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

TECHNICAL FIELD

[0001] The present invention mainly relates to a modified oncolytic virus and a composition thereof. Specifically, the present invention relates to the design, modification of oncolytic viruses and application of compositions thereof. In certain embodiments, the oncolytic virus is a recombinant oncolytic vaccinia virus. In certain embodiments, the recombinant oncolytic vaccinia virus is capable of expressing two full-length antibodies simultaneously. 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 specifically binding to PD-1 and a full-length antibody specifically binding to CTLA-4. In certain embodiments, the target combination of the two full-length antibodies exhibits higher anti-tumor efficacy compared with combinations of other two immune checkpoint inhibitors. In certain embodiments, the combination of the two full-length antibodies exhibits higher anti-tumor efficacy compared with combinations of their antigen-binding fragments. BACKGROUND

[0002] The following description is provided to assist the reader’s understanding. Neither the information provided nor the cited references are admitted to be prior art.

[0003] Tumors are diagnosed in more than 14 million people worldwide each year. Despite numerous advances in medical research, tumors account for approximately 16% of all deaths.

[0004] Malignant tumors are often resistant to conventional therapies and represent substantial therapeutic challenges. For example, micrometastases may arise at a very early stage during primary tumor development. Accordingly, many tumor patients already have micrometastases at the time of diagnosis. Tumor-cytotoxic T-cells can seek out and destroy these micrometastases without injuring surrounding healthy tissues. Nevertheless, naturally-occurring T-cell responses against malignant tumors are generally insufficient to bring about regression of primary or metastatic tumors.

[0005] In recent years, with breakthrough advances in tumor immunotherapy research, immune checkpoint inhibitor (ICI) drugs represented by PD-1 / PD-L1 have been widely applied in clinical treatment. Taking PD-1 / PD-L1 inhibitors as an example, they have become a research hotspot in tumor immunotherapy. Although survival outcomes of cancer patients are improved, these macromolecular biologic agents suffer from poor permeability, which limits their penetration into 1 solid tumor interiors. The single-agent response rate remains limited for common solid tumors. Furthermore, with the clinical adoption of immunotherapy, the therapeutic efficacy of PD-1 / PD-L1 inhibitors against multiple tumors has been validated. However, current data indicate relatively low response rates: most patients show poor or no response to immunotherapy, a small subset of patients develop drug resistance after treatment, and even some patients with initial favorable responses experience disease progression due to acquired resistance.

[0006] In addition, therapeutic monoclonal antibodies targeting another immune checkpoint CTLA-4 have also entered clinical practice. CTLA-4 inhibitors represented by Ipilimumab have been marketed for many years yet have failed to achieve major breakthroughs across multiple solid tumor types. Combination therapy therefore represents a mainstream trend. For first-line treatment of indications such as melanoma, renal cell carcinoma, non-small cell lung cancer, and malignant pleural mesothelioma, the combination of Nivolumab and Ipilimumab has demonstrated significantly superior efficacy compared to monotherapy. Moreover, a bispecific antibody targeting human PD-1 and CTLA-4 (Cadonilimab Injection) has been approved for marketing for treating patients with recurrent or metastatic cervical cancer who have failed prior platinum-containing chemotherapy.

[0007] Oncolytic viruses are a class of viral agents, either naturally occurring or genetically modified, that can specifically infect and kill tumor cells. By employing multiple mechanisms to eliminate tumor cells, they have become a vital branch of current cancer immunotherapy. Recent studies have demonstrated the potential of oncolytic viruses as anti-tumor agents. Distinct from conventional gene therapy, oncolytic viruses propagate within tumor tissues through viral replication and concomitant cytolysis, and their selective replication property confers desirable safety and tumor-targeting profiles. Nevertheless, first-generation oncolytic viruses are inadequate for tumor therapy. Leveraging the innate advantages of viruses as delivery vectors, recombinant oncolytic viruses carrying exogenous genes exhibit promising application prospects, such as oncolytic viruses carrying genes that express diverse immune activators, immune suppressors or cytokines. Combinations among these oncolytic viruses differ with respect to underlying mechanisms of action and clinical potential, and the potential embedded within these strategies is urgently needed to enhance the efficacy of oncolytic viruses and expand tumor therapeutic regimens. SUMMARY OF THE INVENTION

[0008] Current mainstream combination immunotherapies employing oncolytic viruses include direct use of recombinant oncolytic viruses expressing immunotherapeutic genes, and therapeutic 2 combinations of recombinant oncolytic viruses with immune checkpoint inhibitors (e.g., CN117503922, WO2023159102, US20210023151). Nevertheless, designs and applications of recombinant oncolytic viruses and compositions thereof that simultaneously express two or more distinct immune checkpoint inhibitors (e.g., anti-PD-1 antibody, anti-CTLA-4 antibody) in the form of full-length antibodies have not been reported. The present invention fills this gap.

[0009] Disclosed herein are a modified oncolytic virus and a composition thereof for tumor treatment. Said oncolytic virus simultaneously expresses two independent or conjugated distinct immune checkpoint inhibitors (for instance, the combination of a full-length antibody specifically binding PD-1 and a full-length antibody specifically binding CTLA-4. Through extensive screening, the present invention finds that the aforesaid combination exhibits higher anti-tumor efficacy, and / or fewer adverse effects, and / or lower required doses relative to alternative immune checkpoint inhibitor combinations, such as combinations of PD-1 inhibitors with PD-L1 / 2 inhibitors, B7-H3 / 4 inhibitors, LAG3 inhibitors, TIM-3 inhibitors, or CD160 inhibitors, etc., as well as combinations of CTLA-4 inhibitors with other immune checkpoint inhibitors). Said oncolytic virus simultaneously expresses two independent or conjugated distinct full-length antibodies (e.g., a full-length antibody specifically binding PD-1 and a full-length antibody specifically binding CTLA-4. Extensive screening by the present invention demonstrates that the dual full-length-antibody combination exhibits higher anti-tumor efficacy, and / or fewer adverse effects, and / or lower required doses compared with combinations of alternative antibody formats, such as Fab, Fab’, F(ab% Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single-domain antibodies and nanobodies, etc.). Apart from inducing tumor-cell apoptosis via direct viral infection of tumor cells, the two distinct immune checkpoint inhibitors expressed by the modified oncolytic virus can concurrently modulate host immune responses, converting “cold” tumors into “hot” tumors, thereby activating the immune system within the tumor microenvironment and inducing or re-awakening anti-tumor immune responses. Further, immune-system activation initiates systemic host anti-tumor immunity, achieving systemic therapeutic effects via local administration.

[0010] Meanwhile, the oncolytic virus and composition thereof disclosed herein can directly express two different immune checkpoint inhibitors. Compared with separate combined administration of an oncolytic virus plus two monoclonal antibodies, the present invention reduces administration frequency, alleviates patient discomfort caused by medicament injections, and reduces clinical operational complexity. By killing two birds with one stone, it simultaneously achieves the therapeutic effects of both oncolytic virus treatment and monoclonal antibody combination therapy.

[0011] In one aspect, the present invention provides a modified oncolytic virus whose viral genome comprises a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding another distinct 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 virus, parvovirus, chicken anemia virus, measles virus, Coxsackievirus, vesicular stomatitis virus, Seneca Valley Virus, Maraba virus and Newcastle disease virus.

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

[0014] In certain embodiments, the genome of the modified oncolytic virus comprises at least one deletion or disruption that attenuates the virus or enables selective replication of the virus in tumor cells, wherein the deletion or disruption is of the viral thymidine kinase.

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

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

[0017] In certain embodiments, the immune checkpoint inhibitors encoded by the virus are a first antibody or antigen-binding fragment thereof capable of specifically binding one immune checkpoint protein, and a second antibody or antigen-binding fragment thereof capable of specifically binding a 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 are full-length antibodies.

[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 to 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 set forth in SEQ ID NO: 2. In certain embodiments, a Fab region of the first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 3 and a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, CDR regions of the first heavy chain comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 4, HCDR2 as set forth in SEQ ID NO: 5, and HCDR3 as 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 the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, CDR regions of the first light chain comprise the amino acid sequences of 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 an alternative first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, a Fab region of the alternative first heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 12 and a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, CDR regions of the alternative first heavy chain comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 14, and HCDR3 as set forth in SEQ ID NO: 15.

[0024] In certain embodiments, the first antibody or antigen-binding fragment thereof further comprises an alternative first light chain comprising the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, CDR regions of the alternative first light chain comprise the amino acid sequences of 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 to 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 set forth in SEQ ID NO: 21. In certain embodiments, a Fab region of the second heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 22 and a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, CDR regions of the second heavy chain comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 23, HCDR2 as set forth in SEQ ID NO: 24, and HCDR3 as 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 the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, CDR regions of the second light chain comprise the amino acid sequences of LCDR1 as set forth in SEQ ID NO: 27, LCDR2 as set forth in SEQ ID NO: 28, and LCDR3 as set forth in SEQ ID NO: 29.

[0028] In certain embodiments, the first immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof specifically binding PD-1, and the second immune checkpoint 5 inhibitor is an antibody or antigen-binding fragment thereof specifically binding CTLA-4.

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

[0030] In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are configured for expression during the same or different phases of the replication cycle of the modified oncolytic virus.

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

[0032] In certain embodiments, the first early and late promoter is selected from the group consisting of P7.5, H5, poxvirus synthetic early / late promoter (pSE / L), and combinations thereof. In certain embodiments, the first early and late promoter is 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), and combinations 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), and combinations 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), and combinations 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 a chimeric bispecific antibody targeting two different targets.

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

[0038] In another aspect, the present invention provides a method for treating tumors, 6 comprising a 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 non-human mammal (e.g., mouse, rat, rabbit, monkey, and the like).

[0040] In certain embodiments, the tumor is a solid tumor or 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), renal 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, cholangiocarcinoma, pancreatic cancer, prostate cancer, urothelial carcinoma, gastric cancer, brain glioma, and 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 provides use of the modified oncolytic virus of the present invention or the pharmaceutical composition of the present invention in the manufacture of 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 specifically described in the following (such as the examples) can be combined with each other to form a new or preferred technical solution, which is not redundantly repeated one by one herein due to space limitation. DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a schematic structural diagram of WR-301.

[0045] FIG. 2 shows PCR-amplified bands of exogenous genes inserted in WR-301.

[0046] FIG. 3 shows ELISA results for PD-1-binding and CTLA-4-binding antibodies in harvest fluids from WR-301-infected cells.

[0047] FIG. 4 shows replication fold-changes of WR-301 in MC38 and HCC1937 cells.

[0048] FIG. 5 shows viability changes of MC38 and HCC1937 cells infected with WR-301.

[0049] FIG. 6 shows tumor-volume changes in CT26-hPDL1-bearing humanized mice receiving intratumoral WR-301 injection.

[0050] FIG. 7 shows survival-rate changes in CT26-hPDL1-bearing humanized mice receiving intratumoral WR-301 injection.

[0051] FIG. 8 shows replication fold-changes of WR-301 in A549 and HUVEC cells.

[0052] FIG. 9 shows viability changes of eight tumor cell lines after infection with WR-301 at different MOIs.

[0053] FIG. 10 shows in vitro biological activity of proteins expressed by WR-301.

[0054] FIG. 11 shows direct tumor-killing effects of WR-301 in PA-1 tumor-bearing nude mice.

[0055] FIG. 12 shows dose-effect relationships for tumor inhibitory activity of WR-301 in tumor-bearing humanized mice.

[0056] FIG. 13 shows that WR-301 promotes intratumoral immune cell infiltration in tumor-bearing humanized mice.

[0057] FIG. 14 shows that WR-301 promotes expression of anti-tumor cytokines in tumor-bearing humanized mice.

[0058] FIG. 15 shows the abscopal effect of tumor inhibition mediated by WR-301 in tumor-bearing humanized mice.

[0059] FIG. 16 shows that humanized mice cured by WR-301 administration resist re-challenge with the same tumor.

[0060] FIG. 17 shows that WR-301 expressing dual full-length antibodies achieves superior tumor inhibitory efficacy in tumor-bearing humanized mice compared with 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 comprises a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding another distinct immune checkpoint inhibitor.

[0062] Oncolytic Viruses

[0063] As used herein, the term “oncolytic virus” refers to a virus capable of selectively replicating in tumor cells in vitro or in vivo, slowing tumor cell growth or inducing tumor cell death, while exerting no or minimal effects on normal cells. In certain embodiments, an oncolytic virus comprises a viral genome packaged within virions (or viral particles) and is infectious (i.e., capable of infecting and entering host cells or subjects). In certain embodiments, the oncolytic virus may be a DNA virus or RNA virus, and may take any suitable form such as a DNA-virus vector, RNA-virus 8 vector or virion.

[0064] As used herein, the term “selective replication” means that the oncolytic virus exhibits significantly higher replication rates in tumor cells relative to normal somatic cells. In certain embodiments, the oncolytic virus exhibits 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 proliferation rates in tumor cells.

[0065] In certain embodiments, the oncolytic virus 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 carcinoma 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 virus of the present invention may be derived from poxvirus, adenovirus (e.g., Delta-24, Delta-24-RGD, ICOVIR-5, ICOVIR-7, Onyx-015, ColoAdl, H101, and AD5 / 3-D24-GMCSF), reovirus (e.g., Reolysin), measles virus, herpes simplex virus (e.g., HSV, OncoVEX GMCSF), Newcastle disease virus (e.g., 73-T PV701 and HDV-HUJ virus strains, and those described in Phuangsab et al., 2001, Cancer Lett., 172(1):27-36; Lorence et al., 2007, Curr. Cancer Drug Targets, 7(2):157-167; and Freeman et al., 2006, Mol. Ther., 13(1):221-8), retrovirus (e.g., influenza virus), myxoma virus, 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), picornavirus (e.g., Seneca Valley virus; SW-001 and NTX-010), Coxsackievirus or parvovirus.

[0067] In certain embodiments, the oncolytic virus of the present invention is derived from poxvirus. As used herein, the term “poxvirus” denotes viruses belonging to the Poxviridae family. In certain embodiments, the poxvirus is a virus belonging to the Chordopoxviridae subfamily. In certain embodiments, the poxvirus is a virus belonging to the Ortho poxvirus subfamily. The genomic sequences of various poxviruses, such as vaccinia virus, cowpox virus, canarypox virus, ectromelia virus, and myxoma virus, are available in the art and in specialized databases, such as GenBank (Accession Nos. NC_006998, NC_003663, NC_005309, NC_004105, and NC_001132, respectively).

[0068] In certain embodiments, the oncolytic virus of the present invention is derived from vaccinia virus. It is characterized by an approximately 190 kb double-stranded DNA genome that encodes numerous viral enzymes and factors that enable the virus to replicate independently of the host cell machinery. In certain embodiments, the vaccinia virus of the present invention is derived from Elstree, Copenhagen, Western Reserve or Wyeth strains. In certain embodiments, the vaccinia 9 virus of the present invention is the Western Reserve strain. The Western Reserve strain is well-characterized, and its complete genome sequence is accessible via NCBI (www.ncbi.nlm.nih.gov) under accession number AY243312.

[0069] As used herein, the term “modified oncolytic virus” refers to an oncolytic virus that has been modified by introducing a heterologous nucleic acid or protein, or by altering a native nucleic acid or protein. In certain embodiments, modified oncolytic viruses provided herein are genetically altered via deletion and / or addition of nucleic acid sequences. In certain embodiments, modified oncolytic viruses provided herein comprise a deletion of the thymidine kinase (TK) gene. In certain embodiments, modified oncolytic viruses provided herein comprise the addition of a nucleic acid sequence encoding an anti-human PD-1 antibody and an anti-human CTLA-4 antibody.

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

[0071] Immune Checkpoint Inhibitors

[0072] The genome of the modified oncolytic virus 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 “encoding” means capable of being transcribed into mRNA and / or translated into peptides or proteins.

[0075] As used herein, the term “immune checkpoint protein” refers to proteins directly or indirectly involved in immune pathways critical for preventing uncontrolled immune responses, hence essential for maintaining self-tolerance and / or tissue protection. One or more immune checkpoint modulators as used herein may independently act at any step of T cell-mediated immunity, including clonal selection of antigen-specific cells, T cell activation, proliferation, trafficking to antigen-bearing and inflammatory sites, execution of direct effector functions, and signaling via cytokines and membrane ligands.

[0076] As used herein, the term “immune checkpoint inhibitor” denotes a molecule capable of negatively modulating the function of an immune checkpoint protein. Immune checkpoint inhibitors may adopt any molecular modality 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, an immune checkpoint inhibitor is a natural or engineered antagonist of an inhibitory immune checkpoint molecule, including for example ligands of CTLA-4 and ligands of 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, Pidilizumab, 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, and those described in PCT / US2001 / 020964); anti-PD-L2; anti-(both PD-L1 and PD-L2) (e.g., AUR-012 and AMP-224); anti-CTLA-4 (e.g., Ipilimumab, Tremelimumab, or KAHR-102); anti-IDO (e.g., D-1-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., 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 antigen-binding fragment thereof capable of specifically binding immune checkpoint proteins PD-1 and / or CTLA-4.

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

[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” denotes programmed cell death protein 1, a member of the immunoglobulin superfamily that functions as a co-inhibitory receptor to negatively regulate the immune system. PD-1 belongs to the CD28 / CTLA-4 family and has two known ligands: PD-L1 and PD-L2. A representative amino acid sequence for human PD-1 is disclosed under GenBank accession NP_005009.2, and a representative nucleic acid sequence encoding human PD-1 is provided under GenBank accession 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) within its cytoplasmic domain 11 (Parry et al., 2005, Mol. Cell. Biol., 25:9543-53). Disruption of this inhibitory function of PD-1 can lead to autoimmunity. The sustained negative signaling generated by PD-1 is implicated in many pathological conditions, such as tumor immune evasion and T cell dysfunction in chronic viral infections.

[0085] A PD-1 inhibitor may be any agent that inhibits PD-1 activity, for example agents reducing 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., binding between PD-1 and PD-L1), suppression of its biological activation (e.g., PD-1 activation), and / or a decrease in its expression level (e.g., PD-1 expression level).

[0087] In certain embodiments, the PD-1 inhibitor is an antibody (e.g., antagonist antibody) capable of specifically binding PD-1.

[0088] As used herein, the term “specific binding / specifically binds” refers to a non-random binding reaction between two molecules, e.g., between an antibody and its antigen. In certain embodiments, antibodies or antigen-binding fragments provided herein specifically bind human and / or monkey PD-1 with a binding affinity (KD)<10"6M (e.g., <5X10’7M, <2X10’7M, <10’7M, <5X10’8M, <2X10’8M, <10-8M, <5X10’9M, <2X10’9M, <10’9M, or <10’10M). As used herein, KD is defined as the ratio of dissociation rate to association 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 against PD-1.

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

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

[0092] In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises an alternative first heavy chain comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a Fab region of the alternative first heavy chain comprises the amino acid 12 sequence of SEQ ID NO: 12 or a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, CDR regions of the alternative first heavy chain comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 14, and HCDR3 as 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 a reference sequence, after aligning the candidate sequence with the reference sequence and introducing gaps, if necessary, to achieve the maximum number of identical amino acids. Conservative substitutions of amino acid residues are not considered identical residues. Alignments for the purpose of determining the percentage of amino acid sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the U.S. National Center for Biotechnology Information (NCBI); see also Altschul S.F. et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), or ClustalW2 (available on the website of the European Bioinformatics Institute; see also Higgins D.G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by said tools, or may customize parameters suitable for alignment, for example, by selecting an appropriate algorithm.

[0094] In certain embodiments, a 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, an alternative 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, an alternative 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 anti-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 having at least 90% sequence identity thereto. In certain embodiments, the first antibody or antigen-binding fragment thereof comprises first light chain CDR regions, wherein the first light chain CDR regions comprise the amino acid sequences of 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. 13

[0097] In certain embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof further comprises an alternative first light chain comprising the amino acid sequence of SEQ ID NO: 16 or a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the alternative first light chain CDR regions comprise the amino acid sequences of 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.

[00100] As used herein, the term “CTLA-4” denotes cytotoxic T-lymphocyte-associated protein 4, also designated CD152 (cluster of differentiation 152), a protein receptor acting as an immune checkpoint to down-regulate immune responses. CTLA-4 is constitutively expressed in regulatory T cells, but is upregulated in conventional T cells only upon activation, a phenomenon that is particularly pronounced in cancer. It acts as an “off switch” when it binds to CD80 or CD86 on the surface of antigen-presenting cells. The representative amino acid sequence of human CTLA-4 is disclosed in GenBank under Accession No.: NP_033973.2, and the representative nucleic acid sequence encoding human PD-1 is shown in GenBank under Accession No.: NM_005214.5.

[00101] CTLA-4 transmits inhibitory signals to T cells, whereas CD28 delivers stimulatory signals. CTLA-4 is also present in regulatory T cells (Tregs) and contributes to their suppressive functions. T cell activation via the T cell receptor and CD28 leads to upregulated CTLA-4 expression. Anti-CTLA-4 antibodies bind to the CTLA-4 molecule with high affinity, mediating Treg depletion or functional blockade, thereby enhancing T cell activation and anti-cancer immune responses. The effects of CTLA-4 blockade can be mediated through multiple mechanisms: preventing trans-endocytosis to increase CD80 / CD86 levels on antigen-presenting cells (APCs) and enhance T cell activation; directly inducing Treg cytotoxicity; and triggering FcR-IV-mediated antibody-dependent cellular cytotoxicity (ADCC) by intratumoral macrophages.

[00102] A CTLA-4 inhibitor may be any agent inhibiting CTLA-4 activity, for example agents reducing CTLA-4 activity by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

[00103] Factors that can reduce CTLA-4 activity include: inhibition of the binding between the functional protein and its ligand (e.g., the binding between CTLA-4 and CD80 or CD86), suppression of its biological activation (e.g., CTLA-4 activation), and / or a decrease in its expression level (e.g., CTLA-4 expression level).

[00104] In certain embodiments, the CTLA-4 inhibitor is an antibody (e.g., antagonist 14 antibody) capable of specifically binding CTLA-4.

[00105] In certain embodiments, antibodies or antigen-binding fragments provided herein specifically bind human and / or monkey CTLA-4 with a binding affinity (KD)<10-6M (e.g., <5xW7M, <2x10-7M, <10-7M, <5x10"8M, <2x10-8M, <10-8M, <5x10-9M, <2x10-9M, <10-9M, or <10-10M).

[00106] In certain embodiments, the CTLA-4 inhibitor is a full-length monoclonal antibody against CTLA-4.

[00107] In certain embodiments, the anti-CTLA-4 antibody specifically binds SEQ ID NO: 20.

[00108] In certain embodiments, the anti-CTLA-4 antibody or antigen-binding fragment thereof comprises a second heavy chain comprising the amino acid sequence of 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, wherein the second heavy chain Fab region comprises the amino acid sequence of 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 second heavy chain CDR regions, wherein the second heavy chain CDR regions comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 23, HCDR2 as set forth in SEQ ID NO: 24, and HCDR3 as set forth in SEQ ID NO: 25.

[00109] In certain embodiments, a 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.

[00110] In certain embodiments, the anti-CTLA-4 antigen-binding fragment further comprises a second light chain comprising the amino acid sequence of 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 second light chain CDR regions, wherein the second light chain CDR regions comprise the amino acid sequences of LCDR1 as set forth in SEQ ID NO: 27, LCDR2 as set forth in SEQ ID NO: 28, and LCDR3 as set forth in SEQ ID NO: 29.

[00111] Antibodies

[00112] As used herein, the term “antibody” encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody or bispecific (bivalent) antibody that binds a specific antigen. A native intact antibody contains two heavy chains and two light chains. Each 15 heavy chain consists of a variable domain and first, second and third constant domains, while each light chain consists of a variable domain and a constant domain. Mammalian heavy chains are classified as a, 8, s, y and u, and mammalian light chains are classified as X or k. Antibodies exhibit a “Y” shape, wherein the stem of the Y is composed of the second and third constant regions of two heavy chains joined by disulfide bonds. Each arm of the Y comprises the variable region and first constant region of a single heavy chain associated with the variable and constant regions of a single light chain, with the first constant region of the heavy chain being linked to the second constant region via a hinge region. The variable regions of the light and heavy chains are responsible for antigen-binding specificity. The variable regions in both chains generally contain three highly variable loops known as complementarity-determining regions (CDRs) (the light (L) chain CDRs comprise LCDR1, LCDR2, and LCDR3, and the heavy (H) chain CDRs comprise HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified according to the Kabat, Chothia, or Al-Lazikani conventions (see, e.g., Al-Lazikani, B., Chothia, C., Lesk, A.M., 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, A.M., J. Mol. Biol., 196, 901 (1987); Chothia, C., et al., Nature, Dec 21-28; 342(6252):877-83 (1989); Kabat, E.A., et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interspersed between flanking segments known as framework regions (FRs), which are more highly conserved than the CDRs and serve as a structural scaffold supporting the variable region. The constant regions of the heavy and light chains are not involved in antigen-binding specificity but mediate various effector functions. Antibodies are classified based on the amino acid sequence of the heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of a, 8, s, y and u heavy chains, respectively. Several major antibody classes are further divided into subclasses, such as IgG1 (y1 heavy chain), IgG2 (y2 heavy chain), IgG3 (y3 heavy chain), IgG4 (y4 heavy chain), IgA1 (a1 heavy chain), or IgA2 (a2 heavy chain).

[00113] As used herein, the term “antigen-binding fragment” denotes antibody fragments formed from portions of an antibody that contain one or more CDRs, yet lack the full intact-antibody structure. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab’, F(ab% Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single-domain antibodies and nanobodies. Antigen-binding fragments are capable of binding the same antigen bound by the parent antibody.

[00114] As used herein, the term “Fab” refers to a 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 16 variable region and the constant region), which are linked by a disulfide bond.

[00115] As used herein, the term “Fab‘” denotes an Fab fragment containing a portion of the hinge region.

[00116] As used herein, the term “F(ab‘)2” denotes a dimer of Fab’.

[00117] As used herein, the term “Fv” denotes an Fv fragment consisting of the variable region of a single light chain and the variable region of a single heavy chain.

[00118] As used herein, the term “single-chain Fv antibody” or “scFv” denotes an engineered antibody in which light chain variable domain and heavy chain variable domain are linked directly or via a peptide-linker sequence (see e.g., Huston J.S. et al., Proc. Natl. Acad. Sci. USA, 85:5879, 1988).

[00119] As used herein, the term “scFv dimer” denotes a polymer formed by two scFvs.

[00120] The term “camelized single-domain antibody”, also referred to as a “heavy-chain antibody” or “HCAb” (heavy-chain-only antibody), refers to an antibody comprising two heavy-chain variable regions but lacking light chains (see, e.g., Riechmann L. and Muyldermans S., J. Immunol. Methods, Dec. 10; 231(1-2):25-38 (1999); Muyldermans S., J. Biotechnol., June; 74(4):277-302 (2001); WO94 / 04678; WO94 / 25591; and U.S. Patent No. 6,005,079). Heavy-chain antibodies are originally derived from the Camelidae family (camels, dromedaries, and llamas). Although lacking light chains, camelized antibodies possess a genuine 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 of a heavy-chain antibody and two constant regions, CH2 and CH3.

[00121] In certain embodiments, antibodies provided herein are fully-human antibodies, humanized antibodies, chimeric antibodies, mouse antibodies or rabbit antibodies. In certain embodiments, antibodies provided herein are polyclonal, monoclonal or recombinant antibodies. In certain embodiments, antibodies provided herein are monospecific, bispecific or multispecific antibodies. In certain embodiments, antibodies provided herein may be further labeled. In certain embodiments, the antibody or antigen-binding fragment thereof is a fully-human antibody, optionally produced by a transgenic rat, such as a transgenic rat having inactivated endogenous rat immunoglobulin gene expression and carrying a recombinant human immunoglobulin locus with J locus deletion and C-k mutation, and the antibody may also be expressed by engineered cells (e.g., CHO cells).

[00122] As used herein, with respect to an antibody or antigen-binding fragment, the term “fully-human” refers to an antibody or antigen-binding fragment whose amino acid sequence corresponds to that of an antibody produced by a human or a human immune cell, or to the amino acid sequence of an antibody derived from a non-human source, such as a transgenic non-human animal utilizing a human antibody repertoire or other human antibody-encoding sequences.

[00123] As used herein, with respect to an antibody or antigen-binding fragment, the term “humanized” refers to an antibody or antigen-binding fragment comprising CDRs derived from a non-human animal, framework regions (FRs) derived from a human, and, where applicable, a constant region derived from a human. In certain embodiments, a humanized antibody or antigen-binding fragment is suitable for use as a human therapeutic agent because it has 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, a humanized antibody or antigen-binding fragment consists substantially of human sequences except for the CDR sequences, which are non-human sequences.

[00124] As used herein, with respect to an antibody or antigen-binding fragment, the term “chimeric” refers to an antibody or antigen-binding fragment comprising a portion of a heavy chain and / or a light chain derived from one species, and the remaining portion of the heavy chain and / or light chain derived from a different species.

[00125] In certain embodiments, a chimeric antibody may comprise a constant region derived from a human and a variable region derived from a non-human species, such as a mouse or a rabbit.

[00126] As used herein, with respect to an amino acid sequence, the term “conservative substitution” refers to the replacement of an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitutions may be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), among residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), among residues with acidic side chains (e.g., Asp and Glu), among amino acids with basic side chains (e.g., His, Lys, and Arg), or among residues with 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 a protein, and thus the biological activity of the protein can be retained.

[00127] Polynucleotides

[00128] In certain embodiments, the modified oncolytic virus of the present invention contains a first heterologous polynucleotide encoding an inhibitory antibody or antigen-binding fragment 18 thereof that specifically binds PD-1, and a second heterologous polynucleotide encoding an inhibitory antibody or antigen-binding fragment thereof that specifically binds CTLA-4.

[00129] As used herein, the term “polynucleotide” or “nucleic acid” refers to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or hybrid ribonucleic acid-deoxyribonucleic acid, such as a DNA-RNA hybrid. A polynucleotide or nucleic acid may be single-stranded or double-stranded DNA or RNA, or a DNA-RNA hybrid. A polynucleotide or nucleic acid may be linear or circular. In certain embodiments, when the virus is a DNA virus, the first and second heterologous polynucleotides are both DNA; or when the virus is an RNA virus, the first and second heterologous polynucleotides are both RNA. In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are both double-stranded DNA.

[00130] The first heterologous polynucleotide and the second heterologous polynucleotide may be introduced into the modified oncolytic virus by conventional methods known in the art, for example via polymerase chain reaction (PCR) synthesis followed by ligation to a viral genome having compatible restriction ends. For further details, see e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1989), incorporated herein in its entirety by reference.

[00131] In certain embodiments, in the 5’ to 3’ direction on the sense strand, the modified oncolytic virus comprises in-frame the following elements: a polynucleotide encoding the light chain of a CTLA-4-binding antibody - a first early and late promoter - a second early and late promoter - a polynucleotide encoding the heavy chain of a CTLA-4-binding antibody - a polynucleotide encoding the heavy chain of a PD-1-binding antibody - a first late promoter - a second late promoter - a polynucleotide encoding the light chain of a PD-1-binding antibody.

[00132] 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. That is, they are not expressed as fusion proteins and are not linked to each other (neither covalently nor via peptide linkers). In certain embodiments, immune checkpoint inhibitors expressed by the first and second heterologous polynucleotides are not fused to any additional proteins.

[00133] In certain embodiments, other than the first heterologous polynucleotide and the second heterologous polynucleotide, the modified oncolytic virus does not comprise any other heterologous polynucleotide encoding an immune checkpoint inhibitor or an immune activator. In certain embodiments, other than the first heterologous polynucleotide and the second heterologous polynucleotide, the modified oncolytic virus does not comprise any heterologous polynucleotide 19 encoding other proteins.

[00134] The present invention will be further illustrated below with reference to the specific examples. It should be understood that these examples are only to illustrate the present invention, not to limit the scope of the present invention.

[00135] Example 1: Virus Construction

[00136] Wild-type WR virus strain was obtained from ATCC (VR-1354). As shown in FIG. 1, the nucleic acid sequence J1R-antiCTLA4-antiPD1-J3R was synthesized by a biotechnology company and inserted into plasmid vector pUC57. Recombinant plasmids were transfected into HeLa cells pre-infected with the wild-type WR to obtain a recombinant oncolytic virus with dual exogenous gene insertions and a TK deletion, which was designated as WR-301.

[00137] WR-301 was subjected to three rounds of plaque purification in 143B cells, followed by amplification in HeLa cells. Finally, the virus was purified using a sucrose density gradient centrifugation, yielding the WR-301 virus with a titer of approximately 2*10A9 pfu / mL.

[00138] Example 2: Virus Characterization Study

[00139] PCR Sequencing

[00140] To confirm the presence of the designed exogenous sequence in the recombinant viral genome, primers P1 and P2 were designed, and the primer positions are indicated in FIG. 1. This primer pair can amplify and distinguish between the wild-type WR strain sequence containing TK (2.75 kb) and the recombinant virus sequence containing the exogenous gene (7 kb). The PCR amplification results of WR-301 are shown in FIG. 2, which displays a band indicative of WR-301, but no band indicative of wild-type WR.

[00141] Subsequently, the PCR products were sequenced. Sequence alignment revealed that the amplified sequence of WR-301 was identical to the designed nucleic acid sequence.

[00142] ELISA

[00143] To verify whether WR-301 can express functional anti-PD-1 and anti-CTLA-4 antibodies, 96-well plates were coated with recombinant PD-1 or CTLA-4 proteins diluted in PBS at a protein concentration of 1 mg / mL. After blocking with 5% non-fat milk, the harvested supernatants from WR-301-infected cells, diluted 10-, 100-, and 1000-fold, were added. The plates were incubated at 37°C for 1 hour, followed by four washes with PBST. Then, an HRP-conjugated rabbit anti-human secondary antibody was added. After another 1-hour incubation at 37°C and four PBST washes, TMB substrate was added for color development. The reaction was stopped after 20 minutes at room temperature, and the absorbance was measured at 450 nm and 630 nm using a microplate reader.

[00144] The analysis was performed using the supernatant of HeLa cells infected with WR-301 at an MOI of 0.5 for 72 hours. As shown in FIG. 3, WR-301 can express antibodies that specifically bind to PD-1 and CTLA-4, and this binding is concentration-dependent.

[00145] Example 3: In Vitro Viral Studies

[00146] The following studies were conducted to confirm the replication capacity and cytotoxic activity of WR-301 in tumor cells.

[00147] Replication

[00148] 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 post-infection to determine the viral DNA copy number, and the fold increase in viral titer relative to the initial inoculum at different time points was calculated. As shown in FIG. 4, WR-301 replicated significantly in both cancer cell lines, with a stronger replication capacity observed in human cancer cells compared to mouse cancer cells.

[00149] Cytotoxicity

[00150] MC38 (mouse colon cancer cells) and HCC1937 (human breast cancer cells) were infected with WR-301 at MOIs of 0.1, 1, and 10. Samples were collected at 24, 48, 72, and 96 hours post-infection, and cell viability was assessed using the CCK-8 assay, a conventional method known in the art. As shown in FIG. 5, WR-301 exhibited significant cytotoxicity against HCC1937 cells at all tested MOIs. For MC38 cells, WR-301 at an MOI of 0.1 showed no significant cytotoxicity, whereas MOIs of 1 and 10 resulted in favorable cell killing.

[00151] Example 4: In Vivo Viral Studies

[00152] Eighteen humanized mice (BALB / c-hPD1hCTLA4) were implanted with humanized CT26 cells (mouse colon cancer cells, CT26-hPDL1, 1E6 cells per mouse). When the tumor volume reached approximately 100 mm3, the mice were randomly divided into three groups of six: PBS, WR-GFP (where GFP replaces the TK region of the wild-type WR virus), and WR-301. On days 0, 5, and 10, different viruses were administered via intratumoral injection at a dose of 4E8 pfu. Mouse body weight and tumor volume were continuously monitored, with a tumor volume of 2000 mm3 set as the humane endpoint.

[00153] The changes in tumor volume and survival rate of the mice are shown in FIG. 6 and FIG. 7. Compared to the other two groups, the WR-301 group exhibited significantly smaller tumor volumes and a markedly prolonged survival time.

[00154] Example 5: Extended In Vitro Viral Studies

[00155] The following studies were conducted to confirm the replication capacity of WR-301 21 in a broader panel of tumor cell lines, its cytotoxicity against a broader panel of tumor cell lines, and the biological activity of the proteins expressed by WR-301.

[00156] Replication

[00157] Tumor cells A549 (human non-small cell lung cancer cells) and normal cells HUVEC (human umbilical vein endothelial cells) were infected with WR-301 at an MOI of 0.05. Samples were collected 48 hours post-infection, and the viral DNA copy number was determined by qPCR. The fold increase in viral titer in the cells at the time of collection relative to the initial inoculum was calculated. As shown in FIG. 8, WR-301 exhibited a 250.98-fold increase in tumor A549 cells, whereas only a 20.67-fold increase was observed in normal HUVEC cells. This more than 10-fold difference indicates that WR-301 can selectively achieve high replication in tumor cells.

[00158] Cytotoxicity

[00159] Eight tumor cell lines were infected with serially diluted WR-301. Samples were collected 72 hours post-infection, and cell viability was assessed using the CCK-8 assay, a conventional method known in the art. The tumor cell lines 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), respectively. The MOIs used for infection were 0, 0.01, 0.04, 0.12, 0.37, 1.11, 3.33, and 10. As shown in FIG. 9, WR-301 exhibited favorable cytotoxicity against all eight tumor cell lines.

[00160] Biological Activity

[00161] The culture supernatant of HeLa cells infected with WR-301 was collected, and the purified proteins were obtained via Protein A affinity chromatography. The biological activities of the purified anti-PD-1 and anti-CTLA-4 proteins expressed by WR-301 were evaluated using a reporter gene assay, a conventional method known in the art. As shown in FIG. 10, the anti-PD-1 and anti-CTLA-4 proteins expressed by WR-301 exhibited biological activities essentially comparable to those of the positive control proteins.

[00162] Example 6: Extended In Vivo Viral Studies

[00163] The following study was conducted to confirm the pharmacological efficacy of WR-301 in mice in vivo.

[00164] Direct Cytotoxic Effects in Nude Mouse Models

[00165] BALB / c nude mice were subcutaneously inoculated with 1E7 PA-1 cells (human ovarian teratoma cells) per mouse. When the tumor volume reached approximately 100 mm3, 30 mice were randomly divided into five groups of six: a PBS group (negative control), a WR-GFP 22 group (vector control virus: GFP replaces the TK gene of the wild-type WR virus, with a total dose of 1E6 pfu / mouse), a low-dose WR-301 group (total dose of 1E4 pfu / mouse), a medium-dose WR-301 group (total dose of 1E5 pfu / mouse), and a high-dose WR-301 group (total dose of 1E6 pfu / mouse). The corresponding viruses were administered via intratumoral injection on days 0, 5, and 10, respectively. Tumor volume and body weight of mice were monitored 2 to 3 times a week. As shown in FIG. 11, compared to the PBS negative control group, the WR-GFP group and all three WR-301 dose groups effectively inhibited tumor growth. Since BALB / c nude mice lack mature T lymphocytes and cannot mount a normal T-cell immune response, the virus primarily relies on its own replication to achieve the function of directly killing tumor cells. These results indicate that WR-301 can directly kill tumor cells and inhibit tumor growth in nude mice.

[00166] Dose-Effect Relationship in Humanized Mouse Models

[00167] BALB / c-hPD1hCTLA4 mice (humanized mice with dual PD-1 and CTLA-4 targets) were subcutaneously inoculated with CT26-hPDL1 cells (PDL1-humanized mouse colon cancer cells). When the tumor volume reached approximately 100 mm3, 30 mice were randomly divided into five groups of six: a PBS group, a WR-GFP group (total dose of 1E8 pfu / mouse), a low-dose WR-301 group (total dose of 1E6 pfu / mouse), a medium-dose WR-301 group (total dose of 1E7 pfu / mouse), and a high-dose WR-301 group (total dose of 1E8 pfu / mouse). The corresponding viruses were administered via intratumoral injection on days 0, 5, and 10, respectively. Tumor volume and body weight of mice were monitored 2 to 3 times a week. As shown in FIG. 12, there was no significant difference between the WR-GFP group or the low-dose WR-301 group and the PBS group. Both the medium-dose and high-dose WR-301 groups effectively inhibited tumor growth, with the high-dose group demonstrating superior tumor suppression compared to the medium-dose group. These results indicate that the tumor-suppressive efficacy of WR-301 is positively correlated with the administered dose, exhibiting a dose-effect relationship. Furthermore, compared to WR-GFP, WR-301 is capable of effectively inhibiting tumor growth at lower doses.

[00168] Immune Activation Effects

[00169] BALB / c-hPD1hCTLA4 mice were subcutaneously inoculated with CT26-hPDL1 cells. When the tumor volume reached approximately 100 mm3, 18 mice were randomly divided into three groups of six: a PBS group, a WR-GFP group, and a WR-301 group. The corresponding viruses were administered via intratumoral injection on days 0 and 5, respectively, at a total dose of 6.7E7 pfu per mouse. On day 7, the mice were euthanized, and tumors were harvested for flow cytometry and qPCR analysis of tumor immune-related cytokine mRNA. As shown in the flow cytometry results in FIG. 13, the numbers of CD45+ leukocytes, CD3+ T cells, CD4+ T cells, and CD8+ T 23 cells infiltrating the tumors in the WR-301 group were significantly higher than those in the PBS group. As shown in the cytokine analysis results in FIG. 14, the levels of Th1-type cytokines, IL-2 and IFNy, in the mice tumors of the WR-301 group were markedly higher than those in the PBS group, and the levels of Granzyme B and Perforin were also significantly higher than those in the PBS group. However, there was no significant difference in the levels of Th2-type cytokines, IL-10 and TGF01, between the WR-301 and PBS groups. Th1-type cytokines are associated with immune activation, Th2-type cytokines are associated with immune suppression, and Granzyme B and Perforin can reflect the function of cytotoxic lymphocytes. These results indicate that WR-301 can effectively activate anti-tumor immune responses in mice in vivo.

[00170] Abscopal Effect

[00171] C57-hPD1hCTLA4 mice were bilaterally subcutaneously inoculated with MC38-hPDL1 cells. When the bilateral tumors reached approximately 100 mm3, 12 mice were randomly divided into two groups of six: a PBS group and a WR-301 group. The mice in each group received intratumoral injections on days 0, 5, and 10, respectively, at a total dose of 1E8 pfu per mouse, and the bilateral tumor volumes of the mice were detected 2 to 3 times a week. As shown in FIG. 15, the tumor volumes on both the treated and untreated sides of the mice in the WR-301 group were markedly smaller than those in the PBS group, indicating that WR-301 also has the effect of inhibiting the growth of distant tumors that are not at the administration site.

[00172] Tumor Rechallenge Study

[00173] BALB / c-hPD1hCTLA4 mice were subcutaneously inoculated with CT26-hPDL1 cells. When the tumor volume reached approximately 100 mm3, WR-301 was administered via intratumoral injection in three doses, with a total dose of 4E8 pfu per mouse. Forty days after the initial treatment, mice that had achieved complete tumor regression (15 mice obtained in this study) were re-challenged subcutaneously with CT26-hPDL1 cells, while naive mice that had neither been inoculated with tumor cells nor treated with the virus were used as controls. Another 35 days later, mice that remained tumor-free were subcutaneously inoculated with 4T1 cells (mouse breast cancer cells), with naive mice again serving as blank controls. Tumor volumes were monitored 2 to 3 times a week. As shown in FIG. 16, none of the mice with complete tumor regression developed tumors upon re-challenge with CT26-hPDL1 cells, whereas tumors grew normally in the control mice. Following the inoculation with 4T1 cells, tumors grew normally in both the experimental and control groups. These results indicate that while inhibiting tumor growth, WR-301 can stimulate the immune system to mount a long-term specific immune response against this type of tumor, thereby effectively preventing the recurrence of homologous tumors after cure.

[00174] Example 7: Comparison of Pharmacological Efficacy Among Different Viral Constructs

[00175] Two viruses, WR-CF and WR-CFPF, were generated using the construction method of WR-301 as a reference. WR-CF was constructed by replacing the TK region of the wild-type WR virus with the full-length anti-PD-1 antibody and the F(ab')2 region of the anti-CTLA-4 antibody. WR-CFPF was constructed by replacing the TK region of the wild-type WR virus with the F(ab')2 region of the anti-PD-1 antibody and the F(ab')2 region of the anti-CTLA-4 antibody.

[00176] BALB / c-hPD1hCTLA4 mice were subcutaneously inoculated with CT26-hPDL1 cells. When the tumor volume reached approximately 100 mm3, the mice were randomly divided into groups of six. The corresponding viruses were administered via intratumoral injection on days 0, 5, and 10, respectively, at a total dose of 4E8 pfu per mouse, and tumor volumes of mice were monitored 2 to 3 times a week. As shown in FIG. 17, the tumor-suppressive effects of both WR-CF and WR-CFPF were inferior to that of WR-301, indicating that viruses expressing dual full-length antibodies achieve better anti-tumor efficacy in mice.

[00177] All literatures mentioned in the present invention are incorporated herein by reference, as though each one is individually incorporated by reference. In addition, it should be understood that, after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, these equivalents also fall within the scope as defined in the appended claims of the present application.

Claims

1. A modified oncolytic virus, comprising a viral genome, wherein the viral genome has a first heterologous polynucleotide encoding an immune checkpoint inhibitor and a second heterologous polynucleotide encoding another distinct immune checkpoint inhibitor; wherein the immune checkpoint inhibitors are a first antibody or antigen-binding fragment thereof capable of specifically binding to an immune checkpoint protein, and a second antibody or antigen-binding fragment thereof capable of specifically binding to another distinct immune checkpoint protein; preferably, wherein the first antibody or antigen-binding fragment thereof and / or the second antibody or antigen-binding fragment thereof are full-length antibodies.

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 virus, 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 vaccinia virus.

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

5. The modified oncolytic virus according to claim 4, wherein the viral genome comprises at least one deletion or disruption which attenuates the virus or enables selective replication of the virus in tumor cells,wherein the deletion or disruption is located within an open reading frame (ORF) encoding an enzyme essential for viral replication,and 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 according to claim 6, wherein 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.

8. The modified oncolytic virus according to claim 6, wherein the first antibody orantigen-binding fragment thereof specifically binds to SEQ ID NO: 1.

9. The modified oncolytic virus according to claim 8, wherein the first antibody or26antigen-binding fragment thereof comprises a first heavy chain, and the first heavy chain comprises the amino acid sequence set forth in 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 the amino acid sequence set forth in 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 first heavy chain CDR regions, and the first heavy chain CDR regions comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 4, HCDR2 as set forth in SEQ ID NO: 5, and HCDR3 as set forth 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, and the first light chain comprises the amino acid sequence set forth in SEQ ID NO: 7 or a homologous sequence having at least 90% sequence identity thereto.

13. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof comprises first light chain CDR regions, and the first light chain CDR regions comprise the amino acid sequences of 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.

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

16. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof comprises first heavy chain CDR regions, and the first heavy chain CDR regions comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 13, HCDR2 as set forth in SEQ ID NO: 14, and HCDR3 as set forth 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, and the first light chain 27comprises the amino acid sequence set forth in SEQ ID NO: 16 or a homologous sequence having at least 90% sequence identity thereto.

18. The modified oncolytic virus according to claim 8, wherein the first antibody or antigen-binding fragment thereof comprises first light chain CDR regions, and the first light chain CDR regions comprise the amino acid sequences of 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.

19. The modified oncolytic virus according to claim 6, wherein the second antibody or antigen-binding fragment thereof specifically binds to SEQ ID NO: 20.

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

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

22. The modified oncolytic virus according to claim 19, wherein the second antibody or antigen-binding fragment thereof comprises second heavy chain CDR regions, and the second heavy chain CDR regions comprise the amino acid sequences of HCDR1 as set forth in SEQ ID NO: 23, HCDR2 as set forth in SEQ ID NO: 24, and HCDR3 as set forth in SEQ ID NO: 25.

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

24. The modified oncolytic virus according to claim 19, wherein the second antibody or antigen-binding fragment thereof comprises second light chain CDR regions, and the second light chain CDR regions comprise the amino acid sequences of LCDR1 as set forth in SEQ ID NO: 27, LCDR2 as set forth in SEQ ID NO: 28, and LCDR3 as set forth in SEQ ID NO: 29.

25. The modified oncolytic virus according to 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 a chimeric bispecific antibody targeting two different targets.

26. A pharmaceutical composition, comprising the modified oncolytic virus according to any28one of claims 1-25 and a pharmaceutically acceptable carrier.

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

28. The method according to claim 27, wherein the subject is a human or non-human mammal (e.g., mouse, rat, rabbit, monkey, and the like).

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

30. The use according to claim 29, wherein the tumor is a solid tumor or hematological tumor; preferably, wherein the tumor is selected from the group consisting of melanoma, lung cancer (e.g., non-small-cell lung cancer, small-cell lung cancer), renal 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, cholangiocarcinoma, pancreatic cancer, prostate cancer, urothelial carcinoma, gastric cancer, brain glioma, and a combination thereof.

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