrugs with IL-21Rα-ECD as the masking part; PW05-68αPD1-IL21-scFv is a PD-1 antibody-IL-21 prodrug with scFv as the masking part; one of the control molecules, PW04-67αPD1-IL21, is a PD-1 antibody-IL-21 fusion molecule without a masking part, in which IL-21 is wild-type; another control molecule, PW09-02αPD1-IL21 R9ER76A mutant, is a PD-1 antibody-IL-21 fusion molecule without a masking part, in which IL-21 has R9E and R76A amino acid mutations (sequence number and SEQ ID NO:1 is consistent); activated PW04-38αPD1-IL21-a* and activated PW09-16αPD1-IL21-a* are PD-1 antibody-IL-21 fusion molecules without mutations, and their masking parts are cut off by protease.

[0052] Figure 4A and Figure 4B The results show the effects of PD-1-IL-21 prodrug and control molecules on the biological activity of Mino cells before and after MMP2 protease activation. Figure 4A The results are shown after the cytokine fusion molecules and Mino cells were incubated for 72 hours; Figure 4B The results are shown after 120 hours of incubation.

[0053] Figure 5A , Figure 5B and Figure 5C CD4 +Ki67 activity of T cells, wherein PBMCs were pretreated with PD-1 antibody (PD-1), scFv-masked Fc-IL-15 fusion molecule (215β), scFv-masked PD-1 antibody-IL-15 fusion molecule (215γ), Fc-IL-15 fusion molecule without masking part (215βref), and PD-1 antibody-IL-15 fusion molecule without masking part (PD1 / IL15 mutein (M2)). Figure 5A CD4 that does not express PD-1 + Ki67 activity in T cells; Figure 5B CD4 expressing PD-1 + Ki67 activity in T cells; Figure 5C CD4 expressing and not expressing PD-1 treated with different samples + T cell EC 50 The values ​​of CD4 expressing and not expressing PD-1 are also listed. + T cell EC 50 Multiples of value.

[0054] Fig. 6A and Figure 6B CD4 + Ki67 activity of T cells, in which PBMCs were pretreated with PD-1 antibody (PD-1), negative control (IgG1 antibody), and PD-1 antibody-IL-2 mutant protein fusion molecule (PD-1 / IL2V*) with IL-2 receptor β extracellular domain (IL-2Rβ-ECD) as the masking part. Fig. 6A CD4 that does not express PD-1 + Ki67 activity in T cells; Figure 6B CD4 expressing PD-1 + Ki67 activity in T cells.

[0055] Figure 7 The principle of prodrug activation through "cis-binding" is explained. The target cell (right side) expresses both antigens and cytokine receptors that are targeted to the carrier part. The prodrug approaches the cytokine receptor on the cell surface by binding to the cell surface antigen, allowing the cytokine receptor to replace the masking part with a competitive advantage, thereby achieving its signal transduction effect by binding to the cytokine receptor. The prodrug does not require protease cleavage to remove the masking part. In cells that only express cytokine receptors but not antigens (left side), the cytokine of the prodrug is in a masked and inactive state. DETAILED DESCRIPTION OF THE INVENTION

[0057] As used herein and in the claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0058] Reference to a value or parameter "about" herein includes (and describes) variations of the value or parameter itself. For example, a description of "about X" includes a description of "X". In addition, the use of "about" in front of any set of numbers also includes the use of "about" in the set of numbers cited. For example, a description of "about X, Y, or Z" is intended to describe "about X, about Y, or about Z".

[0059] The term "antigen binding portion" refers to a polypeptide or a group of interacting polypeptides that specifically bind to an antigen, including but not limited to antibodies (e.g., monoclonal antibodies, polyclonal antibodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies or bifunctional hybrid antibodies), or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb), diabodies, single chain antibodies and Fc-containing polypeptides (such as immunoadhesins)). In some embodiments, the antibody can be any heavy chain type (e.g., IgG, IgA, IgM, IgE or IgD) or subtype (e.g., IgG1, IgG2, IgG3 or IgG4). In some embodiments, the antibody can be any light chain type (e.g., kappa or lambda). The antibody can be human, non-human (e.g., from mouse, rat, rabbit, goat or other non-human animal), chimeric (e.g., having a non-human variable region and a human constant region) or humanized (e.g., having a non-human CDR, a human framework and a constant region). In some embodiments, the antibody is a derivatized antibody.

[0060] The terms "anti", "binding" and "specific binding" used in the present invention to describe the interaction between two molecules refer to the K measured by conventional means in the art (such as surface plasmon resonance, Elisa, etc.). D The value does not exceed 1000 nM (eg, does not exceed 100, 10, or 1 nM, for example, less than 1 nM, 1-10 nM, 10-100 nM, or 100-1000 nM).

[0061] The term "cytokine agonist polypeptide" refers to a wild-type cytokine or an analog thereof. An analog of a wild-type cytokine has the same biological specificity as the wild-type cytokine (e.g., binds to the same receptor and activates the same target cells), although its activity level may be different from that of the wild-type cytokine. An analog may be, for example, a mutant protein (i.e., a mutant polypeptide) of a wild-type cytokine and may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wild-type cytokine.

[0062] "Cytokine antagonists", "masking moieties", or "cytokine masking moieties" refer to moieties (such as polypeptides) that bind to cytokines. When the antagonist or masking moiety binds to the cytokine, it can inhibit the cytokine from binding to the target cell surface receptor and / or inhibit its biological function. Examples of cytokine antagonists or masking moieties include, but are not limited to, polypeptides that interact with cytokines and are derived from the extracellular domain of the natural receptor of the cytokine.

[0063] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease (e.g., to improve, alleviate, reduce and / or delay one or more symptoms). With respect to diseases such as cancer, an effective amount may be an amount sufficient to delay cancer development or progression (e.g., reduce the growth rate of a tumor, and / or delay or prevent tumor angiogenesis, metastasis, or tumor cell infiltration into peripheral organs), reduce epithelial cells, achieve cancer regression (e.g., reduce or eliminate a tumor), and / or prevent or delay the occurrence or recurrence of cancer. An effective dose may be administered once or more than once.

[0064] A "functional analog" refers to a molecule that has the same biological specificity (eg, binds to the same ligand) and / or activity (eg, activates or inhibits a target cell) as a reference molecule.

[0065] The term "fused" or "fusion" with respect to two polypeptide sequences refers to the connection of two polypeptide sequences through a backbone peptide bond. Two polypeptides can be fused directly or through a peptide connector of one or more amino acids in length. Fusion polypeptides can be prepared by recombinant technology from a coding sequence comprising the respective coding sequences of the two fused moieties, with or without the coding sequences of the peptide connector in between. In some embodiments, fusion encompasses chemical conjugation.

[0066] The term "pharmaceutically acceptable excipient" is used to refer to an ingredient in a composition that is suitable for administration to a subject, including a human, without undue adverse side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0067] The term "prodrug" refers to a therapeutic substance that has no activity or low activity in vitro or in the blood circulation, but has enhanced activity in the environment of the disease site.

[0068] The term "subject" refers to a mammal, including but not limited to a human, a pet (eg, a dog, a cat), a farm animal (eg, a cow, a horse), a rodent, or a primate.

[0069] As used herein, "treatment" or "treating" is a method of obtaining a beneficial or expected clinical outcome. Beneficial or expected clinical outcomes include, but are not limited to, one or more of the following: alleviating one or more symptoms of a disease, reducing the extent of a disease, improving the disease state, stabilizing a disease (e.g., preventing or delaying the deterioration or progression of a disease), preventing or delaying the spread of a disease (e.g., metastasis), preventing or delaying the recurrence of a disease, achieving partial or complete remission of a disease, reducing the dose of one or more other drugs required to treat the disease, improving the patient's quality of life, and / or prolonging survival. The disclosed method contemplates one or more aspects of treatment.

[0070] It should be understood that one, some or all of the attributes in the various embodiments described herein may be combined to form other embodiments of the present invention.The section headings used herein are for organizational purposes and should not be construed as limiting the subject matter described in this application.

[0071] I. Cytokine Prodrugs

[0072] The activity of the cytokine prodrug provided by the present invention is enhanced at the lesion site. The prodrug includes a cytokine agonist polypeptide (cytokine part), a carrier part and a masking part. The cytokine part is connected to the carrier part by or without a peptide connector (such as an uncuttable peptide connector) and is masked (combined) by the masking part. The masking part is connected to the cytokine part or the carrier part by or without a peptide connector (such as an uncuttable peptide connector). Or the cytokine part is connected to the masking part by or without a peptide connector (such as an uncuttable peptide connector), and the masking part is connected to the carrier part by or without a peptide connector (such as an uncuttable peptide connector).

[0073] The carrier portion includes an antigen binding domain that can bind to an antigen on a target cell (e.g., a tumor cell or an immune cell). In some embodiments, the carrier includes an antibody, such as Figure 1A In some embodiments, the carrier portion includes a bispecific antibody that can bind to two different antigens, such as Figure 1B and Figure 1C shown.

[0074] For example, the masking moiety may include a cytokine receptor extracellular domain (ECD), wherein the ECD and the cytokine moiety (e.g. Figure 1D and Figure 1E ) and / or connected to the carrier portion via a non-cleavable peptide linker (e.g. Figure 1A -C and Figure 1E). The masking portion binds to the cytokine portion and inhibits its biological function. The prodrug attracts the target cell by binding to the cytokine receptor and antigen expressed on the cell surface in cis, which can increase the activity of the prodrug without cutting off or removing the masking portion. The activity of the cytokine portion of the prodrug is increased at the target site (such as at the tumor site or the surrounding environment), where the antigen targeted to the carrier and the cytokine receptor are expressed on the same cell. Examples of related prodrugs are described in Figure 1A -E, Figure 7 The principle of the cis-binding is explained.

[0075] A. Cytokine portion of the prodrug

[0076] In some embodiments, the cytokine portion includes an IL-2 agonist polypeptide. In some embodiments, the IL-2 agonist polypeptide is an IL-2 mutant with R38S / F42A / Y45A / E62A or F42A / Y45A / L72G mutations (sequence number and SEQ ID NO: 6 consistent). In some embodiments, the IL-2 mutant disclosed in the present invention includes mutations selected from T3, D20, K35, R38, F42, F44, Y45, E62, E68, L72, A73, N88, N90, C125, and Q126 (sequence number and SEQ ID NO: 6 consistent) sites. In certain embodiments, the new IL-2 mutant includes mutations at R38, F42, Y45, and A73 (sequence number and SEQ ID NO: 6 consistent) sites.

[0077] In some embodiments, the cytokine portion of the prodrug comprises an IL-15 agonist polypeptide. In some embodiments, the human IL-15 polypeptide comprises a polypeptide selected from the group consisting of N1A, N1D, N4A, N4D, I6T, S7A, D8A, D8T, D8E, D8N, K10A, K10D, K11A, K11D, E46, V49, L45, S51, L52, D61A, D61N, T62L, T62A, E64A, E64L, E64K, E64Q, N65A, N65L, N65D, L66D, L66E, I 67D,I67E,I68S,I68E,L69S,L69E,N72A,N72D,V63E,V63D,L66E,L66D,I67E,I67 D,Q108E,N112A,N1D / D61N,N1D / E64Q,N4D / D61N,N4D / E64Q,D8N / D61N,D8N / E64Q , D61N / E64Q, E64Q / Q108E, N1D / N4D / D8N, D61N / E64Q / N65D, N1D / D61N / E64Q, N1D / Q108E, N1D / D61N / E64Q / Q108E, N4D / D61N / E64Q / Q108E, and D30N / E64Q / N65D (sequence number and SEQ ID NO: 7 are consistent). In some embodiments, the IL-15 prodrug further includes a Sushi domain. In some embodiments, the Sushi domain includes SEQ ID NO: 8 or an amino acid sequence having at least 95% homology thereto.

[0078] In some embodiments, the cytokine portion includes an IL-21 agonist polypeptide. In some embodiments, the IL-21 agonist polypeptide can be a wild-type IL-21 polypeptide, such as a wild-type human IL-21 (such as SEQ ID NO: 1), or an IL-21 mutant derived from human IL-21, such as selected from any one of SEQ ID NO: 2-5 amino acid sequences. The IL-21 mutant significantly reduces its affinity for IL-2Rα or IL-21RαRγ compared to wild-type IL-21. In some embodiments, the affinity of the IL-21 mutant for high-affinity IL-2Rα compared to wild-type IL-21 is reduced to 1 / 5, 1 / 10, 1 / 20, 1 / 50, 1 / 100, 1 / 300, 1 / 500, 1 / 1000 or 1 / 10000. Unless otherwise specified, the residue numbers in the IL-21 and IL-21 mutants involved in the present invention are consistent with SEQ ID NO: 1.

[0079] B. Masking Moieties of Prodrugs

[0080] The masking part in the present invention includes a peptide, an antibody or an antibody fragment that binds to the cytokine part of the prodrug, and inhibits its biological function by masking the cytokine. In some embodiments, the masking part is operably connected to the other part of the prodrug through a non-cleavable peptide connector.

[0081] For example, the prodrug includes an IL-2 agonist polypeptide, and the masking portion includes a peptide, an antibody, or an antigen-binding fragment thereof that binds to IL-2 to affect the binding of IL-2 to its cognate receptor. In some embodiments, the masking portion reduces the biological activity of the masked IL-2 portion. In some embodiments, the IL-2 antagonist includes an IL-2Rβ or IL-2Rγ extracellular domain or a functional analog thereof, for example, it can be from human IL-2Rβ or human IL-2Rγ (such as SEQ ID NO: 11 or 13). In some embodiments, the IL-2 masking portion includes a peptide screened from a peptide library. In some embodiments, the IL-2 masking portion includes an antibody or fragment thereof that inhibits the binding of IL-2 or IL-2 mutants to IL-2 receptors. In some embodiments, the masking portion includes an scFv of an anti-IL-2 antibody. In some embodiments, the masking portion includes SEQ ID NO: 22 or 23, or an amino acid sequence having at least 95% homology thereto.

[0082] For example, the prodrug includes an IL-21 agonist polypeptide, and the masking portion includes a peptide, an antibody, or an antigen-binding fragment thereof that binds to IL-21 to affect the binding of IL-21 to its cognate receptor. In some embodiments, the masking portion reduces the biological activity of the masked IL-21 portion. In some embodiments, the IL-21 antagonist includes an IL-21Rα or IL-21Rγ extracellular domain or a functional analog thereof, for example, it can be derived from human IL-21Rα or human IL-21Rγ (such as SEQ ID NO: 12 or 13). In some embodiments, the IL-21 masking portion includes a peptide screened from a peptide library. In some embodiments, the IL-21 masking portion includes an antibody or fragment thereof that inhibits the binding of IL-21 or IL-21 mutants to IL-21 receptors. In some embodiments, the masking portion includes an scFv of an anti-IL-21 antibody. In some embodiments, the masking portion includes SEQ ID NO: 20 or 21, or an amino acid sequence having at least 95% homology thereto.

[0083] For another example, the prodrug includes a masking portion of any cytokine disclosed in the present invention, wherein the cytokine includes but is not limited to an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, an IL-1α agonist polypeptide, an IL-1β agonist polypeptide, an IL-4 agonist polypeptide, an IL-5 agonist polypeptide, an IL-6 agonist polypeptide, an IL-8 agonist polypeptide, an IL-10 agonist polypeptide, an IL-12 agonist polypeptide, an IL-17 agonist polypeptide, an IL-18 agonist polypeptide, an IL-22 agonist polypeptide, an IL-23 agonist polypeptide, an IL-31 agonist polypeptide, an IL-33 agonist polypeptide, an IL-36 agonist polypeptide, an interferon α agonist polypeptide, an interferon γ agonist polypeptide, a 4-1BB ligand, an OX-40 ligand or a CD-40 ligand.

[0084] C. Carrier Moiety of Prodrug

[0085] The carrier portion of the prodrug includes an antigen binding domain and optional other domains. The carrier portion can improve PK, such as the half-life of the cytokine agonist polypeptide in serum, and target the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

[0086] 1. Antigen binding domain of the carrier part

[0087] The carrier portion includes an antigen binding domain, which can be an antibody or an antigen binding fragment thereof, or an immunoadhesin. In some embodiments, the antigen binding carrier portion is a full-length antibody having two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a disulfide-linked Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv). In some embodiments, the antigen binding portion is a bispecific antigen binding portion that can bind to two different antigens or two different epitopes on the same antigen. The antigen binding portion may provide additional and potential synergistic therapeutic effects for the cytokine agonist polypeptide.

[0088] The cytokine agonist polypeptide and its masking part can be fused to the N-terminus or C-terminus of the light chain and / or heavy chain of the antigen binding portion. For example, the IL-21 agonist polypeptide and its masking part can be fused to the antibody heavy chain or its antigen binding fragment, or it can be fused to the antibody light chain or its antigen binding fragment. For example, the IL-21 agonist polypeptide and its masking part can be fused to the antibody heavy chain or its antigen binding fragment, or it can be fused to the antibody light chain or its antigen binding fragment. In some embodiments, one end of the IL-21 agonist polypeptide is fused to the C-terminus of one or two heavy chains of the antibody, and the IL-21 masking is fused to the other end of the IL-21 agonist polypeptide through a non-cut peptide connector. In some embodiments, the IL-21 receptor agonist polypeptide is fused to the C-terminus of one heavy chain of the antibody, and the IL-21 protection unit is fused to the C-terminus of another heavy chain of the antibody through a non-cut peptide connector, wherein the two heavy chains contain mutations, allowing special pairing of two different heavy chains.

[0089] Strategies for forming heterodimers are well known (see, e.g., Spies et al., Mol Imm. 67(2)(A):95-106(2015)). For example, two heavy chain polypeptides in a prodrug can form a stable heterodimer by a "knobs-into-holes" mutation. The "knobs-into-holes" mutation is performed to promote the formation of heterodimers of antibody heavy chains and is commonly used to make bispecific antibodies (see, e.g., U.S. Patent No. 8,642,745). For example, the Fc domain of an antibody may include a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A and / or Y407V mutation in the CH3 domain of the "hole chain". Another interchain disulfide bridge between the CH3 domains can also be used, for example, by introducing a Y349C mutation into the CH3 domain of the "knobs chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain" (see, e.g., Merchant et al., Nature Biotech 16: 677-81 (1998)). In other embodiments, the antibody portion can comprise a Y349C and / or T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A and / or Y407V mutation in the other CH3 domain. In certain specific embodiments, the antibody portion may comprise Y349C and / or T366W mutations in one of the two CH3 domains, and S354C (or E356C), T366S, L368A and / or Y407V mutations in the other CH3 domain; when an additional Y349C mutation on one CH3 domain, and an additional E356C or S354C mutation on the other CH3 domain, an interchain disulfide bond is formed (numbering is always according to the EU index numbering of Kabat; Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Other knobs-in-holes technologies, such as those described in EP1870459A1, may be used alternatively or additionally. Thus, another example of knobs-in-holes mutations in the antibody portion is with R409D / K370E mutations in the CH3 domain of the "knob chain" and D399K / E357K mutations in the CH3 domain of the "hole chain" (EU numbering).

[0090] In some embodiments, the antigen binding portion of the prodrug comprises an antibody having an Fc domain with L234A and L235A ("LALA") mutations. The LALA mutation can abolish complement binding and fixation and Fcγ-dependent ADCC (see Hezareh et al. J. Virol. (2001) 75(24): 12161-8). In further embodiments, the antibody portion has a LALA mutation in addition to the knobs-into-holes mutation.

[0091] In some embodiments, the antigen binding portion includes M252Y / S254T / T256E ("YTE") mutations in the Fc domain. The YTE mutation allows for simultaneous modulation of serum half-life, tissue distribution, and IgG1 activity (see Dall'Acqua et al., J Biol Chem. (2006) 281: 23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57 (12): 6147-53). In further embodiments, in addition to the knobs-into-holes mutation, a YTE mutation is present in the antibody portion. In specific embodiments, the antibody portion has YTE, LALA, and knobs-into-holes mutations or any combination thereof.

[0092] In certain embodiments, the antigen binding portion is an antibody or antigen binding fragment thereof that binds to an antigen on the surface of a target cell (such as an immune cell), wherein immune cells are common knowledge. In non-limiting embodiments, immune cells include T cells, NK cells, and macrophages. The antigen binding portion can activate immune cells and enhance their anti-tumor activity. The antibody may or may not have ADCC activity. The antigen binding portion can further bind to a cytotoxic drug. In some embodiments, the antigen binding portion can bind to PD-1, LAG-3, TIM-3, TIGIT, CTLA-4, or TGF-beta. In other embodiments, the antigen binding portion can bind to an antigen on the surface of a target cell (such as a tumor cell). For example, the antigen binding portion can bind to FAPα, 5T4, Trop-2, PD-L1, HER-2, EGFR, Claudin18.2, DLL-3, GCP3, or carcinoembryonic antigen (CEA). -

[0093] In some embodiments, the antigen binding portion can bind to amidinocyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), proteoglycan-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), nectin-4, Liv-1, glycoprotein N MB (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), 6 transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor alpha (FR-α), SLIT and NTRK-like protein 6 (SLITRK6) carbonic anhydrase VI (CA6), phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, CD47, signal regulatory protein (SIRPα), Claudin 18.2, Claudin 6, BCMA or EPCAM. In some embodiments, the antigen binding portion binds to the epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen binding portion binds to the Delta / Serrate / Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen binding portion binds to an epitope located after the 374th amino acid of GPC3. In some embodiments, the antigen binding portion binds to heparin sulfate glycans of GPC3. In some embodiments, the antigen binding portion binds to Claudin 18.2 but not to Claudin 18.1. In some embodiments, the antigen binding portion has a binding affinity to Claudin 18.1 that is at least 10 times weaker than the binding affinity to Claudin 18.2.

[0094] Exemplary antigen binding portions include trastuzumab, rituximab, bertuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, and antigen binding fragments thereof. In some embodiments, the antigen binding portion is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to trastuzumab, rituximab, bertuximab, cetuximab or panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen binding portion has an antibody heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the antibody heavy chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598 or a fragment thereof. In some embodiments, the antigen binding portion has an antibody light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the antibody light chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598 or a fragment thereof. The antigen binding portion is fused to the IL-2 agonist polypeptide. In some embodiments, the antigen binding portion includes 6 complementary determining regions (CDRs) of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33, anti-EGFR antibody mAb806 or anti-dPNAG antibody F598.

[0095] Many CDR descriptions are known in the art and are covered herein. One skilled in the art can easily determine the CDRs of a given description based on the sequence of the heavy or light chain variable region. "Kabat" CDRs are based on sequence variability and are most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). "Chothia" CDRs refer to the positions of structural loops (Chothia & Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol., vol. 196, pp. 901-917 (1987)). "AbM" CDRs represent a compromise between Kabat CDRs and Chothia structural loops, and are also used by AbM antibody modeling software from Oxford Molecular. "Contact" CDRs are based on analysis of available complex crystal structures. The individual residues of these CDRs are listed in Table 1 below with reference to common antibody numbering schemes. Unless otherwise specified herein, the amino acid numbering in antibodies refers to the Kabat numbering scheme as described by Kabat et al. above, including when CDR descriptions are performed with reference to Kabat, Chothia, AbM or Contact schemes. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to shortening or insertion of the framework region (FR) or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat), and also includes an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of its antibody residues can be determined by comparing the homologous region of the antibody sequence with the "standard" Kabat numbering sequence.

[0096] Table 1: CDR descriptions for different numbering schemes

[0097] CDR Kabat AbM Chothia Contact VL-CDR1 L24 - L34 L24 - L34 L26 - L32 L30 - L36 VL-CDR2 L50—L56 L50—L56 L50—L52 L46—L55 VL-CDR3 L89—L97 L89—L97 L91—L96 L89—L96 VH-CDR1 (Kabat nos.) H31—H35B H26—H35B H26—H32 H30—H35B VH-CDR1 (Chothia nos.) H31—H35 H26 - H35 H26 - H32 H30 - H35 VH-CDR2 H50—H65 H50—H58 H53—H55 H47—H58 VH-CDR3 H95—H102 H95—H102 H95 - H101 H93 - H101

[0098] In some embodiments, the CDRs are "extended CDRs" that include regions that start or end according to different schemes. For example, the extended CDRs may be as follows: L24-L36, L26-L34, or L26-L36 (VL-CDR1); L46-L52, L46-L56, or L50-L55 (VL-CDR2); L91-L97 (VL-CDR3); H47-H55, H47-H65, H50-H55, H53-H58, or H53-H65 (VH-CDR2; and / or H93-H102 (VH-CDR3).

[0099] In some embodiments, the antigen binding portion that binds to PDL1 comprises a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 45 or a fragment thereof, and a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 46 or a fragment thereof. In some embodiments, the antigen binding domain comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 45, and CDR1, CDR2, and CDR3 of SEQ ID NO: 46.

[0100] In some embodiments, the antigen binding portion that binds to PD-1 comprises a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 44 or a fragment thereof, and a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 47 or a fragment thereof. In some embodiments, the antigen binding domain comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 44, and CDR1, CDR2, and CDR3 of SEQ ID NO: 47.

[0101] In some embodiments, the antigen binding portion that binds to PD-1 comprises a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 48 or a fragment thereof, and a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49 or a fragment thereof. In some embodiments, the antigen binding domain comprises CDR1, CDR2, and CDR3 of SEQ ID NO: 48, and CDR1, CDR2, and CDR3 of SEQ ID NO: 49.

[0102] In some embodiments, the antigen binding portion includes one, two or three antigen binding domains. For example, the antigen binding portion is bispecific and can bind to two different antigens, which are selected from CD3, HER2, HER3, EGFR, 5T4, FAP alpha, Trop-2, GPC3, VEGFR2, Claudin 18.2 and PD-L1. In some embodiments, the bispecific antigen binding portion binds to two different epitopes of HER2. In other embodiments, the antigen binding portion is bispecific and can bind to two different antigens, which are selected from PD-1, PD-L1, CTLA-4, CD47, CD3, TIM-3, LAG-3 and TIGIT.

[0103] 2. Other carrier parts

[0104] The carrier moiety may also include other non-antigen binding domains. For example, an antibody Fc domain (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc), a polymer (e.g., PEG), an albumin (e.g., human albumin), or a fragment thereof, or a nanoparticle may be used.

[0105] For example, a cytokine agonist polypeptide (such as IL-2, IL-21, IL-5, or any other cytokine disclosed herein) and its antagonist can be fused to an antibody Fc domain to form an Fc fusion protein. In some embodiments, the cytokine agonist polypeptide is fused to the C-terminus or N-terminus of one of the polypeptide chains of the Fc domain (directly or through a peptide connector), and the cytokine masking portion is fused to the corresponding C-terminus or N-terminus of the other Fc domain polypeptide chain through a cleavable peptide connector, wherein the two Fc domain polypeptide chains contain mutations that allow specific pairing of two different Fc chains. In some embodiments, the Fc domain contains the above-mentioned holes-into-holes mutation. In some further embodiments, the Fc domain may also contain the above-mentioned YTE and / or LALA mutations.

[0106] The carrier portion of the prodrug may comprise albumin (e.g., human serum albumin) or a fragment thereof. In some embodiments, the carrier portion comprises an albumin fragment (e.g., a human serum albumin fragment) having about 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more amino acids in length. In some embodiments, the length of the albumin fragment is between about 10 amino acids and about 584 amino acids (e.g., between about 10 and about 20, about 20 and about 40, about 40 and about 80, about 80 and about 160, about 160 and about 250, about 250 and about 350, about 350 and about 450, or about 450 and about 550 amino acids). In some embodiments, the albumin fragment comprises a Sudlow I domain or a fragment thereof, or a Sudlow II domain or a fragment thereof.

[0107] D. Linking moiety of prodrug

[0108] The cytokine agonist polypeptide can be fused to the carrier portion with or without a peptide linker. The peptide linker can be non-cleavable. In some embodiments, the peptide linker is selected from SEQ ID NOs: 27-34. In some specific embodiments, the peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 30)

[0109] The masking moiety can be fused to the cytokine moiety or the carrier moiety via a non-cleavable peptide linker or without a peptide linker.

[0110] II. Pharmaceutical Compositions

[0111] Pharmaceutical compositions including the prodrugs and mutant proteins described herein (e.g., active pharmaceutical ingredients or APIs) can be prepared by mixing the API of the desired purity with one or more optional pharmaceutically acceptable excipients (e.g., see Remington's Pharmaceutical Sciences, 16th Edition., Osol, A. Ed. (1980)) into lyophilized formulations or aqueous solutions. Pharmaceutically acceptable excipients (or carriers) are generally non-toxic to the administering agent at the dosages and concentrations used, and include, but are not limited to: buffers containing, for example, phosphate, citrate, succinate, histidine, acetate or another inorganic or organic acid or salt thereof; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; resorcinol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including sucrose, glucose, mannose or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0112] Buffer is used to control pH within the optimal therapeutic effect range, especially when stability depends on pH. Buffer concentration is preferably between 50mM and 250mM. Buffers suitable for use with the present invention include organic and inorganic acids and salts thereof, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate and acetate. In addition, the buffer may include histidine and trimethylamine salts, such as tris(hydroxymethyl)aminomethane.

[0113] Preservatives are added to slow the growth of microorganisms and are generally present in the range of 0.2% to 1.0% (w / v). Suitable preservatives for use in the present invention include octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chlorine, bromine, iodine), benzalkonium chloride; thimerosal, phenol, butyl or benzyl alcohol; methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0114] Tonicity agents, sometimes called "stabilizers," are used to adjust or maintain the tonicity of the liquid in the composition. When used with charged biomacromolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions. When considering the relative amounts of the other ingredients, the tonicity agent can be used in any amount between 0.1% and 25% by weight, or more preferably between 1% and 5% by weight. Preferred tonicity agents include polyols, more preferably trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0115] Nonionic surfactants or detergents (also called "wetting agents") are used to help dissolve the therapeutic agent and protect the therapeutic protein from aggregation caused by agitation, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is used in the range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0116] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamer (184, 188, etc.), pluronic polyols, triaton, polyoxyethylene sorbitol monoethers (Tween-20, Tween-80, etc.), lauromacrogol 400, polyoxyalkylene stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, monostearate, sucrose fatty acid esters, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0117] The choice of pharmaceutical carrier, excipient or diluent can be based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may also include a suitable binder, lubricant, suspending agent, coating agent or solubilizer.

[0118] Depending on the drug delivery system, different composition / formulation requirements may be required. For example, the pharmaceutical ingredients useful in the present invention can be formulated to be suitable for micropumps or delivered via mucosal routes, for example, as inhalers or absorbable solutions for nasal sprays or aerosols, or as injectable forms of ingredients for parenteral administration, which are delivered by, for example, intravenous, intramuscular or subcutaneous routes.

[0119] In some embodiments, the pharmaceutical composition of the present invention is a lyophilized protein preparation. In other embodiments, the pharmaceutical composition can be an aqueous liquid preparation.

[0120] III. Treatment Methods

[0121] Cytokine prodrugs and fusion molecules can be used to treat diseases, depending on the antigen to which the antigen binding domain binds. In some embodiments, cytokine prodrugs or fusion molecules are used to treat cancer. In some embodiments, cytokine prodrugs or fusion molecules are used to treat infection.

[0122] In some embodiments, a method of treating a disease (eg, cancer, parasitic infection, viral infection, or bacterial infection) comprises administering to a subject an effective amount of a cytokine prodrug or fusion molecule.

[0123] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer or a solid tumor. Exemplary cancers that can be treated include, but are not limited to, leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer (e.g., non-small cell lung cancer), bile duct cancer, breast cancer, and ovarian cancer.

[0124] In some embodiments, the cytokine prodrug or fusion molecule is used to treat viral infection. In some embodiments, the virus causing the viral infection is hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV) or human papillomavirus (HPV). In some embodiments, the antigen binding portion binds to a viral antigen.

[0125] In some embodiments, the cytokine prodrug or fusion molecule is used to treat bacterial infection, such as sepsis. In some embodiments, the bacteria causing the bacterial infection are drug-resistant bacteria. In some embodiments, the antigen binding portion binds to a bacterial antigen.

[0126] In general, the dosage and route of administration of existing drugs are determined according to the conditions and conditions of the subject in accordance with standard pharmaceutical practice. In some embodiments, the drug composition is administered by a route of administration, including oral, subcutaneous, inhaled, intravenous, intraarterial, intramuscular, directly applied to the wound, applied to the surgical site, intraperitoneal, suppository, subcutaneous, intradermal injection, transdermal, aerosol, intrapleural, intraventricular, intraarticular, intraocular, intracranial or intraspinal. In some embodiments, the drug is intravenously administered to the subject.

[0127] In certain embodiments, the dosage of the pharmaceutical composition is a single dose or multiple doses. In certain embodiments, the dosage is once a day, twice a day, three times a day, or four times a day or more. In some embodiments, about 1 or more (e.g., about 2, 3, 4, 5, 6 or 7 or more) doses are administered in a week. In certain embodiments, the pharmaceutical composition is administered once a week, once every two weeks, once every three weeks, once every four weeks, once a week (weekly in two weeks of three weeks), or once a week (weekly in three weeks of four weeks). In certain embodiments, multiple doses are administered in a few days, weeks, months or years. In certain embodiments, the course of treatment is about 1 or more doses (e.g., about 2, 3, 4, 5, 7, 10, 15 or 20 or more doses).

[0128] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. In the event of a conflict, the specification description, including definitions, shall prevail. Generally, the terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid chemistry, as well as hybridization and mixing described herein are well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as generally achieved in the art or as described herein. In addition, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout the specification description and embodiments, the words "having" and "comprising", or their synonyms, alternatives and variants, will be understood to imply that the integer or integer group is included, but any other integer or integer group is not excluded. It will be appreciated that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0129] IV. Exemplary Embodiments

[0130] Other specific embodiments of the present invention are described below. These embodiments are intended to illustrate the components and methods described in the present invention, but are not intended to limit the scope of the present invention.

[0131] 1. A prodrug comprising a cytokine portion, a masking portion and a carrier portion, wherein:

[0132] a) the masking moiety binds to the cytokine moiety and inhibits the biological activity of the cytokine;

[0133] b) the carrier portion includes an antigen binding portion that can bind to an antigen expressed on the cell surface, and

[0134] c) the masking moiety is directly or indirectly linked to the carrier moiety via a non-cleavable peptide linker or without a peptide linker;

[0135] Among them, the prodrug has a higher activity on the activation of cells that express both the antigen targeting the carrier portion and the cytokine receptor than on the activation of cells that only express the cytokine receptor but not the antigen targeting the carrier portion.

[0136] 2. A prodrug comprising a cytokine portion, a masking portion and a carrier portion, wherein:

[0137] a) the masking portion binds to the cytokine portion and inhibits the biological activity of the cytokine;

[0138] b) the carrier portion includes an antigen binding portion that can bind to an antigen expressed on the cell surface; and,

[0139] c) the masking moiety is directly or indirectly linked to the carrier moiety via a non-cleavable peptide linker or without a peptide linker;

[0140] Wherein, the prodrug is activated at the lesion site, or the biological function of the prodrug at the lesion site is increased by at least 2 times, at least 5 times or at least 10 times, and the cells at the lesion site simultaneously express antigens and cytokine receptors that are targeted to the vector portion.

[0141] 3. The prodrug of embodiment 1 or 2, wherein the receptor of the cytokine portion comprises two or more subunits.

[0142] 4. The prodrug of embodiment 1 or 2, wherein the carrier portion comprises an antigen binding portion of an immune cell expressing an antigen,

[0143] 5. The prodrug described in Example 1 or 2, wherein the carrier portion includes an antigen binding portion, and the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD38 and BCMA.

[0144] 6. The prodrug described in any one of Examples 1-5, wherein the cytokine portion comprises a cytokine selected from an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide and an IL-21 agonist polypeptide.

[0145] 7. The prodrug of any one of Examples 1-5, wherein the cytokine is selected from IL-1α agonist polypeptide, IL-1β agonist polypeptide, IL-4 agonist polypeptide, IL-5 agonist polypeptide, IL-6 agonist polypeptide, IL-8 agonist polypeptide, IL-10 agonist polypeptide, IL-12 agonist polypeptide, IL-17 agonist polypeptide, IL-18 agonist polypeptide, IL-22 agonist polypeptide, IL-23 agonist polypeptide, IL-31 agonist polypeptide, IL-33 agonist polypeptide, IL-36 agonist polypeptide, interferon-α agonist polypeptide, interferon-γ agonist polypeptide, 4-1BB ligand, OX-40 ligand and CD-40 ligand.

[0146] 8. The prodrug of any one of Examples 1-7, wherein the masking moiety is the extracellular domain of a cytokine receptor.

[0147] 9. The prodrug of any one of Examples 1-6, wherein the cytokine is an IL-7 agonist polypeptide, and the masking portion is an IL-7 receptor α extracellular domain (IL-7RαECD) or a functional analog thereof.

[0148] 10. The prodrug of any one of Examples 1-6, wherein the cytokine is an IL-21 agonist polypeptide, and the masking portion is an IL-21 receptor α extracellular domain (IL-21RαECD) or a functional analog thereof.

[0149] 11. The prodrug of any one of Examples 1-6, wherein the cytokine is an IL-2 agonist polypeptide or an IL-15 agonist polypeptide, and the masking portion is an IL-2 receptor β extracellular domain (IL-2RβECD).

[0150] 12. The prodrug of any one of Examples 1-6, wherein the cytokine is an IL-21 agonist polypeptide, and the masking portion is an anti-IL-21 Fab, a single-chain Fv (scFv), or a single-domain antibody.

[0151] 13. The prodrug of any one of Examples 1-6, wherein the cytokine is an IL-2 agonist polypeptide, and the masking portion is an anti-IL-2 Fab, a single-chain Fv (scFv), or a single-domain antibody.

[0152] 14. The prodrug of any one of Examples 1-6, wherein the cytokine is an IL-15 agonist polypeptide, and the masking portion is an anti-IL-15 Fab, a single-chain Fv (scFv), or a single-domain antibody.

[0153] 15. The prodrug of any one of Examples 1-6, 11 or 14, wherein the cytokine comprises an IL-15 agonist polypeptide, and the fusion molecule further comprises the sushi domain of the IL-15 receptor α (IL-15Rαsushi domain).

[0154] 16. The prodrug of any one of embodiments 10 or 12, wherein the IL-21 agonist polypeptide comprises SEQ ID NO: 1 or an amino acid sequence having at least 90% homology thereto.

[0155] 17. The prodrug of embodiment 10 or 16, wherein the masking moiety is IL-21RαECD or a functional analog thereof, which includes SEQ ID NO: 12, 63-72 and 73, or an amino acid sequence having at least 90% homology with SEQ ID NO: 12.

[0156] 18. The prodrug of embodiment 12 or 16, wherein the masking moiety is a scFv that binds to human IL-21, and the scFv comprises the amino acid sequence of SEQ ID NO: 20 or 21.

[0157] 19. The prodrug of embodiment 11 or 13, wherein the IL-2 agonist polypeptide comprises SEQ ID NO: 6 or 61, or an amino acid sequence having at least 95% homology thereto.

[0158] 20. The prodrug of embodiment 11 or 14, wherein the IL-15 agonist polypeptide comprises SEQ ID NO: 7 or an amino acid sequence having at least 95% homology thereto.

[0159] 21. The prodrug of embodiment 11, 19 or 20, wherein the masking moiety comprises IL-2Rβ-ECD or a functional analog thereof, and IL-2Rβ-ECD comprises SEQ ID NO: 11 or an amino acid sequence having at least 95% homology thereto.

[0160] 22. The prodrug of any one of Examples 6, 13 or 19, wherein the masking portion is a scFv that binds to IL-2, and the scFv inhibits or interferes with the interaction between IL-2 and IL-2Rγ, the interaction between IL-2 and IL-2Rβ, and / or the interaction between IL-2 and IL-2Rγ.

[0161] 23. The prodrug of any one of Examples 6, 14, 15 or 20, wherein the masking portion is a scFv that binds to IL-15, and the scFv inhibits or interferes with the interaction between IL-15 and IL-2Rβ, and / or IL-2 and IL-2Rγ.

[0162] 24. The prodrug of any one of Examples 6, 12 or 16, wherein the masking portion is a scFv that binds to IL-21, and the scFv inhibits or interferes with the interaction between IL-21 and IL-21Rα, and / or IL-21 and IL-2Rγ.

[0163] 25. The prodrug of Example 6, wherein the masking portion is a scFv that binds to a cytokine selected from IL-2, IL-7, IL-9, IL-15, or IL-21, and the scFv inhibits or interferes with the interaction between the cytokine and IL-2Rγ, and the half-life of the prodrug in non-human primates or humans is increased by at least 20 times, at least 50 times, at least 100 times, at least 150 times, or at least 200 times compared to the wild-type cytokine.

[0164] 26. A prodrug comprising a carrier portion, a cytokine portion, and a masking portion, wherein:

[0165] a) cytokine is selected from IL-2 agonist polypeptide, IL-7 agonist polypeptide, IL-9 agonist polypeptide, IL-15 agonist polypeptide, IL-21 agonist polypeptide;

[0166] b) the masking portion is a scFv that binds to the cytokine portion;

[0167] c) the scFv inhibits or interferes with the interaction between cytokines and IL-2Rγ; and

[0168] d) the half-life of the prodrug in non-human primates or humans is increased by at least 20 times, at least 50 times, at least 100 times, at least 150 times, or at least 200 times compared to the wild-type cytokine.

[0169] 27. The prodrug of any one of embodiments 22-25, wherein the scFv is K D 1-10nM and cytokine binding.

[0170] 28. The prodrug of any one of embodiments 22-25, wherein the scFv is K D It is 10-100nM and binds to cytokines.

[0171] 29. The prodrug of any one of embodiments 22-25, wherein the scFv is K D It is 100-1000nM and binds to cytokines.

[0172] 30. The prodrug of embodiment 13 or 19, wherein the masking moiety is a scFv that binds IL-2, wherein the scFv comprises the same heavy and light chain CDRs as hybridoma 4E12B2D10.

[0173] 31. The prodrug of embodiment 13 or 19, wherein the masking moiety is an scFv that binds to IL-2, and the scFv comprises SEQ ID NO: 22 or 23, or an amino acid sequence having at least 95% homology thereto.

[0174] 32. The prodrug of embodiment 14 or 20, wherein the masking portion is a scFv that binds IL-15, and the scFv includes the same heavy chain and light chain CDRs as the IL-15 antibody 146B7, 146H5, or 404E4.

[0175] 33. The prodrug of embodiment 14 or 20, wherein the masking moiety is a scFv that binds IL-15, and the scFv comprises the amino acid sequence of SEQ ID NO: 18 or 19.

[0176] 34. The prodrug of any one of embodiments 1-32, wherein the prodrug does not contain a cleavable peptide linker.

[0177] 35. The prodrug of Example 1, wherein the amino acid sequence of the two light chains included in the prodrug is SEQ ID NO: 44, and the amino acid sequences of the heavy chain polypeptide chains are:

[0178] SEQ ID NO:33 and 36;

[0179] SEQ ID NO:37 and 36;

[0180] SEQ ID NO:37 and 38;

[0181] SEQ ID NO:39 and 41; or

[0182] SEQ ID NO:42 and 43.

[0183] 36. The prodrug of Example 1, wherein the amino acid sequence of the two light chains included in the prodrug is SEQ ID NO: 50, and the amino acid sequences of the heavy chain polypeptide chains are:

[0184] SEQ ID NO: 51 and 54;

[0185] SEQ ID NO: 51 and 55;

[0186] SEQ ID NO: 51 and 56;

[0187] SEQ ID NO: 52 and 54;

[0188] SEQ ID NO: 53 and 58;

[0189] SEQ ID NO: 53 and 59; or

[0190] SEQ ID NO:52 and 57.

[0191] 37. A pharmaceutical composition comprising the prodrug described in any one of Examples 1-36 and a pharmaceutically acceptable excipient.

[0192] 38. A polynucleotide encoding the prodrug described in any one of Examples 1-36.

[0193] 39. An expression vector comprising the polynucleotide described in Example 38.

[0194] 40. A host cell comprising the expression vector described in Example 39.

[0195] 41. A method for preparing the prodrug of any one of Examples 1-36, comprising:

[0196] Culturing the host cell of Example 46 under conditions that allow expression of the prodrug, wherein the host cell is a mammalian cell, and

[0197] and isolating the prodrug.

[0198] 42. A method for treating cancer, autoimmune disease or infectious disease or stimulating the immune system in a patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition described in Example 37.

[0199] 43. Use of the prodrug described in any one of Examples 1-36 in treating cancer, autoimmune diseases, infectious diseases or stimulating the immune system in patients in need.

[0200] 44. Use of the prodrug described in any one of Examples 1-36 in the preparation of a drug for treating cancer, autoimmune diseases, infectious diseases or stimulating the immune system for a patient in need thereof.

[0201] 45. The method described in Example 41, the use of the prodrug in claim 42 or the use of the fusion molecule in claim 43, or the use of the prodrug in claim 44, wherein the patient suffers from an infectious disease such as HIV, HBV, HCV or HPV; an autoimmune system disease selected from lupus erythematosus, type I diabetes, psoriasis, dermatomyositis, GvHD, rheumatoid disease; or a cancer selected from breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer and gastric cancer.

[0202] 46. ​​A prodrug comprising a carrier portion, a cytokine portion and a masking portion, wherein:

[0203] a) the cytokine portion is selected from an IL-2 agonist polypeptide, an IL-7 agonist polypeptide, an IL-9 agonist polypeptide, an IL-15 agonist polypeptide, an IL-21 agonist polypeptide;

[0204] b) the masking portion is a scFv that binds to a cytokine;

[0205] c) the scFv inhibits or interferes with the interaction between cytokines and IL-2Rγ, and

[0206] d) the area under the curve (AUC) of the prodrug in non-human primates or humans is increased by at least 20 times, at least 50 times, at least 100 times, at least 150 times or at least 200 times compared to the wild-type cytokine.

[0207] In order that the present invention can be better understood, the following examples are described herein. These examples are for illustrative purposes only and should not be interpreted in any way as limiting the scope of the present invention. Example

[0208] The materials and methods used in Examples 1 and 2 are as follows:

[0209] SEC-HPLC analysis

[0210] SEC-HPLC analysis was performed on an Agilent 1100 series HPLC system equipped with a TSKgel G3000SWXL column (7.8 mmID x 30 cm, particle size 5 μm) from Tosoh Bioscience. A maximum of 100 μl was loaded, and the mobile phase of the column was 200 mM K3PO4, 250 mM KCl, pH 6.5 buffer. The flow rate was set at 0.5 ml / min. The column temperature was room temperature.

[0211] Proteolytic treatment

[0212] Proteases, human MMP2, human MMP9, mouse MMP2 and mouse MMP9 were purchased from R&D systems. The steps of protease digestion were as follows: 10 μg-50 μg of prodrug protein and 1 μg of human MMP2, human MMP9, mouse MMP2 or mouse MMP9 were added to HBS buffer (20 mM HEPES, 150 mM NaCl2, pH 7.4) containing 2 mM CaCl2 and 10 μM ZnCl2, and incubated at 37 degrees for 12 hours.

[0213] Cell viability assay

[0214] Prodrugs and control samples before and after protease digestion were tested using a cell viability assay. Briefly, NK92 cells were grown in PRMI-1640 medium supplemented with L-glutamine, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, and 55 μM mercaptoethanol. NK92 cells were non-adherent and maintained at 1 x 10 in medium containing 100 ng / ml IL-2. 5 -1x 10 6 cells / ml. In general, cells were split twice a week. For bioassays, it is best to use cells no less than 48 hours after passaging. NK92 cells were cultured at 5×10 4The samples were serially diluted and cultured for 2 days in the presence of a fixed amount of IL-2 to determine the functional activity of IL-21. The supernatant was then tested for interferon-γ by ELISA.

[0215] Mino IL-21 activity assay

[0216] Mino cell viability assay was performed as follows:

[0217] a) Add the sample to be tested, serially diluted in 50 μL of assay medium (RPMI 1640, 10% fetal bovine serum, NEAA, sodium pyruvate, 55 μM b-mercaptoethanol) to a 96-well plate;

[0218] b) Add 50 μL of assay medium containing 20,000 Mino cells to each well;

[0219] c) culturing for 3 to 5 days;

[0220] d) Add 100 μL of Cell Titer Glo (Promega) to each well. Cell Titer Glo provides a measure of cell viability by quantitatively evaluating ATP.

[0221] e) Measuring fluorescence.

[0222] Analysis of Ki67 activity in PBMC primary cells

[0223] Human PBMCs were activated with anti-CD3 antibody (OKT3) at a concentration of 100 ng / mL for 2 days, washed 3 times, and then placed in a 37°C, 5% CO2 incubator with regular cell culture medium for 3 days. The activated / rested PBMCs were seeded into 96-well plates at a density of 200K cells / well, and the above PBMCs were treated with different concentrations of the test samples. After five days of culture, they were stained and analyzed by flow cytometry for PD-1. + CD4 + Cells and PD-1 - CD4 + Ki67 in cells (all mAbs used for cell staining were purchased from BD).

[0224] Example 1: Construction of anti-PD-1 antibody-IL-21 prodrug fusion molecule

[0225] An IL-21 prodrug based on an anti-PD-1 antibody was constructed using two identical light chains (amino acid sequence as shown in SEQ ID NO: 44), a first heavy chain polypeptide chain (amino acid sequence as shown in SEQ ID NO: 42) and a second heavy chain polypeptide chain (amino acid sequence as shown in SEQ ID NO: 76). The molecule was transiently expressed and purified (lot # PW04-38).

[0226] The second anti-PD-1 antibody-based IL-21 prodrug was masked with a scFv moiety, which was expressed and purified (Lot#PW05-68).

[0227] In addition, the control was an anti-PD-1 antibody-IL-21 fusion molecule without the masking moiety, which was expressed and purified (Lot#PW05-67).

[0228] Furthermore, the second control was an anti-PD-1 antibody-IL-21 mutant (R9ER76A) fusion molecule without the masking moiety, which was expressed and purified (Lot#PW09-02).

[0229] Example 2. Biological activity of anti-PD-1 antibody-IL-21 prodrug fusion molecules

[0230] The binding of the prodrug molecules and various control molecules to Mino cells was analyzed by FACS detection. Figure 2 The results shown show that both the PD-1 antibody and the Fc-IL-21 fusion molecule can bind to Mino cells, indicating that Mino cells can express PD-1 and IL-21 receptors. The results show that the Fc-IL-21 prodrug molecule does not bind to the cells, indicating that the IL-21 cytokine is masked by the corresponding masking part. However, the PD-1 antibody-IL-21 prodrug molecule and fusion molecule can bind to Mino cells.

[0231] The cellular activity of the molecules was analyzed using NK92 cells and natural killer (NK) cells. Figure 3 The results shown show that, under non-activated conditions, the prodrug molecule with IL-21α ECD as the masking part (Lot#PW04-38) has minimal activity, while the activity of the scFv as the masking part (Lot#PW05-68) is about 1 / 1000 of that without the masking part (PW04-67). The results show that the biological activity of the prodrug after treatment with the protease MMP2 is significantly enhanced.

[0232] Mino cells were used to detect the tumor-killing activity of the fusion molecule. Mino is a mantle cell lymphoma cell line that can express PD-1 and IL-21 receptors (Harington et al., Leuk Lymphoma (2019) 60 (10): 2498-2507 and Gelebart et al., Leukemia (2009) 23: 1836-1846). Figure 4A and Figure 4BResults of Mino cell viability analysis. Surprisingly, anti-PD-1 antibody-IL-21 prodrugs (Lots#PW04-38 and PW05-68) had significant activity before activation, while the control molecule (PD-1 antibody-IL-21R9E / R76A fusion molecule, Lot#PW09-02) had no or negligible activity. The results showed that the anti-PD-1-IL-21 fusion molecule was activated by "cis-binding", such as by binding to PD-1 and IL-21 receptors at the same time. The cis-binding of PD-1 antibody and cell surface PD-1 antigen and cytokine and cytokine receptor on the same cell surface can remove the masking effect of the masking part. Since the corresponding local immune cells simultaneously express antigens that target the carrier and receptors that bind to the cytokine part, the prodrug without a cleavable peptide linker is activated at the lesion site (such as a tumor).

[0233] Example 3. Construction of anti-PD-1 antibody-IL-15 prodrug fusion molecule

[0234] A group of anti-PD-1 antibody-IL-15 prodrugs were constructed, wherein the IL-15 molecule (i) was masked by scFv 215β; (ii) was masked by scFv 215γ; (iii) did not contain a masking portion (215βRef); or (iv) included a mutation but did not contain a masking portion (PD1 / IL15 mutant (M2)). The anti-PD-1 antibody of the prodrug includes two identical light chains, the amino acid sequence of which is SEQ ID NO: 44, and two identical heavy chains, the amino acid sequence of which is SEQ ID NO: 47.

[0235] The Fc-based prodrug 215β is a heterodimer comprising the amino acid sequences of SEQ ID NOs: 16 and 17.

[0236] The anti-PD-1 antibody-based prodrug 215γ includes two identical light chains (SEQ ID NO: 44), the amino acid sequence of the first heavy chain fusion polypeptide chain is SEQ ID NO: 37, and the amino acid sequence of the second heavy chain fusion polypeptide chain is SEQ ID NO: 75.

[0237] 215βRef is an IL-15 mutant (D30N / E64Q / N65D) Fc fusion molecule.

[0238] PD1 / IL15 mutant (M2) is an anti-PD-1 antibody fused to an IL-15 mutant, wherein the IL-15 agonist includes E46G, V49R, E64Q, D30N, and N1G mutations.

[0239] The above substances are all expressed by CHO cells and purified in sequence by protein A column affinity chromatography and additional chromatography steps.

[0240] Example 4. Biological activity of anti-PD-1 antibody-IL-15 prodrug fusion molecules

[0241] To evaluate and analyze the effect of IL-15 prodrug on activating Ki67 expression in human PBMCs. Figures 5A-5C CD4 + Ki67 activity of T cells; Figure 5A CD4 that does not express PD-1 + Ki67 activity of T cells; Figure 5B CD4 expressing PD-1 + Ki67 activity of T cells; Figure 5C CD4 expressing and not expressing PD-1 treated with different samples + T cell EC 50 The values ​​of CD4 expressing and not expressing PD-1 are also listed. + T cell EC 50 The data showed that 215γ has a multiple relationship with CD4 + The activity of Ki67 in cells was significantly higher than that in CD4 + The Ki67 activity of cells indicated that the prodrug 215γ was activated in cells expressing both the PD-1 antigen and the IL-15 cytokine receptor without the need for protease hydrolysis.

[0242] Example 5. Anti-PD-1 Antibody-IL-2 Prodrug Fusion Molecule

[0243] An anti-PD-1 antibody-IL-2 prodrug was constructed in a similar manner, wherein the prodrug PD-1 / IL-2v* includes two identical light chains (SEQ ID NO: 44) and two heavy chain fusion polypeptides (SEQ ID NO: 24 and 25, respectively). The IL-2 portion is an IL-2 mutant, and the masking portion in the prodrug is an IL-2 receptor β extracellular domain. The activity of the IL-2 prodrug on activating the expression of Ki67 in human PBMC was analyzed.

[0244] Fig. 6A and Figure 6B The CD4 + Ki67 activity of T cells. Negative control was IgG1 antibody against human Claudin 18.2. The above substances were expressed by CHO cells and purified by protein A column affinity chromatography and additional chromatography steps. Fig. 6A CD4 that does not express PD-1 + Ki67 activity of T cells; Figure 6BCD4 expressing PD-1 + Ki67 activity of T cells. The data show that the prodrug PD-1 / IL2V* has an inhibitory effect on CD4 + The Ki67 induction activity of the cells was significantly higher than that of cells that did not express PD-1, indicating that PD-1 / IL-2v* was activated in cells that simultaneously expressed PD-1 antigen and IL-2 cytokine receptor without the need for protease hydrolysis.

[0245] The above data indicate that cytokine prodrugs can be activated in cells that express both cytokine receptors and antigens that target the carrier portion. Prodrugs with antigen binding moieties as carriers can be activated without the need for protease hydrolysis. Although not wishing to be bound by theory, it can be predicted that the antigen binding domain of the carrier portion targets the prodrug molecule to the surface of cells expressing the antigen, thereby allowing the cytokine receptors on the cell surface and the masking portion of the prodrug to competitively bind to the cytokine. Figure 7 As shown, the prodrug that does not undergo proteolysis replaces the masking moiety through the cytokine receptor on the cell surface to complete the entire activation process.

[0246] Cells, such as exhausted Teff cells, express both antigens (such as PD-1) and cytokine receptors (such as IL-2Rβ and IL-2Rγ). Exhausted Teff cells are generally present in the tumor microenvironment rather than normal tissues. The differences in the distribution of cells expressing antigens and cytokine receptors in the body enable the prodrug to be specifically activated at the lesion site. The target specificity also further enhances the safety of cytokine therapy.

[0247] Sequence Listing

[0248] SEQ ID NO: 1—Human IL-21

[0249]

[0250] SEQ ID NO:2—IL-21 mutant A

[0251]

[0252] SEQ ID NO:3—IL-21 mutant B

[0253]

[0254] SEQ ID NO:4—IL-21 mutant C

[0255]

[0256] SEQ ID NO:5—IL-21 mutant D

[0257]

[0258] SEQ ID NO:6—Human IL-2

[0259]

[0260] SEQ ID NO:7—Human IL-15

[0261]

[0262] SEQ ID NO:8—Human IL-15Rαsushi domain

[0263]

[0264] SEQ ID NO:9—Human IL-7

[0265]

[0266] SEQ ID NO: 10—Human IL-9

[0267]

[0268] SEQ ID NO:11 - IL-2RβECD

[0269]

[0270] SEQ ID NO:12-IL-21RαECD (Source: uniprot.org / uniprot / Q9HBE5)

[0271]

[0272] SEQ ID NO: 13—Human IL-21RγECD

[0273]

[0274] SEQ ID NO:14-IL-7α receptor ECD (Source: https: / / www.uniprot.org / uniprot / P16871)

[0275]

[0276] SEQ ID NO:15-IL-9α receptor ECD (Source: )

[0277]

[0278] SEQ ID NO: 16 Fc-anti-IL-15 scFv2

[0279]

[0280] SEQ ID NO: 17Fc-IL-15N65D

[0281]

[0282] SEQ ID NO: 18 Anti-IL-15 scFv1 (italic parts: VH and VL)

[0283]

[0284] SEQ ID NO: 19 Anti-IL-15 scFv2 (italic parts: VH and VL)

[0285]

[0286] SEQ ID NO: 20 Anti-IL-21 scFv1 (italic parts: VH and VL)

[0287]

[0288] SEQ ID NO: 21 Anti-IL-21 scFv2 (italic parts: VH and VL)

[0289]

[0290] SEQ ID NO: 22 Anti-IL-2 scFv1 (underlined: HCDR1-3 and LCDR1-3; italics: VH and VL)

[0291]

[0292] SEQ ID NO: 23 Anti-IL-2 scFv2 (underlined: LCDR1-3 and HCDR1-3; italics: VL and VH)

[0293]

[0294] SEQ ID NO:24PD1-HC-IL-2v(CX3.58.1)

[0295]

[0296] SEQ ID NO:25PD1-HC-IL-2Rβ-ECD(CX3.58.3)

[0297]

[0298] SEQ ID NO: 26 PD1-HC-IL-21RαECD containing a non-cleavable peptide linker

[0299]

[0300] SEQ ID NO:27-34 Peptide connector (GGGGS) n (SEQ ID NO:27), wherein n=1, 2, 3, and 4

[0301] GGGGS (SEQ ID NO: 28)

[0302] GGGGSGGGGS (SEQ ID NO: 29)

[0303] GGGGSGGGGSGGGGS (SEQ ID NO:30)

[0304] GGGGSGGGGSXXGGGGSGGGGS (SEQ ID NO:31),X=A or N

[0305] GGGGSGGGGSGGGGSXXGGGGSGGGGS (SEQ ID NO:32),X=A or N

[0306] GGGGSGGGGSGGGGSXXGGGGSGGGGSGGGGS (SEQ ID NO:33),X=A or N

[0307] GGGGSGGGGXGGGGYGGGGS (SEQ ID NO:34), X=S, A or N, and Y=A or N

[0308] SEQ ID NO:35PD1-HC-IL-2v

[0309]

[0310] wherein Xaa3 is N or A; Xaa125 is A or S; Xaa35 is selected from K and N; Xaa42 is selected from A, G, S, T, Q, E, N, D, R and K; Xaa45 is selected from A, G, S, T, Q, E, N, D, R and K; Xaa72 is selected from A, GS, T, Q, E, N, D, R and K; Xaa73 is selected from A and T; n1=0, 1, 2 or 3; n2=0, 1, 2 or 3; X1 is alanine or is missing; X2 is alanine or is missing.

[0311] SEQ ID NO:36-PD1-HC-IL-2Rβ-ECD

[0312]

[0313] Wherein n1=0, 1, 2, or 3; n2=0, 1, 2, or 3; X1 is alanine or is missing; X2 is alanine or is missing.

[0314] SEQ ID NO:37PD1-HC with-sushi-IL-15v

[0315]

[0316] SEQ ID NO:38 PD1-HC-anti-IL-15 scFV2

[0317]

[0318] Wherein n1=0, 1, 2 or 3; n2=0, 1, 2 or 3; X1 is alanine or is missing; X2 is alanine or is missing.

[0319] SEQ ID NO:39PD1-HC-IL-21

[0320]

[0321] SEQ ID NO:40 PD1-HC-IL-21RαECD containing a cleavable peptide linker

[0322]

[0323] SEQ ID NO:41PD1-HC-anti-IL-21 scFv21

[0324]

[0325] SEQ ID NO:42 PD1-HC-IL-21 including knob mutation

[0326]

[0327] SEQ ID NO: 43 PD1-HC-IL-21RαECD including hole mutation

[0328]

[0329] SEQ ID NO:44PD1-LC

[0330]

[0331] SEQ ID NO:45 Anti-PDL1 Atezolizumab LC

[0332]

[0333] SEQ ID NO:46 Anti-PDL1 Atezolizumab HC

[0334]

[0335] SEQ ID NO:47 Anti-PD-1 Nivolumab HC

[0336]

[0337] SEQ ID NO:48 Anti-PD-1 Pembrolizumab LC

[0338]

[0339] SEQ ID NO:49 Anti-PD-1 Pembrolizumab HC

[0340]

[0341] SEQ ID NO:50 CTLA-4 antibody Ipilimumab LC

[0342] SEQ ID NO:51 CTLA-4 antibody Ipilimumab HC-IL2v

[0343]

[0344] wherein Xaa3 is N or A; Xaa125 is A or S; Xaa35 is selected from K and N; Xaa42 is selected from A, G, S, T, Q, E, N, D, R and K; Xaa45 is selected from A, G, S, T, Q, E, N, D, R and K;

[0345] Xaa72 is selected from A, G, S, T, Q, E, N, D, R and K; Xaa73 is selected from A and T; n1=0, 1, 2 or 3; n2=0, 1, 2 or 3; and X1 is alanine or is missing; X2 is alanine or is missing.

[0346] SEQ ID NO:52 CTLA-4 antibody Ipilimumab HC-IL15v

[0347]

[0348] SEQ ID NO:53 CTLA-4 antibody Ipilimumab HC-IL-21

[0349]

[0350] Wherein n1=0, 1, 2 or 3; n2=0, 1, 2 or 3; X1 is alanine or missing; X2 is alanine or missing. SEQ ID NO: 54 CTLA-4 antibody Ipilimumab HC-IL-2Rβ-ECD

[0351]

[0352] Wherein n1=0, 1, 2 or 3; n2=0, 1, 2 or 3; X1 is alanine or is missing; X2 is alanine or is missing.

[0353] SEQ ID NO:55 CTLA-4 antibody Ipilimumab HC-anti-IL-2 scFv1 (in scFV, the underlined parts are: HCDR1-3 and LCDR1-3; the italic parts are: VH and VL)

[0354]

[0355] Wherein n1=0, 1, 2 or 3; n2=0, 1, 2 or 3; X1 is alanine or is missing; X2 is alanine or is missing.

[0356] SEQ ID NO: 56 CTLA-4 antibody Ipilimumab HC-anti-IL-2 scFv2 (underlined parts: LCDR1-3 and HCDR1-3; italic parts: VL and VH)

[0357]

[0358] Wherein n1=0, 1, 2, or 3; n2=0, 1, 2, or 3; X1 is alanine or missing; X2 is alanine or missing. SEQ ID NO: 57 CTLA-4 antibody Ipilimumab HC-anti-IL-15 scFv2

[0359]

[0360] Wherein n1=0, 1, 2 or 3; n2=0, 1, 2 or 3; X1 is alanine or is missing; X2 is alanine or is missing.

[0361] SEQ ID NO:58CTLA-4 antibody Ipilimumab HC-IL-21Rα-ECD

[0362]

[0363] SEQ ID NO:59 CTLA-4 antibody Ipilimumab HC-anti-IL-21 scFv2

[0364]

[0365] SEQ ID NO:604E12B2D10 Anti-IL2 HC variable region (underlined portion: HCDR1-3)

[0366]

[0367] SEQ ID NO:614E12B2D10 anti-IL2LC variable region (underlined portion: LCDR1-3)

[0368]

[0369] SEQ ID NO:62 IL-2 agonist polypeptide

[0370]

[0371] wherein Xaa3 is N or A; Xaa125 is C, A or S; Xaa35 is selected from K and N; Xaa42 is selected from A, G, S, T, Q, E, N, D, R and K; Xaa45 is selected from A, G, S, T, Q, E, N, D, R and K; Xaa72 is selected from A, G, S, T, Q, E, N, D, R and K; and Xaa73 is selected from A and T.

[0372] SEQ ID NO:63 IL-21 receptor α ECD mutant 1 (the mutant part is underlined)

[0373]

[0374] SEQ ID NO:64 IL-21 receptor α ECD mutant 2 (the mutant part is underlined)

[0375]

[0376] SEQ ID NO:65 IL-21 receptor α ECD mutant 3 (the mutant part is underlined)

[0377]

[0378] SEQ ID NO:66 IL-21 receptor α ECD mutant 4 (the mutant part is underlined)

[0379]

[0380] SEQ ID NO:67 IL-21 receptor α ECD mutant 5 (the mutant part is underlined)

[0381]

[0382] SEQ ID NO:68 IL-21 receptor α ECD mutant 6 (the mutant part is underlined)

[0383]

[0384] SEQ ID NO:69 IL-21 receptor α ECD mutant 7 (the mutant part is underlined)

[0385]

[0386] SEQ ID NO:70 IL-21 receptor α ECD mutant 8 (the mutant part is underlined)

[0387]

[0388] SEQ ID NO:71 IL-21 receptor α ECD mutant 9 (the mutant part is underlined)

[0389]

[0390] SEQ ID NO:72 IL-21 receptor α ECD mutant 10 (the mutant part is underlined)

[0391]

[0392] SEQ ID NO:73 IL-21 receptor α ECD mutant 11

[0393]

[0394] SEQ ID NO: 74 PD1-HC-IL-21RαECD containing hole mutation

[0395]

[0396] SEQ ID NO: 75 Anti-IL-15 scFv2-PD1-HC fusion polypeptide

[0397] SEQ ID NO:76 Anti-IL-15 antibody 146B7 HC CDR1 (protein sequence)

[0398] TYWIG

[0399] SEQ ID NO:77 Anti-IL-15 antibody 146B7 HC CDR2 (protein sequence)

[0400] IIYPGDSDTR YSPSFQG

[0401] SEQ ID NO:78 Anti-IL-15 antibody 146B7 HC CDR3 (protein sequence)

[0402] GNWNCFDY

[0403] SEQ ID NO:79 Anti-IL-15 antibody 146B7 LC CDR1 (protein sequence)

[0404] RASQSVSSSY LA

[0405] SEQ ID NO:80 Anti-IL-15 antibody 146B7 LC CDR2 (protein sequence)

[0406] GASRRAT

[0407] SEQ ID NO:81 Anti-IL-15 antibody 146B7 LC CDR3 (protein sequence)

[0408] QRYGSSHT

[0409] SEQ ID NO:82 Version 2 of anti-IL-15 antibody 146B7 HC CDR3 (protein sequence) GNWNSFDY

[0410] SEQ ID NO: 83 Anti-IL-15 antibody 146B7 HC variable region (underlined portion: HCDR1-3)

[0411]

[0412] SEQ ID NO: 84 Anti-IL-15 antibody 146B7 LC variable region (underlined portion: LCDR1-3)

[0413]

Claims

1. A prodrug, characterized in that The prodrug comprises two identical light chains and two heavy chains, the amino acid sequence of the light chain is SEQ ID NO:44, the amino acid sequence of the first heavy chain fusion polypeptide chain is SEQ ID NO:37, and the amino acid sequence of the second heavy chain fusion polypeptide chain is 75.

2. A prodrug, characterized in that The prodrug is a heterodimer comprising the amino acid sequences of SEQ ID NOs: 16 and 17.

3. A pharmaceutical composition comprising the prodrug according to claim 1 or 2 and a pharmaceutically acceptable excipient.

4. A polynucleotide encoding the prodrug according to claim 1 or 2.

5. An expression vector comprising the polynucleotide according to claim 4.

6. A host cell comprising the expression vector according to claim 5, wherein the host cell is a mammalian cell.

7. A method for preparing the prodrug according to claim 1 or 2, comprising: a) culturing the host cell according to claim 6 under conditions that allow expression of the prodrug, wherein the host cell is a mammalian cell, and b) Isolating the prodrug from the cell culture.

8. Use of the prodrug according to claim 1 or 2 in the preparation of a drug for treating cancer, autoimmune diseases, or infectious diseases, or for stimulating the immune system of a patient in need thereof.

9. The use according to claim 8, wherein the patient suffers from: HIV, HBV, HCV, or HPV infection; An autoimmune disease selected from lupus erythematosus, type I diabetes, psoriasis, dermatomyositis, GvHD, rheumatoid disease; or A cancer selected from the group consisting of breast cancer, lung cancer, pancreatic cancer, esophageal cancer, medullary thyroid cancer, ovarian cancer, uterine cancer, prostate cancer, testicular cancer, colorectal cancer, and gastric cancer.

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