Modified oncolytic virus and composition thereof

Abstract

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

Description

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 different immune checkpoint inhibitors located on the surface of T cells. In certain embodiments, the two different immune checkpoint inhibitors are PD-1 antibody and CTLA-4 antibody, respectively. 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 cancer immunotherapy research, immune checkpoint inhibitors (ICIs), represented by PD-1 / PD-L1, have been widely used in clinical treatment. PD-1 / PD-L1 inhibitors, for example, have become a research hotspot in cancer immunotherapy. While they have improved survival in cancer patients, their poor permeability as large-molecule biologics restricts their penetration into solid tumors, resulting in limited single-agent efficacy against most solid tumors. Furthermore, with the clinical application of immunotherapy, the efficacy of PD-1 / PD-L1 inhibitors has been demonstrated in a variety of tumor types. However, current data suggest that their efficacy remains relatively low, with most patients experiencing poor or no response to immunotherapy. A small number of patients also develop resistance to the drug after treatment, and even some patients who initially respond well develop acquired resistance, leading to disease progression.

[0006] In addition, therapeutic monoclonal antibodies targeting another immune checkpoint, CTLA-4, are also being used clinically. CTLA-4 inhibitors, such as ipilimumab, have been on the market for many years but have failed to achieve significant breakthroughs in various solid tumors. Therefore, combination therapy is becoming a mainstream trend. Several combinations of nivolumab and ipilimumab have shown significant efficacy compared to monotherapy for first-line treatment of melanoma, renal cell carcinoma, non-small cell lung cancer, and malignant pleural mesothelioma. Furthermore, a bispecific antibody targeting human PD-1 and CTLA-4 (cardunilimab injection) has been approved for the treatment of patients with recurrent or metastatic cervical cancer who have failed prior platinum-based 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] Currently, mainstream oncolytic virus combined immunotherapy approaches include direct use of recombinant oncolytic viruses expressing immunotherapy genes, and combinations of recombinant oncolytic viruses and immune checkpoint inhibitors (e.g., CN117503922, WO2023159102, US20210023151). However, there are no reports of recombinant oncolytic viruses and their combinations that simultaneously express two or more different immune checkpoint inhibitors (e.g., PD-1 antibodies, CTLA-4 antibodies, etc.) located on the surface of T cells. This 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. 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 the body's 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 virally encoded immune checkpoint inhibitor is a first antibody or an antigen-binding fragment thereof that specifically binds to an immune checkpoint protein, and a second antibody or an antigen-binding fragment thereof that specifically binds to a different immune checkpoint protein, both of which are located on the surface of T cells.

[0016] In certain embodiments, the two immune checkpoint proteins are PD-1 and CTLA-4.

[0017] In certain embodiments, the first antibody or antigen-binding fragment thereof specifically binds SEQ ID NO: 1.

[0018] 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.

[0019] 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.

[0020] In certain embodiments, the first antibody or antigen-binding fragment thereof comprises another first heavy chain comprising an amino acid sequence having SEQ ID NO: 11. In certain embodiments, the another first heavy chain Fab region comprises an amino acid sequence having SEQ ID NO: 12 and a homologous sequence having at least 90% sequence identity thereto. In certain embodiments, the another first heavy chain CDR region comprises an amino acid sequence having 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.

[0021] 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 LCDR1 having an amino acid sequence set forth in SEQ ID NO: 17, an LCDR2 having an amino acid sequence set forth in SEQ ID NO: 18, and an LCDR3 having an amino acid sequence set forth in SEQ ID NO: 19.

[0022] In certain embodiments, the second antibody or antigen-binding fragment thereof specifically binds SEQ ID NO:20.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the recombination steps of WR-301.

[0030] Figure 2 It is the PCR amplification band of the exogenous gene inserted into WR-301.

[0031] Figure 3 Shown are the ELISA results for PD1 and CTLA4 binding antibodies in cell harvests infected with WR-301.

[0032] Figure 4 The proliferation folds of WR-301 in MC38 and HCC1937 cells are shown.

[0033] Figure 5 Shown are the changes in viability of MC38 and HCC1937 cells infected with WR-301.

[0034] Figure 6 Shown are the changes in tumor volume in CT26-hPDL1 humanized mice injected intratumorally with WR-301.

[0035] Figure 7 Shown are the changes in survival rate of CT26-hPDL1 humanized mice injected intratumorally with WR-301. DETAILED DESCRIPTION

[0036] 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.

[0037] Oncolytic viruses 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.

[0038] 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.

[0039] 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.

[0040] 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, 7(2):157-67; and Freeman et al., 2006, Mol Therapy. .Ther.) 13(1):221-8), retroviruses (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.

[0041] 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 (Orthopoxvirus). 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).

[0042] 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 virus strain. In certain embodiments, the vaccinia virus of the present invention is a Western Reserve virus 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.

[0043] 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.

[0044] 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.

[0045] Immune checkpoint inhibitors The modified oncolytic virus genome provided herein comprises a first heterologous polynucleotide encoding an immune checkpoint inhibitor.

[0046] As used herein, the term "heterologous" means that the sequence is not endogenous to the wild-type virus.

[0047] As used herein, the term "encoding" means capable of being transcribed into mRNA and / or translated into a peptide or protein.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In certain embodiments, the immune checkpoint inhibitor is an antibody (e.g., an antagonist antibody) selected from the group consisting of anti-PD-1 (e.g., Nivolumab, Pililizumab, Pembrolizumab, BMS-936559, BMS-936558, atezolizumab, Lambrolizumab, MK-3475, AMP-224, AMP-514, ST I-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-CTLA-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., 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.

[0052] In certain embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof that can specifically bind to immune checkpoint proteins such as PD-1 and / or CTLA-4.

[0053] 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.

[0054] PD-1 inhibitors In certain embodiments, the first heterologous polynucleotide of the present invention encodes a PD-1 inhibitor.

[0055] 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.

[0056] 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 lead to autoimmunity. Persistent negative signals generated by PD-1 are implicated in T cell dysfunction in many pathological conditions, such as tumor immune evasion and chronic viral infection.

[0057] 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.

[0058] Factors that can reduce PD-1 activity: the binding between the functional protein and its ligand (e.g., the binding between PD-1 and PD-L1) is inhibited, its biological activation (e.g., the activation of PD-1) is inhibited, and / or its expression level (e.g., the expression level of PD-1) is reduced.

[0059] In certain embodiments, the PD-1 inhibitor is an antibody (eg, an antagonist antibody) that can specifically bind to PD-1.

[0060] 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) ≤ 10-6M (e.g., ≤ 5 × 10-7M, ≤ 2 × 10-7M, ≤ 10-7M, ≤ 5 × 10-8M, ≤ 2 × 10-8M, ≤ 10-8M, ≤ 5 × 10-9M, ≤ 2 × 10-9M, ≤ 10-9M, ≤ 10-10M). As used herein, KD refers to the ratio of the dissociation rate to the association rate (koff / kon), which can be determined using a surface plasmon resonance method, such as an instrument such as Biacore.

[0061] In certain embodiments, the PD-1 inhibitor is a full-length monoclonal antibody directed against PD-1.

[0062] In certain embodiments, the PD-1 antibody specifically binds to SEQ ID NO: 1.

[0063] 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.

[0064] 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 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.

[0065] 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 U.S. 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 MA et al., Methods in Enzymology, 267:383-402 (1997)). 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] CTLA-4 inhibitors In certain embodiments, the second heterologous polynucleotide of the present invention encodes a CTLA-4 inhibitor.

[0071] 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 functions as an immune checkpoint and downregulates immune responses. CTLA-4 is constitutively expressed in regulatory T cells but is only upregulated in conventional T cells upon activation, a phenomenon that is particularly prominent 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 as GenBank Accession No. NP_033973.2, and the representative nucleic acid sequence encoding human PD-1 is shown as GenBank Accession No. NM_005214.5.

[0072] 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 suppressive 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 against cancer. The effects of CTLA-4 blockade can be mediated through 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.

[0073] 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.

[0074] 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) In certain embodiments, the CTLA-4 inhibitor is an antibody (eg, an antagonist antibody) that specifically binds to CTLA-4.

[0075] 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).

[0076] In certain embodiments, the CTLA-4 inhibitor is a full-length monoclonal antibody directed against CTLA-4.

[0077] In certain embodiments, the CTLA-4 antibody specifically binds SEQ ID NO:20.

[0078] 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. 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.

[0079] 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.

[0080] Antibody 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 of 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 may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (for details, see 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., J. Mol. Biol., 273(4), 927 (1997); Chothia, C ... et al., National Institutes of Health, Bethesda, MD (1991). The three CDRs are interposed 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 based on 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).

[0081] 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.

[0082] 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.

[0083] As used herein, the term "Fab'" refers to the Fab fragment comprising a portion of the hinge region.

[0084] As used herein, the term "F(ab')2" refers to a dimer of Fab'.

[0085] 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.

[0086] 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)).

[0087] As used herein, the term "scFv dimer" refers to a polymer formed by two scFvs.

[0088] 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. Pat. No. 6,005,079). Heavy chain antibodies were originally derived from Camelidae (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 a heavy chain variable region from a heavy chain antibody and two constant regions, CH2 and CH3.

[0089] 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).

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] polynucleotides 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.

[0096] 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.

[0097] 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: Experimental Guide (Molecular Cloning: A Laboratory Manual)" (Cold Spring Harbor Laboratory, New York (Cold Spring Harbor Laboratory, N.Y.) (1989)), which is incorporated herein by reference in its entirety.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] Example 1: Virus Construction The wild-type WR virus strain was obtained from ATCC (VR-1354). Figure 1 As shown, 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 double exogenous genes and TK deletion, named WR-301.

[0102] 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.

[0103] Example 2: Virus characterization study PCR sequencing In order to ensure that the recombinant virus genome carries the designed exogenous sequence, primers P1 and P2 are set. The positions of the primers are shown in Figure 1 This pair of primers can amplify and distinguish the wild-type WR strain sequence containing TK (2.75kb) and the recombinant virus sequence containing the exogenous gene (7kb). The PCR amplification results of WR-301 are as follows: Figure 2 As shown, a band indicating WR-301 appeared, but a band indicating wild-type WR did not appear.

[0104] The PCR products were then sequenced, and alignment showed that the WR-301 amplified sequence was identical to the designed nucleic acid sequence.

[0105] ELISA To verify that WR-301 can express functional antibodies anti-PD1 and anti-CTLA4, a 96-well plate was coated with PBS-diluted PD-1 or CTLA-4 recombinant protein at a protein concentration of 1 mg / ml. After blocking with 5% skim milk powder, WR-301-infected cell harvests diluted 10-fold, 100-fold, and 1000-fold 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.

[0106] The supernatant of HeLa cells infected with WR-301 at 0.5 MOI for 72 hours was used for analysis. Figure 3 As shown, WR-301 can express antibodies that specifically bind to PD-1 and CTLA-4, and this binding is concentration-dependent.

[0107] Example 3: In vitro viral studies The following studies were conducted to confirm the proliferation ability of WR-301 in tumor cells and its ability to kill tumor cells.

[0108] proliferation MC38 (mouse colorectal 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 to detect the viral DNA copy number and obtain the multiple of the viral amount at different times relative to the initial inoculation amount. Figure 4 As shown, WR-301 significantly promoted the proliferation of both cancer cells, and its proliferation ability in human cancer cells was stronger than that in mouse cancer cells.

[0109] Killing MC38 (mouse colorectal 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 detected using CCK-8, a conventional method known in the art. The results are shown in Figure 2. Figure 5 As shown, WR-301 at various 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. This suggests that WR-301 is more potent against human cancer cells than against mouse cancer cells.

[0110] Example 4: Virus in vivo studies Eighteen humanized mice (BALB / c-hPD1hCTLA4) were implanted with humanized CT26 cells (murine colorectal cancer cells, CT26-hPDL1, 1E6 per mouse). When the tumors grew to 100 mm 3 At around 4 dpi, mice were randomly divided into three groups: PBS, WR-GFP (the TK region of the wild-type WR virus was replaced by GFP), and WR-301, with 6 mice in each group. 4E8 pfu of each virus were injected intratumorally on days 0, 5, and 10, and the body weight and tumor volume of the mice were continuously monitored. The tumor volume was 2000 mm. 3 Be kind.

[0111] The changes in tumor volume and survival rate of mice Figure 6 、 7 As shown, compared with the other two groups, the tumor volume of the WR-301 group was much smaller and the survival time was significantly prolonged.

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.

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 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, in, Both immune checkpoint proteins are located on the surface of T cells.

7. The modified oncolytic virus according to claim 6, wherein the immune checkpoint protein is selected from PD-1 and 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 comprising 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 of 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 proteins.

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

Images