Modified oncolytic virus and composition thereof

Abstract

The present invention provides a modified oncolytic virus, the oncolytic virus having a first heterologous polynucleotide encoding a PD-1 immune checkpoint inhibitor first molecule, and a second heterologous polynucleotide encoding a CTLA-4 immune checkpoint inhibitor second molecule. Further provided is a pharmaceutical composition, the pharmaceutical composition comprising the modified oncolytic virus.

Description

A modified oncolytic virus and its composition Technical Field

[0001] The present invention generally relates to a modified oncolytic virus and a composition thereof. Specifically, the present invention relates to the design, modification and application of an oncolytic virus and a composition thereof. In certain embodiments, the oncolytic virus is a recombinant oncolytic vaccinia virus. In certain embodiments, the recombinant oncolytic vaccinia virus can simultaneously express two full-length antibodies; in certain embodiments, the two full-length antibodies expressed by the recombinant oncolytic vaccinia virus are different immune checkpoint inhibitors; in certain embodiments, the two different immune checkpoint inhibitors are a combination of a full-length antibody that specifically binds to PD-1 and a full-length antibody that specifically binds to CTLA-4; in certain embodiments, the target combination of the two full-length antibodies exhibits a higher anti-tumor effect than the combination of the other two immune checkpoint inhibitors; in certain embodiments, the combination of the two full-length antibodies exhibits a higher anti-tumor effect than the combination of their antigen-binding fragments. Background Art

[0002] The following description is provided to aid the reader's understanding. None of the information provided or references cited are admitted to be prior art.

[0003] Cancer is diagnosed in more than 14 million people worldwide each year and, despite numerous advances in medical research, accounts for approximately 16% of all deaths.

[0004] Malignant tumors are often resistant to conventional therapies and represent a significant therapeutic challenge. For example, micrometastases can develop very early in the development of a primary tumor. Therefore, many tumor patients already have micrometastases at the time of diagnosis. Tumor cytotoxic T cells can seek out and destroy these micrometastases without harming surrounding healthy tissue. However, naturally occurring T cell responses against malignant tumors are generally insufficient to cause regression of primary or metastatic tumors.

[0005] In recent years, with breakthroughs in tumor immunotherapy research, immune checkpoint inhibitors (ICIs), represented by PD-1 / PD-L1, have been widely used in clinical treatment. Taking PD-1 / PD-L1 inhibitors as an example, they have become a research hotspot in tumor immunotherapy. Although they have improved the survival of cancer patients, their poor permeability as large-molecule biologics restricts their entry into solid tumors, and their single-agent efficacy is limited for general solid tumors. Furthermore, with the clinical application of immunotherapy, the efficacy of PD-1 / PD-L1 inhibitors in various tumors has been confirmed, but current data show that their efficacy remains relatively low. Most patients have poor or no effect on immunotherapy, and a small number of patients develop drug resistance after use. Even some patients who initially respond well develop acquired drug resistance, leading to disease progression.

[0006] In addition, therapeutic monoclonal antibodies targeting another immune checkpoint, CTLA-4, have also been used in clinical practice. CTLA-4 inhibitors, represented by Ipilimumab, have been on the market for many years but have failed to make major breakthroughs in a variety of solid tumors. Therefore, the use of combination therapy is the mainstream trend. The combination of multiple Nivolumab and Ipilimumab for the first-line treatment of indications such as melanoma, renal cell carcinoma, non-small cell lung cancer, and malignant pleural mesothelioma is more effective than single-agent therapy. In addition, a bispecific antibody targeting human PD-1 and CTLA-4 (cardunilimab injection) has been approved for marketing for the treatment of patients with recurrent or metastatic cervical cancer who have previously failed platinum-containing chemotherapy.

[0007] Oncolytic viruses are a class of naturally occurring or genetically modified viral products that can specifically infect and kill tumor cells. They can kill tumor cells through a variety of mechanisms and are an important branch of current tumor immunotherapy. Recent studies have shown the potential of oncolytic viruses as anti-tumor agents. Unlike conventional gene therapy, oncolytic viruses can spread in tumor tissues by virtue of viral replication and accompanying cell lysis, and their selective replication characteristics give them ideal safety and targeting. However, the first generation of oncolytic viruses is not sufficient for tumor treatment. By utilizing the inherent advantages of viruses as vectors, recombinant oncolytic viruses carrying exogenous genes have shown great application prospects. For example, oncolytic viruses carrying different immune activators, immunosuppressants, and cytokines have different mechanisms of action and application prospects. The potential contained therein is also urgently needed to enhance the efficacy of oncolytic viruses and increase tumor treatment therapies. Summary of the Invention

[0008] The current mainstream oncolytic virus combined immunotherapy includes the direct use of recombinant oncolytic viruses expressing immunotherapy genes, recombinant oncolytic viruses and immune checkpoint inhibitors (CN117503922, WO2023159102, US20210023151, etc.), however, there is no design and application of recombinant oncolytic viruses and their compositions that simultaneously express two or more full-length antibodies with different immune checkpoint inhibitors (such as PD-1 antibodies, CTLA-4 antibodies, etc.). The present invention fills this gap.

[0009] The present invention discloses a modified oncolytic virus and a composition thereof for use in tumor treatment. The oncolytic virus simultaneously expresses two independent or coupled different immune checkpoint inhibitors (e.g., a combination of a full-length antibody that specifically binds to PD-1 and a full-length antibody that specifically binds to CTLA-4. The present invention has been found through extensive screening to have a higher anti-tumor effect and / or lower side effect effect and / or lower required dose than a combination of two other immune checkpoint inhibitors (such as a combination of a PD-1 inhibitor and a PD-L1 / 2 inhibitor, a B7-H3 / 4 inhibitor, a LAG3 inhibitor, a TIM-3 inhibitor, a CD160 inhibitor, etc., and a combination of a CTLA-4 inhibitor and other immune checkpoint inhibitors). The oncolytic virus simultaneously expresses two independent or coupled different full-length antibodies (for example, a full-length antibody that specifically binds to PD-1 and a full-length antibody that specifically binds to CTLA-4. After extensive screening, the present invention found that the combination of dual full-length antibodies exhibits higher anti-tumor effects and / or lower side effects and / or requires a lower dose than other antibody combinations (such as Fab, Fab', F(ab')2, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single-domain antibodies and nanobodies, etc.). In addition to inducing tumor cell apoptosis by directly infecting tumor cells through the virus, the modified oncolytic virus expresses two different immune checkpoint inhibitors that can simultaneously regulate the body's immune response, turning "cold" tumors into "hot" tumors, thereby activating the immune system of the tumor microenvironment, inducing or reawakening the immune system's anti-tumor immune response, and further activating the immune system to initiate a systemic anti-tumor immune response, thereby achieving the purpose of obtaining a systemic therapeutic effect through local administration.

[0010] At the same time, the oncolytic virus and its composition disclosed in the present invention can directly express two different immune checkpoint inhibitors. Compared with the combination of oncolytic virus and two monoclonal antibodies, it can reduce the number of dosing times, reduce the pain caused by drug injection to patients, and reduce the complexity of clinical operations. It kills two birds with one stone and achieves the effects of oncolytic virus treatment and monoclonal antibody combination treatment at the same time.

[0011] In one aspect, the present invention relates to a modified oncolytic virus whose viral genome has a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding another different immune checkpoint inhibitor.

[0012] In certain embodiments, the oncolytic virus is selected from the group consisting of vaccinia virus, adenovirus, reovirus, measles virus, herpes simplex virus, Semliki Forest virus, Venezuelan equine encephalitis, parvovirus, chicken anemia virus, measles virus, coxsackie virus, vesicular stomatitis virus, Seneca Valley virus, Maraba virus, and Newcastle disease virus.

[0013] In certain embodiments, the oncolytic virus is selected from vaccinia virus. In certain embodiments, the oncolytic vaccinia virus is derived from a Western Reserve strain.

[0014] In certain embodiments, the modified oncolytic viral genome comprises at least one deletion or disruption that renders the virus attenuated or selectively replicates in tumor cells, wherein the deletion or disruption is of viral thymidine kinase.

[0015] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are inserted at the location of the deletion or disruption.

[0016] In certain embodiments, the first heterologous polynucleotide is immediately upstream of the second heterologous polynucleotide or immediately downstream of the second heterologous polynucleotide.

[0017] In certain embodiments, the virally encoded immune checkpoint inhibitor is a first antibody or an antigen-binding fragment thereof that can specifically bind to an immune checkpoint protein, and a second antibody or an antigen-binding fragment thereof that can specifically bind to another different immune checkpoint protein.

[0018] In certain embodiments, the first antibody or antigen-binding fragment thereof and / or the second antibody or antigen-binding fragment thereof is a full-length antibody.

[0019] In certain embodiments, the two immune checkpoint proteins are PD-1 and CTLA-4.

[0020] In certain embodiments, the first antibody or antigen-binding fragment thereof specifically binds SEQ ID NO: 1.

[0021] In certain embodiments, the first antibody or antigen-binding fragment thereof comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the first heavy chain Fab region comprises the amino acid sequence of SEQ ID NO: 3 and a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the first heavy chain CDR region comprises the amino acid sequence of HCDR1 set forth in SEQ ID NO: 4, the HCDR2 set forth in SEQ ID NO: 5, and the HCDR3 set forth in SEQ ID NO: 6.

[0022] In certain embodiments, the first antibody or antigen-binding fragment thereof further comprises a first light chain comprising an amino acid sequence having SEQ ID NO: 7. In certain embodiments, the first light chain CDR region comprises an amino acid sequence having LCDR1 as set forth in SEQ ID NO: 8, LCDR2 as set forth in SEQ ID NO: 9, and LCDR3 as set forth in SEQ ID NO: 10.

[0023] In certain embodiments, the first antibody or antigen-binding fragment thereof comprises another first heavy chain comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the Fab region of the another first heavy chain comprises the amino acid sequence of SEQ ID NO: 12 and a homologous sequence thereof having at least 90% sequence identity. In certain embodiments, the CDR region of the another first heavy chain comprises the amino acid sequence of HCDR1 set forth in SEQ ID NO: 13, the HCDR2 set forth in SEQ ID NO: 14, and the HCDR3 set forth in SEQ ID NO: 15.

[0024] In certain embodiments, the first antibody or antigen-binding fragment thereof further comprises another first light chain comprising an amino acid sequence having SEQ ID NO: 16. In certain embodiments, the another first light chain CDR region comprises an amino acid sequence having LCDR1 as set forth in SEQ ID NO: 17, LCDR2 as set forth in SEQ ID NO: 18, and LCDR3 as set forth in SEQ ID NO: 19.

[0025] In certain embodiments, the second antibody or antigen-binding fragment thereof specifically binds SEQ ID NO:20.

[0026] In certain embodiments, the second antibody or antigen-binding fragment thereof comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the second heavy chain Fab region comprises the amino acid sequence of SEQ ID NO: 22 and a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the second heavy chain CDR region comprises the amino acid sequence of HCDR1 set forth in SEQ ID NO: 23, the HCDR2 set forth in SEQ ID NO: 24, and the HCDR3 set forth in SEQ ID NO: 25.

[0027] In certain embodiments, the second antibody or antigen-binding fragment thereof further comprises a second light chain comprising an amino acid sequence having SEQ ID NO: 26. In certain embodiments, the second light chain CDR region comprises an amino acid sequence having LCDR1 set forth in SEQ ID NO: 27, LCDR2 set forth in SEQ ID NO: 28, and LCDR3 set forth in SEQ ID NO: 29.

[0028] In certain embodiments, the first immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to PD-1, and the second immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4.

[0029] In certain embodiments, the first immune checkpoint inhibitor is a full-length antibody that specifically binds PD-1, and the second immune checkpoint inhibitor is a full-length antibody that specifically binds CTLA-4.

[0030] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are configured such that they are expressed during the same or different stages of the replication cycle of the modified oncolytic virus.

[0031] In certain embodiments, the modified oncolytic virus comprises the following elements in frame in the 5′ to 3′ direction of the sense strand: a polynucleotide encoding the light chain of an antibody that binds to CTLA-4 - first early and late promoters - second early and late promoters - a polynucleotide encoding the heavy chain of an antibody that binds to CTLA-4 - a polynucleotide encoding the heavy chain of an antibody that binds to PD-1 - a first late promoter - a second late promoter - a polynucleotide encoding the light chain of an antibody that binds to PD-1.

[0032] In certain embodiments, the first early and late promoters are selected from the group consisting of P7.5, H5, poxvirus synthetic early / late promoter (pSE / L), or a combination thereof. In certain embodiments, the first early and late promoters are P7.5.

[0033] In certain embodiments, the second early and late promoter is selected from the group consisting of P7.5, H5, poxvirus synthetic early / late promoter (pSE / L), or a combination thereof. In certain embodiments, the second early and late promoter is pSE / L.

[0034] In certain embodiments, the first late promoter is selected from the group consisting of P11, P28, poxvirus synthetic late promoter (pSL), or a combination thereof. In certain embodiments, the first late promoter is P11.

[0035] In certain embodiments, the second late promoter is selected from the group consisting of P11, P28, poxvirus synthetic late promoter (pSL), or a combination thereof. In certain embodiments, the second late promoter is pSL.

[0036] In certain embodiments, the immune checkpoint inhibitor expressed by the first heterologous polynucleotide and the immune checkpoint inhibitor expressed by the second heterologous polynucleotide are expressed as separate antibodies or as chimeric bispecific antibodies of two different target antibodies.

[0037] In another aspect, the present invention relates to a pharmaceutical composition comprising the modified oncolytic virus of the present invention and a pharmaceutically acceptable carrier.

[0038] In another aspect, the present invention relates to a method for treating a tumor, comprising the step of administering to a subject an effective amount of the modified oncolytic virus of the present invention or the pharmaceutical composition of the present invention.

[0039] In certain embodiments, the subject is a human or a non-human mammal (eg, mouse, rat, rabbit, monkey, etc.).

[0040] In certain embodiments, the tumor is a solid tumor or a hematological tumor. In certain embodiments, the tumor is selected from the group consisting of melanoma, lung cancer (e.g., non-small cell lung cancer, small cell lung cancer), kidney cancer (e.g., renal cell carcinoma), lymphoma (e.g., Hodgkin's lymphoma), head and neck cancer (e.g., head and neck squamous cell carcinoma), bladder cancer, colorectal cancer, liver cancer (e.g., hepatocellular carcinoma), ovarian cancer, breast cancer (e.g., triple-negative breast cancer), cervical cancer, osteosarcoma, soft tissue sarcoma, follicular soft tissue sarcoma, neuroendocrine tumor, neuroendocrine carcinoma, malignant peripheral nerve sheath tumor, gallbladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, urothelial carcinoma, gastric cancer, brain glioma, or a combination thereof.

[0041] In certain embodiments, the route of administration is local administration. In certain embodiments, the route of administration is intratumoral injection.

[0042] In another aspect, the present invention relates to use of the modified oncolytic virus of the present invention or the pharmaceutical composition of the present invention in preparing a medicament for treating tumors.

[0043] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of the structure of WR-301.

[0045] FIG2 is a PCR amplification band of the exogenous gene inserted into WR-301.

[0046] FIG3 shows the ELISA results of PD-1 and CTLA-4 binding antibodies in cell harvest fluid infected with WR-301.

[0047] FIG4 shows the proliferation folds of WR-301 in MC38 and HCC1937 cells.

[0048] FIG5 shows the changes in viability of MC38 and HCC1937 cells infected with WR-301.

[0049] FIG6 shows the changes in tumor volume in CT26-hPDL1 humanized mice injected intratumorally with WR-301.

[0050] FIG7 shows the changes in the survival rate of CT26-hPDL1 humanized mice injected intratumorally with WR-301.

[0051] FIG8 shows the proliferation folds of WR-301 in A549 and HUVEC cells.

[0052] FIG9 shows the changes in the viability of 8 tumor cells after infection with WR-301 at different MOIs.

[0053] FIG10 shows the in vitro biological activity of the WR-301 expressed protein.

[0054] FIG11 shows the direct killing effect of WR-301 on tumors in PA-1 tumor-bearing nude mice.

[0055] FIG12 shows the dose-effect relationship of the tumor inhibition effect of WR-301 in tumor-bearing humanized mice.

[0056] FIG13 shows that WR-301 promotes intratumoral immune cell infiltration in tumor-bearing humanized mice.

[0057] FIG14 shows that WR-301 promotes anti-tumor cytokine expression in tumor-bearing humanized mice.

[0058] FIG15 shows the abscopal effects of WR-301 in tumor-bearing humanized mice.

[0059] Figure 16 shows that humanized mice cured after WR-301 administration are resistant to rechallenge of the same tumor.

[0060] FIG17 shows that WR-301 expressing dual full-length antibodies has better tumor inhibition effect in tumor-bearing humanized mice than recombinant viruses expressing non-full-length antibodies. DETAILED DESCRIPTION

[0061] In one aspect, the present invention relates to a modified oncolytic virus whose viral genome has a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding another different immune checkpoint inhibitor.

[0062] Oncolytic viruses

[0063] As used herein, the term "oncolytic virus" refers to a virus that can selectively replicate in tumor cells in vitro or in vivo and slow tumor cell growth or induce tumor cell death while having no effect or minimal effect on normal cells. In certain embodiments, the oncolytic virus contains a viral genome packaged into a virion (or viral particle) and is infectious (ie, capable of infecting and entering a host cell or subject). In certain embodiments, the oncolytic virus can be a DNA virus or an RNA virus, and can be in any suitable form, such as a DNA viral vector, an RNA viral vector, or a virion.

[0064] As used herein, the term "selective replication" refers to a significantly higher replication rate of an oncolytic virus in tumor cells compared to normal somatic cells. In certain embodiments, an oncolytic virus exhibits a proliferation rate in tumor cells that is at least 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold higher than that in normal somatic cells.

[0065] In certain embodiments, the oncolytic viruses of the present invention can selectively replicate in the following cells: liver tumor cells (e.g., Hepal-6 cells, Hep3B cells), breast tumor cells (e.g., MCF- cells, MDA-MB-231 cells, HCC1937 cells), tongue tumor cells (e.g., TCa8113 cells), adenoid cystic tumor cells (e.g., ACC-M cells), prostate tumor cells (e.g., LNCaP cells), immortalized human embryonic kidney cells (e.g., HEK293 cells), lung tumor cells (e.g., A549 cells), or cervical tumor cells (e.g., Hela cells), etc.

[0066] The oncolytic viruses of the present invention can be derived from poxviruses, adenoviruses (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAdl, H101, and AD5 / 3-D24-GMCSF), reoviruses (e.g., Reolysin), measles virus, herpes simplex virus (e.g., HSV, OncoVEX GMCSF), Newcastle disease virus (e.g., 73-T PV701 and HDV-HUJ strains, and those described in Phuangsab et al., 2001, Cancer Lett. 172(1):27-36; Lorence et al., 2007, Curr. Cancer Drug Targets. Targets 7(2):157-67; and Freeman et al., 2006, Mol. Ther. 13(1):221-8), a retrovirus (e.g., influenza virus), a myxoma virus, a rhabdovirus (e.g., vesicular stomatitis virus; those described in Stojdl et al., 2000, Nat. Med. 6(7):821-5 and Stojdl et al., 2003, Cancer Cell 4(4):263-75), a picornavirus (e.g., Seneca Valley virus; SW-001 and NTX-010), a coxsackievirus, or a parvovirus.

[0067] In certain embodiments, the oncolytic virus of the present invention is derived from poxvirus. As used herein, the term "poxvirus" refers to a virus belonging to the Poxviridae family (Poxviridae). In certain embodiments, poxvirus is a virus belonging to the Chordopoxviridae subfamily (Chordopoxviridae). In certain embodiments, poxvirus is a virus belonging to the Orthopoxvirinae subfamily (Ortho poxvirus). The genomes of various poxviruses, such as vaccinia virus (vaccinia virus), cowpox virus (cowpox virus), canarypox (Canarypox) virus, mousepox (Ectromelia) virus, myxoma virus genome sequences can be obtained in this area and dedicated databases, such as gene banks (Genbank) (accession numbers are NC_006998, NC_003663, NC_005309, NC_004105, NC_001132, respectively).

[0068] In certain embodiments, the oncolytic virus of the present invention is derived from a vaccinia virus. It is characterized by encoding a large number of viral enzymes and factors that enable the virus to replicate independently of the host cell mechanism. In certain embodiments, the vaccinia virus of the present invention is derived from Elstree, Copenhagen, Western Reserve strain or Wyeth strain. In certain embodiments, the vaccinia virus of the present invention is a Western Reserve strain. The Western Reserve strain has been well characterized, and its complete sequence is available on the NCBI website (www.ncbi.nlm.nih.gov) with accession number AY243312.

[0069] As used herein, the term "modified oncolytic virus" refers to an oncolytic virus that has been modified by introducing heterologous nucleic acids or proteins or changing natural nucleic acids or proteins. In certain embodiments, the modified oncolytic virus provided herein is genetically altered by the deletion and / or addition of nucleic acid sequences. In certain embodiments, the modified oncolytic virus provided herein includes a deletion of a thymidine kinase (TK) gene. In certain embodiments, the modified oncolytic virus provided herein includes the addition of nucleic acid sequences encoding anti-human PD-1 antibodies and anti-human CTLA-4 antibodies.

[0070] In certain embodiments, the modified oncolytic virus of the present invention is attenuated. In certain embodiments, in normal somatic cells, the modified oncolytic virus has a reduced (e.g., at least 90%, 80%, 70%, 60%, 50%) or undetectable toxicity compared to its wild-type counterpart.

[0071] Immune checkpoint inhibitors

[0072] The modified oncolytic virus genome provided herein comprises a first heterologous polynucleotide encoding an immune checkpoint inhibitor.

[0073] As used herein, the term "heterologous" means that the sequence is not endogenous to the wild-type virus.

[0074] As used herein, the term "encoded for" means capable of being transcribed into mRNA and / or translated into a peptide or protein.

[0075] As used herein, the term "immune checkpoint protein" refers to a protein that is directly or indirectly involved in an immune pathway that is important for preventing uncontrolled immune responses and, therefore, for maintaining self-tolerance and / or tissue protection. As used herein, one or more immune checkpoint regulators can independently act at any step of T cell-mediated immunity, including clonal selection of antigen-specific cells, T cell activation, proliferation, trafficking to sites of antigen and inflammation, execution of direct effector functions, and signaling through cytokines and membrane ligands.

[0076] As used herein, the term "immune checkpoint inhibitor" refers to a molecule that can negatively regulate the function of immune checkpoint proteins. Immune checkpoint inhibitors can be any of the molecular modalities known in the art, including but not limited to aptamers, mRNA, siRNA, microRNA, shRNA, peptides, antibodies, spherical nucleic acids, TALENs, zinc finger nucleases, and CRISPR / Cas9.

[0077] In certain embodiments, immune checkpoint inhibitors are natural or engineered antagonists of inhibitory immune checkpoint molecules, including, for example, ligands for CTLA-4 and ligands for PD-1.

[0078] In certain embodiments, the immune checkpoint inhibitor is an antibody (e.g., antagonist antibody) selected from the group consisting of anti-PD-1 (e.g., Nivolumab, Pilizumab, Pembrolizumab, BMS-936559, BMS-936558, atezolizumab, Lambrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, TSR- 042 or ANB011), anti-PD-L1 (e.g., KY-1003, MCLA-145, atezolizumab, MEDI-4736, MSB0010718C, STI-A1010, MPDL3280A, Dapirolizumab CDP-7657, MEDI-4920, or those described in PCT / US2001 / 020964), anti-PD-L2, anti-(both PD-L1 and PD-L2) (e.g., AUR-012 and AMP-224), anti-CT LA-4 (e.g., Ipilimumab, Tremelimumab, or KAHR-102), anti-IDO (e.g., D1-methyl-tryptophan (Lunate)), anti-KIR (e.g., Lirilumab, IPH2101, or IPH4102), anti-LAG3 (e.g., BMS-986016, IMP701, IMP321, or C9B7W), anti-TIM3 (e.g., F38-2E2, or ENUM005), anti-VISTA (e.g., Such as VA.F6) anti-BTLA (e.g., AF3354), anti-CD73 (e.g., OSU-HDAC42 or MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a or 8H9), anti-A2aR, anti-B7-1, anti-B7-H3 (e.g., MGA271), anti-B7-H4, anti-(both B7-H3 and B7-H4), anti-CD52 (e.g., alemtuzumab), anti-IL-10, anti-IL-35, anti-MICA (e.g., IPH43), and anti-CD39.

[0079] In certain embodiments, the immune checkpoint inhibitor is a full-length antibody or an antigen-binding fragment thereof that is capable of specifically binding to an immune checkpoint protein such as PD-1 and / or CTLA-4.

[0080] In certain embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or a PD-1 inhibitor. In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or an inhibitor of CTLA-4.

[0081] PD-1 inhibitors

[0082] In certain embodiments, the first heterologous polynucleotide of the present invention encodes a PD-1 inhibitor.

[0083] As used herein, the term "PD-1" refers to a programmed cell death protein that belongs to the immunoglobulin superfamily and acts as a co-inhibitory receptor to negatively regulate the immune system. PD-1 is a member of the CD28 / CTLA-4 family and has two known ligands, including PD-L1 and PD-L2. The representative amino acid sequence of human PD-1 is disclosed in GenBank Accession No. NP_005009.2, and the representative nucleic acid sequence encoding human PD-1 is shown in GenBank Accession No. NM_005018.2.

[0084] PD-1 negatively regulates T cell activation, and this inhibitory function is associated with the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (Parry et al., 2005, Mol. Cell. Biol. 25:9543-53). Disruption of this inhibitory function of PD-1 can cause autoimmunity. The persistent negative signal generated by PD-1 is implicated in many pathological conditions, such as tumor immune evasion and T cell dysfunction in chronic viral infection.

[0085] A PD-1 inhibitor can be any agent that inhibits PD-1 activity, such as those that reduce PD-1 activity by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

[0086] Factors that can reduce PD-1 activity include: inhibition of the binding between the functional protein and its ligand (e.g., the binding between PD-1 and PD-L1), inhibition of its biological activation (e.g., activation of PD-1), and / or reduction of its expression level (e.g., PD-1 expression level).

[0087] In certain embodiments, the PD-1 inhibitor is an antibody (eg, an antagonist antibody) that can specifically bind to PD-1.

[0088] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human and / or monkey PD-1 with a binding affinity (KD) ≤ 10 -6 M (e.g. ≤5×10 -7 M, ≤2×10 - 7 M, ≤10-7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤2×10 -9 M, ≤10 -9 M, ≤10 -10 As used herein, KD refers to the ratio of the off-rate to the on-rate (koff / kon), which can be determined using surface plasmon resonance methods, for example, using an instrument such as Biacore.

[0089] In certain embodiments, the PD-1 inhibitor is a full-length monoclonal antibody directed against PD-1.

[0090] In certain embodiments, the PD-1 antibody specifically binds to SEQ ID NO: 1.

[0091] In certain embodiments, the PD-1 antibody or antigen-binding fragment thereof comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 2, or a homologous sequence thereof with at least 90% sequence identity. In certain embodiments, the first heavy chain Fab region comprises the amino acid sequence of SEQ ID NO: 3, or a homologous sequence thereof with at least 90% sequence identity. In certain embodiments, the first antibody or antigen-binding fragment thereof comprises a first heavy chain CDR region comprising the amino acid sequence of HCDR1 set forth in SEQ ID NO: 4, the amino acid sequence of HCDR2 set forth in SEQ ID NO: 5, and the amino acid sequence of HCDR3 set forth in SEQ ID NO: 6.

[0092] In certain embodiments, the PD-1 antibody or antigen-binding fragment thereof comprises another first heavy chain comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the Fab region of the another first heavy chain comprises the amino acid sequence of SEQ ID NO: 12 and a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the CDR region of the another first heavy chain comprises the amino acid sequence of HCDR1 set forth in SEQ ID NO: 13, the HCDR2 set forth in SEQ ID NO: 14, and the HCDR3 set forth in SEQ ID NO: 15.

[0093] As used herein with respect to amino acid sequences, the term "identity" refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the candidate sequence with the reference sequence and introducing gaps, if necessary, to maximize the number of identical amino acids. Conservative substitutions of amino acid residues are not considered identical residues. Alignment for the purpose of determining percent amino acid sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997)). MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software. Those skilled in the art can use the default parameters provided by the tools, or can customize the parameters suitable for the alignment, for example, by selecting an appropriate algorithm.

[0094] In certain embodiments, the first heavy chain Fab region comprises SEQ ID NO: 3 or a homologous sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In certain embodiments, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity thereto.

[0095] In certain embodiments, the first heavy chain Fab region comprises SEQ ID NO: 12 or a homologous sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In certain embodiments, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 11 or a homologous sequence having at least 90% sequence identity thereto.

[0096] In certain embodiments, the PD-1 antibody or antigen-binding fragment thereof further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 7 or a homologous sequence thereof having at least 90% sequence identity. In certain embodiments, the first antibody or antigen-binding fragment thereof comprises a first light chain CDR region comprising the amino acid sequence of LCDR1 set forth in SEQ ID NO: 8, LCDR2 set forth in SEQ ID NO: 9, and LCDR3 set forth in SEQ ID NO: 10.

[0097] In certain embodiments, the PD-1 antibody or antigen-binding fragment thereof further comprises another first light chain comprising an amino acid sequence having SEQ ID NO: 16 or a homologous sequence thereof having at least 90% sequence identity. In certain embodiments, the another first light chain CDR region comprises an amino acid sequence having LCDR1 as set forth in SEQ ID NO: 17, LCDR2 as set forth in SEQ ID NO: 18, and LCDR3 as set forth in SEQ ID NO: 19.

[0098] CTLA-4 inhibitors

[0099] In certain embodiments, the second heterologous polynucleotide of the present invention encodes a CTLA-4 inhibitor.

[0100] As used herein, the term "CTLA-4" refers to cytotoxic T lymphocyte-associated protein 4, also known as CD152 (cluster of differentiation 152), a protein receptor that acts as an immune checkpoint and downregulates the immune response. CTLA-4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation, a phenomenon that is particularly significant in cancer. When bound to CD80 or CD86 on the surface of antigen-presenting cells, it acts as an "off" switch. The representative amino acid sequence of human CTLA-4 is disclosed in Gene Bank Accession No.: NP_033973.2, and the representative nucleic acid sequence encoding human PD-1 is shown in Gene Bank Accession No.: NM_005214.5.

[0101] CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. CTLA-4 is also found on regulatory T cells (Tregs) and contributes to their inhibitory function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4. Anti-CTLA-4 antibodies bind to CTLA-4 molecules with high affinity, mediating Treg depletion or functional blockade, thereby enhancing T cell activation and immune responses to cancer. The effects of CTLA-4 blockade can be mediated by multiple mechanisms: preventing transendocytosis, increasing CD80 / CD86 levels on antigen-presenting cells (APCs), and enhancing T cell activation responses; directly causing Treg cytotoxicity; and triggering FcR-IV-mediated antibody-dependent cellular cytotoxicity (ADCC) of intratumoral macrophages.

[0102] A CTLA-4 inhibitor can be any agent that inhibits CTLA-4 activity, such as those that reduce CTLA-4 activity by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

[0103] Factors that can reduce CTLA-4 activity: Inhibition of binding between the functional protein and its ligand (e.g., binding between CTLA-4 and CD80 or CD86), inhibition of its biological activation (e.g., activation of CTLA-4), and / or reduction of its expression level (e.g., CTLA-4 expression level)

[0104] In certain embodiments, the CTLA-4 inhibitor is an antibody (eg, an antagonist antibody) that specifically binds to CTLA-4.

[0105] In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human and / or monkey CTLA-4 with a binding affinity (KD) ≤ 10 -6 M (e.g. ≤5×10 -7 M, ≤2×10 -7 M, ≤10 -7 M, ≤5×10 -8 M, ≤2×10 -8 M, ≤10 -8 M, ≤5×10 -9 M, ≤2×10 -9 M, ≤10 -9 M, ≤10 -10 M).

[0106] In certain embodiments, the CTLA-4 inhibitor is a full-length monoclonal antibody directed against CTLA-4.

[0107] In certain embodiments, the CTLA-4 antibody specifically binds SEQ ID NO:20.

[0108] In certain embodiments, the CTLA-4 antibody or antigen-binding fragment thereof comprises a second heavy chain, wherein the second antibody or antigen-binding fragment thereof comprises a second heavy chain comprising an amino acid sequence having SEQ ID NO: 21 or a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the second antibody or antigen-binding fragment thereof comprises a second heavy chain Fab region comprising an amino acid sequence having SEQ ID NO: 22 or a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the second antibody or antigen-binding fragment thereof comprises a second heavy chain CDR region comprising the HCDR1 set forth in SEQ ID NO: 23, the HCDR2 set forth in SEQ ID NO: 24, and the HCDR3 amino acid sequence set forth in SEQ ID NO: 25.

[0109] In certain embodiments, the second heavy chain Fab region comprises SEQ ID NO: 22 or a homologous sequence having at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In certain embodiments, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 21 or a homologous sequence having at least 90% sequence identity thereto.

[0110] In certain embodiments, the CTLA-4 antibody antigen-binding fragment further comprises a second light chain, wherein the second light chain comprises an amino acid sequence having SEQ ID NO: 26 or a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the second antibody or antigen-binding fragment thereof comprises a second light chain CDR region comprising the LCDR1 set forth in SEQ ID NO: 27, the LCDR2 set forth in SEQ ID NO: 28, and the LCDR3 amino acid sequence set forth in SEQ ID NO: 29.

[0111] Antibody

[0112] As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds to a specific antigen. A natural complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ and μ, and mammalian light chains are classified as λ or κ. Antibodies are "Y" shaped, wherein the stem of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises the variable region and the first constant region of a single heavy chain combined with the variable region and constant region of a single light chain, wherein the first constant region of the heavy chain is connected to the second constant region by a hinge region. The variable regions of the light and heavy chains are responsible for generating antigen binding specificity. The variable regions in both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (the light (L) chain CDRs include LCDR1, LCDR2, and LCDR3, and the heavy (H) chain CDRs include HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (for details, see Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol. Biol. Dec 5;186(3):651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature Dec 21-28;342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold that supports the structure of the variable region. The constant regions of the heavy and light chains are not associated with antigen binding specificity, but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0113] As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed by a portion of an antibody comprising one or more CDRs, but does not comprise the entire antibody structure. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single-domain antibodies, and nanobodies. Antigen-binding fragments are capable of binding to the same antigen as the parent antibody.

[0114] As used herein, the term "Fab" refers to the portion of an antibody consisting of a single heavy chain (both the variable region and the first constant region) and a single light chain (both the variable region and the constant region) bound by disulfide bonds.

[0115] As used herein, the term "Fab'" refers to the Fab fragment comprising a portion of the hinge region.

[0116] As used herein, the term "F(ab')2" refers to a dimer of Fab'.

[0117] As used herein, the term "Fv" refers to an Fv fragment consisting of the variable region of a single light chain and the variable region of a single heavy chain.

[0118] As used herein, the term "single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region and a heavy chain variable region linked to each other directly or through a peptide linker sequence (see, e.g., Huston JS et al., Proc Natl Acad Sci USA, 85:5879 (1988)).

[0119] As used herein, the term "scFv dimer" refers to a polymer formed by two scFvs.

[0120] The term "camelized single domain antibody", also called "heavy chain antibody" or "HCAb" (heavy chain antibody only) refers to an antibody that contains two heavy chain variable regions but no light chain (see, e.g., Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; and U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae family (camels, dromedaries, and llamas). Although they do not contain light chains, camelized antibodies have a true antigen-binding repertoire (see Hamers-Casterman C. et al., Nature 363(6428):446-8 (1993); Nguyen VK. et al., "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation," Immunogenetics. 54(1):39-47 (2002); and Nguyen VK. et al., Immunology. 109(1):93-101 (2003), which are incorporated herein by reference in their entirety). As used herein, the term "nanobody" refers to an antibody consisting of the heavy chain variable region from a heavy chain antibody and two constant regions, CH2 and CH3.

[0121] In certain embodiments, the antibodies provided herein are fully human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies or rabbit antibodies. In certain embodiments, the antibodies provided herein are polyclonal antibodies, monoclonal antibodies or recombinant antibodies. In certain embodiments, the antibodies provided herein are monospecific antibodies, bispecific antibodies or multispecific antibodies. In certain embodiments, the antibodies provided herein may be further labeled. In certain embodiments, the antibodies or their antigen-binding fragments are fully human antibodies, which are optionally produced by transgenic rats, such as transgenic rats with inactivated endogenous rat immunoglobulin gene expression, and carrying recombinant human immunoglobulin loci with J locus deletion and C-κ mutations, and the antibodies may also be expressed by engineered cells (e.g., CHO cells).

[0122] As used herein, with respect to an antibody or antigen-binding fragment, the term "fully human" means that the amino acid sequence of the antibody or antigen-binding fragment corresponds to the amino acid sequence of an antibody produced by a human or human immune cell, or derived from a non-human source, such as a transgenic non-human animal utilizing a human antibody repertoire or other human antibody encoding sequences.

[0123] As used herein, with respect to antibodies or antigen-binding fragments, the term "humanized" refers to an antibody or antigen-binding fragment that comprises CDRs derived from non-human animals, FR regions derived from humans, and, where applicable, constant regions derived from humans. In certain embodiments, humanized antibodies or antigen-binding fragments are suitable for use as therapeutic agents in humans because they have reduced immunogenicity. In certain embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In certain embodiments, the humanized antibody or antigen-binding fragment consists essentially entirely of human sequences, except for the CDR sequences, which are non-human sequences.

[0124] As used herein, with reference to antibodies or antigen-binding fragments, the term "chimeric" refers to antibodies or antigen-binding fragments that have a portion of the heavy and / or light chains derived from one species and the remainder of the heavy and / or light chains derived from a different species.

[0125] In certain embodiments, a chimeric antibody may comprise a constant region derived from a human and a variable region from a non-human species, such as from a mouse or rabbit.

[0126] As used herein, with respect to amino acid sequences, the term "conservative substitution" refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues having acidic side chains (e.g., Asp, Glu), between amino acids having basic side chains (e.g., His, Lys, and Arg), or between residues having aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore, the biological activity of the protein can be retained.

[0127] polynucleotides

[0128] In certain embodiments, the modified oncolytic virus of the present invention contains a first heterologous polynucleotide encoding an inhibitory antibody or an antigen-binding fragment thereof that specifically binds to PD-1, and a second heterologous polynucleotide encoding an inhibitory antibody or an antigen-binding fragment thereof that specifically binds to CTLA-4.

[0129] As used herein, the term "polynucleotide" or "nucleic acid" refers to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or mixed ribonucleic acid-deoxyribonucleic acid, such as a DNA-RNA hybrid. A polynucleotide or nucleic acid can be single-stranded or double-stranded DNA or RNA or a DNA-RNA hybrid. A polynucleotide or nucleic acid can be linear or circular. In certain embodiments, when the virus is a DNA virus, both the first and second heterologous polynucleotides are DNA; or when the virus is an RNA virus, both the first and second heterologous polynucleotides are RNA. In certain embodiments, both the first heterologous polynucleotide and the second heterologous polynucleotide are double-stranded DNA.

[0130] The first heterologous polynucleotide and the second heterologous polynucleotide can be introduced into the modified oncolytic virus using conventional methods known in the art, for example, by being synthesized by polymerase chain reaction (PCR) and connected to the viral genome with compatible restriction ends. For more details, see, for example, Sambrook et al. "Molecular Cloning: A Laboratory Manual" (Cold Spring Harbor Laboratory, NY) (1989), which is incorporated herein by reference in its entirety.

[0131] In certain embodiments, the modified oncolytic virus comprises the following elements in frame in the 5′ to 3′ direction of the sense strand: a polynucleotide encoding the light chain of an antibody that binds to CTLA-4 - first early and late promoters - second early and late promoters - a polynucleotide encoding the heavy chain of an antibody that binds to CTLA-4 - a polynucleotide encoding the heavy chain of an antibody that binds to PD-1 - a first late promoter - a second late promoter - a polynucleotide encoding the light chain of an antibody that binds to PD-1.

[0132] In certain embodiments, the immune checkpoint inhibitor expressed by the first heterologous polynucleotide and the immune checkpoint inhibitor expressed by the second heterologous polynucleotide are expressed as separate proteins. In other words, they are not expressed as fusion proteins and are not connected to each other (whether covalently linked or through a linker). In certain embodiments, the immune checkpoint inhibitor expressed by the first and second heterologous polynucleotides is not fused with any other protein.

[0133] In certain embodiments, in addition to the first heterologous polynucleotide and the second heterologous polynucleotide, the modified oncolytic virus does not include any other heterologous polynucleotides encoding immune checkpoint inhibitors or immune activators. In certain embodiments, in addition to the first heterologous polynucleotide and the second heterologous polynucleotide, the modified oncolytic virus does not include any heterologous polynucleotides encoding other proteins.

[0134] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0135] Example 1: Virus Construction

[0136] The wild-type WR virus strain was obtained from ATCC (VR-1354). As shown in Figure 1, the J1R-antiCTLA4-antiPD1-J3R nucleic acid sequence was synthesized by a biotechnology company and inserted into the pUC57 vector plasmid. The recombinant plasmid was transfected into HeLa cells infected with wild-type WR to obtain a recombinant oncolytic virus with two exogenous genes inserted and TK deleted, named WR-301.

[0137] WR-301 was subjected to three rounds of single plaque screening using 143B cells, then amplified using HeLa cells, and finally purified using sucrose density gradient centrifugation to obtain a WR-301 virus with a titer of approximately 2*10^9 pfu / mL.

[0138] Example 2: Virus characterization study

[0139] PCR sequencing

[0140] To ensure that the recombinant viral genome carries the designed exogenous sequence, primers P1 and P2 were set up; their positions are shown in Figure 1. This primer pair can amplify and distinguish between the wild-type WR strain sequence containing TK (2.75 kb) and the recombinant viral sequence containing the exogenous gene (7 kb). The PCR amplification results for WR-301 are shown in Figure 2. The band representing WR-301 is visible, while the band representing wild-type WR is absent.

[0141] The PCR products were then sequenced, and alignment showed that the WR-301 amplified sequence was identical to the designed nucleic acid sequence.

[0142] ELISA

[0143] To verify that WR-301 can express functional anti-PD-1 and anti-CTLA-4 antibodies, 96-well plates were coated with PBS-diluted PD-1 or CTLA-4 recombinant proteins at a concentration of 1 mg / ml. After blocking with 5% skim milk powder, WR-301-infected cell harvests at 10-, 100-, and 1000-fold dilutions were added. The plates were incubated at 37°C for one hour, washed four times with PBST, and HRP-labeled rabbit anti-human secondary antibody was added. The plates were incubated at 37°C for one hour, washed four times with PBST, and TMB was added for color development. The reaction was terminated after 20 minutes at room temperature, and the absorbance at 450 nm and 630 nm was read using a microplate reader.

[0144] The analysis was performed using supernatants from HeLa cells infected with WR-301 at a 0.5 MOI for 72 hours. As shown in Figure 3, WR-301 can express antibodies that specifically bind to PD-1 and CTLA-4, and this binding is concentration-dependent.

[0145] Example 3: In vitro viral studies

[0146] The following studies were conducted to confirm the proliferation ability of WR-301 in tumor cells and its ability to kill tumor cells.

[0147] proliferation

[0148] MC38 (mouse colon cancer cells) and HCC1937 (human breast cancer cells) were infected with WR-301 at an MOI of 0.05. Samples were collected at 24, 48, and 72 hours, and viral DNA copy number was measured. The viral load at different times relative to the initial inoculum was calculated. As shown in Figure 4, WR-301 significantly proliferated in both cancer cells, with a stronger proliferation ability in human cancer cells than in mouse cancer cells.

[0149] Killing

[0150] MC38 (mouse colon cancer cells) and HCC1937 (human breast cancer cells) were infected with WR-301 at MOIs of 0.1, 1, and 10, respectively. Samples were collected at 24, 48, 72, and 96 hours, and cell viability was assessed using CCK-8 assays, a standard method known in the art. As shown in Figure 5, WR-301 at different MOIs showed significant cytotoxicity against HCC1937 cells. WR-301 at an MOI of 0.1 showed no significant cytotoxicity against MC38 cells, while MOIs of 1 and 10 showed significant cytotoxicity.

[0151] Example 4: Virus in vivo studies

[0152] Eighteen humanized mice (BALB / c-hPD1hCTLA4) were implanted with humanized CT26 cells (mouse colon cancer cells, CT26-hPDL1, 1E6 per mouse). 3 Around 24 h, mice were randomly divided into three groups: PBS, WR-GFP (TK region of wild-type WR virus was replaced by GFP), and WR-301, with 6 mice in each group. 4E8 pfu of different viruses were injected intratumorally on days 0, 5, and 10, and the weight and tumor volume of mice were continuously monitored. The tumor volume was 2000 mm. 3 Be kind.

[0153] The changes in tumor volume and survival rate of mice are shown in Figures 6 and 7. Compared with the other two groups, the tumor volume of the WR-301 group was much smaller and the survival time was significantly prolonged.

[0154] Example 5: In vitro virus expansion studies

[0155] The following studies were conducted to confirm the proliferation ability of WR-301 in more types of tumor cells, the killing ability of WR-301 on more types of tumor cells, and the biological activity of the WR-301 expressed protein.

[0156] proliferation

[0157] WR-301 was used at an MOI of 0.05 to infect tumor cells A549 (human non-small cell lung cancer cells) and normal cells HUVEC (human umbilical vein endothelial cells). Samples were collected 48 hours later and the viral DNA copy number was determined by qPCR. The viral load in the cells at the time of collection was calculated as the multiple of the initial inoculum. The results, as shown in Figure 8, show that WR-301 increased 250.98-fold in tumor cells A549, while it only increased 20.67-fold in normal cells HUVEC, a difference of more than 10-fold, indicating that WR-301 can selectively replicate at high levels in tumor cells.

[0158] Killing

[0159] Eight tumor cell types were infected with gradient dilutions of WR-301. Samples were collected 72 hours later and cell viability was detected using CCK-8 (a conventional method known in the art). The tumor cells were MC38 (mouse colon cancer cells), CT26 (mouse colon cancer cells), 4T1 (mouse breast cancer cells), HeLa (human cervical cancer cells), U2OS (human osteosarcoma cells), HCC1937 (human breast cancer cells), A549 (human non-small cell lung cancer cells), and PA-1 (human ovarian teratoma cells). The MOI of infection was 0, 0.01, 0.04, 0.12, 0.37, 1.11, 3.33, and 10. As shown in Figure 9, WR-301 had a significant killing effect on all eight tumor cell types.

[0160] Biological activity

[0161] The culture supernatant of HeLa cells infected with WR-301 was collected and purified by Protein A affinity chromatography. The biological activities of anti-PD-1 and anti-CTLA-4 in the purified proteins expressed by WR-301 were respectively detected using reporter gene activity assays (conventional methods known in the art). As shown in Figure 10, the anti-PD-1 and anti-CTLA-4 expressed by WR-301 had biological activities that were substantially consistent with those of the positive control proteins.

[0162] Example 6: Virus expansion studies in vivo

[0163] The following studies were conducted to confirm the pharmacological efficacy of WR-301 in mice.

[0164] Direct killing effect in nude mouse model

[0165] Each BALB / c nude mouse was subcutaneously inoculated with 1E7 PA-1 cells (human ovarian teratoma cells). 3 Around 6:00 pfu (1E6 pfu / mouse), 30 mice were randomly divided into five groups: PBS (negative control), WR-GFP (vector control virus: wild-type WR virus with the TK gene replaced by GFP), low-dose WR-301 (1E4 pfu / mouse), medium-dose WR-301 (1E5 pfu / mouse), and high-dose WR-301 (1E6 pfu / mouse), with six mice in each group. Each group received intratumoral injections of the corresponding virus on days 0, 5, and 10. Tumor volume and body weight were measured 2-3 times per week. As shown in Figure 11, compared to the PBS negative control, all three WR-GFP and WR-301 dose groups effectively inhibited tumor growth. Because BALB / c nude mice lack mature T lymphocytes and cannot generate normal T cell immunity, the virus relies primarily on its own replication to directly kill tumor cells. These results demonstrate that WR-301 can directly kill tumor cells and inhibit tumor growth in nude mice.

[0166] Dose-effect relationship in humanized mouse models

[0167] BALB / c-hPD1hCTLA4 mice (humanized mice with dual PD1 and CTLA4 targets) were subcutaneously inoculated with CT26-hPDL1 cells (humanized mouse colon cancer cells expressing PDL1). 3 Around 10 days post-inoculation, 30 mice were randomly divided into five groups: PBS, WR-GFP (total amount of 1E8 pfu / mouse), low-dose WR-301 (total amount of 1E6 pfu / mouse), medium-dose WR-301 (total amount of 1E7 pfu / mouse), and high-dose WR-301 (total amount of 1E8 pfu / mouse), with 6 mice in each group. Each group received intratumoral injections of the corresponding virus on days 0, 5, and 10. Tumor volume and body weight were measured 2-3 times per week. As shown in Figure 12, the WR-GFP and low-dose WR-301 groups showed no significant differences compared to the PBS group. Both the medium-dose and high-dose WR-301 groups effectively inhibited tumor growth, with the high-dose group exhibiting a superior inhibitory effect compared to the medium-dose group. This result demonstrates that the tumor-suppressing effect of WR-301 is positively correlated with the administered dose, demonstrating a dose-response relationship. Furthermore, compared to WR-GFP, WR-301 can effectively inhibit tumor growth at a lower dose.

[0168] Immune activation effect

[0169] BALB / c-hPD1hCTLA4 mice were subcutaneously inoculated with CT26-hPDL1 cells. When the tumors grew to 100 mm 3 Around 6:00 pfu (approximately 100 μg / mL) of 18 mice were randomly divided into three groups: PBS, WR-GFP, and WR-301, with 6 mice in each group. Each group received intratumoral injections of the corresponding virus on days 0 and 5, respectively, at a total volume of 6.7E7 pfu / mouse. The mice were euthanized on day 7, and their tumors were harvested for flow cytometry and quantitative PCR analysis of tumor-immune cytokine mRNA. The flow cytometry results, shown in Figure 13, showed that the numbers of infiltrating CD45+ leukocytes, CD3+ T cells, CD4+ T cells, and CD8+ T cells in the tumors of mice in the WR-301 group were significantly higher than those in the PBS group. Cytokine analysis, shown in Figure 14, showed that the levels of the Th1 cytokines IL-2 and IFNγ in the tumors of mice in the WR-301 group were significantly higher than those in the PBS group. Granzyme B and perforin levels were also significantly higher than those in the PBS group. However, the levels of the Th2 cytokines IL-10 and TGFβ1 did not differ significantly from those in the PBS group. Th1 cytokines are associated with immune activation, Th2 cytokines are associated with immunosuppression, and granzyme B and perforin reflect the function of cytotoxic lymphocytes. These results indicate that WR-301 can effectively activate anti-tumor immune responses in mice.

[0170] distal effects

[0171] C57-hPD1hCTLA4 mice were subcutaneously inoculated with MC38-hPDL1 cells on both sides. When the tumors on both sides grew to 100 mm, 3 Around 6:00 pfu (1E8 pfu) was injected intratumorally in each group of mice on days 0, 5, and 10, respectively. Tumor volumes were measured bilaterally two to three times weekly. As shown in Figure 15, tumor volumes on both the treated and non-treated sides of the WR-301 group were significantly smaller than those in the PBS group, indicating that WR-301 also inhibits the growth of distal tumors, not at the administration site.

[0172] Tumor re-stimulation studies

[0173] BALB / c-hPD1hCTLA4 mice were subcutaneously inoculated with CT26-hPDL1 cells. When the tumors grew to 100 mm 3Around 2:00 pfu / mouse, WR-301 was injected intratumorally three times at a total dose of 4E8 pfu / mouse. Forty days after the initial administration, mice with completely regressed tumors (15 mice in this experiment) were subcutaneously inoculated with CT26-hPDL1 again. Blank mice, which had not been previously inoculated with tumor cells or given the drug, were used as controls. Thirty-five days later, mice without tumors were subcutaneously inoculated with 4T1 cells (mouse breast cancer cells), and blank controls were also used. Tumor volume in the mice was measured 2-3 times per week. As shown in Figure 16, all mice with completely regressed tumors did not develop tumors after re-inoculation with CT26-hPDL1 cells, while tumors in the control mice grew normally. Following inoculation with 4T1 cells, tumors in both the experimental and control groups grew normally. This result demonstrates that WR-301, while inhibiting tumor growth, stimulates the mouse immune system to produce a long-term, specific immune response to this tumor, effectively preventing recurrence of the same tumor after cure.

[0174] Example 7: Comparison of drug efficacy of different virus configurations

[0175] Two viruses, WR-CF and WR-CFPF, were constructed using the same method as WR-301. WR-CF replaces the TK region of wild-type WR with the full-length anti-PD-1 and anti-CTLA-4 F(ab')2 regions. WR-CFPF replaces the TK region of wild-type WR with the F(ab')2 regions of anti-PD-1 and anti-CTLA-4 F(ab')2 regions.

[0176] BALB / c-hPD1hCTLA4 mice were subcutaneously inoculated with CT26-hPDL1 cells. When the tumors grew to 100 mm 3 Around 400 mice were randomly divided into groups of 6. The corresponding viruses were injected intratumorally on days 0, 5, and 10, with a total of 4E8 pfu per mouse. Tumor volume was measured 2-3 times per week. As shown in Figure 17, both WR-CF and WR-CFPF were less effective at inhibiting tumor growth than WR-301, indicating that viruses expressing dual full-length antibodies have a more potent anti-tumor effect in mice.

[0177] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A modified oncolytic virus comprising a viral genome having a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding another different immune checkpoint inhibitor; wherein the immune checkpoint inhibitor is a first antibody or an antigen-binding fragment thereof that can specifically bind to an immune checkpoint protein, and a second antibody or an antigen-binding fragment thereof that can specifically bind to another different immune checkpoint protein; preferably, wherein the first antibody or its antigen-binding fragment and / or the second antibody or its antigen-binding fragment is a full-length antibody.

2. The modified oncolytic virus according to claim 1, wherein the oncolytic virus is selected from the group consisting of vaccinia virus, adenovirus, reovirus, measles virus, herpes simplex virus, Semliki Forest virus, Venezuelan equine encephalitis, parvovirus, chicken anemia virus, measles virus, coxsackievirus, vesicular stomatitis virus, Seneca Valley virus, Maraba virus, and Newcastle disease virus.

3. The modified oncolytic virus according to claim 2, wherein the oncolytic virus is a vaccinia virus.

4. The modified oncolytic virus according to claim 3, wherein the oncolytic virus is derived from a Western Reserve strain.

5. The modified oncolytic virus according to claim 4, wherein the viral genome comprises at least one deletion or disruption that renders the virus attenuated or selectively replicates in tumor cells, in, The deletion or disruption is in an open reading frame (ORF) encoding an enzyme essential for viral replication, Wherein, the enzyme is thymidine kinase.

6. The modified oncolytic virus according to claim 1, wherein the immune checkpoint proteins are PD-1 and CTLA-4.

7. The modified oncolytic virus of claim 6, wherein the first immune checkpoint inhibitor is a full-length antibody that specifically binds to PD-1, and the second immune checkpoint inhibitor is a full-length antibody that specifically binds to CTLA-4.

8. The modified oncolytic virus according to claim 6, wherein the first antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO:

1.

9. The modified oncolytic virus of claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain comprising an amino acid sequence having SEQ ID NO: 2 or a homologous sequence having at least 90% sequence identity thereto.

10. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain Fab region, and the first heavy chain Fab region comprises an amino acid sequence having SEQ ID NO: 3 or a homologous sequence having at least 90% sequence identity thereto.

11. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain CDR region comprising the HCDR1 specified in SEQ ID NO: 4, the HCDR2 specified in SEQ ID NO: 5, and the HCDR3 amino acid sequence specified in SEQ ID NO:

6.

12. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof further comprises a first light chain comprising an amino acid sequence having SEQ ID NO: 7 or a homologous sequence having at least 90% sequence identity thereto.

13. The modified oncolytic virus of claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first light chain CDR region comprising LCDR1 as specified in SEQ ID NO: 8, LCDR2 as specified in SEQ ID NO: 9, and LCDR3 as specified in SEQ ID NO:

10.

14. The modified oncolytic virus of claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain comprising an amino acid sequence having SEQ ID NO: 11 or a homologous sequence having at least 90% sequence identity thereto.

15. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain Fab region comprising an amino acid sequence having SEQ ID NO: 12 or a homologous sequence having at least 90% sequence identity thereto.

16. The modified oncolytic virus of claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain CDR region comprising the HCDR1 specified in SEQ ID NO: 13, the HCDR2 specified in SEQ ID NO: 14, and the HCDR3 amino acid sequence specified in SEQ ID NO:

15.

17. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof further comprises a first light chain comprising an amino acid sequence having SEQ ID NO: 16 or a homologous sequence having at least 90% sequence identity thereto.

18. The modified oncolytic virus of claim 8, wherein the first antibody or antigen-binding fragment thereof comprises a first light chain CDR region comprising LCDR1 as specified in SEQ ID NO: 17, LCDR2 as specified in SEQ ID NO: 18, and LCDR3 as specified in SEQ ID NO:

19.

19. The modified oncolytic virus of claim 6, wherein the second antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO:

20.

20. The modified oncolytic virus of claim 19, wherein the second antibody or antigen-binding fragment thereof comprises a second heavy chain comprising an amino acid sequence having SEQ ID NO: 21 or a homologous sequence having at least 90% sequence identity thereto.

21. The modified oncolytic virus of claim 19, wherein the second antibody or antigen-binding fragment thereof comprises a second heavy chain Fab region comprising an amino acid sequence having SEQ ID NO: 22 or a homologous sequence having at least 90% sequence identity thereto.

22. The modified oncolytic virus of claim 19, wherein the second antibody or antigen-binding fragment thereof comprises a second heavy chain CDR region comprising a HCDR1 specified in SEQ ID NO: 23, a HCDR2 specified in SEQ ID NO: 24, and a HCDR3 amino acid sequence specified in SEQ ID NO:

25.

23. The modified oncolytic virus of claim 19, wherein the second antibody or antigen-binding fragment thereof further comprises a second light chain comprising an amino acid sequence having SEQ ID NO: 26 or a homologous sequence having at least 90% sequence identity thereto.

24. The modified oncolytic virus of claim 19, wherein the second antibody or antigen-binding fragment thereof comprises a second light chain CDR region comprising the LCDR1 specified in SEQ ID NO: 27, the LCDR2 specified in SEQ ID NO: 28, and the LCDR3 amino acid sequence specified in SEQ ID NO:

29.

25. The modified oncolytic virus of claim 1, wherein the immune checkpoint inhibitor expressed by the first heterologous polynucleotide and the immune checkpoint inhibitor expressed by the second heterologous polynucleotide are expressed as separate antibodies or chimeric bispecific antibodies of two different target antibodies.

26. A pharmaceutical composition comprising the modified oncolytic virus according to any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

27. A method for treating a tumor, comprising the step of administering to a subject an effective amount of the modified oncolytic virus according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26.

28. The method of claim 27, wherein the subject is a human or a non-human mammal (such as a mouse, rat, rabbit, monkey, etc.).

29. Use of the modified oncolytic virus according to any one of claims 1 to 25 or the pharmaceutical composition according to claim 26 in the preparation of a medicament for treating tumors.

30. The method of claim 29, wherein the tumor is a solid tumor or a blood tumor; preferably, the tumor is selected from the group consisting of melanoma, lung cancer (such as non-small cell lung cancer, small cell lung cancer), kidney cancer (such as renal cell carcinoma), lymphoma (such as Hodgkin lymphoma), head and neck cancer (such as head and neck squamous cell carcinoma), bladder cancer, colorectal cancer, liver cancer (such as hepatocellular carcinoma), ovarian cancer, breast cancer (such as triple-negative breast cancer), cervical cancer, osteosarcoma, soft tissue sarcoma, follicular soft tissue sarcoma, neuroendocrine tumor, neuroendocrine carcinoma, malignant peripheral nerve sheath tumor, gallbladder cancer, bile duct cancer, pancreatic cancer, prostate cancer, urothelial carcinoma, gastric cancer, brain glioma, or a combination thereof.

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