Patent Application
20250152644
The contents of the electronic sequence listing (SequenceListing.xml; Size: 53,365 bytes; and Date of Creation: Jan. 13, 2025) is herein incorporated by reference.
The present application relates to a technical field of biomedicine and, in particular, to a recombinant oncolytic virus and a use thereof.
The oncolytic virus is a tumor-killing virus with an ability to replicate and has been accepted by general public as an important branch of tumor immunotherapy. The oncolytic virus can specifically target and infect a tumor cell, for example, by exploiting an inactivation or a defect of a tumor suppressor gene in the tumor cell to selectively infect the tumor cell; after infecting the tumor cell, the oncolytic virus will replicate in large numbers within the tumor cell and eventually destroy the tumor cell, thereby killing the tumor cell. At the same time, the oncolytic virus can further provide an immune stimulation signal necessary to improve a host's own anti-cancer response, thereby attracting more immune cells to continue killing a residual tumor cell.
Although the oncolytic virus has a good application prospect in tumor immunotherapy, a wild-type oncolytic virus often causes an inflammation of a body's nervous system and other problems. A large pathogenic risk exists are in a process of using the wild-type virus to infect the tumor cell. Therefore, in order to further promote a clinical application of the oncolytic virus, the wild-type oncolytic virus needs to be engineered to obtain an attenuated oncolytic virus. The clinical application of the attenuated oncolytic virus can reduce a risk of the oncolytic virus and improve a safety of the oncolytic virus.
However, in a process of engineering the oncolytic virus, if only the wild-type oncolytic virus is randomly genetically engineered, although a toxicity of the wild-type oncolytic virus can be reduced, an engineered oncolytic virus may have a poor cure rate, or even the engineered oncolytic virus cannot be packaged, which is not conducive to promoting the clinical application of the oncolytic virus.
In order to further improve the in vitro and in vivo killing ability of oncolytic viruses on tumor cells in vitro and in vivo, and ensure the safety of oncolytic viruses on normal cells, the present application provides a recombinant oncolytic virus and a use thereof.
The recombinant oncolytic virus of the present application adopts the following technical solutions.
A recombinant oncolytic virus, the recombinant oncolytic virus including an M protein and an antigen encoded by an exogenous gene, wherein the M protein includes the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 1: mutating of methionine at position 51 into arginine (M51R); mutating of valine at position 221 into phenylalanine (V221F); and mutating of serine at position 226 into arginine (S226R).
Further, the antigen is selected from solid tumor antigens or hematological tumor antigens.
Further, the solid tumor antigens include but are not limited to 5T4, RORI, EGFR, FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), CD28, CD137 (4-1BB), CTLA-4, HER-2, FAS, FAP (fibroblast activation protein), LGR5, C5aR1, A2AR, fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid-induced TNFR-related (GITR) protein, lymphotoxin-β receptor (LTβR), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL receptor 1), TRAIL receptor 2, prostate-specific membrane antigen (PSMA) protein, prostate stem cell antigen (PSCA) protein, tumor-related protein carbonic anhydrase IX (CAIX), epidermal growth factor receptor 1 (EGFR1), EGFRvIII, ErbB3 (HER3), folate receptor, ephrin receptor, PDGFRa, ErbB-2, CD2, CD40, CD74, CD80, CD86, CCAM5 (CD66e), CCAM6 (CD66c), p53, cMet (tyrosine protein kinase Met), HGFR, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BACE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, BRCA1, BRCA2, MART-1, MCIR, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, Cyp-B, hTERT, hTRT, iCE, MUC2, P-cadherin, myostatin (GDF8), Cripto (TDGF1), MUC5AC, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, β-catenin/m, caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, annexin II, CDC27/m, TPI/mbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML1, CD28, CD137, CanAg, mesothelin (MSLN), DR5, PD-1, PD-L1, IGF-1R, CXCR4, neuropilin 1 (NRP-1), glypican2/3 (GPC2/3), EphA2, B7-H3, B7-H4, gpA33, GPC3, SSTR2, GD2, VEGF-A, VEGFR-2, PDGFR-a, ANKL, RANKL, MSLN, EBV, TROP2, FOLR1, and AXL.
Further, the hematological tumor antigens include but are not limited to BCMA (TNFRSF17), CD4, CD5 (Leu-1), CD7, CD10, FcγRIIIa (CD16a), FcγRIIIb (CD16b), CD19, CD20 (MS4A1), CD22 (Siglec-2), CD23, CD30 (TNFRSF8), CD33 (Siglec-3), CD34, CD37, CD38, CD44, CD47, CD56 (NCAM1), CD70, CD117, CD123 (IL3RA), CD138 (SDC1), CD174, CLL-1, ROR1, NKG2DL1/2 (ULBP1/2), IL1R3 (IL-1-RAP), FCRL5, GPRC5D, CLEC12A, WT1, FLT3, TLR8, SHP2, KAT6A/B, CSNK1A1, FLI1, IKZF1/3, PI3K, c-Kit, SLAMF3 (CD229), SLAMF7 (CD319), TCRB-chain, ITGB7, k-1gG, TACI, TRBCI, LeY, and MUC1.
Further, the antigen is one or more selected from a group consisting of: CD19, BCMA, NY-ESO-1, MUC-1, MSLN, EGFR, VEGFR2, MAGE A4, cMet, HGFR, and Claude18.2.
Further, the recombinant oncolytic virus further includes a cytokine encoded by an exogenous gene.
Further, the cytokine is selected from interleukins (IL), interferons (IFN), tumor necrosis factors (TNF), colony stimulating factors (CSF), transforming growth factor-β family (TGF-β family), and chemokine family.
Further, the cytokine is one or more selected from a group consisting of: GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, IFN-β, TGF-β, and TNF-α.
Further, the cytokine is one or more selected from a group consisting of: GM-CSF, IL-2, IL-12, IL-15, IL-18, TNF-α, and IFN-β.
Further, the M protein further includes one or more of the following site mutations: mutating of asparagine at position 32 into serine (N32S); and/or mutating of asparagine at position 49 into aspartic acid (N49D); and/or mutating of histidine at position 54 into tyrosine (H54Y); and/or mutating of valine at position 225 into isoleucine (V225I).
Further, the M protein further includes one or more of the following site mutations: knocking out of leucine at position 111; or, mutating of leucine at position 111 into alanine (L111A).
Further, the M protein further includes one or more of the following site mutations: mutating of glycine at position 21 into alanine (G21E); and/or mutating of methionine at position 33 into alanine (M33A); and/or mutating of alanine at position 133 into threonine (A133T).
In a specific embodiment, the site mutation of the M protein includes mutating of methionine at position 51 into arginine (M51R).
In a specific embodiment, the site mutation of the M protein includes mutating of valine at position 221 into phenylalanine (V221F).
In a specific embodiment, the site mutation of the M protein includes mutating of serine at position 226 into arginine (S226R).
In a specific embodiment, the site mutation of the M protein includes mutating of asparagine at position 32 into serine (N32S).
In a specific embodiment, the site mutation of the M protein includes mutating of asparagine at position 49 into aspartic acid (N49D).
In a specific embodiment, the site mutation of the M protein includes mutating of histidine at position 54 into tyrosine (H54Y).
In a specific embodiment, the site mutation of the M protein includes mutating of valine at position 225 into isoleucine (V225I).
In a specific embodiment, the site mutation of the M protein includes knocking out of leucine at position 111.
In a specific embodiment, the site mutation of the M protein includes mutating of leucine at position 111 into alanine (L111A).
In a specific embodiment, the site mutation of the M protein includes mutating of glycine at position 21 into alanine (G21E).
In a specific embodiment, the site mutation of the M protein includes mutating of methionine at position 33 into alanine (M33A).
In a specific embodiment, the site mutation of the M protein includes mutating of alanine at position 133 into threonine (A133T).
In a specific embodiment, the M protein has amino acid substitutions of M51R, V221F and S226R.
In a specific embodiment, the M protein has amino acid substitutions of N32S, N49D, M51R, H54Y, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of N32S, N49D, M51R, H54Y, knocking out of leucine at position 111, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of N32S, N49D, M51R, H54Y, L111A, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of G21E, N32S, N49D, M51R, H54Y, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of G21E, N32S, M33A, N49D, M51R, H54Y, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of G21E, N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of N32S, M33A, N49D, M51R, H54Y, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of N32S, M33A, N49D, M51R, H54Y, A133T, V221F, V225I and S226R.
In a specific embodiment, the M protein has amino acid substitutions of N32S, N49D, M51R, H54Y, A133T, V221F, V225I and S226R.
In the present application, an M protein in a wild-type Indiana MuddSummer subtype strain of vesicular stomatitis virus (VSV) includes an amino acid sequence as shown in SEQ ID NO 1.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 2.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 3.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 4.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 5.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 6.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 7.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 8.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 9.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 10.
In a specific embodiment, the M protein includes an amino acid sequence as shown in SEQ ID NO 11.
In a specific embodiment, the antigen is CD19.
In a specific embodiment, the antigen is BCMA.
In a specific embodiment, the antigen is NY-ESO-1.
In a specific embodiment, the antigen is MUC-1.
In a specific embodiment, the antigen is MSLN.
In a specific embodiment, the antigen is EGFR.
In a specific embodiment, the antigen is VEGFR2.
In a specific embodiment, the antigen is MAGE A4.
In a specific embodiment, the antigen is cMet.
In a specific embodiment, the antigen is Claude 18.2.
In a specific embodiment, the antigen is CD22.
In a specific embodiment, the antigen CD19 includes an amino acid sequence as shown in SEQ ID NO 28.
In a specific embodiment, the antigen BCMA includes an amino acid sequence as shown in SEQ ID NO 29.
In a specific embodiment, the antigen NY-ESO-1 includes an amino acid sequence as shown in SEQ ID NO 30.
In a specific embodiment, the antigen MUC-1 includes an amino acid sequence as shown in SEQ ID NO 31.
In a specific embodiment, the antigen MSLN includes an amino acid sequence as shown in SEQ ID NO 32.
In a specific embodiment, the antigen EGFR includes an amino acid sequence as shown in SEQ ID NO 33.
In a specific embodiment, the antigen VEGFR2 includes an amino acid sequence as shown in SEQ ID NO 34.
In a specific embodiment, the antigen MAGE A4 includes an amino acid sequence as shown in SEQ ID NO 35.
In a specific embodiment, the antigen cMet includes an amino acid sequence as shown in SEQ ID NO 36.
In a specific embodiment, the antigen Claude 18.2 includes an amino acid sequence as shown in SEQ ID NO 37.
In a specific embodiment, the antigen CD22 includes an amino acid sequence as shown in SEQ ID NO 38.
In a specific embodiment, the antigen CD19 includes an amino acid sequence as shown in SEQ ID NO 39.
In a specific embodiment, the antigen MAGE A4 includes an amino acid sequence as shown in SEQ ID NO 40.
In a specific embodiment, the antigen Claude 18.2 includes an amino acid sequence as shown in SEQ ID NO 41.
In a specific embodiment, the cytokine is GM-CSF.
In a specific embodiment, the cytokine is IL-2.
In a specific embodiment, the cytokine is IL-12.
In a specific embodiment, the cytokine is IL-15.
In a specific embodiment, the cytokine is IL-18.
In a specific embodiment, the cytokine is TNF-α.
In a specific embodiment, the cytokine is IFN-β.
In a specific embodiment, the cytokine GM-CSF includes an amino acid sequence as shown in SEQ ID NO 20.
In a specific embodiment, the cytokine IL-2 includes an amino acid sequence as shown in SEQ ID NO 21.
In a specific embodiment, the cytokine IL-12-A includes an amino acid sequence as shown in SEQ ID NO 22.
In a specific embodiment, the cytokine IL-12-B includes an amino acid sequence as shown in SEQ ID NO 23.
In a specific embodiment, the cytokine IL-15 includes an amino acid sequence as shown in SEQ ID NO 24.
In a specific embodiment, the cytokine IL-18 includes an amino acid sequence as shown in SEQ ID NO 25.
In a specific embodiment, the cytokine TNF-α includes an amino acid sequence as shown in SEQ ID NO 26.
In a specific embodiment, the cytokine IFN-β includes an amino acid sequence as shown in SEQ ID NO 27.
A recombinant oncolytic virus, including the M protein as described above; the recombinant oncolytic virus further includes a G protein, wherein the G protein includes one or more of the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 12: mutating of valine at position 53 into isoleucine (V53I); and/or, mutating of alanine at position 141 into valine (A141V); and/or, mutating of aspartic acid at position 172 into tyrosine (D172Y); and/or, mutating of lysine at position 217 into glutamic acid (K217E); and/or, mutating of aspartic acid at position 232 into glycine and/or, mutating of valine at position 331 to alanine (V331A); and/or, mutating of valine at position 371 into glutamic acid (V371E); and/or, mutating of glycine at position 436 into aspartic acid (G436D); and/or, mutating of threonine at position 438 into serine (T438S); and/or, mutating of phenylalanine at position 453 into leucine (F453L); and/or, mutating of threonine at position 471 into isoleucine (T471I); and/or, mutating of tyrosine at position 487 into histidine (Y487H).
In a specific embodiment, the site mutation of the G protein includes mutating of valine at position 53 into isoleucine (V53I).
In a specific embodiment, the site mutation of the G protein includes mutating of alanine at position 141 into valine (A141V).
In a specific embodiment, the site mutation of the G protein includes mutating of aspartic acid at position 172 into tyrosine (D172Y).
In a specific embodiment, the site mutation of the G protein includes mutating of lysine at position 217 into glutamic acid (K217E).
In a specific embodiment, the site mutation of the G protein includes mutating of aspartic acid at position 232 into glycine (D232G).
In a specific embodiment, the site mutation of the G protein includes mutating of valine at position 331 into alanine (V331A).
In a specific embodiment, the site mutation of the G protein includes mutating of valine at position 371 into glutamic acid (V371E).
In a specific embodiment, the site mutation of the G protein includes mutating of glycine at position 436 into aspartic acid (G436D).
In a specific embodiment, the site mutation of the G protein includes mutating of threonine at position 438 into serine (T438S).
In a specific embodiment, the site mutation of the G protein includes mutating of phenylalanine at position 453 into leucine (F453L).
In a specific embodiment, the site mutation of the G protein includes mutating of threonine at position 471 into isoleucine (T471I).
In a specific embodiment, the site mutation of the G protein includes mutating of tyrosine at position 487 into histidine (Y487H).
In a specific embodiment, the G protein has an amino acid substitution of V53I.
In a specific embodiment, the G protein has amino acid substitutions of V53I and A141V.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V and D172Y.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y and K217E.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E and D232G.
In a specific embodiment, the M protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G and V331A.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A and V371E.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E and G436D.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D and T438S.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S and F453L.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L and T471I.
In a specific embodiment, the G protein has amino acid substitutions of V53I, A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of A141V, D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of D172Y, K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of K217E, D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of D232G, V331A, V371E, G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of V331A, V371E, G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of V371E, G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of G436D, T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of T438S, F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of F453L, T471I and Y487H.
In a specific embodiment, the G protein has amino acid substitutions of T471I and Y487H.
In a specific embodiment, the G protein has an amino acid substitution of Y487H.
In the present application, a G protein in a wild-type Indiana MuddSummer subtype strain of the VSV includes an amino acid sequence as shown in SEQ ID NO 12.
In a specific embodiment, the G protein includes an amino acid sequence as shown in SEQ ID NO 13.
A recombinant oncolytic virus, including the M protein as described above or the M protein and G protein as described above; the recombinant oncolytic virus further includes an N protein, wherein the N protein includes one or more of the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 14: mutating of isoleucine at position 14 into valine (I14V); and/or, mutating of arginine at position 155 into lysine (R155K); and/or, mutating of serine at position 353 into asparagine (S353N).
In a specific embodiment, the site mutation of the N protein includes mutating of isoleucine at position 14 into valine (I14V).
In a specific embodiment, the site mutation of the N protein includes mutating of arginine at position 155 into lysine (R155K).
In a specific embodiment, the site mutation of the N protein includes mutating of serine at position 353 into asparagine (S353N).
In a specific embodiment, the N protein has an amino acid substitution of I14V.
In a specific embodiment, the N protein has amino acid substitutions of I14V and R155K.
In a specific embodiment, the N protein has amino acid substitutions of I14V, R155K and S353N.
In a specific embodiment, the N protein has amino acid substitutions of R155K and S353N. In a specific embodiment, the N protein has an amino acid substitution of S353N.
In the present application, an N protein in a wild-type Indiana MuddSummer subtype strain of the VSV includes an amino acid sequence as shown in SEQ ID NO 14.
In a specific embodiment, the N protein includes an amino acid sequence as shown in SEQ ID NO 15.
A recombinant oncolytic virus, including the M protein as described above; or the M protein and G protein as described above; or the M protein, G protein and N protein as described above; the recombinant oncolytic virus further includes a P protein, wherein the P protein includes one or more of the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 16: mutating of arginine at position 50 into lysine (R50K); and/or mutating of valine at position 76 into alanine (V76A); and/or mutating of asparagine at position 99 into glutamic acid (D99E); and/or mutating of leucine at position 126 into serine (L126S); and/or mutating of leucine at position 140 into serine (L140S); and/or mutating of histidine at position 151 into tyrosine (H151Y); and/or mutating of isoleucine at position 168 into methionine (T168M); and/or mutating of lysine at position 170 into glutamic acid (K170E); and/or mutating of tyrosine at position 189 into serine (Y189S); and/or mutating of asparagine at position 237 into aspartic acid (N237D).
In a specific embodiment, the site mutation of the P protein includes mutating of arginine at position 50 into lysine (R50K).
In a specific embodiment, the site mutation of the P protein includes mutating of valine at position 76 into alanine (V76A).
In a specific embodiment, the site mutation of the P protein includes mutating of asparagine at position 99 into glutamic acid (D99E).
In a specific embodiment, the site mutation of the P protein includes mutating of leucine at position 126 into serine (L126S).
In a specific embodiment, the site mutation of the P protein includes mutating of leucine at position 140 into serine (L140S).
In a specific embodiment, the site mutation of the P protein includes mutating of histidine at position 151 into tyrosine (H151Y).
In a specific embodiment, the site mutation of the P protein includes mutating of isoleucine at position 168 into methionine (1168M).
In a specific embodiment, the site mutation of the P protein includes mutating of lysine at position 170 into glutamic acid (K170E).
In a specific embodiment, the site mutation of the P protein includes mutating of tyrosine at position 189 into serine (Y189S).
In a specific embodiment, the site mutation of the P protein includes mutating of asparagine at position 237 into aspartic acid (N237D).
In a specific embodiment, the P protein has an amino acid substitution of R50K.
In a specific embodiment, the P protein has amino acid substitutions of R50K and V76A.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A and D99E.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A, D99E and L126S.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A, D99E, L126S and L140S.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S and H151Y.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y and I168M.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y, 1168M and K170E.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y, 1168M, K170E and Y189S.
In a specific embodiment, the P protein has amino acid substitutions of R50K, V76A, D99E, L126S, L140S, H151Y, 1168M, K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of V76A, D99E, L126S, L140S, H151Y, 1168M, K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of D99E, L126S, L140S, H151Y, 1168M, K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of L126S, L140S, H151Y, 1168M, K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of L140S, H151Y, I168M, K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of H151Y, 1168M, K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of I168M, K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of K170E, Y189S and N237D.
In a specific embodiment, the P protein has amino acid substitutions of Y189S and N237D.
In a specific embodiment, the P protein has an amino acid substitution of N237D.
In the present application, a P protein in in a wild-type Indiana MuddSummer subtype strain of the VSV includes an amino acid sequence as shown in SEQ ID NO 16.
In a specific embodiment, the P protein includes an amino acid sequence as shown in SEQ ID NO 17.
A recombinant oncolytic virus, including the M protein as described above; or the M protein and G protein as described above; or the M protein, G protein and N protein as described above; or the M protein, G protein, N protein and P protein as described above; the recombinant oncolytic virus further includes an L protein, wherein the L protein includes one or more of the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 18: mutating of serine at position 87 into proline (S87P); and/or, mutating of isoleucine at position 487 into threonine (1487T).
In a specific embodiment, the site mutation of the L protein includes mutating of serine at position 87 into proline (S87P).
In a specific embodiment, the site mutation of the L protein includes mutating of isoleucine at position 487 into threonine (1487T).
In a specific embodiment, the L protein has amino acid substitutions of S87P and I487T.
In the present application, an L protein in a wild-type Indiana MuddSummer subtype strain of the VSV includes an amino acid sequence as shown in SEQ ID NO 18.
In a specific embodiment, the L protein includes an amino acid sequence as shown in SEQ ID NO 19.
In some specific embodiments, the recombinant oncolytic virus is obtained by site-directed mutagenesis based on a rhabdovirus.
In some specific embodiments, the recombinant oncolytic virus is obtained by site-directed mutagenesis based on a vesicular stomatitis virus (VSV).
In some specific embodiments, the recombinant oncolytic virus is obtained by site-directed mutagenesis based on an Indiana MuddSummer subtype strain of the VSV.
In some specific embodiments, the recombinant oncolytic virus further includes or expresses an exogenous target protein.
In some specific embodiments, the recombinant oncolytic virus includes a nucleic acid molecule; the nucleic acid molecule includes a nucleic acid sequence encoding the M protein with the site mutation(s), and/or a nucleic acid sequence encoding the G protein with the site mutation(s), and/or a nucleic acid sequence encoding the N protein with the site mutation(s), and/or a nucleic acid sequence encoding the P protein with the site mutation(s), and/or a nucleic acid sequence encoding the L protein with the site mutation(s), and a nucleic acid sequence encoding the antigen and a nucleic acid sequence encoding the cytokine.
In a specific embodiment, in the nucleic acid molecule, the nucleic acid sequence encoding the antigen is located between the nucleic acid sequence encoding the G protein with the site mutation(s) and the nucleic acid sequence encoding the L protein with the site mutation(s).
In a specific embodiment, in the nucleic acid molecule, the nucleic acid sequence encoding the antigen is located between the nucleic acid sequence encoding the M protein with the site mutation(s), the nucleic acid sequence encoding the N protein with the site mutation(s) or the nucleic acid sequence encoding the P protein with the site mutation(s) and the nucleic acid sequence encoding the L protein with the site mutation(s).
In a specific embodiment, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the G protein with the site mutation(s) and the nucleic acid sequence encoding the L protein with the site mutation(s).
In a specific embodiment, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the M protein with the site mutation(s), the nucleic acid sequence encoding the N protein with the site mutation(s) or the nucleic acid sequence encoding the P protein with the site mutation(s) and the nucleic acid sequence encoding the L protein with the site mutation(s).
In a specific embodiment, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the antigen and the nucleic acid sequence encoding the L protein with the site mutation(s).
In a specific embodiment, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the G protein with the site mutation(s) and the nucleic acid sequence encoding the antigen.
In a second aspect, the present application provides an expression vector of the recombinant oncolytic virus and adopts the following technical solution.
An expression vector of the recombinant oncolytic virus capable of expressing the recombinant oncolytic viruses as described above.
In a third aspect, the present application provides a virus-producing cell and adopts the following technical solution.
A virus-producing cell capable of producing the recombinant oncolytic virus as described above.
In a fourth aspect, the present application provides a vaccine and adopts the following technical solution.
A vaccine prepared by using the recombinant oncolytic virus as described above.
In a fifth aspect, the present application provides a pharmaceutical composition and adopts the following technical solution.
A pharmaceutical composition, including the recombinant oncolytic virus or the vaccine as described above, and optionally a pharmaceutically acceptable carrier.
In a sixth aspect, the present application provides a method for preparing the recombinant oncolytic virus, the expression vector of the recombinant oncolytic virus, the virus-producing cell, the vaccine, or the pharmaceutical composition as described above.
In a seventh aspect, the present application provides a use of the recombinant oncolytic virus, the expression vector of the recombinant oncolytic virus, the virus-producing cell, the vaccine or the pharmaceutical composition in preparing a medicine for preventing and/or treating a disease and/or disorder.
In some specific embodiment, the recombinant oncolytic virus, the expression vector of the recombinant oncolytic virus, the virus-producing cell, the vaccine and/or the pharmaceutical composition are used in a method for sustained killing of an abnormally proliferative cell.
In some specific embodiments, the abnormally proliferative cell is selected from a group consisting of: a tumor cell and a cell related to a tumor tissue.
In an eighth aspect, the present application provides a use of the recombinant oncolytic virus, the vaccine or the pharmaceutical composition as described above in preparing a medicine for treating a tumor.
In some specific embodiments, the tumor includes a solid tumor or a hematological tumor.
In some specific embodiments, the tumor includes but is not limited to acute lymphoblastic leukemia, acute B-cell leukemia, chronic non-lymphocytic leukemia, non-Hodgkin's lymphoma, anal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, mastocarcinoma, breast cancer, cervical cancer, chronic myeloproliferative tumors, colorectal cancer, endometrial cancer, ependymoma, esophageal cancer, diffuse large B-cell lymphoma (DLBCL), sensorineuroblastoma, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, hepatocellular carcinoma, hypopharyngeal cancer, Kaposi sarcoma, kidney cancer, Langerhans cell hyperplasia, laryngeal cancer, liver cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, neuroblastoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, pharyngeal cancer, pituitary tumors, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, skin cancer, small cell lung cancer, small intestine cancer, squamous neck cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, vaginal cancer and vascular tumors.
In summary, the present application has the following beneficial effects:
The recombinant oncolytic viruses of the present application all have better killing ability to abnormally proliferative (tumor) LLC cell and are not easy to eliminate in the LLC cell. In addition, the recombinant oncolytic viruses of the present application have poor killing ability to a normal MEF cell in vitro and are all easy to eliminate in the normal MEF cell. In view of this, the recombinant oncolytic viruses of the present application can be well used for infecting and killing tumor, cancer and other cells, and are not easy to eliminate in tumor, cancer and other cells, further improving the cure rate of the recombinant oncolytic viruses for tumor, cancer and other cells. Moreover, the recombinant oncolytic viruses of the present application can not damage normal cells and are easier to eliminate in the normal cells, further ensuring the safety of the normal cells.
In the above figures, number 0 on the horizontal axis represents the wild-type oncolytic virus; numbers 1-130 on the horizontal axis represent the recombinant oncolytic viruses prepared in Preparation Examples 1-130, respectively.
OD570 on the vertical axis represents the OD value of the cell. The larger the value of OD570, the poorer the killing ability of a recombinant oncolytic virus to the cell; the smaller the value of OD570, the better the killing ability of the recombinant oncolytic virus against the cell.
The IFN-β level on the vertical axis represents the expression level of the IFN-β gene. The larger the value of the IFN-β level, the weaker the reproduction ability of a recombinant oncolytic virus in the cell and the easier of the recombinant oncolytic virus to eliminate in the cell; the smaller the value of the IFN-β level, the stronger the reproduction ability of the recombinant oncolytic virus in the cell, and the more difficult of the recombinant oncolytic virus to eliminate in the cell.
Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will appreciate, the teachings of the present application enable those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the drawings and descriptions in the specification of the present application are merely illustrative and not restrictive.
The implementation of the invention will be described below by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the invention from the contents described herein.
The term “oncolytic virus”, as used herein, generally refers to a virus that can replicate in tumor cells and kill tumor cells. Oncolytic viruses include, but are not limited to, vesicular stomatitis virus (VSV), poxvirus, herpes simplex virus (HSV), measles virus, Semliki Forest virus, poliovirus, reovirus, Seneca Valley virus (SVV), Echo-type enterovirus, coxsackievirus, Newcastle disease virus (NDV), and Maraba virus. In certain embodiments, the oncolytic virus is engineered to improve the selectivity for tumor cells. In certain embodiments, the oncolytic virus is engineered to reduce its immunogenicity.
In some embodiments, the oncolytic virus described herein is a VSV.
In some embodiments, the VSV is a mutant of the Indiana MuddSummer subtype strain of the VSV and can be used to treat a tumor. This virus does not interact with endogenous IFN-β in normal cells and can only selectively amplify and grow in tumor cells.
The VSV can express a variety of cell surface molecules, including low-density lipoprotein receptors, phosphatidylserine, sialolipids, and heparan sulfate, and can attach to the cell surface by these molecules. Compared with other oncolytic virus platforms currently under development, the VSV has the following advantages: (1) small genome, short replication time, and fast transsynaptic speed; (2) extremely high expression of exogenous genes, resulting in high titers and a possibility of large-scale production; (3) independent cell cycle and no risk of transformation in the cytoplasm of host cells. This oncolytic virus will not integrate into DNA, and after being attenuated, this oncolytic virus can avoid the nervous system inflammation caused by wild-type viruses. In view of the above characteristics, the VSV has great potential in tumor immunotherapy.
In some embodiments, the M protein, and/or the G protein, and/or the N protein, and/or the P protein, and/or the L protein of the VSV may be subjected to site-directed mutagenesis.
In certain embodiments, the recombinant oncolytic virus described herein may be an oncolytic virus that has been genetically modified, such as modified by modification of one or more genes, so as to improve its tumor selectivity and/or preferentially replicate in dividing cells. The “genetically modified” may refer to modification of genes involved in DNA/RNA replication, nucleic acid metabolism, host tropism, surface attachment, virulence, lysis and diffusion, or modification of integrating exogenous genes. The exogenous genes may include exogenous immune regulatory genes, exogenous screening genes and exogenous reporter genes. The oncolytic virus that has been modified may also be an oncolytic virus modified at an amino acid level, such as, by insertion, deletion, or substitution of one or more amino acids.
The term “M protein”, as used herein, generally refers to a matrix protein of the VSV. The M protein is an important virulence factor of the VSV and is also a protein in the VSV that is known to interfere with the natural immune response of mice. The term “M protein” also encompasses homologs, orthologs, variants and functionally active fragments thereof. In the present application, the M protein in a wild-type Indiana MuddSummer subtype strain of the VSV may include an amino acid sequence as shown in SEQ ID NO 1. In the present application, the M protein of the oncolytic virus may include amino acid sequences as shown in SEQ ID NOs 2-11.
The term “G protein”, as used herein, generally refers to a glycoprotein of the VSV, also known as the envelope protein. The term “G protein” also encompasses homologs, orthologs, variants and functionally active fragments thereof. In the present application, the G protein in a wild-type Indiana MuddSummer subtype strain of the VSV may include an amino acid sequence as shown in SEQ ID NO 12. In the present application, the G protein of the oncolytic virus may include an amino acid sequence as shown in SEQ ID NO 13.
The term “N protein”, as used herein, generally refers to a nucleocapsid protein of the VSV. The term “N protein” also encompasses homologs, orthologs, variants and functionally active fragments thereof. In the present application, the N protein in Indiana MuddSummer subtype of a wild-type VSV may include an amino acid sequence as shown in SEQ ID NO 14. In the present application, the N protein of the oncolytic virus may include an amino acid sequence as shown in SEQ ID NO 15.
The term “P protein”, as used herein, generally refers to a phosphoprotein of the VSV. The term “P protein” also encompasses homologs, orthologs, variants and functionally active fragments thereof. In the present application, the P protein in a wild-type Indiana MuddSummer subtype strain of the VSV may include an amino acid sequence as shown in SEQ ID NO 16. In the present application, the P protein of the oncolytic virus may include an amino acid sequence as shown in SEQ ID NO 17.
The term “L protein”, as used herein, generally refers to an RNA poly E protein of the VSV. An L gene of the VSV encodes the RNA poly E protein. The term “L protein” also encompasses homologs, orthologs, variants and functionally active fragments thereof. In the present application, the L protein in a wild-type Indiana MuddSummer subtype strain of the VSV may include an amino acid sequence as shown in SEQ ID NO 18. In the present application, the L protein of the oncolytic virus may include an amino acid sequence as shown in SEQ ID NO 19.
In the present application, the mutation site of a protein is generally expressed as “amino acid+amino acid position+amino acid after mutation”. In the present application, the mutation may include but is not limited to the addition, substitution absence and/or deletion of an amino acid. For example, the term “M51R” generally refers to the mutation of methionine M at position 51 to arginine R.
The term “amino acid substitution”, as used herein, generally refers to the substitution of an amino acid residue present in a parent sequence with another amino acid residue. The amino acid in the parent sequence may be substituted, for example, via chemical synthesis or by recombinant methods known in the art. Therefore, “substitution at position xx” generally refers to the substitution of an amino acid present at position xx with an alternative amino acid residue. In the present application, the amino acid substitution may include an amino acid mutation.
In the present application, the term “mutation” generally refers to a change in the nucleotide or amino acid sequence of a wild-type molecule. Amino acid changes may include the substitution, deletion, absence, insertion, addition or truncation of an amino acid, or the processing or cleavage of a protein.
In the present application, the recombinant oncolytic virus means integration of an exogenous gene at the same time of an site-directed gene mutation on the M protein, and/or G protein, and/or N protein, and/or P protein, and/or L protein of the VSV. The exogenous gene specifically refers to a gene encoding a cytokine.
In this application, the term “antigen” refers to a substance that can cause the production of antibodies or immune cells, and is any substance that can induce an immune response in the body. That is, the antigen is a substance that can be specifically recognized and bound by the antigen receptor (TCR/BCR) on the surface of T/B lymphocytes, activate T/B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro. Therefore, antigenic substances have two important characteristics: immunogenicity and immunoreactivity. Immunogenicity refers to the ability of antigens to induce specific immune responses in the body and produce antibodies and/or sensitized lymphocytes. Immunoreactivity refers to the ability to specifically bind to corresponding immune effector substances (antibodies or sensitized lymphocytes) in vivo and in vitro.
In a specific embodiment, the oncolytic virus is engineered to carry a coding sequence for an antigen that can be recognized by CART cells.
In some specific embodiments, the antigen is exogenous, meaning that the antigen comes from a different species.
In some specific embodiments, the antigen is an endogenous antigen. Specifically, the antigen is an antigen that is usually expressed on tumor cells.
In a specific embodiment, the antigen is a tumor associated antigen (TAA) or a tumor specific antigen (TSA).
In a specific embodiment, the TAA or TSA encompasses a molecule or a portion thereof that is presented on the cell surface or present in a tumor environment (e.g., in a tumor microenvironment).
In some specific embodiments, the cell is a tumor cell.
In some specific embodiments, the TAA or TSA includes a tumor associated antigen or a tumor specific antigen on the cell surface or in the cell membrane.
In some specific embodiments, the cell is a non-tumor cell present in a tumor environment. The cell is, for example, but not limited to, a cell present in the vasculature tissue associated with a tumor or cancer.
In some specific embodiments, the TAA or TSA is an angiogenic antigen in a tumor microenvironment. In some specific embodiments, the TAA or TSA is an antigen on a blood vessel in a tumor microenvironment.
In some specific embodiments, the cell is a stromal cell present in a tumor environment.
In some specific embodiments, the TAA or TSA is a stromal cell antigen in a tumor microenvironment.
In some specific embodiments, the TAA or TSA includes an extracellular epitope of a tumor cell surface antigen, a tetramer inside or outside the tumor cell membrane, or other structures that can be recognized by antibodies or immune cells.
In some specific embodiments, the TAA or TSA includes an extracellular matrix antigen.
In some specific embodiments, the TAA or TSA includes an antigen present in a tumor microenvironment (TME). In some specific embodiments, the TAA or TSA includes a molecule secreted by a tumor cell into a TME.
In some specific embodiments, the TAA or TSA includes an effector molecule secreted by a tumor cell into a TME.
In some specific embodiments, the TAA or TSA includes an effector molecule secreted by a tumor cell into a TME to downregulate or inhibit the activity of cytotoxic natural killer (NK) cells or T cells.
In some specific embodiments, the TAA or TSA includes a soluble activating receptor ligand that is secreted by a tumor cell into a TME to block recognition of tumor cells by NK cells or T cells.
In some specific embodiments, examples of TAA or TSA include but are not limited to 5T4, ROR1, EGFR, FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, CD28, CD137, CTLA-4, FAS, FAP (fibroblast activation protein), LGR5, C5aR1, A2AR, fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid-induced TNFR-related (GITR) protein, lymphotoxin-β receptor (LTβR), toll-like receptor (TLR), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL receptor 1), TRAIL receptor 2, prostate-specific membrane antigen (PSMA) protein, prostate stem cell antigen (PSCA) protein, tumor-related protein carbonic anhydrase IX (CAIX), epidermal growth factor receptor 1 (EGFR1), EGFRvIII, human epidermal growth factor receptor 2 (Her2/neu; Erb2), ErbB3 (Her3), folate receptor, ephrin receptor, PDGFRa, ErbB2, CD2, CD20, CD22, CD30, CD33, CD40, CD37, CD38, CD70, CD74, CD56, CD80, CD86, CD123, CCAM5, CCAM6, BCMA, p53, cMet (tyrosine protein kinase Met), hepatocyte growth factor receptor (HGFR), MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, BRCA1, BRCA2, MART-1, MCIR, Gp100, PSA, PSM, tyrosinase, Wilms tumor antigen (WT1), TRP-1, TRP-2, ART-4, CAMEL, Cyp-B, hTERT, hTRT, ICE, MUC1, MUC2, P-cadherin, myostatin (GDF8), Cripto (TDGF1), MUC5AC, PRAME, P15, RU1, RU2, SART-1, SART-3, WT1, AFP, β-catenin/m, caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, annexin II, CDC27/m, TPI/mbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML1, CD28, CD137, CanAg, mesothelin, DR5, PD-1, PD-L1, HER-2, IGF-1R, CXCR4, neuropilin 1, glypicans, EphA2, CD138, B7-H3, B7-H4, gpA33, GPC3, SSTR2 or VEGF-R2.
The term “cytokines”, as used herein, refers to biologically active substances synthesized and secreted by immune cells (such as lymphocytes, monocytes and macrophages) and their related cells (such as vascular endothelial cells and fibroblasts) to regulate the functions of other immune cells or target cells. The cytokines belong to small molecule polypeptides or glycoproteins. Cytokines with immune regulation effects can be expressed by recombinant oncolytic viruses. According to their main functions, cytokines are divided into: interleukins (IL), interferons (IFN), tumor necrosis factors (TNF), colony stimulating factors (CSF), transforming growth factor-β family (TGF-β family), growth factors (GF), and chemokine family.
Interleukins include IL-1, IL-2, IL-7, IL-9, IL-15, IL-21, IL-4, IL-12 and IL-18.
Specifically, interleukin 1 (IL-1): IL-1 is a pleiotropic cytokine involved in cortical inflammatory response, cell growth and tissue repair. The IL-1 superfamily has 11 members, such as IL-1A, IL-1B, IL-1Ra, and IL-18. IL-1 is a drug target for some cancers and is also used in cell therapy. In terms of cellular immunotherapy, IL-1 can stimulate the proliferation of CD4+ T cells in vitro, induce the production of IL-2, co-stimulate the activation of CD8+/IL1R+ T cells, and stimulate the proliferation of mature B cell and the secretion of immunoglobulin.
Specifically, interleukin 2 (IL-2): IL-2, also known as T cell growth factor, is produced by T cells in response to antigens or mitotic stimulation and is widely used to promote the activation and proliferation of T cells and NK cells. IL-2 can stimulate proliferation of NK cells, increase cytotoxicity, and stimulate NK cells to secrete a variety of cytokines. However, further studies have found that IL-2 can cause over-differentiation of T cells and induce apoptosis of activated T cells, and can also activate CD4+ FoxP3 Treg regulatory cells, thereby inhibiting activation of T cells and tumor killing activity. Therefore, IL-2 is considered to be a T cell regulatory factor rather than just an activation factor, so some studies have now used IL-7, IL-15, and IL-21 to replace IL-2.
Specifically, interleukin 7 (IL-7): IL-7 is a hematopoietic growth factor, secreted by stromal cells in the bone marrow and thymus, and shares the γc receptor subunit with IL-2 to stimulate the proliferation of lymphocyte progenitor cells. IL-7 can provide continuous stimulation signals for Naïve T cells and memory T cells. As mentioned above, IL-7 will not activate CD4+ FoxP3+ Treg cells during the activation of CD8+ T cells. In clinical practice, IL-7 can also be used to restore the number of T cells after chemotherapy or hematopoietic stem cell transplantation. And IL-7 plays an important role in certain stages of maturation and can affect the proliferation of B cells. IL-7 can also act as a regulatory factor for intestinal mucosal lymphocytes.
Specifically, interleukin 15 (IL-15): IL-15 has a similar structure to IL-2, shares the γc receptor subunit, and belongs to the family with 4 α-helix bundles (others include, such as, IL-2, IL-4, IL-7, IL-9, G-CSF and GM-CSF). IL-15 regulates the activation and proliferation of T cells and NK cells. IL-15 mainly kills virus-infected cells in the innate immune system. Also, IL-15 can activate NKT cells and γδT cells. In immune cell therapy, IL-15 does not cause apoptosis of activated T cells, but activates CD8+ effector T cells. IL-15 maintains the survival of memory T cells, thus playing an important role in long-term anti-tumor activity.
Specifically, interleukin 21 (IL-21): IL-21 also belongs to the IL-2 family, shares the γc receptor subunit, has a strong regulatory effect on the cells of the immune system, and can induce cell division and proliferation in its target cells. In cellular immunotherapy, IL-21 can promote the proliferation of CD4+ and CD8+ T cells, enhance the cytotoxicity of CD8+ T cells and NK cells, and will not cause cell apoptosis due to activation. IL-21 will preferentially expand “young” CD8+ T cells with CD27+ and CD28+, which have stronger cytotoxicity. Of course, IL-21 will not cause the expansion of Treg, so IL-21 is increasingly used in cell immunotherapy.
Specifically, interleukin 4 (IL-4): IL-4 activates the proliferation of activated B cells and T cells, and regulates the expression of Fc receptors on lymphocytes and monocytes. IL-4 induces the transformation of Th1 cells to Th2 cells. IL-4 stimulates Th2 cells to secrete IL-4, IL-5, IL-6, IL-10 and IL-13. IL-4 guides monocytes to differentiate into DCs by inhibiting the growth of macrophages. When IL-4 is not added to a culture system, monocytes will differentiate into macrophages. IL-4 plays a key role in regulating humoral immunity and adaptive immunity, inducing B cell antibody class switching to IgE and upregulating the production of MHC class II molecules. The combined action of IL-4 and GM-CSF can direct the differentiation of monocytes into immature DCs. At this time, DCs have stronger antigen uptake and processing capabilities, but weaker antigen presentation capabilities. The sequential use of IL-4 and TNF-α can promote DC maturation.
Specifically, interleukin 12 (IL-12): IL-12 acts on activated T and NK cells, has a wide range of biological activities, and acts on lymphocytes through the mediation of the activator of the transcription protein STAT4. IL-12 is required for the T cell-independent induction of IFN-γ and plays an important role in the differentiation of Th1 and Th2 cells. IL12B combines with IL23A to form IL-23 interleukin, which has innate and adaptive immune functions. IL-12 is a drug target. In cellular immunotherapy, IL-12 promotes the differentiation of CD4+ T cells into CD4+ Th1 T cells and enhances the activity of CD8+ CTLs cells. The therapeutic effect of IL-12 is related to its dose, duration of action, and other interacting cytokines, and promotes the tumor-killing activity of immune cells through multiple mechanisms. In the mouse anti-melanoma model, high-dose IL-12 acts through NK cells, while low-dose IL-12 has a tumor-killing effect through NKT.
Specifically, interleukin 18 (IL-18): IL-18 is also called interferon-γ inducing factor, which is a proinflammatory cytokine produced by macrophages and other cells. IL-18 can stimulate NK cells and CD8+ T cells to secrete IFN-γ, and enhance the cytotoxic effect of NK cells and CD8+ T cells. IL-18 can also activate macrophages, promote the development of Th1 CD4+ T cells and promote the expression of FasL by lymphocytes. IL-18 can provide a potential therapeutic target for allergic diseases. In addition, the synergistic effect of IL-18, IL-12 and IL-15 can maintain Th1 response and monokine production in autoimmune diseases.
Interferon-γ (IFN-γ) belongs to type II interferon, which is mainly produced by NK and NKT cells, has antiviral, antitumor and immunomodulatory effects and includes IFN-γ and IFN-β. IFN-γ has an anti-proliferative effect on transformed cells and can enhance the antiviral and anti-tumor effects of type I interferon. By activating macrophages, IFN-γ induces the expression of MHCI, MHCIII and co-activator molecules on antigen presenting cells (APCs). In addition, IFN-hages induce changes in the expression of proteasomes to enhance antigen presentation ability. IFN-γ can also promote the differentiation of CD4+ T cells into Th1 cells and inhibit the subtype switching of B cells that depends on IL-4. IFN-γ activates the JAK-STAT cell pathway by phosphorylating JAK1 and JAK2 proteins. In cellular immunotherapy, IFN-γ acts on host immune cells and has certain effects on macrophages, T cells, B cells and NK cells. IFN-γ enhances antigen presentation ability by promoting the expression of MHC class II molecules in macrophages or by causing some cells that normally do not express MHC class II molecules (such as vascular endothelial cells, some epithelial cells and connective tissue cells) to express MHC class II molecules. IFN-γ can promote the differentiation of B cells and CD8+ T cells, but cannot promote their proliferation. IFN-γ can enhance the activity and immune function of TH1 cells. IFN-γ enhances the phagocytic ability of neutrophils and activates NK cells to enhance their cytotoxic effect. Abnormal expression of IFN-γ is associated with many autoinflammatory and autoimmune diseases.
Tumor necrosis factor belongs to the TNF superfamily cytokines and is a multifunctional molecule that regulates biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism and coagulation. TNF-α participates in anti-tumor. In terms of cellular immunotherapy, TNF-α causes immature DC to differentiate into mature DC. This process is achieved by TNF-α downregulating the macropinocytosis of immature DC and the expression of surface Fc receptors, and upregulating the expression of MHCI class, class II molecules and B7 family molecules (CD80, CD86, etc.) on cell surface. The antigen uptake and processing capabilities of mature DCs are significantly weakened, but their antigen presentation capabilities are significantly enhanced, which can strongly activate T cells. TNF-α can also affect the production of other cytokines, such as stimulating the secretion of IL-1 by monocytes and macrophages, enhancing the proliferation of IL-2-dependent thymocytes and T cells, promoting the production of lymphokines such as IL-2, CSF and IFN-γ, and enhancing the proliferation and Ig secretion of B cells stimulated by mitogens or foreign antigens.
Granulocyte macrophage colony-stimulating factor (GM-CSF) plays a key role in embryo transfer and development. GM-CSF is one of the earliest cytokines discovered to have an effect on DCs. In DC culture, GM-CSF promotes the differentiation of monocytes into large macrophage-like cells and promotes the expression of MHC class II molecules on the cell surface, thereby enhancing the antigen presentation function of cells. In addition, GM-CSF can also promote DC survival. In terms of cellular immunotherapy, GM-CSF can activate immune responses and produce anti-tumor activity by activating macrophages and DCs. In terms of antigen presentation, GM-CSF can promote DC cell maturation, promote the upregulation of costimulatory molecules and the expression of CD1d receptors. Recent studies have found that GM-CSF stimulates the differentiation of hematopoietic progenitor cells into monocytes and neutrophils, thereby reducing the risk of febrile neutropenia in cancer patients. Other studies have shown that GM-CSF can induce the differentiation of bone marrow DCs, promote Th1 cell-biased immune responses and angiogenesis, and affect the development of allergic inflammation and autoimmune diseases. Therefore, GM-CSF is used clinically to treat malignant tumors.
The term “nucleic acid molecule”, as used herein, generally refers to nucleotides of any length. In the present application, the term “nucleic acid molecule” may encode a protein included by the oncolytic virus. In the present application, the nucleic acid molecule may include DNA and/or RNA. In some cases, the RNA may include single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA). The single-stranded RNA may include sense RNA or anti-sense RNA, or it may include ambisense RNA.
In the present application, the term “expression vector” generally refers to a nucleic acid vector. Under appropriate conditions, it is usually capable of expressing a target gene and/or target protein. In certain embodiments of the present application, the expression vector includes a nucleic acid molecule for expressing one or more components of a virus (e.g., an oncolytic virus). For example, the expression vector may include at least one viral genomic element and may be packaged into a virus or into virions.
The term “virus-producing cell”, as used herein, generally refers to cells, cell lines or cell cultures that can or have contained nucleic acid molecules or expression vectors described herein, or are capable of expressing recombinant oncolytic viruses described herein. The cell may include progeny of a single host cell. The cells may be obtained by in vitro transfection using the expression vector described herein.
The term “pharmaceutical composition”, as used herein, generally refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the formulation is to be administered. In certain embodiments, these formulations may include active ingredients of a medicine and a pharmaceutically acceptable carrier. In certain embodiments, the medicine product encompasses medicine products that are administered parenterally, transdermally, intracavitary, intraarterially, intrathecally, and/or intranasally, or injected directly into a tissue. The medicine product may be administered by different routes, such as an intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal or intratissue route.
The term “prevention/preventing”, as used herein, generally refers to preventing the occurrence and onset, recurrence, and/or spread of a disease or one or more symptoms of the disease by taking certain measures in advance. The term “treatment”, as used herein, generally refers to eliminating or ameliorating a disease, or one or more symptoms associated with the disease. In certain embodiments, treatment generally refers to administering one or more drugs to a patient suffering from the disease so that the disease is eliminated or alleviated. In certain embodiments, “treatment” may be the administration of the pharmaceutical composition and/or drug product in the presence or absence of other drugs after the onset of symptoms of a particular disease. For example, it may be the use of the pharmaceutical composition and/or drug product described herein to prevent the occurrence, development, recurrence and/or metastasis of a tumor.
The term “tumor”, as used herein, generally refers to any new pathological growth of tissue. The tumor may be benign or malignant. In the present application, the tumor may be a solid tumor or a hematological tumor. For research use, these tissues can be isolated from readily available sources by methods well known to those skilled in the art.
In some specific embodiments, the tumor includes but is not limited to acute lymphoblastic leukemia, acute B-cell leukemia, chronic non-lymphocytic leukemia, non-Hodgkin's lymphoma, anal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, mastocarcinoma, breast cancer (BRCA), cervical cancer, chronic myeloproliferative tumors, colorectal cancer, endometrial cancer, ependymoma, esophageal cancer, diffuse large B-cell lymphoma (DLBCL), sensorineuroblastoma, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, hepatocellular carcinoma, hypopharyngeal cancer, Kaposi sarcoma, kidney cancer, Langerhans cell hyperplasia, laryngeal cancer, liver cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, neuroblastoma, non-small cell lung cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors, pharyngeal cancer, pituitary tumors, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, skin cancer, small cell lung cancer, small intestine cancer, squamous neck cancer, testicular cancer, thymoma, thyroid cancer, uterine cancer, vaginal cancer and vascular tumors.
A wild-type VSV, in particular, an Indiana strain of the VSV, Indiana MuddSummer subtype strain of the VSV. The Indiana MuddSummer subtype strain of the VSV includes: an M protein with an amino acid sequence as shown in SEQ ID NO 1; a G protein with an amino acid sequence as shown in SEQ ID NO 12; an N protein with an amino acid sequence as shown in SEQ ID NO 14; a P protein with an amino acid sequence as shown in SEQ ID NO 16; an L protein with an amino acid sequence as shown in SEQ ID NO 18. In the present application, the M protein, G protein, N protein, P protein, and L protein all can be modified.
An oncolytic virus, the oncolytic virus, based on the wild-type VSV as described above, being obtained by mutating the sites on the amino acid sequences of the M protein, G protein, N protein, P protein, and L protein.
The present application provides a recombinant oncolytic virus. The recombinant oncolytic virus is obtained by introducing an exogenous gene encoding an antigen into the oncolytic virus as described above.
The recombinant oncolytic virus includes an M protein and an antigen encoded by an exogenous gene, wherein the M protein includes the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 1: mutating of methionine at position 51 into arginine (M51R); mutating of valine at position 221 into phenylalanine (V221F); and mutating of serine at position 226 into arginine (S226R).
Further, the recombinant oncolytic virus is obtained by introducing an exogenous gene encoding an antigen into the recombinant oncolytic virus as described above.
Further, the antigen is selected from hematological tumor antigens or solid tumor antigens.
Further, the solid tumor antigens include but are not limited to 5T4, ROR1, EGFR, FcγRI (CD64), FcγRIIa (CD32a), FcγRIIb (CD32b), CD28, CD137 (4-1BB), CTLA-4, FAS, FAP (fibroblast activation protein), LGR5, C5aR1, A2AR, fibroblast growth factor receptor 1 (FGFR1), FGFR2, FGFR3, FGFR4, glucocorticoid-induced TNFR-related (GITR) protein, lymphotoxin-β receptor (LTβR), tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL receptor 1), TRAIL receptor 2, prostate-specific membrane antigen (PSMA) protein, prostate stem cell antigen (PSCA) protein, tumor-related protein carbonic anhydrase IX (CAIX), epidermal growth factor receptor 1 (EGFR1), EGFRvIII, human epidermal growth factor receptor 2 (Her2/neu; Erb2), ErbB3 (HER3), folate receptor, ephrin receptor, PDGFRa, ErbB-2, CD2, CD40, CD74, CD80, CD86, CCAM5 (CD66e), CCAM6 (CD66c), p53, cMet (tyrosine protein kinase Met), HGFR, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BACE, DAM-6, DAM-10, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, NA88-A, NY-ESO-1, BRCA1, BRCA2, MART-1, MCIR, Gp100, PSA, PSM, tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, Cyp-B, hTERT, hTRT, iCE, MUC2, P-cadherin, myostatin (GDF8), Cripto (TDGF1), MUC5AC, PRAME, P15, RU1, RU2, SART-1, SART-3, AFP, β-catenin/m, caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, annexin II, CDC27/m, TPI/mbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, TEL/AML1, CD28, CD137, CanAg, mesothelin (MSLN), DR5, PD-1, PD-L1, IGF-1R, CXCR4, neuropilin 1 (NRP-1), glypican2/3 (GPC2/3), EphA2, B7-H3, B7-H4, gpA33, GPC3, SSTR2, GD2, VEGF-A, VEGFR-2, PDGFR-a, ANKL, RANKL, MSLN, EBV, TROP2, FOLR1, and AXL.
Further, the hematological tumor antigens include but are not limited to BCMA (TNFRSF17), CD4, CD5 (Leu-1), CD7, CD10, FcγRIIIa (CD16a), FcγRIIIb (CD16b), CD19, CD20 (MS4A1), CD22 (Siglec-2), CD23, CD30 (TNFRSF8), CD33 (Siglec-3), CD34, CD37, CD38, CD44, CD47, CD56 (NCAM1), CD70, CD117, CD123 (IL3RA), CD138 (SDC1), CD174, CLL-1, ROR1, NKG2DL1/2 (ULBP1/2), IL1R3 (IL-1-RAP), FCRL5, GPRC5D, CLEC12A, WT1, FLT3, TLR8, SHP2, KAT6A/B, CSNK1A1, FLI1, IKZF1/3, PI3K, c-Kit, SLAMF3 (CD229), SLAMF7 (CD319), TCRB-chain, ITGB7, k-1gG, TACI, TRBCI, LeY, and MUC1.
Further, the antigen is one or more selected from a group consisting of: CD19, BCMA, NY-ESO-1, MUC-1, MSLN, EGFR, VEGFR2, MAGE A4, cMet, HGFR, and Claude18.2.
Further, the recombinant oncolytic virus further includes a cytokine encoded by an exogenous gene.
Further, the cytokine is selected from interleukins (IL), interferons (IFN), tumor necrosis factors (TNF), colony stimulating factors (CSF), transforming growth factor-β family (TGF-β family), chemokine family, and growth factors.
Further, the cytokine is one or more selected from a group consisting of: GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-23, IL-27, IFN-α, IFN-β, IFN-γ, IFN-β, TGF-β, and TNF-α.
Further, the cytokine one or more is selected from a group consisting of: GM-CSF, IL-2, I-12, IL-15, IL-18, TNF-α, and IFN-β.
The M protein further includes one or more of the following site mutations: mutating of asparagine at position 32 into serine (N32S); and/or mutating of asparagine at position 49 into aspartic acid (N49D); and/or mutating of histidine at position 54 into tyrosine (H54Y); and/or mutating of valine at position 225 into isoleucine (V225I).
The M protein further includes one or more of the following site mutations: knocking out of leucine at position 111; or, mutating of leucine at position 111 into alanine (L111A).
The M protein further includes one or more of the following site mutations: mutating of glycine at position 21 into alanine (G21E); and/or mutating of methionine at position 33 into alanine (M33A); and/or mutating of alanine at position 133 into threonine (A133T). For example, the M protein includes amino acid sequences as shown in SEQ ID NOs 2-11.
In the present application, the M protein includes amino acid substitutions at positions 51, 221, and 226.
In the present application, the M protein includes amino acid substitutions at positions 32, 49, 51, 54, 221, 225, and 226.
In the present application, the M protein includes amino acid substitutions at positions 32, 49, 51, 54, 111, 221, 225, and 226.
In the present application, the M protein includes amino acid substitutions at positions 21, 32, 49, 51, 54, 221, 225, and 226.
In the present application, the M protein includes amino acid substitutions at positions 21, 32, 33, 49, 51, 54, 221, 225, and 226.
In the present application, the M protein includes amino acid substitutions at positions 21, 32, 33, 49, 51, 54, 133, 221, 225, and 226.
In the present application, the M protein includes amino acid substitutions at positions 32, 33, 49, 51, 54, 221, 225, and 226.
In the present application, the M protein includes amino acid substitutions at positions 32, 33, 49, 51, 54, 133, 221, 225, and 226.
In the present application, the M protein includes amino acid substitutions at positions 32, 49, 51, 54, 133, 221, 225, and 226.
In the present application, the M protein may further include amino acid substitutions at other positions.
A recombinant oncolytic virus, including the M protein as described above; the recombinant oncolytic virus further included a G protein, wherein the G protein includes one or more of the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 12: mutating of valine at position 53 into isoleucine (V53I); and/or, mutating of alanine at position 141 into valine (A141V); and/or, mutating of aspartic acid at position 172 into tyrosine (D172Y); and/or, mutating of lysine at position 217 into glutamic acid (K217E); and/or, mutating of aspartic acid at position 232 into glycine and/or, mutating of valine at position 331 to alanine (V331A); and/or, mutating of valine at position 371 into glutamic acid (V371E); and/or, mutating of glycine at position 436 into aspartic acid (G436D); and/or, mutating of threonine at position 438 into serine (T438S); and/or, mutating of phenylalanine at position 453 into leucine (F453L); and/or, mutating of threonine at position 471 into isoleucine (T471I); and/or, mutating of tyrosine at position 487 into histidine (Y487H). For example, the G protein includes an amino acid sequence as shown in SEQ ID NO 13.
In the present application, the G protein may include amino acid mutations at positions 53, 141, 172, 217, 232, 331, 371, 436, 438, 453, 471 and 487.
In the present application, the G protein may further include amino acid substitutions at other positions.
In some embodiments, the G protein includes at least one or more amino acid substitutions in a conserved region. For example, the conserved region may include amino acids at positions 437-461 of the G protein. In some embodiments, the G protein includes at least one or more amino acid substitutions in a truncated region of a cytoplasmic domain. For example, the truncated region of the cytoplasmic domain may include amino acids at positions 483-511 of the G protein.
A recombinant oncolytic virus, including the M protein as described above or the M protein and G protein as described above; the recombinant oncolytic virus further includes an N protein, wherein the N protein includes one or more of the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 14: mutating of isoleucine at position 14 into valine (I14V); and/or, mutating of arginine at position 155 into lysine (R155K); and/or, mutating of serine at position 353 into asparagine (S353N). For example, the N protein includes an amino acid sequence as shown in SEQ ID NO 15.
In the present application, the N protein may include amino acid mutations at positions 14, 155, and 353.
In the present application, the N protein may further include amino acid substitutions at other positions.
A recombinant oncolytic virus, including the M protein as described above; or the M protein and G protein as described above; or the M protein, G protein and N protein as described above; the recombinant oncolytic virus further includes a P protein, wherein the P protein includes one or more of the following site mutations compared to an amino acid sequence as shown in SEQ ID NO 16: mutating of arginine at position 50 into lysine (R50K); and/or mutating of valine at position 76 into alanine (V76A); and/or mutating of asparagine at position 99 into glutamic acid (D99E); and/or mutating of leucine at position 126 into serine (L126S); and/or mutating of leucine at position 140 into serine (L140S); and/or mutating of histidine at position 151 into tyrosine (H151Y); and/or mutating of isoleucine at position 168 into methionine (T168M); and/or mutating of lysine at position 170 into glutamic acid (K170E); and/or mutating of tyrosine at position 189 into serine (Y189S); and/or mutating of asparagine at position 237 into aspartic acid (N237D). For example, the P protein includes an amino acid sequence as shown in SEQ ID NO 17.
In the present application, the G protein may include amino acid mutations at positions 50, 76, 99, 126, 140, 151, 168, 170, 189 and 237.
In the present application, the P protein may further include amino acid substitutions at other positions.
A recombinant oncolytic virus, including the M protein as described above; or the M protein and G protein as described above; or the M protein, G protein and N protein as described above; or the M protein, G protein, N protein and P protein as described above; the recombinant oncolytic virus further includes an L protein, wherein the L protein includes one or more of the following site mutations compared to an amino acid sequence shown in SEQ ID NO 18: mutating of serine at position 87 into proline (S87P); and/or, mutating of isoleucine at position 487 into threonine (1487T). For example, the L protein includes an amino acid sequence as shown in SEQ ID NO 19.
In the present application, the L protein may include amino acid mutations at positions 87 and 487.
In the present application, the L protein may further include amino acid substitutions at other positions.
The recombinant oncolytic virus includes a nucleic acid molecule; the nucleic acid molecule includes a nucleic acid sequence encoding the M protein with the site mutation(s), and/or a nucleic acid sequence encoding the G protein with the site mutation(s), and/or a nucleic acid sequence encoding the N protein with the site mutation(s), and/or a nucleic acid sequence encoding the P protein with the site mutation(s), and/or a nucleic acid sequence encoding the L protein with the site mutation(s), and a nucleic acid sequence encoding the antigen and a nucleic acid sequence encoding the cytokine.
Further, the nucleic acid sequence encoding the antigen is located between the nucleic acid sequence encoding the G protein with the site mutation(s) and the nucleic acid sequence encoding the L protein with the site mutation(s).
Further, the nucleic acid sequence encoding the antigen is located between the nucleic acid sequence encoding the M protein with the site mutation(s), the nucleic acid sequence encoding the N protein with the site mutation(s) or the nucleic acid sequence encoding the P protein with the site mutation(s) and the nucleic acid sequence encoding the L protein with the site mutation(s).
Further, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the G protein with the site mutation(s) and the nucleic acid sequence encoding the L protein with the site mutation(s).
Further, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the M protein with the site mutation(s), and/or the nucleic acid sequence encoding the N protein with the site mutation(s), and/or the nucleic acid sequence encoding the P protein with the site mutation(s), and/or the nucleic acid sequence encoding the L protein with the site mutation(s).
Further, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the antigen and the nucleic acid sequence encoding the L protein with the site mutation(s).
Further, in the nucleic acid molecule, the nucleic acid sequence encoding the cytokine is located between the nucleic acid sequence encoding the G protein with the site mutation(s) and the nucleic acid sequence encoding the antigen.
In the present application, the recombinant oncolytic virus described herein can be obtained through a virus packaging process and a virus rescue process. The specific process may include infecting and inoculating BSR-T7 cells with poxvirus vTF7-3 expressing T7 RNA polymerase, and transfecting with lipofectamine using expression plasmids and backbone plasmids that clone VSV N, VSV P, and VSV L genes, respectively, to obtain a target oncolytic virus.
The present application further provides an expression vector of the recombinant oncolytic virus, a virus-producing cell, a vaccine, and a pharmaceutical composition.
The expression vector of the recombinant oncolytic virus may include nucleic acid sequences encoding the M protein and G protein of the recombinant oncolytic virus; the expression vector of the recombinant oncolytic virus may further include nucleic acid sequences encoding the N protein, P protein, and L protein of the recombinant oncolytic virus.
The virus-producing cell is capable of producing the recombinant oncolytic virus as described above; the virus-producing cell may include BSR-T7 cells, Vero cells, 293 cells, MRC-5 cells, and WI38 cells.
The vaccine is prepared by using the recombinant oncolytic virus as described above.
The pharmaceutical composition includes the recombinant oncolytic virus as described above, and optionally a pharmaceutically acceptable carrier.
In certain embodiments, the pharmaceutical composition may include a suitable formulation of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the pharmaceutical composition are preferably non-toxic to a recipient at the dose and concentration used. The pharmaceutical compositions of the present application include but are not limited to liquid, frozen and lyophilized compositions.
In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with drug administration, and are generally safe and non-toxic.
The pharmaceutical composition includes the recombinant oncolytic virus as described above, and optionally other pharmaceutically acceptable drugs. The pharmaceutical composition described above can be used for combined treatment of a disease, including but not limited to the treatment of a tumor.
In certain embodiments, the pharmaceutical composition may be useful for administration including parenteral, subcutaneous, intracavitary, intravenous, intrathecal, and/or intranasal administration or may be directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be performed in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal or intratissue administration. In certain embodiments, the pharmaceutical composition may be administered uninterruptedly. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by a patient to measure a therapeutic agent flowing into the patient's body, as described in WO2015/036583.
In addition, the present application provides a method for preparing the recombinant oncolytic virus, the expression vector of the recombinant oncolytic virus, the virus-producing cell, the vaccine or the pharmaceutical composition as described above. Any method suitable for producing oncolytic viruses may be used to produce the recombinant oncolytic viruses of the present application. For example, a poxvirus expressing T7 RNA polymerase can be added to a cell for transfection, a plasmid and a backbone plasmid expressing the N protein, L protein, and P protein of the recombinant oncolytic virus can be added for transfection, and the recombinant oncolytic virus of the present application can be obtained through a virus rescue process.
The present application provides a use of the recombinant oncolytic virus, the expression vector of the recombinant oncolytic virus, the virus-producing cell, the vaccine or the pharmaceutical composition in preparing a medicine for preventing and/or treating a disease and/or disorder.
The recombinant oncolytic viruses of the present application are obtained by performing site-directed mutations on the amino acids on the M protein, G protein, N protein, P protein, and L protein of an oncolytic virus, and inserting an antigen and/or cytokine encoded by an exogenous gene, thereby further improving the infective ability of the oncolytic viruses to the abnormally proliferative (tumor) LLC cell. Moreover, the recombinant oncolytic viruses prepared have poor infective ability to normal cells (normal MEF cells), indicating that the recombinant oncolytic viruses prepared herein can be well used for infecting tumors, cancers and other cells without damaging normal cells, and have broad application prospects.
The recombinant oncolytic viruses of the present application are obtained by performing site-directed mutagenesis on the amino acids on the M protein, G protein, N protein, P protein and L protein of an oncolytic virus, respectively, thereby further improving the killing ability of the oncolytic viruses to the abnormally proliferative (tumor) LLC cell in vitro. Moreover, the recombinant oncolytic viruses prepared almost have no effect on normal MEF cells, indicating that the recombinant oncolytic viruses prepared herein can be well used for damaging and killing abnormal cells such as tumor and cancer cells, without causing damage to normal cells.
The recombinant oncolytic viruses of the present application are not easy to eliminate in the abnormally proliferative (tumor) LLC cell. In contrast, wild-type oncolytic viruses are easier to eliminate in the LLC cell. The recombinant oncolytic viruses of the present application are obtained by performing site-directed mutagenesis on the amino acids on the M protein, G protein, N protein, P protein, and L protein of an oncolytic virus, respectively and further inserting an antigen and/or cytokine encoded by an exogenous gene, so that the oncolytic viruses can be more difficult to eliminate in the LLC cell, thereby further ensuring that the oncolytic viruses can better exert their infective ability and killing ability to the LLC cell. Moreover, the recombinant oncolytic viruses of the present application are easier to eliminate in normal MEF cells and the safety of the normal MEF cells is further ensured, thereby improving the safety of the recombinant oncolytic viruses.
The present disclosure is further described in detail below in combination with Preparation Examples 1-131 and Examples 1-2.
Preparation Examples 1-10 respectively provide a recombinant oncolytic virus. The recombinant oncolytic virus includes an M protein and an antigen. These preparation examples differ in the type of the antigen contained, as shown in Table 1.
A construction method for the recombinant oncolytic virus provided by each of the above preparation examples is as follows.
Using a pRV-core plasmid (BioVector NTCC Plasmid Vector Cell Gene Collection Center) as a template, the mutation sites of the M protein as shown in Table 1 were introduced by a PCR technology.
A gene fragment containing XbaI and MluI restriction sites and including the protein mutation sites was synthesized. PCR amplification was performed using the gene fragment as a template. The PCR product was subjected to 1% agarose gel electrophoresis and double-digested with XbaI and MluI, and a gene fragment with the protein mutation sites was obtained by gel extraction using a gel extraction kit.
The pRV-core plasmid was double-digested with XbaI and Mlu I, and a pRV-core restriction digestion extraction skeleton fragment was obtained by gel extraction using a gel extraction kit.
The gene fragment with the protein mutation sites and the pRV-core restriction digestion extraction backbone fragment were connected and transformed and then plated, and monoclones were selected and shaken for identification by PCR. The plasmid was extracted to obtain the constructed plasmid pRV-core Mut, which was sequenced by a sequencing company.
The plasmid pRV-core Mut obtained in step (1) was double-digested with Xho I and Mlu I to extract a long fragment.
An exogenous gene encoding the antigen was synthesized by a gene synthesis company and amplified with corresponding primers. The amplification product was double-digested with XhoI and NheI and a target gene fragment was extracted. The double-digested pRV-core Mut and the exogenous gene fragment were connected and transformed. Monoclones were selected and identified by PCR or enzyme digestion, and then sequenced by a sequencing company, as shown in Table 1, to obtain a plasmid pRV-core Mut carrying the exogenous gene.
The plasmid pRV-core Mut carrying the exogenous gene was transfected into a BSR-T7 cell (purchased from ATCC, American Type Culture Collection) by cell transfection technology using a calcium phosphate transfection kit (Thermo Fisher Scientific).
Four plasmids, i.e., the pRV-core Mut, pP, pN, and pL, were mixed at a ratio of 10:5:4:1, and the total amount of the plasmids was 5 μg; the plasmid mixture was then diluted with 200 μl of an Opti-MEM (Thermo Fisher Scientific), and 7.5 μl of a transfection reagent Plus Reagent (Life Technologies) was added to obtain a transfection plasmid premix, where the pP was a plasmid carrying the phosphoprotein genes of a rhabdovirus, the pN was a plasmid carrying the nucleoprotein genes of a rhabdovirus, and the pL was a plasmid carrying the polymerase protein genes of a rhabdovirus; parent vectors corresponding to the three plasmids pN, pP, and pL were all pCAGGS (purchased from ATCC).
10 μl of Lipofectamine LTX (Thermo Fisher Scientific) was diluted with 200 μl of the Opti-MEM to obtain an LTX mixed solution.
Plasmid transfection was performed according to the method described in the instruction manual of Lipofectamine LTX. Six hours later, the BSR-T7 cell was washed twice with PBS and then inoculated in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and incubated for three days.
The cell supernatant of the BSR-T7 cell culture solution was transferred to a Vero cell (Thermo Fisher Scientific), and the Vero cell was incubated at 37° C. for three days. The cells were observed for green fluorescence under a fluorescence microscope to determine the result of virus rescue. The rescued mutant rhabdovirus library was further passaged through the Vero cell, and monoclonal virus strains were selected in an established plaque screening system.
(4) Gene sequencing. The viral genomic RNA was extracted using a Trizol kit, and reverse transcription was performed using random primers. The reverse transcribed cDNA was subjected to PCR using primers designed for the gene sequence of the M protein and primers designed for the gene sequence encoding the antigen.
The primer sequences designed for the gene sequence of the M protein were:
The primer sequences designed for the gene sequence encoding the antigen were:
The product was extracted after 1% agarose gel electrophoresis and sequenced by a sequencing company. The sequencing results are shown in Table 1.
Preparation Examples 11-20 respectively provide a recombinant oncolytic virus. The recombinant oncolytic virus includes an M protein, a G protein and an antigen.
The mutation sites of the M protein are the same as the corresponding mutation sites in Preparation Example 1, and the G protein includes an amino acid sequence as shown in SEQ ID NO 13, as shown in Table 2. These preparation examples differ in the type of the antigen contained, as shown in Table 2.
The construction method of the recombinant oncolytic viruses provided in the above preparation examples is the same as the construction method in Preparation Example 2, except that: the recombinant oncolytic viruses further include a G protein with mutation sites as shown in Table 2. The differences in the construction method are specifically as follows:
The product was extracted after 1% agarose gel electrophoresis and sequenced by a sequencing company. The sequencing results are shown in Table 2.
Preparation Examples 21-30 respectively provide a recombinant oncolytic virus. The recombinant oncolytic virus includes an M protein, a G protein, an N protein and an antigen.
The mutation sites of the M protein and the G protein are the same as the corresponding mutation sites in Preparation Example 11, and the N protein includes an amino acid sequence as shown in SEQ ID NO 15, as shown in Table 3. These preparation examples differ in the type of the antigen contained, as shown in Table 3.
The construction method of the recombinant oncolytic viruses provided in the above preparation examples is the same as the construction method in Preparation Example 11, except that: the recombinant oncolytic viruses further include an N protein with mutation sites as shown in Table 3. The differences in the construction method are specifically as follows:
The product was extracted after 1% agarose gel electrophoresis and sequenced by a sequencing company. The sequencing results are shown in Table 3.
Preparation Examples 31-40 respectively provide a recombinant oncolytic virus. The recombinant oncolytic virus includes an M protein, a G protein, an N protein, a P protein and an antigen.
The mutation sites of the M protein, the G protein and the P protein are the same as the corresponding mutation sites in Preparation Example 21, and the P protein includes an amino acid sequence as shown in SEQ ID NO 17, as shown in Table 4. These preparation examples differ in the type of the antigen contained, as shown in Table 4.
The construction method of the recombinant oncolytic viruses provided in the above preparation examples is the same as the construction method in Preparation Example 21, except that: the recombinant oncolytic viruses further include a P protein with mutation sites as shown in Table 4. The differences in the construction method are specifically as follows:
The product was extracted after 1% agarose gel electrophoresis and sequenced by a sequencing company. The sequencing results are shown in Table 4.
Preparation Examples 41-50 respectively provide a recombinant oncolytic virus. The recombinant oncolytic virus includes an M protein, a G protein, an N protein, a P protein, an L protein and an antigen.
The mutation sites of the M protein, the G protein, the P protein and the N protein are the same as the corresponding mutation sites in Preparation Example 31, and the L protein includes an amino acid sequence as shown in SEQ ID NO 19, as shown in Table 5. These preparation examples differ in the type of the antigen contained, as shown in Table 5.
The construction method of the recombinant oncolytic viruses provided in the above preparation examples is the same as the construction method in Preparation Example 31, except that: the recombinant oncolytic viruses further include an L protein with mutation sites as shown in Table 5. The differences in the construction method are specifically as follows:
The product was extracted after 1% agarose gel electrophoresis and sequenced by a sequencing company. The sequencing results are shown in Table 5.
Preparation Examples 51-120 respectively provide a recombinant oncolytic virus. The recombinant oncolytic virus includes an M protein, a G protein, an N protein, an L protein, a P protein, an antigen and a cytokine.
The recombinant oncolytic viruses provided in the above preparation examples differ from those in Preparation Examples 41-50 in that: the recombinant oncolytic viruses further include a cytokine.
The cytokine included in the recombinant oncolytic virus provided in each of Preparation Examples 51-60 is GM-CSF which includes an amino acid sequence as shown in SEQ ID NO 20, as shown in Table 6.
The cytokine included in the recombinant oncolytic virus provided in each of Preparation Examples 61-70 is IL-2 which includes an amino acid sequence as shown in SEQ ID NO 21, as shown in Table 6.
The cytokine included in the recombinant oncolytic virus provided in each of Preparation Examples 71-80 is IL-12 which includes amino acid sequences as shown in SEQ ID NOs 22 and 23, as shown in Table 6.
The cytokine included in the recombinant oncolytic virus provided in each of Preparation Examples 81-90 is IL-15 which includes an amino acid sequence as shown in SEQ ID NO 24, as shown in Table 6.
The cytokine included in the recombinant oncolytic virus provided in each of Preparation Examples 91-100 is IL-18 which includes an amino acid sequence as shown in SEQ ID NO 25, as shown in Table 6.
The cytokine included in the recombinant oncolytic virus provided in each of Preparation Examples 101-110 is TNF-α which includes an amino acid sequence as shown in SEQ ID NO 26, as shown in Table 6.
The cytokine included in the recombinant oncolytic virus provided in each of Preparation Examples 111-120 is IFN-β which includes an amino acid sequence as shown in SEQ ID NO 27, as shown in Table 6.
The construction method of the recombinant oncolytic viruses provided in the above preparation examples is the same as the construction method in Preparation Example 1, except that: the construction method further includes insertion of an exogenous gene encoding the cytokine. The differences in the construction method are specifically as follows.
The mutation sites as shown in Table 6 were introduced by PCR technology in construction of plasmid in step (1), the same as Preparation Examples 41-50.
Step (2) further included insertion of an exogenous gene encoding the cytokine.
Step (4) further included sequencing of the cytokine. The viral genomic RNA was extracted using a Trizol kit, and reverse transcription was performed using random primers. The reverse transcribed cDNA was subjected to PCR using primers designed for the gene sequence of the cytokine; and
Preparation Examples 121-130 respectively provide a recombinant oncolytic virus. The recombinant oncolytic virus is obtained by introducing an exogenous gene encoding the antigen into a wild-type oncolytic virus.
A construction method for the recombinant oncolytic virus provided by each of the above preparation examples is as follows.
Using a pRV-core plasmid (BioVector NTCC Plasmid Vector Cell Gene Collection Center) as a template, double restriction digestion with XhoI and Mlu I was performed and then an exogenous gene encoding the antigen was inserted (the exogenous gene encoding the antigen was synthesized by a gene synthesis company and amplified with corresponding primers). The amplification product was double-digested with XhoI and NheI and a target gene fragment was extracted. The double-digested pRV-core and the exogenous gene fragment were connected and transformed. Monoclones were selected and identified by PCR or enzyme digestion, and then sequenced by a sequencing company, as shown in Table 7, to obtain a plasmid pRV-core Mut carrying the exogenous gene.
The plasmid pRV-core Mut carrying the exogenous gene was transfected into a BSR-T7 cell (purchased from ATCC, American Type Culture Collection) by cell transfection technology using a calcium phosphate transfection kit (Thermo Fisher Scientific).
Four plasmids, i.e., the pRV-core Mut, pP, pN, and pL, were mixed at a ratio of 10:5:4:1, and the total amount of the plasmids was 5 μg; the plasmid mixture was then diluted with 200 μl of an Opti-MEM (Thermo Fisher Scientific), and 7.5 μl of a transfection reagent Plus Reagent (Life Technologies) was added to obtain a transfection plasmid premix, where the pP was a plasmid carrying the phosphoprotein genes of a rhabdovirus, the pN was a plasmid carrying the nucleoprotein genes of a rhabdovirus, and the pL was a plasmid carrying the polymerase protein genes of a rhabdovirus; parent vectors corresponding to the three plasmids pN, pP, and pL were all pCAGGS (purchased from ATCC).
10 μl of Lipofectamine LTX (Thermo Fisher Scientific) was diluted with 200 μl of the Opti-MEM to obtain an LTX mixed solution.
Plasmid transfection was performed according to the method described in the instruction manual of Lipofectamine LTX. Six hours later, the BSR-T7 cell was washed twice with PBS and then inoculated in DMEM (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and incubated for three days.
The cell supernatant of the BSR-T7 cell culture solution was transferred to a Vero cell (Thermo Fisher Scientific), and the Vero cell was incubated at 37° C. for three days. The cells were observed for green fluorescence under a fluorescence microscope to determine the result of virus rescue. The rescued mutant rhabdovirus library was further passaged through the Vero cell, and monoclonal virus strains were selected in an established plaque screening system.
(3) Gene sequencing. The viral genomic RNA was extracted using a Trizol kit, and reverse transcription was performed using random primers. The reverse transcribed cDNA was subjected to PCR using primers (same as the primer sequences shown in Example 1) designed for the gene sequence encoding the antigen.
The product was extracted after 1% agarose gel electrophoresis and sequenced by a sequencing company. The sequencing results are shown in Table 7.
This preparation example provides a packaging process of the recombinant oncolytic virus prepared in any one of the Preparation Examples 1-120 as described above, specifically including the following steps.
1) Infection and inoculation of a BSR-T7 cell (purchased from ATCC) with a poxvirus vTF7-3 (BioVector NTCC Plasmid Vector Cell Gene Collection Center) expressing T7 RNA polymerase.
Specifically, the BSR-T7 cell was plated on a 6-well plate, with 3×105 cells/well; 14-16 hours after plating, the poxvirus vTF7-3 expressing the T7 RNA polymerase was added to infect the BSR-T7 cell; 6 hours after infection, the BSR-T7 cell was rinsed once with DPBS for transfection later.
Specifically: four plasmids, the pRV-core Mut, pP, pN, and pL, were mixed at a ratio of 10:5:4:1, and the total amount of the plasmids was 5 μg; the plasmid mixture was then diluted with 200 μl of an Opti-MEM (Thermo Fisher Scientific), and 7.5 μl of a transfection reagent Plus Reagent (Life Technologies) was added to obtain a transfection plasmid premix, where the pP was a plasmid carrying the phosphoprotein genes of a rhabdovirus, the pN was a plasmid carrying the nucleoprotein genes of a rhabdovirus, and the pL was a plasmid carrying the polymerase protein genes of a rhabdovirus; parent vectors corresponding to the three plasmids pN, pP, and pL were all pCAGGS (purchased from ATCC).
10 μl of Lipofectamine LTX (Thermo Fisher Scientific) was diluted with 200 μl of the Opti-MEM to obtain an LTX mixed solution.
200 μl of the LTX mixture was mixed with 200 μl of the transfection plasmid premix, followed by incubation at room temperature for 15 min, to obtain an LTX-DNA mixed solution.
The DPBS in the 6-well plate in step 1) was replaced with the Opti-MEM, the LTX-DNA mixed solution was dropwise added to the 6-well plate in which the BSR-T7 cell was incubated, and the 6-well plate was then shaken gently to evenly distribute the LTX-DNA mixed solution in the 6-well plate; 6-8 hours after transfection, the transfection reagent was removed and 3 ml of fresh complete medium (Thermo Fisher Scientific) was then added; 72 hours later, the cell supernatant of the BSR-T7 cell was harvested and filtered with a 0.22 μm filter to obtain the recombinant oncolytic viruses respectively corresponding to Preparation Examples 1-130.
In this example, a killing ability of the recombinant oncolytic viruses prepared in Preparation Examples 1-130 and the wild-type oncolytic virus to different cells in vitro were respectively detected.
The detection method used was an MTT detection method, that is, 200 pfu of each of the recombinant oncolytic viruses prepared in Preparation Examples 1-130 and 200 pfu of the wild-type oncolytic virus were added to culture solutions of different cells, and and a cell activity was detected using the MTT detection method after 24 hour 24 hours later.
The cells tested included: LLC cell and MEF cell. Specifically, the detection method included the following steps.
Test results are shown in
It can be seen from the above figures that the recombinant oncolytic viruses prepared in Preparation Examples 1-120 of the present application have better killing ability to the LLC cell in vitro and are all superior to the killing ability of the recombinant oncolytic viruses prepared in Preparation Examples 121-130 using wild-type oncolytic viruses combined with antigens to the LLC cell in vitro. And especially the recombinant oncolytic viruses provided in Preparation Examples 111-120 have better killing ability to the LLC cell in vitro than the wild-type oncolytic virus.
From the above test results, it can be seen that the recombinant oncolytic viruses of the present application have better killing ability to the LLC cell in vitro. It can be concluded that the recombinant oncolytic viruses of the present application all have better killing ability to cancer cells (such as 4T1 cell, MC38 cell and Hela cell) in vitro. Moreover, the recombinant oncolytic viruses prepared almost have no effect on MEF cells, indicating that the recombinant oncolytic viruses prepared herein can be well used for damaging and killing abnormal cells such as tumor and cancer cells, without causing damage to normal cells.
Although the wild-type oncolytic virus has better killing ability to the LLC cell in vitro, while damaging and killing these cells, the wild-type oncolytic virus may also damage and kill the MEF cell to a large extent, thereby limiting the clinical application of the wild-type oncolytic virus. Therefore, the modification of the wild-type oncolytic virus in the present application not only ensures the safety of oncolytic viruses to normal cells, but also ensures the killing ability of oncolytic viruses to tumor and cancer cells, thereby achieving broad clinical application prospects.
In this example, the expression of IFN-β induced by the recombinant oncolytic viruses prepared in Preparation Examples 1-130 and the wild-type oncolytic virus in different cells was detected.
The test indicator was the expression of the gene IFN-β in different cells. The gene IFN-β is a soluble glycoprotein gene produced by cells and having a wide range of antiviral, anti-tumor and immunomodulatory effects. The expression of the gene IFN-β can be used to determine the ability of cells to clear a recombinant oncolytic virus: when the expression of the gene IFN-β is high, it indicates that the recombiant oncolytic virus can be easily cleared from cells; when the expression of the gene IFN-β is low, it indicates that the recombinant oncolytic virus is difficultly cleared from cells.
The cells tested included: LLC cell and MEF cell.
Specifically, the detection method included the following steps.
Test results are shown in
It can be seen from the above figures that the recombinant oncolytic viruses prepared in Preparation Examples 1-120 of the present application and the wild-type virus have good reproduction ability in the LLC cell and are difficultly eliminated, and are all superior to the reproduction ability of the recombinant oncolytic viruses prepared in Preparation Examples 121-130 using wild-type oncolytic viruses combined with antigens in the LLC cell. And especially the recombinant oncolytic viruses provided in Preparation Examples 111-120 are more difficultly eliminated from the LLC cell, thereby further ensuring that these oncolytic viruses can better exert their ability to infect and kill the LLC cell.
From the above test results, it can be seen that the recombinant oncolytic viruses of the present application have good reproduction ability in the LLC cell and are not easily eliminated. It can be concluded that the recombinant oncolytic viruses of the present application all have good reproduction ability in cancer cells (such as 4T1 cell, MC38 cell, and Hela cell) and are not easily eliminated. Moreover, the recombinant oncolytic viruses of the present application are more easily eliminated in the MEF cell, thereby further ensuring the safety of the MEF cell and improving the safety of the oncolytic viruses.
The specific examples are merely an explanation of the present application and not for limiting the present application. Those skilled in the art may make modifications, without creative contribution, to the examples as needed after reading this specification. Any of the modifications made within the scope of the claims of the present application shall be protected by the Patent Law.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210838779.9 | Jul 2022 | CN | national |
The present application is a continuation application of PCT application No. PCT/CN2023/105020, filed on Jun. 30, 2023, which claims priority to Chinese patent application No. 202210838779.9, filed on Jul. 14, 2022. The entireties of PCT application No. PCT/CN2023/105020 and Chinese patent application No. 202210838779.9 are hereby incorporated by reference herein and made a part of this specification.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2023/105020 | Jun 2023 | WO |
| Child | 19019594 | US |
