CN117222436A

CN117222436A - Modified oncolytic virus, compositions comprising modified oncolytic virus and uses thereof

Info

Publication number: CN117222436A
Application number: CN202180083706.7A
Authority: CN
Inventors: 梁旻
Original assignee: Shanghai Jinsi Yuanchuang Biotechnology Co ltd; Shanghai Jinsi Biotechnology Co ltd
Current assignee: Shanghai Jinsi Yuanchuang Biotechnology Co ltd; Shanghai Jinsi Biotechnology Co ltd
Priority date: 2020-12-11
Filing date: 2021-12-10
Publication date: 2023-12-12
Also published as: WO2022122026A1

Abstract

The present invention provides a modified oncolytic virus having a first heterologous polynucleotide encoding a first molecule capable of inhibiting the interaction between PD-1 and PD-L1 and a second heterologous polynucleotide encoding a second molecule capable of inhibiting TGF-beta signaling. The invention also provides a pharmaceutical composition comprising a modified oncolytic virus and uses of the pharmaceutical composition, when in use, to administer the modified oncolytic virus or pharmaceutical composition to a subject. The modified oncolytic virus can selectively replicate in tumor cells, and the replication rate of the modified oncolytic virus is obviously higher than that of non-tumor cells in the tumor cells, so that the efficacy of the oncolytic virus is enhanced, and metastatic tumor cells are better cleared.

Description

Modified oncolytic virus, compositions comprising modified oncolytic virus and uses thereof

Technical Field

The invention relates to the technical field of tumor treatment, in particular to a modified oncolytic virus, a composition containing the modified oncolytic virus and application thereof.

Background

Over 1400 ten thousand people worldwide are diagnosed with tumors each year. Despite many advances in medical research, tumors account for about 16% of all deaths.

Malignant tumors are often resistant to conventional therapies and represent a rather difficult therapeutic challenge. For example, micrometastases can be established at a very early stage of primary tumor development. Thus, at the time of diagnosis, many tumor patients already have micrometastases. Tumor-reactive T cells can locate and destroy micrometastases and protect surrounding healthy tissue. However, naturally occurring anti-malignant T cell responses are often insufficient to cause regression of primary or metastatic tumors.

Oncolytic viruses have shown potential as antitumor agents. Unlike conventional gene therapy, oncolytic viruses are able to spread through tumor tissue by viral replication and concomitant cell lysis. However, oncolytic viruses alone are not sufficient to treat primary or metastatic tumors.

Thus, there is a particular urgent need to enhance the efficacy of oncolytic viruses and to clear metastatic tumor cells.

Disclosure of Invention

In one aspect, the disclosure relates to a modified oncolytic virus comprising a viral genome having a first heterologous polynucleotide encoding a first molecule capable of inhibiting an interaction between PD-1 and PD-L1 and a second heterologous polynucleotide encoding a second molecule capable of inhibiting TGF- β signaling.

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 anaemia virus, measles virus, coxsackie virus, vesicular stomatitis virus, saikovirus, malabar virus, newcastle disease virus, and myxoma virus. In certain embodiments, the oncolytic virus is a vaccinia virus.

In certain embodiments, the modified oncolytic virus is an attenuated virus and can replicate in tumor cells.

In certain embodiments, the viral genome comprises at least one deletion or disruption, such that the virus is capable of selective replication in tumor cells. In certain embodiments, the deletion or disruption is located in an Open Reading Frame (ORF) encoding at least a portion of an enzyme that is both critical for viral replication and preferentially expressed in tumor cells over non-tumor cells. In certain embodiments, the enzyme is a kinase. In certain embodiments, the enzyme is thymidine kinase.

In certain embodiments, the oncolytic virus is derived from a Western Reserve strain.

In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are inserted in place of the deletion.

In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are configured such that they are expressed in the same or different phases of the replication cycle of the modified oncolytic virus.

In certain embodiments, the first molecule is a first fusion protein and the second molecule is a second fusion protein. In certain embodiments, the first heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: first promoter-polynucleotide encoding first fusion protein-first stop codon, and second heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: second promoter-polynucleotide encoding a second fusion protein-second stop codon.

In certain embodiments, the first fusion protein expressed from the first heterologous polynucleotide and the second fusion protein expressed from the second heterologous polynucleotide are expressed as separate proteins. In certain embodiments, the first heterologous polynucleotide is immediately upstream or immediately downstream of the second heterologous polynucleotide. In certain embodiments, wherein the first promoter is capable of driving expression of the first fusion protein and the second promoter is capable of driving expression of the second fusion protein, wherein the first promoter and the second promoter are in a head-to-head orientation.

In certain embodiments, the first promoter and the second promoter are the same or different. In certain embodiments, the first promoter and the second promoter are both early and late promoters. In certain embodiments, the early and late promoters are pSE/L.

In certain embodiments, the first stop codon and the second stop codon are the same or different.

In certain embodiments, the first fusion protein comprises a PD-1 extracellular domain (PD-1 ECD). In certain embodiments, the first fusion protein further comprises a first immunoglobulin Fc region. In certain embodiments, the first immunoglobulin Fc region is a first human IgG1 Fc region.

In certain embodiments, the PD-1ECD is operably linked to the first immunoglobulin Fc region at the C-terminus of the PD-1 ECD.

In certain embodiments, the first fusion protein further comprises a signal peptide.

In certain embodiments, the signal peptide is operably linked to the PD-1ECD at the C-terminus of the signal peptide. In certain embodiments, the signal peptide is a CD33 signal peptide.

In certain embodiments, the PD-1ECD comprises the amino acid sequence of SEQ ID NO. 1 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO. 7 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the first human IgG1 Fc region comprises the amino acid sequence of SEQ ID NO. 3 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO. 9 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the CD33 signal peptide comprises the amino acid sequence of SEQ ID NO. 4 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID NO. 11 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the first fusion protein comprises the amino acid sequence of SEQ ID NO. 5 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID NO. 13 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the first molecule is an anti-PD-1 antibody and the second molecule is a second fusion protein.

In certain embodiments, an anti-PD-1 antibody comprises: HCDR1 having the amino acid sequence of SEQ ID No. 23 or a homologous sequence thereof having at least 80% sequence identity, HCDR2 having the amino acid sequence of SEQ ID No. 24 or a homologous sequence thereof having at least 80% sequence identity, HCDR3 having the amino acid sequence of SEQ ID No. 25 or a homologous sequence thereof having at least 80% sequence identity, LCDR1 having the amino acid sequence of SEQ ID No. 26 or a homologous sequence thereof having at least 80% sequence identity, LCDR2 having the amino acid sequence of SEQ ID No. 27 or a homologous sequence thereof having at least 80% sequence identity and LCDR3 having the amino acid sequence of SEQ ID No. 28 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, HCDR1 is encoded by the nucleic acid sequence of SEQ ID NO. 31 or a homologous sequence thereof having at least 80% sequence identity, HCDR2 is encoded by the nucleic acid sequence of SEQ ID NO. 32 or a homologous sequence thereof having at least 80% sequence identity, HCDR3 is encoded by the nucleic acid sequence of SEQ ID NO. 33 or a homologous sequence thereof having at least 80% sequence identity, LCDR1 is encoded by the nucleic acid sequence of SEQ ID NO. 34 or a homologous sequence thereof having at least 80% sequence identity, LCDR2 is encoded by the nucleic acid sequence of SEQ ID NO. 35 or a homologous sequence thereof having at least 80% sequence identity, and LCDR3 is encoded by the nucleic acid sequence of SEQ ID NO. 36 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, an anti-PD-1 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO. 29 or a homologous sequence thereof having at least 80% sequence identity and a light chain variable region having the amino acid sequence of SEQ ID NO. 30 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the anti-PD-1 antibody heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO. 37 or a homologous sequence thereof having at least 80% sequence identity, and the anti-PD-1 antibody light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO. 38 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, an anti-PD-1 antibody comprises a full length heavy chain having the amino acid sequence of SEQ ID NO. 21 or a homologous sequence thereof having at least 80% sequence identity and a full length light chain having the amino acid sequence of SEQ ID NO. 20 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the full length heavy chain of the anti-PD-1 antibody is encoded by the nucleic acid sequence of SEQ ID NO. 39 or a homologous sequence thereof having at least 80% sequence identity, and the full length light chain of the anti-PD-1 antibody is encoded by the nucleic acid sequence of SEQ ID NO. 40 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the first heterologous polynucleotide further comprises a third heterologous polynucleotide and a fourth heterologous polynucleotide, wherein the third heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: third promoter-polynucleotide encoding heavy chain of anti-PD-1 antibody-third stop codon, and wherein the fourth heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: fourth promoter-polynucleotide encoding anti-PD-1 antibody light chain-fourth stop codon; and the second heterologous polynucleotide further comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: second promoter-polynucleotide encoding a second fusion protein-second stop codon.

In certain embodiments, the third heterologous polynucleotide is immediately upstream or immediately downstream of the fourth heterologous polynucleotide. In certain embodiments, the first heterologous polynucleotide is immediately upstream or immediately downstream of the second heterologous polynucleotide.

In certain embodiments, the third promoter is capable of driving expression of the anti-PD-1 antibody heavy chain and the fourth promoter is capable of driving expression of the anti-PD-1 antibody light chain, wherein the third promoter and the fourth promoter are in a head-to-head orientation.

In certain embodiments, the anti-PD-1 antibody expressed from the first heterologous polynucleotide and the second fusion protein expressed from the second heterologous polynucleotide are expressed as separate proteins.

In certain embodiments, the second promoter, the third promoter, and the fourth promoter are the same or different. In certain embodiments, the second promoter, the third promoter, and the fourth promoter are all early and late promoters. In certain embodiments, the early and late promoters are pSE/L.

In certain embodiments, the second stop codon, the third stop codon, and the fourth stop codon are the same or different.

In certain embodiments, the second fusion protein comprises a TGF- β receptor II extracellular domain (TGFBRII ECD). In certain embodiments, the second fusion protein further comprises a second immunoglobulin Fc region. In certain embodiments, the immunoglobulin Fc region is a second human IgG1 Fc region.

In certain embodiments, the TGFBRII ECD is operably linked to the second immunoglobulin Fc region at the C-terminus of the TGFBRII ECD.

In certain embodiments, the second fusion protein further comprises a signal peptide. In certain embodiments, the signal peptide is operably linked to TGFBRII ECD at the C-terminus of the signal peptide. In certain embodiments, the signal peptide is a CD33 signal peptide.

In certain embodiments, the TGFBRII ECD comprises the amino acid sequence of SEQ ID NO. 2 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO. 8 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the second human IgG1 Fc region comprises the amino acid sequence of SEQ ID NO. 3 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO. 10 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the CD33 signal peptide comprises the amino acid sequence of SEQ ID NO. 4 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID NO. 12 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the second fusion protein comprises the amino acid sequence of SEQ ID NO. 6 or a homologous sequence thereof having at least 80% sequence identity. In certain embodiments, the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID NO. 14 or a homologous sequence thereof having at least 80% sequence identity.

In certain embodiments, the first fusion protein and the second fusion protein are capable of forming a dimer. In certain embodiments, the dimer is formed by association of a first immunoglobulin Fc region with a second immunoglobulin Fc region.

In certain embodiments, the modified oncolytic viruses provided herein have the nucleic acid sequence of SEQ ID NO. 17 or SEQ ID NO. 22.

In one aspect, the invention provides a pharmaceutical composition comprising a modified oncolytic virus provided herein and a pharmaceutically acceptable carrier.

In one aspect, the invention provides the use of a pharmaceutical composition for the treatment of a tumor, in which an effective amount of a modified oncolytic virus or pharmaceutical composition is administered to a subject, wherein the subject is a human, the route of administration is topical, and the route of administration is intratumoral injection;

Wherein the tumor is a solid tumor, which is melanoma, non-small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, head and neck squamous cell carcinoma, bladder cancer, colorectal cancer, triple negative breast cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, colon cancer, pharyngeal squamous cell carcinoma, or ovarian teratoma.

Compared with the prior art, the invention has the following beneficial effects:

the present invention introduces the first heterologous polynucleotide and the second heterologous polynucleotide into the modified oncolytic virus such that the oncolytic virus is modified, the modified oncolytic virus being capable of selectively replicating in tumor cells where the replication rate of the modified oncolytic virus is significantly higher than the replication rate in non-tumor cells, thereby enhancing the efficacy of the oncolytic virus and better clearing metastatic tumor cells.

Drawings

FIG. 1 shows a WR-GO-001 vaccinia virus construct comprising the insertion of nucleic acid sequences encoding human PD-1G22-170ECD Fc fusion protein and TGF-beta RII 23-166ECD Fc fusion protein.

FIG. 2 shows a WR-GO-002 vaccinia virus construct comprising the insertion of a nucleic acid sequence encoding the TGF-beta RII 23-166ECD Fc fusion protein.

FIG. 3 shows a WR-GO-003 vaccinia virus construct comprising the insertion of a nucleic acid sequence encoding a human PD-1G22-170ECD Fc fusion protein.

FIG. 4 shows a WR-GO-004 vaccinia virus construct comprising an insertion of nucleic acid sequences encoding anti-PD-1 antibodies and TGF-beta RII 23-166ECD Fc fusion proteins.

FIGS. 5A-5C show the expression of human PD-1Fc chimeric proteins, human TGF-beta receptor II Fc chimeric proteins and anti-human PD-1 antibodies in both supernatant and intracellular samples of WR-GO-002, WR-GO-003 or WR-GO-004 infected Hela cells measured using ELISA methods.

FIGS. 6A-6H show cytotoxicity of WR-GO-001, WR-GO-002, WR-GO-003 and WR-GO-004 in MC38 and CT26 cell lines.

FIGS. 7A to 7D show cytotoxicity assays of WR-GO-003 in human tumor cell lines (FaDu, PA1, PANC-1) and human normal cell lines (HUVEC).

FIGS. 8A to 8D show cytotoxicity assays of WR-GO-004 in human tumor cell lines (FaDu, PA1, PANC-1) and human normal cell lines (HUVEC).

Fig. 9A and 9B show the fold change in VP of WR-GO-003 or WR-GO-004 in FaDu.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Oncolytic viruses

The term "oncolytic virus" as used in this patent refers to a virus that is capable of selectively replicating and slowing the growth of or inducing death of tumor cells in vitro or in vivo with minimal or no impact on normal cells. In certain embodiments, the oncolytic virus contains a viral genome packaged into a viral particle (or virion) and is infectious (i.e., capable of infecting and entering a host cell or subject). In certain embodiments, the oncolytic virus may be a DNA virus or an RNA virus, and may be in any suitable form, such as a DNA virus vector, an RNA virus vector, or a viral particle.

As used in this patent, the term "selective replication" refers to the replication rate of oncolytic viruses in tumor cells being significantly higher than the replication rate in non-tumor cells (e.g., healthy cells). In certain embodiments, the oncolytic virus exhibits a lysis 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 in non-tumor cells (e.g., healthy cells).

In certain embodiments, the oncolytic viruses of the present disclosure can selectively replicate in liver tumor cells (e.g., hepal-6 cells, hep3B cells, 7402 cells, and 7721 cells), breast tumor cells (e.g., MCF-7 cells), tongue tumor cells (e.g., TCa8113 cells), adenoid cystic tumor cells (e.g., ACC-M cells), prostate tumor cells (e.g., LNCaP cells), human embryonic kidney cells (e.g., HEK293 cells), lung tumor cells (e.g., a549 cells), or cervical tumor cells (e.g., hela cells).

Oncolytic viruses of the present disclosure may be derived from poxviruses (e.g., vaccinia virus), adenoviruses (e.g., delta-24-RGD, ICOVIR-5, ICOVIR-7, onyx-015, coloAdl, H101, and AD 5/3-D24-GMCSF), reoviruses (e.g., reolysin), measles viruses, herpes simplex viruses (e.g., HSV, oncoveX GMCSF), newcastle viruses (e.g., 73-T PV701 and HDV-HUJ strains, and those described in Phungsab et al, 2001, cancer Lett., vol.172:1, pages 27-36; lorence et al, 2007, curr Drug Targets, volume 7, page 2: 157-167; and Freeman et al, 2006, mol. Ther, volume 13, page 1: 221-228), retrovirus (e.g., influenza virus), myxoma virus, rhabdovirus (e.g., vesicular stomatitis virus; those described in Stojdl et al, 2000, nat. Med., volume 6, page 7: 821-825; and Stojdl et al, 2003, cancer Cell, volume 4, pages 263-275), picornaviruses (e.g., seca valley virus; SW-001 and NTX-010), coxsackie virus or parvovirus.

In certain embodiments, the oncolytic viruses of the present disclosure are derived from poxviruses. The term "poxvirus" as used in this patent refers to a virus belonging to the subfamily poxviridae. In certain embodiments, the poxvirus is a virus belonging to the subfamily vertebrate poxviridae. In certain embodiments, the poxvirus is a virus belonging to the subfamily orthopoxvirus. The sequences of the genomes of various poxviruses (e.g., vaccinia virus, canary pox virus, murine poxvirus, myxoma virus) are available in the art and in specialized databases such as Genbank (accession numbers nc_006998, nc_003663, nc_005309, nc_004105, nc_001132, respectively).

In certain embodiments, the oncolytic viruses of the present disclosure are derived from vaccinia virus. Vaccinia virus is a member of the poxviridae family, characterized by a double stranded DNA genome of about 190kb, which encodes a number of viral enzymes and factors that enable replication of the virus independent of host cell mechanisms. In certain embodiments, the vaccinia virus of the present disclosure is derived from an Elstree strain, a Copenhagen strain, a Western Reserve strain, or a Wyeth strain. In certain embodiments, a vaccinia virus of the disclosure is a Western Reserve (WR) strain. The Western Reserve strain is well characterized and its complete sequence is available on the NCBI website under accession number AY243312 (www.ncbi.nlm.nih.gov).

The term "modified oncolytic virus" as used in this patent refers to oncolytic viruses that have been modified by the introduction of a heterologous nucleic acid or protein or by altering the native nucleic acid or protein. In certain embodiments, the modified oncolytic viruses provided herein are genetically altered by the deletion and/or addition of nucleic acid sequences. In certain embodiments, the modified oncolytic viruses provided herein comprise a deletion of the Thymidine Kinase (TK) gene. In certain embodiments, the modified oncolytic viruses provided herein comprise the addition of a nucleic acid sequence encoding an anti-human PD-1 antibody, a PD-1ECD-IgG1 Fc fusion protein, and/or a TGFBRII ECD-IgG1 Fc fusion protein.

In certain embodiments, the modified oncolytic viruses of the present disclosure are attenuated viruses. In certain embodiments, the modified oncolytic virus has reduced toxicity (e.g., at least 90%, 80%, 70%, 60%, 50%) or undetectable toxicity compared to its wild-type counterpart in a normal cell (e.g., a healthy cell).

The modified oncolytic viruses of the present disclosure may be derived from any oncolytic virus known in the art that is oncolytic due to its propensity to selectively replicate and kill tumor cells compared to non-tumor cells. Oncolytic viruses may be naturally oncolytic or may be made oncolytic by genetic engineering, such as by modifying one or more genes to increase tumor selectivity and/or preferential replication in tumor cells. Examples of such genes for modification include those involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, toxicity, host cell lysis, and viral transmission (see, e.g., kim et al, 2001, nat. Med., volume 7: page 781; wong et al, 2010, viruses, volume 2: pages 78-106).

In certain embodiments, the viral genome of the modified oncolytic viruses of the present disclosure comprises at least one deletion or disruption, such that the virus is capable of selective replication in tumor cells. For example, a deletion or disruption may reduce the expression or function of an enzyme critical for viral replication such that the ability of the virus to replicate in the absence of such an enzyme is reduced. In some embodiments, viral replication is dependent on the presence and/or level of such enzymes in the cell, the higher the level of enzymes, the more replication capacity or replication rate the virus.

In certain embodiments, the deletion or disruption is in an Open Reading Frame (ORF). The term "open reading frame" or "ORF" or "coding sequence" as used in this patent refers to a DNA sequence capable of translation into an amino acid sequence. The ORF typically begins with a start codon (e.g., ATG), followed by an amino acid encoding codon, and ends with a stop codon (e.g., TGA, TAA, TAG).

In certain embodiments, the ORF encodes at least a portion of an enzyme that is critical for replication of the virus and is preferentially expressed in tumor cells over non-tumor cells. The term "expression" as used in this patent refers to a process in which a protein or peptide sequence is produced from its coding DNA or RNA sequence. In certain embodiments, the enzyme is a kinase.

In certain embodiments, the deletion in the ORF constitutes 100%, more than 99%, more than 98%, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%, more than 70%, more than 65%, more than 60%, more than 55%, more than 50%, more than 45%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, or more than 10% of the full length of the ORF. In certain embodiments, the deletions in the ORF constitute at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 500, 800, 1000, 1200, 1500, 1800, 2000, 2200, 2400, 2500 or more nucleotides (optionally contiguous).

In certain embodiments, the ORF for Thymidine Kinase (TK) is deleted or disrupted. TK is involved in the synthesis of deoxyribonucleotides. TK is necessary for the replication of the virus in normal cells, as these cells usually have low concentrations of nucleotides, whereas it is not necessary in tumor cells containing high concentrations of nucleotides. In poxviruses, the thymidine kinase encoding gene is located at the locus J2R. In certain embodiments, TK is completely absent.

In certain embodiments, the ORF of Ribonucleotide Reductase (RR) is deleted or disrupted. RR catalyzes the reduction of ribonucleotides to deoxyribonucleotides, a key step in DNA biosynthesis. The viral enzyme consists of two heterologous subunits, designated R1 and R2, encoded by the I4L locus and the F4L locus, respectively. The sequences of the I4L gene and F4L gene and their positions in the genomes of various poxviruses can be obtained in public databases, for example by GenBank accession numbers DQ437594, DQ437593, DQ377804, AH015635, AY313847, AY313848, nc_003391, nc_003389, nc_003310, M-35027, AY243312, DQ011157, DQ011156, DQ011155, DQ011154, DQ011153, Y16780, X71982, AF438165, U60315, AF410153, AF380138, U86916, L22579, nc_006998, DQ121394 and nc_008291. In the context of the present invention, the I4L gene (encoding the R1 large subunit) or the F4L gene (encoding the R2 small subunit) or both may be deleted or disrupted.

In certain embodiments, the modified oncolytic virus viral genome further comprises additional deletions or disruptions that further increase the tumor specificity of the virus. In certain embodiments, the additional deletion or disruption is located in an ORF encoding at least a portion of a tumor specific protein that is preferentially or specifically expressed in tumor cells. A representative example of a tumor-specific protein is VGF. VGF is a secreted protein expressed early after infection of cells by a virus, whose function appears to be important for the transmission of the virus in normal cells. Another example is the A56R gene encoding hemagglutinin (Zhang et al, 2007, cancer Res., volume 67: pages 10038-10046). Another example is the F2L gene encoding viral dUTPase, which is involved in both maintaining the accuracy of DNA replication and providing a precursor for TMP production by thymidylate synthase (Broyles et al, 1993, virol. Vol.195:863-865). The sequence of the vaccinia virus F2L gene is available in GenBank under accession number M25392.

The term "fused" or "fused" when used with respect to an amino acid sequence (e.g., a peptide, polypeptide, or protein) refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur naturally, e.g., by chemical bonding or recombinant means. The fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences and may be expressed by introducing a construct comprising the recombinant polynucleotide into a host cell.

The term "heterologous" as used in this patent means that the sequence is not endogenous to the wild-type virus.

The term "encode" or "encode … …" as used in this patent refers to a polypeptide that is capable of transcription into mRNA and/or translation into a peptide or protein.

PD-1 extracellular Domain (PD-1 ECD)

In certain embodiments, the first molecule is a first fusion protein and the second molecule is a second fusion protein. In certain embodiments, the first fusion protein is capable of binding PD-L1. In certain embodiments, the first fusion protein is capable of blocking PD-L1 function in a tumor cell.

The term "PD-1" as used in this patent refers to a programmed cell death protein that belongs to the immunoglobulin superfamily and serves as a co-inhibitory receptor for down-regulation of 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. Representative amino acid sequences of human PD-1 are identified in GenBank accession numbers: np_005009.2, and a representative nucleic acid sequence encoding human PD-1 is disclosed in GenBank accession No.: shown under NM 005018.3.

The term "PD-1" as used in this patent is intended to include any form of PD-1 that retains useful activity, e.g., 1) naturally unprocessed PD-1 molecules, "full length" PD-1 chains, or naturally occurring variants of PD-1, including, e.g., splice variants or allelic variants; 2) Any form of PD-1 resulting from processing in a cell; or 3) full length forms, fragment forms (e.g., truncated forms, extracellular domain/transmembrane domain) or modified forms (e.g., mutant forms, glycosylated/pegylated forms, his-tag/immunofluorescent fusion forms) of PD-1 subunits produced by recombinant methods. Preferably, the PD-1 polypeptides as described in this patent, as well as protein complexes or fusion proteins comprising said polypeptides, are soluble.

Ligands for PD-1: PD-L1 (also known as B7-H1/CD 274) is expressed on antigen presenting cells and tumor cells. Binding of PD-L1 to PD-1, such as B7 to CD28, inhibits T cell activation and counteracts T cell stimulation signals. PD-L1 is not expressed by normal epithelial tissue, but it is abnormally expressed in a wide range of human cancers. In this context, activation of PD-L1 signaling may promote cancer progression by disabling host anti-tumor responses. Its expression on tumor cells is associated with a poor prognosis in renal cell carcinoma, lung carcinoma, breast carcinoma, pancreatic carcinoma, ovarian carcinoma, urothelial carcinoma, gastric carcinoma, esophageal carcinoma, colon carcinoma, pharyngeal squamous cell carcinoma, ovarian teratoma and hepatocellular carcinoma. Representative amino acid sequence of human PD-L1 is identified in NCBI accession number: np_054862.1, and a representative nucleic acid sequence encoding human PD-L1 is disclosed under NCBI accession No.: shown under NM 014143.3.

Binding of the PD-1 ECDs of the present disclosure to PD-L1 inhibits PD-L1 function, such as reducing the activity of PD-L1 by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

The activity or function (e.g., of PD-L1) may be reduced due to, for example, inhibition of binding between the functional protein and its ligand (e.g., binding between PD-1 and PD-L1), inhibition of its biological activation (e.g., activation of PD-L1), and/or reduction of the level (e.g., PD-L1 level).

In certain embodiments, the first fusion protein comprises a PD-1 protein truncation capable of specifically binding to PD-L1. In certain embodiments, the PD-1 protein truncations comprise the extracellular domain of a PD-1 protein (PD-1 ECD). In certain embodiments, the PD-1ECD is residues 22-170 of the PD-1 protein ECD (PD-1 ECD G22-170) having the amino acid sequence of SEQ ID NO. 1, corresponding to the nucleic acid sequence of SEQ ID NO. 7.

PD-1ECD G22-170 amino acid sequence (SEQ ID NO: 1)

PD-1ECD G22-170 nucleic acid sequence (SEQ ID NO: 7)

As used in this patent, the term "specific binding" or "specifically binding" refers to a non-random binding reaction between two molecules, such as, for example, a non-random binding reaction between 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) of 10-6M (e.g., 5X10-7M, 2X10-7M, 5X10-8M, 2X10-8M, 5X10-9M, 2X10-9M, 10-10M). KD as used in this patent refers to 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.

The term "identity" as used in this patent with respect to an amino acid sequence (or nucleic acid sequence) refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to reach the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues are not considered to be identical residues. To determine the percent amino acid (or Nucleic acid) sequence identity, an alignment can be accomplished, for example, using a publicly available tool such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), see also Altschul S.F. et al, J.mol.biol., volume 215: pages 403-410 (1990); stephen F. Et al, nucleic Acids Res., volume 25: pages 3389-3402 (1997)), clustalW2 (available on the website of the European Bioinformatics institute, see also Higgins D.G. et al, methods in Enzymology, volume 266: pages 383-402 (1996); larkin M.A. et al, bioinformation (Oxford, england), volume 23: pages 21: 2947-2948 (2007)), and ALIGN or Megalign (DNASTAR) software. The person skilled in the art may use default parameters provided by the tool or may customize parameters suitable for alignment, such as by selecting an appropriate algorithm.

In certain embodiments, the PD-1ECD has the amino acid sequence of SEQ ID NO. 1 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the PD-1ECD is encoded by the nucleic acid sequence of SEQ ID NO. 7 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

anti-PD-1 antibodies

The term "antibody" as used in this patent includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds a particular antigen. A natural intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and a first, second and third constant region, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ and p, while mammalian light chains are classified as λ or κ. The antibody has a "Y" shape, and the backbone of Y consists of a second constant region and a third constant region of two heavy chains bonded together via disulfide bonds. Each arm of Y comprises a variable region and a first constant region of a single heavy chain joined to a variable region and a constant region of a single light chain, wherein the first constant region of the heavy chain is linked to the second constant region by a hinge region. The variable regions of the light and heavy chains are responsible for antigen binding specificity. The variable region in both chains typically contains three highly variable loops, known as Complementarity Determining Regions (CDRs) (light (L) chain CDRs, including LCDR1, LCDR2 and LCDR3, and heavy (H) chain CDRs, including HCDR1, HCDR2 and HCDR 3). The CDR boundaries of antibodies and antigen binding fragments disclosed in this patent can be defined or divided according to the convention of Rabat, chothia or Al-Lazikani (see for details Al-Lazikani, B., chothia, C., lesk, A.M., J.Mol.Biol., vol.273, p.927 (1997)), chothia, C., et Al, J Mol biol., vol.186, vol.3, pp.651-663 (12, 5, 1985), chothia, C., and Lesk, A.M., J.Mol.Biol., vol.196, p.901 (1987)), chothia, C., et Al, nature, vol.342, 6252, pp.877-883 (21-28, 1989), rabat E.A. et Al, national Institutes of Health, bethesda, md. (1991)). The three CDRs are inserted between flanking stretches called Framework Regions (FR), which are more highly conserved than the CDRs and form a scaffold to support the structure of the variable region. The constant regions of the heavy and light chains are not associated with antigen binding specificity, but exhibit various effector functions. Antibodies are classified according to the amino acid sequence of the heavy chain constant region of the antibody. The five main 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 of the main antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), igG2 (gamma 2 heavy chain), igG3 (gamma 3 heavy chain), igG4 (gamma 4 heavy chain), igA1 (alpha 1 heavy chain) or IgA2 (alpha 2 heavy chain).

The term "antigen-binding fragment" as used in this patent refers to an antibody fragment formed from a portion of an antibody that comprises one or more CDRs but does not comprise the complete antibody structure. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') 2, fv fragments, single chain antibody molecules (scFv), scFv dimers, camelbody single domain antibodies, and nanobodies. The antigen binding fragment is capable of binding to the same antigen to which the parent antibody binds.

The term "Fab" as used in this patent refers to the portion of an antibody consisting of a single light chain (both variable and constant regions) that is bound by disulfide bonds to the variable and first constant regions of a single heavy chain.

The term "Fab'" as used in this patent refers to a Fab fragment comprising a portion of a hinge region.

The term "F (ab ') 2" as used in this patent refers to the dimer of Fab'.

The term "Fv" as used in this patent refers to an Fv fragment consisting of a variable region of a single light chain and a variable region of a single heavy chain.

The term "single chain Fv antibody" or "scFv" as used in this patent refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked to each other either directly or through a peptide linker sequence (see, e.g., huston JS et al, proc Natl Acad Sci USA, vol. 85: p. 5879 (1988)).

The term "scFv dimer" as used in this patent refers to a polymer formed from two scFv.

The term "camelized single domain antibody", also known as "heavy chain antibody" or "HCAb" (heavy chain-only antibody), 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, volume 231, stages 1-2, pages 25-38 (12, 10, 1999), muyldermans s., J biotechnol, volume 74, stages 4, pages 277-302 (6, 2001), WO94/04678, WO94/25591, and U.S. Pat. No. 6,005,079). Heavy chain antibodies were originally derived from camelidae (camel, dromedary and llama). In spite of the lack of light chains, camelized antibodies have a true antigen binding repertoire (see Hamers-Casterman C. Et al., nature, volume 363, 6428: pages 446-448 (1993); nguyen VK. et al, "Heavy-chain antibodies in Camelidae; a case of evolutionary innovation", immunogenetics, volume 54, page 1: pages 39-47 (2002); and Nguyen VK. et al, immunology, volume 109, page 1: pages 93-101 (2003), which are incorporated by reference in their entirety into this patent).

The term "nanobody" as used in this patent refers to an antibody consisting of a heavy chain variable region and two constant regions CH2 and CH3 from a chain antibody.

In certain embodiments, the antibodies provided herein are fully human, humanized, chimeric, mouse, or rabbit antibodies. In certain embodiments, the antibodies provided herein are polyclonal, monoclonal, 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 antibody or antigen-binding fragment thereof is a fully human antibody, which is optionally produced by a transgenic rat, e.g., a transgenic rat in which expression of endogenous rat immunoglobulin genes is inactivated and carries a recombinant human immunoglobulin locus with a J locus deletion and a C- κ mutation, and which may also be expressed by an engineered cell (e.g., CHO cell).

The term "fully human" as used in this patent with respect to an antibody or antigen binding fragment refers to an amino acid sequence of an antibody or antigen binding fragment that corresponds to an amino acid sequence of an antibody produced by a human or human immune cell, or an amino acid sequence derived from a non-human source such as a transgenic non-human animal utilizing a human antibody repertoire, or other human antibody coding sequence.

The term "humanized" as used in this patent with respect to an antibody or antigen binding fragment refers to an antibody or antigen binding fragment that comprises CDRs derived from a non-human animal, human-derived FR regions, and, when applicable, human-derived constant regions. Humanized antibodies or antigen binding fragments may be used as human therapeutics in certain embodiments because of their reduced immunogenicity. In certain embodiments, the non-human animal is a mammal, e.g., a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In certain embodiments, the humanized antibody or antigen binding fragment consists of substantially all human sequences except the human CDR sequences.

The term "chimeric" as used herein with respect to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment in which a portion of the heavy and/or light chain is derived from one species and the remainder of the heavy and/or light chain is derived from a different species. In certain embodiments, chimeric antibodies may comprise constant regions derived from humans and variable regions derived from non-human species, such as from mice or rabbits.

The term "conservative substitution" as used in this patent with respect to an amino acid sequence refers to the substitution of an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions may be made between amino acid residues with hydrophobic side chains (e.g., met, ala, val, leu and Ile), between residues with neutral hydrophilic side chains (e.g., cys, ser, thr, asn and gin), between residues with acidic side chains (e.g., asp, glu), between amino acids with basic side chains (e.g., his, lys, and Arg), or between residues with aromatic side chains (e.g., trp, tyr, and Phe). As known in the art, conservative substitutions typically do not cause a significant change in the conformational structure of the protein, and thus may preserve the biological activity of the protein.

The CDR sequences of anti-PD-1 antibodies are shown in Table 1. Heavy chain variable region sequences and light chain variable region sequences are also provided in table 2 below.

TABLE 1 CDR amino acid sequences and nucleotide sequences

TABLE 2 amino acid and nucleotide sequences of the variable regions

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In certain embodiments, an anti-PD-1 antibody comprises:

HCDR1 having the amino acid sequence of SEQ ID No. 23 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;

HCDR2 having the amino acid sequence of SEQ ID No. 24 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;

HCDR3 having the amino acid sequence of SEQ ID No. 25 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;

LCDR1 having the amino acid sequence of SEQ ID No. 26 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity;

LCDR2 having the amino acid sequence of SEQ ID No. 27 or a homologous sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and

LCDR3 having the amino acid sequence of SEQ ID No. 28 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, HCDR1 is encoded by the nucleic acid sequence of SEQ ID NO. 31 or a homologous sequence thereof having at least 80% sequence identity,

HCDR2 is encoded by the nucleic acid sequence of SEQ ID NO. 32 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,

HCDR3 is encoded by the nucleic acid sequence of SEQ ID NO. 33 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,

LCDR1 is encoded by the nucleic acid sequence of SEQ ID NO. 34 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity,

LCDR2 is encoded by the nucleic acid sequence of SEQ ID NO. 35 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and

LCDR3 is encoded by the nucleic acid sequence of SEQ ID NO:36 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, an anti-PD-1 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 29 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and a light chain variable region having the amino acid sequence of SEQ ID No. 30 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the anti-PD-1 antibody heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO. 37 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the anti-PD-1 antibody light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO. 38 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, an anti-PD-1 antibody comprises a full length heavy chain having the amino acid sequence of SEQ ID NO. 21 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity and a full length light chain having the amino acid sequence of SEQ ID NO. 20 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the anti-PD-1 antibodies and fragments thereof provided herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain constant region and/or a light chain constant region. The heavy chain constant region comprises a CH1 region, a hinge region, and/or a CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises ck or cλ.

In some embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein have an immunoglobulin (Ig), optionally a human Ig, optionally a constant region of human IgG. In certain embodiments, the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein comprise a constant region of an IgG1 isotype that induces ADCC or CDC, or a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function. Effector function can be assessed using various assays such as Fc receptor binding assays, C1q binding assays, and cell lysis assays.

Table 3 shows the full length sequence of the anti-PD-1 antibodies provided by this patent.

TABLE 3 full-length amino acid and nucleotide sequences of anti-PD-1 antibodies

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In certain embodiments, the full length heavy chain of an anti-PD-1 antibody is encoded by the nucleic acid sequence of SEQ ID NO. 39 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, and the full length light chain of an anti-PD-1 antibody is encoded by the nucleic acid sequence of SEQ ID NO. 40 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

The binding of the anti-PD-1 antibodies and antigen-binding fragments thereof provided herein to PD-1 inhibits the interaction between PD-1 and PD-L1, thereby reducing the activity of PD-L1, e.g., by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

The activity or function (e.g., of PD-L1) may be reduced due to, for example, inhibition of binding between a functional protein and its ligand (e.g., binding between an anti-PD-1 antibody and PD-1), inhibition of its biological activation (e.g., activation of PD-L1), and/or reduction of the level (e.g., PD-L1 level).

TGF-beta receptor extracellular domain (TGFBRII ECD)

In certain embodiments, the second molecule is a second fusion protein. In certain embodiments, the second fusion protein comprises a TGF- β receptor II extracellular domain (TGFBRII ECD).

The "transforming growth factor-beta" or "TGF-beta" superfamily is a protein composed of extracellular cytokines present in most human cells. TGF-beta refers to any protein in the TGF-beta family of proteins having any full length, native amino acid sequence of any TGF-beta from a subject (e.g., human), including latent forms and associative or non-associative complexes of precursor and mature TGF-beta. TGF-. Beta.has been shown to play a major role in the regulation of immune responses primarily through its inhibitory function on cells of the immune system. TGF- β inhibits antigen-specific T cell proliferation, at least by decreasing cell cycle rate, as opposed to inducing apoptosis. In particular, TGF- β acts on Cytotoxic T Lymphocytes (CTLs) to specifically inhibit expression of at least five cytolytic gene products important for CTL-mediated tumor cytotoxicity: perforin, granzyme a, granzyme B, fas ligand and interferon gamma (Thomas and Massague, cancer Cell, 11 month 2005, 8/5: pages 369-380). All about 40 TGF- β superfamily ligands share the same overall architecture, and each region of the protein has general features. Such TGF-beta referred to in this patent is understood to refer to any of the presently divided forms, including TGF-beta 1, TGF-beta 2, TGF-beta 3 isoforms and latent forms thereof, and to future divided human TGF-beta species, including polypeptides derived from and at least about 75%, preferably at least about 80%, more preferably at least about 85%, still more preferably at least about 90%, even more preferably at least about 95% homologous to any known TGF-beta sequence. The terms "transforming growth factor-beta", "transforming growth factor beta", "tgfβ", "TGF-beta" and "TGFB" are used interchangeably in this disclosure.

As used herein, the term "human TGF- β1" refers to a TGF- β1 protein encoded by a human TGFB1 gene (e.g., a wild-type human TGFB1 gene). An exemplary wild-type human tgfβ1 protein is provided under GenBank accession number np_ 000651.3. As used herein, the term "human TGF- β2" refers to a TGF- β2 protein encoded by a human TGFB2 gene (e.g., a wild-type human TGFB2 gene). Exemplary wild-type human TGF- β2 proteins are provided under GenBank accession numbers NP-001129071.1 and NP-003229.1. As used herein, the term "human TGF- β3" refers to a TGF- β3 protein encoded by a human TGFB3 gene (e.g., a wild-type human TGFB3 gene). Exemplary wild-type human TGF-beta 3 proteins are provided under GenBank accession numbers NP-003230.1, NP-001316868.1, and NP-001316867.1.

As used herein, the term "TGF- β receptor type II" or "TGFBRII" refers to the transforming growth factor- β receptor II (TGFBRII) protein family. Members of the TGFBRII family are typically transmembrane proteins, consisting of a ligand-binding extracellular domain with a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity. The nucleic acid sequence encoding the TGFBRII isoform a precursor protein is shown under Genbank reference sequence nm_001024847.2 and the amino acids sequenced for human TGFBRII are shown under Genbank accession No. np_ 001020018.1. The nucleic acid sequence encoding the TGFBRII isoform B precursor protein is shown under Genbank reference sequence nm_003242.6 and the amino acids sequenced for human TGFBRII are shown under Genbank accession No. np_ 003233.4. The nucleic acid sequence encoding the TGFBRII isoform X1 protein is shown under Genbank reference sequence xm_011534043.2 and the amino acids sequenced from human TGFBRII are shown under Genbank accession number xp_ 011532345.1. The nucleic acid sequence encoding the TGFBRII isoform X2 protein is shown under Genbank reference sequence xm_011534045.3 and the amino acids sequenced from human TGFBRII are shown under Genbank accession number xp_ 011532347.1. The nucleic acid sequence encoding the TGFBRII isoform X2 protein is shown under Genbank reference sequence xm_017007106.1 and the amino acids sequenced from human TGFBRII are shown under Genbank accession number xp_ 016862595.1. The expressed TGFBRII polypeptide lacks a signal sequence.

The term "TGF- β receptor type II" or "TGFBRII" as used in this patent is intended to include any form of TGF- β receptor type II that retains useful activity, e.g., 1) naturally unprocessed TGF- β receptor type II molecules, "full length" TGF- β receptor type II chains, or naturally occurring variants of TGF- β receptor type II, including, e.g., splice variants or allelic variants; 2) Any form of TGF- β receptor type II that results from processing in a cell; or 3) full length forms, fragment forms (e.g., truncated forms, extracellular domain/transmembrane domain) or modified forms (e.g., mutant forms, glycosylated/pegylated forms, his tag/immunofluorescent fusion forms) of TGF-beta receptor type II subunits produced by recombinant methods. Preferably, a TGF-beta type II polypeptide as described in this patent is soluble in a protein complex or fusion protein comprising the polypeptide.

Binding of TGFBRII of the present disclosure to TGF- β inhibits TGF- β function, such as reducing activity of TGF- β by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.

The activity or function (e.g., of TGF- β) may be reduced due to, for example, inhibition of binding between a functional protein and its ligand (e.g., binding between TGFBRII and TGF- β), inhibition of its biological activation (e.g., activation of TGF- β), and/or reduction of the level (e.g., of TGF- β).

In certain embodiments, the second fusion protein comprises a TGFBRII protein truncation capable of specifically binding to TGF- β. In certain embodiments, the TGFBRII protein truncates an extracellular domain comprising TGFBRII protein (TGFBRII ECD). In certain embodiments, TGFBRII ECD is residues 23-166 of TGFBRII ECD protein (TGFBRII ECD G23-166) having the amino acid sequence of SEQ ID NO. 2, corresponding to the nucleic acid sequence of SEQ ID NO. 8.

The amino acid sequence of TGFBRII ECD G23-166 (SEQ ID NO: 2)

TGFBRII ECD G23-166 nucleic acid sequence (SEQ ID NO: 8)

In certain embodiments, the TGFBRII ECD has the amino acid sequence of SEQ ID NO. 2 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the TGFBRII ECD is encoded by the nucleic acid sequence of SEQ ID NO. 8 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

Immunoglobulin Fc region

In certain embodiments, the first fusion protein further comprises an immunoglobulin Fc region.

In certain embodiments, the second fusion protein further comprises an immunoglobulin Fc region.

As used herein, an immunoglobulin "Fc" region refers to a portion of an antibody that consists of a second constant region and a third constant region of a first heavy chain that are bound to the second constant region and the third constant region of a second heavy chain via disulfide bonding. The Fc portion of antibodies is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not play a role in antigen binding.

In certain embodiments, the first fusion protein comprises a first immunoglobulin Fc region.

In certain embodiments, the second fusion protein comprises a second immunoglobulin Fc region.

In certain embodiments, the first immunoglobulin Fc region or the second immunoglobulin Fc region comprises one or more amino acid substitutions that improve pH-dependent binding to neonatal Fc receptor (FcRn). This variant may have an extended pharmacokinetic half-life because it binds FcRn at acidic pH, which allows it to escape degradation in lysosomes, and then translocate and be released from the cell.

In certain embodiments, the first immunoglobulin Fc region or the second immunoglobulin Fc region comprises one or more amino acid substitutions that alter Antibody Dependent Cellular Cytotoxicity (ADCC). Certain amino acid residues at the CH2 domain of the Fc region may be substituted to provide enhanced ADCC activity. Alternatively or in addition, the carbohydrate structure on the antibody may be altered to enhance ADCC activity. In certain embodiments, the first immunoglobulin Fc region or the second immunoglobulin Fc region comprises one or more amino acid substitutions that alter Complement Dependent Cytotoxicity (CDC), e.g., by increasing or decreasing C1q binding and/or CDC.

When the first heterologous polynucleotide encodes a first fusion protein and the second heterologous polynucleotide encodes a second fusion protein, the first immunoglobulin Fc region and the second immunoglobulin Fc region are capable of binding into dimers, for example, via formation of a knob-to-socket structure, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, or increased flexibility.

In certain embodiments, the first immunoglobulin Fc region or the second immunoglobulin Fc region comprises one or more amino acid substitutions in the interface of the Fc regions to facilitate/promote heterodimerization. Such modifications include introducing a protuberance into a first Fc polypeptide and introducing a cavity into a second Fc polypeptide (or introducing a cavity into a first Fc polypeptide and introducing a protuberance into a second Fc polypeptide), wherein the protuberance may be positioned in the cavity so as to facilitate interaction of the first Fc polypeptide and the second Fc polypeptide to form a heterodimer or complex.

The PD-1ECD of the present disclosure is operably linked to the first immunoglobulin Fc region at the C-terminus of the PD-1 ECD. The TGFBRII ECD of the present disclosure is operably linked to a second immunoglobulin Fc region at the C-terminus of the TGFBRII ECD.

The PD-1ECD or TGFBRII ECD of the present disclosure may be directly or indirectly linked to the first immunoglobulin Fc region or the second immunoglobulin Fc region, respectively. In certain embodiments, the PD-1ECD is linked to the first immunoglobulin Fc region through a linker. In certain embodiments, the TGFBRII ECD is linked to the second immunoglobulin Fc region through a linker. In certain embodiments, the linker is a polypeptide linker. In certain embodiments, the linker is a bifunctional cross-linking agent, such as disuccinimidyl glutarate or 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride, which may be linked at one end to the amino group of the first polypeptide and at the other end to the carboxy terminus of the second polypeptide. The skilled artisan can select an appropriate linker from among those known in the art, provided that the linked fusion protein retains sufficient biological activity.

In certain embodiments, the first immunoglobulin Fc region or the second immunoglobulin Fc region is derived from a human IgG Fc region. In certain embodiments, the first immunoglobulin Fc region and the second immunoglobulin Fc region are identical. In certain embodiments, the human IgG Fc region is a human IgG1, igG2, igG3, or IgG4 Fc region.

In certain embodiments, the first immunoglobulin Fc region and the second immunoglobulin Fc region are human IgG1Fc regions and have the amino acid sequence of SEQ ID NO:3 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

Amino acid sequence of human IgG1Fc region (SEQ ID NO: 3)

In certain embodiments, the human IgG1Fc region is encoded by the nucleic acid sequence of SEQ ID NO 9 or 10 or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

Nucleic acid sequence of human IgG1Fc region (SEQ ID NO: 9)

The nucleic acid sequence of the Fc region of human IgG1 (SEQ ID NO: 10),

In certain embodiments, the PD-1ECD is operably linked to a human IgG1Fc region at the C-terminus of the PD-1 ECD. In certain embodiments, the human IgG1Fc region is encoded by the nucleic acid sequence of SEQ ID NO. 9.

In certain embodiments, the TGFBRII ECD is operably linked to a human IgG1 Fc region at the C-terminus of the TGFBRII ECD. In certain embodiments, the human IgG1 Fc region is encoded by the nucleic acid sequence of SEQ ID NO. 10.

The term "operably linked" refers to two or more biological sequences of interest being juxtaposed with or without a spacer or linker in a manner such that they are in a relationship that allows them to function in the intended manner. When used with respect to a polypeptide, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have the intended biological function. The term may also be used with respect to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that allows the polypeptide to regulate expression from the polynucleotide.

Signal peptides

Transport of the protein may be mediated by a signal peptide located at the amino terminus of the protein itself. The signal peptide consists of about ten to twenty hydrophobic amino acids, which target nascent proteins from the ribosome to specific membrane-bound compartments such as the Endoplasmic Reticulum (ER). The ER-targeting proteins may be carried out either by the secretory pathway or may reside in any secretory organelle such as the ER, golgi apparatus or lysosomes. Proteins transported by the secretory pathway are either secreted into the extracellular space or remain in the plasma membrane. Secreted proteins are typically synthesized as inactive precursors that are activated by post-translational processing events during transport through the secretory pathway. These events include glycosylation, phosphorylation, proteolysis, and removal of signal peptides by signal peptidases. Other events that may occur during protein transport include chaperone-dependent unfolding and folding of nascent proteins and interactions of proteins with receptors or pore complexes.

In certain embodiments, the first fusion protein further comprises a signal peptide. In certain embodiments, the signal peptide of the first fusion protein is operably linked to the PD-1ECD at the C-terminus of the signal peptide.

In certain embodiments, the second fusion protein further comprises a signal peptide. In certain embodiments, the signal peptide of the second fusion protein is operably linked to TGFBRII ECD at the C-terminus of the signal peptide.

In certain embodiments, the signal peptide of the first fusion protein is a CD33 signal peptide having the amino acid sequence of SEQ ID No. 4 (MPLLLLLPLLWAGALAM) or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the signal peptide of the second fusion protein is a CD33 signal peptide having the amino acid sequence of SEQ ID No. 4 (MPLLLLLPLLWAGALAM) or a homologous sequence thereof having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the CD33 signal peptide consists of SEQ ID NO. 11

(ATGCCACTGCTCCTCCTGCTGCCACTGCTCTGGGCCGGCGCCCTCG CTATG)

Or SEQ ID NO. 12

(ATGCCTCTGCTGCTGCTGCTGCCTCTGCTGTGGGCCGGCGCCCTGG CCAT G) or a homologous sequence encoding at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

In certain embodiments, the CD33 signal peptide is operably linked to TGFBRII ECD at the C-terminus of the CD33 signal peptide. In certain embodiments, the CD33 signal peptide is encoded by the nucleic acid sequence of SEQ ID NO. 11.

In certain embodiments, the CD33 signal peptide is operably linked to TGFBRII ECD at the C-terminus of the CD33 signal peptide. In certain embodiments, the CD33 signal peptide is encoded by the nucleic acid sequence of SEQ ID NO. 12.

Polynucleotide

In certain embodiments, the modified oncolytic viruses of the present disclosure comprise a viral genome having a first heterologous polynucleotide encoding a first fusion protein capable of binding to PD-L1 and a second heterologous polynucleotide encoding a second fusion protein capable of binding to TGF- β.

The term "polynucleotide" or "nucleic acid" as used in this patent refers to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or mixed ribonucleic acid-deoxyribonucleic acid such as DNA-RNA hybrids. The polynucleotide or nucleic acid may be single-or double-stranded DNA or RNA or DNA-RNA hybrids. The polynucleotide or nucleic acid may be linear or circular. In certain embodiments, wherein when the virus is a DNA virus, both the first heterologous polynucleotide and the second heterologous polynucleotide are DNA, or when the virus is an RNA virus, both the first heterologous polynucleotide and the second heterologous polynucleotide are RNA. In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are both double stranded DNA. The polynucleotides of the present disclosure are double stranded DNA and the nucleic acid sequences are represented by coding sequences such as those shown in SEQ ID NOS 7-19, 22 and 31-40.

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 synthesized by Polymerase Chain Reaction (PCR) and ligated to a viral genome having compatible restriction ends. See, for example, sambrook et al, molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, N.Y. (1989)), which manual is incorporated by reference in its entirety.

In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are introduced in place of the deletion in the ORF. In certain embodiments, the first heterologous polynucleotide is immediately upstream or immediately downstream of the second heterologous polynucleotide. The term "immediately upstream or immediately downstream" as used in this patent means that the first heterologous polynucleotide and the second heterologous polynucleotide are positioned sufficiently close on the viral genome that they are no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides apart from each other. For example, if the 3 'end of an upstream polynucleotide is separated from the 5' end of a downstream polynucleotide by no more than 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, the 3 'end of the upstream polynucleotide is immediately adjacent to the 5' end of the downstream polynucleotide. In certain embodiments, there is no ORF between the first heterologous polynucleotide and the second heterologous polynucleotide. In certain embodiments, a restriction site exists between the first heterologous polynucleotide and the second heterologous polynucleotide.

In certain embodiments, the first heterologous polynucleotide encodes a first molecule that is a first fusion protein and the second heterologous polynucleotide encodes a second molecule that is a second fusion protein. In certain embodiments, the first heterologous polynucleotide further comprises a first promoter capable of driving expression of the first fusion protein. In certain embodiments, the second heterologous polynucleotide further comprises a second promoter capable of driving expression of the second fusion protein. In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are arranged in opposite directions with respect to protein translation. In certain embodiments, the first promoter and the second promoter are in a head-to-head orientation.

The term "head-to-head orientation" as used in this patent refers to two promoters that are directly adjacent to each other on the viral genome and that drive protein expression in opposite directions. Illustrative examples are shown in fig. 1 and 4.

The term "promoter" as used in this patent refers to a polynucleotide sequence that controls transcription of a coding sequence. Promoter sequences include specific sequences sufficient for RNA polymerase recognition, binding, and transcription initiation. In addition, the promoter sequence may include sequences that regulate such recognition, binding, and transcription initiation activity of the RNA polymerase. Promoters can affect the transcription of genes located on the same nucleic acid molecule as themselves or on different nucleic acid molecules than themselves. Depending on the nature of the regulation, the function of the promoter sequence may be constitutive or stimulus-inducible. "constitutive" promoter as used in this patent refers to a promoter that functions to continuously activate expression of a gene in a host cell. An "inducible" promoter as used in this patent refers to a promoter that activates gene expression in a host cell in the presence of certain stimuli.

In certain embodiments, the promoters of the present disclosure are eukaryotic promoters, such as promoters from CMV (e.g., CMV immediate early promoter (CMV promoter)), epstein-Barr virus (EBV) promoters, human Immunodeficiency Virus (HIV) promoters (e.g., HIV Long Terminal Repeat (LTR) promoters), moloney virus promoters, mouse Mammary Tumor Virus (MMTV) promoters, rous Sarcoma Virus (RSV) promoters, SV40 early promoters, promoters from human genes, such as the human myoglobin promoter, the human hemoglobin promoter, the human muscle creatine promoter, the human metallothionein beta-actin promoter, the human ubiquitin C (UBC) promoter, the mouse phosphoglycerate kinase (PGK) 1 promoter, the human Thymidine Kinase (TK) promoter, the human elongation factor 1 alpha (EF 1A) promoter, the cauliflower mosaic virus (CaMV) 35S promoter, the E2F-1 promoter (E2F 1 transcription factor 1 promoter), the alpha-fetoprotein promoter, the cholecystokinin promoter, the carcinoembryonic antigen promoter, the C-erbB2/neu oncogene promoter, the cyclooxygenase gene promoter, the CXC chemokine receptor 4 (CXCR 4) promoter, the human epididymal protein 4 (HE 4) promoter, the hexokinase type II promoter, the L-wire bundle protein (L-stin) promoter, the mucin-like glycoprotein (MUC 1) promoter, the Prostate Specific Antigen (PSA) promoter, the survivin promoter, tyrosinase related protein (TRP 1) promoter and tyrosinase promoter.

In certain embodiments, the promoters of the present disclosure may be tumor-specific promoters. The term "tumor-specific promoter" as used in this patent refers to a promoter that is used to preferentially or exclusively activate gene expression in tumor cells, and that is inactive or has reduced activity in non-tumor cells. Illustrative examples of tumor specific promoters include, but are not limited to, E2F-1 promoter, alpha fetoprotein promoter, cholecystokinin promoter, carcinoembryonic antigen promoter, C-erbB2/neu oncogene promoter, cyclooxygenase promoter, CXCR4 promoter, HE4 promoter, type II hexokinase promoter, L-silk bundle protein promoter, MUC1 promoter, PSA promoter, survivin promoter, TRP1 promoter, and tyrosinase promoter.

In certain embodiments, the first promoter and the second promoter are the same or different. In certain embodiments, the first promoter and the second promoter are both early and late promoters. In certain embodiments, the early and late promoters are pSE/L. In certain embodiments, the pSE/L promoter has the nucleic acid sequence of SEQ ID NO. 15 (AAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATAAG).

In certain embodiments, the first heterologous polynucleotide further comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: first promoter-polynucleotide encoding first fusion protein-first stop codon. The second heterologous polynucleotide further comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: second promoter-polynucleotide encoding a second fusion protein-second stop codon. In certain embodiments, the first heterologous polynucleotide is immediately upstream or immediately downstream of the second heterologous polynucleotide.

In certain embodiments, the first stop codon and the second stop codon are the same or different. In certain embodiments, the first stop codon and the second stop codon have the nucleic acid sequence of SEQ ID NO. 16 (TTTTTNT, where N is A, T, C or G).

In certain embodiments, the first fusion protein expressed from the first heterologous polynucleotide and the second fusion protein expressed from the second heterologous polynucleotide are expressed as separate proteins. In other words, they are not expressed as fusion proteins, nor are they linked to each other (whether covalently linked or via a linker).

In certain embodiments, the first heterologous polynucleotide and the second heterologous polynucleotide are configured such that they are expressed in the same or different phases of the replication cycle of the modified oncolytic virus. For example, both polynucleotides may be driven by an early promoter that is induced early in viral replication; or by a late promoter induced late in viral replication; or one driven by an early promoter and the other driven by a late promoter.

In certain embodiments, the modified oncolytic virus does not comprise any other heterologous polynucleotide encoding a protein in addition to the first heterologous polynucleotide and the second heterologous polynucleotide.

In certain embodiments, the first heterologous polynucleotide encodes a first molecule that is an anti-PD-1 antibody, and the second heterologous polynucleotide encodes a second molecule that is a second fusion protein. In certain embodiments, the first heterologous polynucleotide further comprises a third heterologous polynucleotide and a fourth heterologous polynucleotide. In certain embodiments, the third heterologous polynucleotide further comprises a third promoter capable of driving expression of the heavy chain of the anti-PD-1 antibody. In certain embodiments, the fourth heterologous polynucleotide further comprises a fourth promoter capable of driving expression of the anti-PD-1 antibody light chain.

In certain embodiments, the third heterologous polynucleotide further comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: third promoter-polynucleotide encoding heavy chain of anti-PD-1 antibody-third stop codon. In certain embodiments, the fourth heterologous polynucleotide further comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: fourth promoter-polynucleotide encoding anti-PD-1 antibody light chain-fourth stop codon. In certain embodiments, the third promoter and the fourth promoter are in a head-to-head orientation.

In certain embodiments, the second heterologous polynucleotide further comprises a second promoter capable of driving expression of the second fusion protein. In certain embodiments, the second heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: second promoter-polynucleotide encoding a second fusion protein-second stop codon.

In certain embodiments, the first heterologous polynucleotide comprising a third heterologous polynucleotide and a fourth heterologous polynucleotide therein is immediately upstream or immediately downstream of the second heterologous polynucleotide. In certain embodiments, the third heterologous polynucleotide is immediately upstream or immediately downstream of the fourth heterologous polynucleotide.

In certain embodiments, the second promoter, the third promoter, and the fourth promoter are the same or different. In certain embodiments, the second promoter, the third promoter, and the fourth promoter are all early and late promoters. In certain embodiments, the early and late promoters are pSE/L. In certain embodiments, the pSE/L promoter has the nucleic acid sequence of SEQ ID NO. 15.

In certain embodiments, the second stop codon, the third stop codon, and the fourth stop codon are the same or different. In certain embodiments, the second stop codon, the third stop codon, and the fourth stop codon have the nucleic acid sequence of SEQ ID NO. 16.

In certain embodiments, the anti-PD-1 antibody heavy chain expressed by the third heterologous polynucleotide and the anti-PD-1 antibody light chain expressed by the fourth heterologous polynucleotide are expressed as separate proteins. In other words, they are not expressed as fusion proteins, nor are they linked to each other (whether covalently linked or via a linker).

In certain embodiments, the anti-PD-1 antibody expressed by the first heterologous polynucleotide comprising the third heterologous polynucleotide and the fourth heterologous polynucleotide and the second fusion protein expressed by the second heterologous polynucleotide are expressed as separate proteins. In other words, they are not expressed as fusion proteins, nor are they linked to each other (whether covalently linked or via a linker).

When the third heterologous polynucleotide and the fourth heterologous polynucleotide encode the heavy and light chain, respectively, of an anti-PD-1 antibody, the third heterologous polynucleotide, the fourth heterologous polynucleotide, and the second heterologous polynucleotide are arranged in sequence for protein translation.

In certain embodiments, the third heterologous polynucleotide, the fourth heterologous polynucleotide, and the second heterologous polynucleotide are configured such that they are expressed in the same or different phases of the replication cycle of the modified oncolytic virus. For example, the polynucleotide may be driven by an early promoter that is induced early in viral replication; or by a late promoter induced late in viral replication; or one driven by an early promoter and the other two driven by a late promoter; or one driven by the late promoter and the other two driven by the early promoter.

In certain embodiments, the modified oncolytic virus does not comprise any other heterologous polynucleotide encoding a protein other than the third heterologous polynucleotide, the fourth heterologous polynucleotide, and the second heterologous polynucleotide.

Pharmaceutical composition

In another aspect, the present disclosure provides a pharmaceutical composition comprising a modified oncolytic virus described in the present disclosure and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" as used in this patent is directed to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In certain embodiments, pharmaceutically acceptable compounds, materials, compositions, and/or dosage forms refer to those compounds, materials, compositions, and/or dosage forms for animals, more particularly for humans, approved by a regulatory agency, such as the united states food and drug administration, chinese food and drug administration, or european medicines administration, or listed in a recognized pharmacopeia, such as the united states pharmacopeia, chinese pharmacopeia, or european pharmacopeia.

Pharmaceutically acceptable carriers for pharmaceutical compositions of the invention can include, but are not limited to, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles (e.g., sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactate ringer's injection), non-aqueous vehicles (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (such as sodium chloride or dextrose), buffers (such as phosphate or citrate buffers), antioxidants (such as sodium bisulfate), anesthetics (such as procaine hydrochloride), suspending/dispersing agents (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agents (such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifying agents (such as polysorbate 80 (tween 80)), diluents, adjuvants, excipients or nontoxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavouring agents, thickening agents, colouring agents or emulsifying agents.

In certain embodiments, the pharmaceutical composition is an oral formulation. Oral formulations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (for taste bases, typically sucrose and acacia or tragacanth), powders, granules or aqueous or non-aqueous solutions or suspensions, or water-in-oil or oil-in-water emulsions, or elixirs or syrups, or candy lozenges (for inert bases such as gelatin and glycerin, or sucrose or acacia) and/or mouthwashes and the like.

In certain embodiments, the pharmaceutical composition may be an injectable formulation, including a sterile aqueous solution or dispersion, suspension or emulsion. In all cases, the injectable formulation should be sterile and should be liquid to facilitate injection. It should be stable under the conditions of manufacture and storage and should be resistant to infection by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof and/or vegetable oils. The injectable formulation should maintain proper fluidity, which can be maintained in a variety of ways, for example, by the use of coatings such as lecithin, by the use of surfactants, and the like. Antimicrobial contamination can be achieved by the addition of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like).

In certain embodiments, the unit dose parenteral formulations are packaged in ampules, vials or needled syringes. As known and practiced in the art, all formulations for parenteral administration should be sterile and not cause fever.

Application method

In another aspect, the present disclosure provides a use of a pharmaceutical composition for treating a tumor and a method of use comprising administering to a subject an effective amount of a modified oncolytic virus of the present disclosure or a pharmaceutical composition of the present disclosure.

The term "subject" as used in this patent refers to both human and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals. The "subject" may also be a livestock animal (e.g., cow, pig, goat, chicken, rabbit, or horse), or a rodent (e.g., rat or mouse), or a primate (e.g., gorilla or monkey), or a domestic animal (e.g., dog or cat). The "subject" may be male or female, or may have different ages. In certain embodiments, the subject is a human. The human "subject" may be caucasian, african, asian, sumer or other race, or a mixed blood of different race. The human "subject" may be an elderly person, adult, adolescent, child or infant.

The term "tumor" as used in this patent refers to any medical condition mediated by neoplastic or malignant cell growth, proliferation or metastasis, including solid tumors and non-solid tumors such as leukemia. In the present disclosure, "tumor" is used interchangeably with the terms "cancer," malignant tumor, "" hyperproliferative, "and" neoplasm. The term "tumor cell" is interchangeable with the terms "cancer cell", "malignant cell", "hyperproliferative cell" and "neoplastic cell" unless explicitly stated otherwise. In certain embodiments, the tumor is selected from the group consisting of head and neck tumors, breast tumors, colorectal tumors, liver tumors, pancreatic adenocarcinoma, gall bladder and bile duct tumors, ovarian tumors, cervical tumors, small cell lung tumors, non-small cell lung tumors, renal cell carcinoma, bladder tumors, prostate tumors, bone tumors, mesothelioma, brain tumors, soft tissue sarcomas, uterine tumors, thyroid tumors, nasopharyngeal carcinoma, and melanoma. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is melanoma, non-small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, head and neck squamous cell carcinoma, bladder cancer, colorectal cancer, triple negative breast cancer, or hepatocellular carcinoma. In certain embodiments, the tumor is pancreatic cancer, ovarian cancer, colon cancer, pharyngeal squamous cell carcinoma, ovarian teratoma. In certain embodiments, the tumor is refractory to prior treatment (e.g., administration of oncolytic viruses, immune checkpoint inhibitors, and/or immune activators alone).

The term "treating" a disorder as used herein includes preventing or alleviating the disorder, slowing the onset or rate of progression of the disorder, reducing the risk of developing the disorder, preventing or delaying the progression of symptoms associated with the disorder, alleviating or terminating symptoms associated with the disorder, causing complete or partial regression of the disorder, curing the disorder, or some combination thereof. With respect to a tumor, "treating" may refer to inhibiting or slowing the growth, proliferation, or metastasis of a neoplastic or malignant cell, preventing or delaying the progression of the growth, proliferation, or metastasis of a neoplastic or malignant cell, or some combination thereof. With respect to tumors, "treatment" includes eradicating all or part of the tumor, inhibiting or slowing the growth and metastasis of the tumor, preventing or delaying the progression of the tumor, or some combination thereof.

The modified oncolytic viruses and pharmaceutical compositions may be administered via any suitable route known in the art including, but not limited to, parenteral, oral, enteral, buccal, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary, and subcutaneous routes of administration. In certain embodiments, the route of administration is topical. In certain embodiments, the route of administration is intratumoral injection.

In certain embodiments, the modified oncolytic viruses and pharmaceutical compositions are administered in a therapeutically effective dose. The term "therapeutically effective dose" as used in this patent refers to an amount of a drug that is capable of ameliorating or eliminating a disease or condition in a subject, or prophylactically inhibiting or preventing the occurrence of a disease or condition. A therapeutically effective amount may be an amount of a drug that ameliorates one or more diseases or symptoms of a subject to some extent; an amount of drug capable of partially or completely restoring one or more physiological or biochemical parameters associated with the etiology of the disease or condition to normal; and/or an amount of a drug capable of reducing the likelihood of occurrence of a disease or symptom.

The therapeutically effective dose of the modified oncolytic viruses and pharmaceutical compositions depends on various factors known in the art, such as, for example, body weight, age, pre-existing medical conditions, currently accepted treatment, the health of the subject, the strength of drug interactions, allergies, hyper-allergies and side effects, as well as the route of administration and the extent of disease progression. Those skilled in the art (e.g., a physician or veterinarian) can reduce or increase the dosage according to these or other conditions or requirements.

In certain embodiments, the modified oncolytic viruses and pharmaceutical compositions may be administered at a dose of about 104PFU to about 1014PFU (e.g., about 104PFU, about 2 x 104PFU, about 5 x 104PFU, about 105PFU, about 2 x 105PFU, about 5 x 105PFU, about 106PFU, about 2 x 106PFU, about 5 x 106PFU, about 107PFU, about 2 x 107PFU, about 5 x 107PFU, about 108PFU, about 2 x 108PFU, about 5 x 108PFU, about 109PFU, about 2 x 109PFU, about 5 x 109PFU, about 1010PFU, about 2 x 1o10 u, about 5 x 1010PFU, about 1011PFU, about 2 x 1011 u, about 1012PFU, about 2 x 1012PFU, about 5 x 1012PFU, about 1013PFU, about 2 x 101u, about 1014PFU, or about 3 x PFU). In certain of these embodiments, the modified oncolytic viruses and pharmaceutical compositions are administered at a dose of about 1011PFU or less. In the above embodiments, the dosage is 5 x 1010PFU or less, 2 x 1010PFU or less, 5 x 109PFU or less, 4 x 109PFU or less, 3 x 109PFU or less, 2 x 109PFU or less, or 109PFU or less. The particular dose may be divided and administered multiple times at intervals, such as once a day, two or more times a day, twice a month or more, once a week, once every two weeks, once every three weeks, once a month, or once every two months or more. In certain embodiments, the dosage administered may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than the subsequently administered dose. In certain embodiments, the dosage administered is adjusted during the course of treatment according to the response of the subject to whom it is administered.

The term "PFU" as used in this patent refers to plaque forming units, which is a measure of the number of particles capable of forming plaques.

The dosing regimen may be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

Combination of two or more kinds of materials

In certain embodiments, the pharmaceutical composition may be used in combination with one or more other drugs. In certain embodiments, the composition comprises at least one additional drug.

In certain embodiments, the other drug is an antineoplastic agent. Any agent known to have antitumor activity can be used as the antitumor agent. In certain embodiments, the anti-neoplastic agent is selected from a chemical agent, a polynucleotide, a peptide, a protein, or any combination thereof.

In certain embodiments, the antineoplastic agent is a chemical agent. Illustrative examples of anti-tumor chemicals include, but are not limited to, mitomycin C, daunorubicin, doxorubicin, etoposide, tamoxifen, paclitaxel, vincristine, and rapamycin.

In certain embodiments, the anti-neoplastic agent is a polynucleotide. Illustrative examples of anti-tumor polynucleotides include, but are not limited to, antisense oligonucleotides such as bcl-2 antisense oligonucleotides, clusterin antisense oligonucleotides, and c-myc antisense oligonucleotides; and RNAs capable of RNA interference, including small interfering RNAs (sirnas), short hairpin RNAs (shrnas), and micro-interfering RNAs (mirnas), such as anti-VEGF sirnas, shrnas or mirnas, anti-bcl-2 sirnas, shrnas or mirnas, and anti-claudin-3 sirnas, shrnas or mirnas.

In certain embodiments, the antineoplastic agent is a peptide or protein. Illustrative examples of anti-tumor peptides or proteins include, but are not limited to, antibodies such as trastuzumab, rituximab, alemtuzumab, darizumab, nituzumab, gemtuzumab, ibritumomab and ibritumomab, protein therapeutics such as endostatin, angiostatin K1-3, leuprorelin, sex hormone binding globulin, and Bikunin.

Medical application

In another aspect, the present disclosure provides the use of a modified oncolytic virus of the present disclosure or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a tumor.

In another aspect, the present disclosure provides a modified oncolytic virus of the present disclosure or a pharmaceutical composition of the present disclosure for use in treating a tumor.

Examples

The following examples are shown to aid in the understanding of the present disclosure and should not be construed to limit in any way the scope of the invention as defined in the claims following the examples.

Example 1: virus construction

The initial Western Reserve (WR) strain of vaccinia virus was obtained from ATCC (www.atcc.org: VR-1354). Due to the involvement of multiple genes, the viral WR-GO-001 (see FIG. 1) with the genomic sequence SEQ ID NO:17 has been established by stepwise engineering methods. Briefly, in a first step, WR DNA was recombined with the modified pJS1175 vector to insert a selection gene into the Thymidine Kinase (TK) locus. Then, recombinant plasmids having HR and J3R flanking sequences and encoding recombinant human PD-1Fc chimeric proteins and recombinant human TGF- βrii Fc chimeric proteins were transfected into WR-infected CV-1 cells to completely delete TK and insert chimeric protein sequences (see table 4 below).

TABLE 4 WR-GO-001 plaque production and purification methods

The amino acid sequences of the recombinant human PD-1Fc chimeric protein and the recombinant human TGF-beta RII Fc chimeric protein are shown in SEQ ID NO. 5 and 6 respectively. The nucleic acid sequence encoding the recombinant human PD-1Fc chimeric protein is shown as SEQ ID NO. 13 and the recombinant human TGF- βRII Fc chimeric protein is shown as SEQ ID NO. 14 (see Table 5).

WR-GO-002 (FIG. 2 shows the insertion of the gene encoding the recombinant human TGF- βRII Fc chimeric protein, wherein the genomic sequence SEQ ID NO:18 is shown in Table 5), WR-GO-003 (FIG. 3 shows the insertion of the gene encoding the recombinant human PD-1Fc chimeric protein, wherein the genomic sequence SEQ ID NO:19 is shown in Table 5) and WR-GO-004 (the insertion of the gene encoding the recombinant human TGF- βRII Fc chimeric protein and the anti-human PD-1 heavy and light chains, wherein the genomic sequence SEQ ID NO:22 is shown in Table 5). The amino acid sequences of the anti-human PD-1 light and heavy chains shown in SEQ ID Nos. 20 and 21, respectively, and the amino acid sequence of the recombinant human TGF- βRII Fc chimeric protein shown in SEQ ID No. 6 were prepared by the same protocol as WR-GO-001 (see Table 5).

TABLE 5 sequences in WR-GO-001, WR-GO-002, WR-GO-003, WR-GO-004

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Example 2: characterization of WR-GO-001, WR-GO-002, WR-GO-003 and WR-GO-004

During engineering of these new viruses, their genomic integrity and protein expression are closely monitored.

PCR and sequencing

The identity of the viruses (WR-GO-001, WR-GO-002, WR-GO-003 and WR-GO-004) was confirmed by qPCR (TaqMan). TK deletion was also verified by sanger sequencing. Mulberry sequencing of the WR-GO-001 viral genome was performed with an alignment of DNA sequences designed for expression of recombinant human PD-1Fc chimeric protein (rPD-1 Fc) and recombinant human TGF- βRII Fc chimeric protein (rTGF- βRII Fc) in WR-GO-001. Alignment shows that the viral genome of WR-GO-001 is identical to the designed DNA sequence.

Confirmation of expression of human PD-1Fc chimeric proteins, human TGF-beta RII Fc chimeric proteins and anti-human PD-1 antibodies using ELISA

Recombinant human PD-1Fc chimeric proteins (rPD-1 Fc) expressed by WR-GO-001 and WR-GO-003 and WR-WT infected supernatants and intracellular samples were tested by ELISA (R & D Systems). Recombinant human TGF-beta RII Fc chimeric proteins expressed by WR-GO-001, WR-GO-002 and WR-GO-004 (rTGF-beta RII Fc) and WR-WT infected supernatants and intracellular samples were tested by ELISA (R & D Systems). The WR-GO-004 infected supernatants and intracellular samples were tested for anti-human PD-1 antibodies by ELISA (R & D Systems). The capture antibodies were diluted in PBS without carrier protein. The 96-well microwell plates were immediately coated with 100 μl per well of diluted capture antibody. Seal plate and incubate overnight at room temperature. Each well was aspirated and washed with wash buffer, and the process was repeated twice for a total of three washes. Wash by filling each well with wash buffer (400 μl) using a spray bottle, manifold dispenser, or automatic washer. Complete removal of liquid in each step is critical for good performance. After the last wash, any residual wash buffer was removed by aspiration or by inverting the plate and blotting with a clean paper towel. Each well of the microplate is closed. Incubate at room temperature for at least 1 hour. To each well 100 μl of reagent diluent or appropriate diluent for the sample or standard is added, the plate is covered with adhesive tape and incubated for 2 hours at room temperature. The aspiration/washing steps were repeated as previously described. To each well, 100 μl of detection antibody was added, the plate was covered with fresh adhesive tape, and incubated for 2 hours at room temperature. The aspiration/washing steps were repeated as previously described. To each well was added 100. Mu.L of working dilution of streptavidin-HRP, covered with plate, and incubated for 20 min at room temperature. Avoiding placing the plate under direct light. The aspiration/washing steps were repeated as previously described. To each well 100 μl of substrate solution was added and incubated for 20 minutes at room temperature. Avoiding placing the plate under direct light. To each well 50. Mu.L of stop solution was added. The plate was gently tapped to ensure adequate mixing. The optical density of each well was measured immediately using a microplate reader set at 450 nm.

Expression of human PD-1Fc chimeric proteins, human TGF-beta receptor II Fc chimeric proteins, and anti-human PD-1 antibodies in both supernatant and intracellular samples of Hela cells infected with WR-GO-002, WR-GO-003, or WR-GO-004 was measured using ELISA methods.

The results of the expression levels are shown in fig. 5A, 5B and 5C. FIG. 5A shows that WR-GO-001 stably expresses the PD-l-Fc protein, which is detected mainly in WR-GO-003 supernatant. FIG. 5B shows that WR-GO-004 stably expressed anti-PD-1 antibodies were detected primarily in the WR-GO-004 supernatant. FIG. 5C shows that all WR-GO-001, WR-GO-002 and WR-GO-004 stably expressed the TGFBRII-Fc protein, which was detected mainly in the supernatant.

Example 3: in vitro and in vivo studies of WR-GO-001, WR-GO-002, WR-GO-003 and WR-GO-004 recombinant viruses

The following in vitro and in vivo studies were performed to test the selectivity and anti-tumor effects of the novel viral constructs.

Stage 1. Determination of in vitro killing effect on cancer cells and transgene expression.

1. A sufficient amount of each of the four recombinant viruses was prepared and purified with sucrose buffer for in vitro assays and in vivo experiments.

2. Viral identity was confirmed by transgene using qPCR (TaqMan) and ELISA (R & D Systems).

3. Mycoplasma testing was performed on genetically engineered vaccinia viruses and cell lines used above to confirm sterility.

4. Cell killing effects of Hepal-6, 4T1, faDu and a549 at four different time points (24 hours, 48 hours, 72 hours and 96 hours) and three different infectious titers (MOI) were measured using MTS (3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazol-e). Hepal-6 (ATCC, CRL-1830) and 4T1 (ATCC, CRL-2539) are mouse tumor cell lines, faDu (ATCC, HTB-43) and A549 (ATCC, CCL-185) are human tumor cell lines.

5. ELISA (R & D Systems) measurements of in vitro expression of recombinant PD-1Fc (rPD-1 Fc) and recombinant TGF- βRII Fc (rTGF- βRII Fc) in FaDu human tumor cell lines.

6. Four recombinant viruses were formulated at 108PFU/ml and 107PFU/ml, respectively, in buffers compatible with in vivo administration.

7. Viral replication assays in the 4 different tumor cell lines described above were tested by q-RT-PCR (Taqman).

Stage 2. Testing of viral vector safety and biodistribution.

BALB/C mice and BALB/C nude mice were used at 1.5 weeks of age. BALB/C mice were distributed among 5 treatment groups (i.e., PBS control group, WR-WT control group, low, medium and high dose WR-GO-001 group). BALB/C nude mice were treated in the same manner as BALB/C mice.

2. Oncolytic virus therapy is initiated when the tumor size of the tumor group reaches 100mm3-300mm 3. The recombinant virus is administered in a single intratumoral injection.

3. Body weight, tumor volume and health status of recombinant virus treated mice were monitored.

4. On day 9 post virus injection, mice were sacrificed and tissues from heart, liver, spleen, stomach, kidneys, lungs and tumors were collected. Vaccinia genome copies in different tissues were quantified by qPCR.

5. And re-exciting. After tumor regression, tumors were implanted into the other side of the mice. Tumor volume and health status of treated mice were monitored.

Stage 3 anti-tumor Activity in tumor model

1.5 week old BALB/C mice were used for implantation of the 4T1 tumor cell line (day 1). Mice were further distributed among 6 treatment groups (i.e., PBS control group, WR-WT control group, WR-GO-001 group, WR-GO-002 group, WR-GO-003 group, and WR-GO-004 group).

Treatment is started when the tumor size reaches 100mm3-300mm 3.

2. Viruses (1X 107 pfu) were injected into each tumor twice on day 7 and day 10 from the day of tumor implantation, respectively.

3. Mice were monitored for body weight and tumor size.

4. On day 28, mice were sacrificed and PBMC cells were collected.

5. Antigen specific spleen cell responses were determined by ELISPOT and CTL.

Stage 4 measurement of tumor-specific cytotoxicity

Cytotoxicity of WR-GO 001, WR-GO 002, WR-GO-003 and WR-GO 004 in two classes of mouse tumor cell lines (CT 26 and MC 38), three classes of human tumor cell lines (FaDu, PA1 and PANC-1) and one class of human normal cell lines (HUVEC) was measured using the CCK-8 method.

With WR-GO-001, WR-GO-002, WR-GO-003 and WR-GO-004 respectively at 10.0,

MOI of 1.0 and 0.1 infects specific cells. Cytotoxicity was tested 24 hours, 48 hours and 72 hours after virus infection.

2.1 cell culture:

200. Mu.L of medium containing 2.5X104 specific cells was inoculated in 96-well plates and incubated overnight. Cell count prior to viral infection.

2.2 viral infection:

cells in 96-well plates were infected with WR-GO-001, WR-GO-002, WR-GO-003 and WR-GO-004 at three different MOI (0.1, 1.0 and 10.0 PFU/cell), respectively.

2.3 cell viability experiments:

at the indicated time points (24, 48 and 72 hours after virus infection), 10 μl CCK-8 was added to the test wells and incubated in the incubator for 2 hours. Then, cell viability was measured by uv spectrophotometer at 450nm (n=3).

FIGS. 6A-6H show the apparent cytotoxicity of these recombinant viruses against MC38 and CT26 cells in vitro 72 hours after viral infection at MOI of 10.0 and 1.0.

FIGS. 7A to 7D show cytotoxicity assays of WR-GO-003 in human tumor cell lines (FaDu, PA1, PANC-1) and human normal cell lines (HUVEC). Shows that WR-GO-003 has obvious cytotoxicity to human tumor cells in vitro, and that the cytotoxicity of WR-GO-003 to FaDu and PANC-1 is more obvious than that to PA 1. Meanwhile, cytotoxicity to HUVEC was not obvious, which suggests that WR-GO-003 has tumor-specific cytotoxicity.

FIGS. 8A to 8D show cytotoxicity assays of WR-GO-004 in human tumor cell lines (FaDu, PA1, PANC-1) and human normal cell lines (HUVEC). The apparent cytotoxicity of WR-GO-004 against human tumor cells in vitro was shown, and the cytotoxicity of WR-GO-004 against FaDu, PANC-1 was more pronounced than that of PA1 24 hours after viral infection. Meanwhile, cytotoxicity to HUVEC was not obvious, which suggests that WR-GO-004 has tumor-specific cytotoxicity.

Viral replication (viral particles; VP) curves in FaDu, measured by q-RT-PCR.

5.0X105 tumor cells inoculated overnight in 6-well plates were infected with WR-GO-003 or WR-GO-004 at a MOI of 0.1 in serum-free medium. Cell lysates and supernatants were harvested at the indicated time points (24 hours, 48 hours, 72 hours after virus infection). Genomic DNA was extracted using tissue and blood DNA extraction kits. VP was finally evaluated by q-RT-PCR system.

The results are shown in FIGS. 9A and 9B, and demonstrate that WR-GO-003 and WR-GO-004 replicate efficiently in FaDu cells.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Sequence listing

<110> Shanghai brocade biotechnology Co., ltd

Shanghai Jinzhiyuan biotechnology Co., ltd

<120> modified oncolytic viruses, compositions and uses thereof

<130> 068615-8002WO01

<160> 40

<170> patent version 3.5

<210> 1

<211> 149

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> PD-1 ECD G22-170

<222> (1)..(149)

<400> 1

Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe

1 5 10 15

Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr

20 25 30

Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg

35 40 45

Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp

50 55 60

Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro

65 70 75 80

Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp

85 90 95

Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln

100 105 110

Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala

115 120 125

Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln

130 135 140

Phe Gln Thr Leu Val

145

<210> 2

<211> 144

<212> PRT

<213> Chile person

<220>

<221> TGF-beta receptor II ECD23-166

<222> (1)..(144)

<400> 2

Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val

1 5 10 15

Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys

20 25 30

Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn

35 40 45

Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala

50 55 60

Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His

65 70 75 80

Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser

85 90 95

Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe

100 105 110

Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser

115 120 125

Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe Gln

130 135 140

<210> 3

<211> 232

<212> PRT

<213> Chile person

<220>

<221> human IgG1-Fc region

<222> (1)..(232)

<400> 3

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 4

<211> 17

<212> PRT

<213> Chile person

<220>

<221> CD33 Signal peptide

<222> (1)..(17)

<400> 4

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met

<210> 5

<211> 398

<212> PRT

<213> artificial sequence

<220>

<223> CD33 sp-PD-1-Fc

<400> 5

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

20 25 30

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

35 40 45

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

50 55 60

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

65 70 75 80

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

85 90 95

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

100 105 110

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

115 120 125

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

130 135 140

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

145 150 155 160

Gln Phe Gln Thr Leu Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

165 170 175

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

180 185 190

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

195 200 205

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

210 215 220

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

225 230 235 240

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

245 250 255

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

260 265 270

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

275 280 285

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

290 295 300

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

305 310 315 320

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

325 330 335

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

340 345 350

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

355 360 365

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

370 375 380

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

385 390 395

<210> 6

<211> 393

<212> PRT

<213> artificial sequence

<220>

<223> CD33 sp-TGF-beta receptor II-Fc

<400> 6

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met Thr Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

20 25 30

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

35 40 45

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

50 55 60

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

65 70 75 80

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

85 90 95

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

100 105 110

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

115 120 125

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

130 135 140

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Leu Leu Leu Val Ile Phe

145 150 155 160

Gln Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

165 170 175

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

180 185 190

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

195 200 205

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

210 215 220

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

225 230 235 240

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

245 250 255

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

260 265 270

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

275 280 285

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

290 295 300

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

305 310 315 320

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

325 330 335

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

340 345 350

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

355 360 365

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

370 375 380

Lys Ser Leu Ser Leu Ser Pro Gly Lys

385 390

<210> 7

<211> 447

<212> DNA

<213> Chile person

<220>

<221> PD-1 ECD G22-170

<222> (1)..(447)

<400> 7

ggctggttcc tcgactctcc cgacagacct tggaacccac ctacattcag ccccgccctc 60

ctcgtggtga ccgagggcga caacgccacc ttcacatgca gcttcagcaa cacatccgag 120

tctttcgtgc tgaactggta tcggatgtct ccatctaacc agaccgacaa gctcgccgcc 180

ttccccgagg accgctcgca gcctggccag gattgcagat tcagagtgac ccagctccca 240

aacggcaggg acttccacat gagcgtggtg cgcgcccgga gaaacgactc tggcacatac 300

ctgtgcggcg ccattagcct cgcccctaag gcccagatta aggagtccct gcgggccgag 360

ctgagagtga cagagcggag agccgaggtg cctaccgccc acccatctcc atctcctaga 420

cccgccggcc agttccagac actcgtg 447

<210> 8

<211> 432

<212> DNA

<213> Chile person

<220>

<221> TGF-beta receptor II ECD23-166

<222> (1)..(432)

<400> 8

acaattcctc ctcacgtgca gaagagcgtg aataatgata tgattgtgac cgataataac 60

ggcgccgtga aatttcccca gctgtgtaag ttttgtgatg tgagattcag cacctgtgat 120

aatcagaagt cttgcatgtc taactgttcc atcacatcca tctgtgagaa gccacaggag 180

gtgtgcgtgg ccgtgtggag aaagaatgat gagaatatca cacttgagac cgtgtgccac 240

gacccaaaac tgccatacca cgattttatt ctggaggatg ccgcctctcc taagtgcatt 300

atgaaggaga agaagaagcc aggcgagacc ttctttatgt gtagctgttc ctctgatgag 360

tgtaatgata atattatctt cagcgaagag tataacacca gtaatcctga cctgctgctt 420

gtgattttcc ag 432

<210> 9

<211> 696

<212> DNA

<213> Chile person

<220>

<221> human IgG1-Fc region of PD-1

<222> (1)..(696)

<400> 9

gagcctaagt cttgcgacaa gacccacaca tgcccacctt gccccgcccc tgagctgctg 60

ggcggcccat ccgtgttcct gttcccacca aagccaaagg acacactcat gattagcagg 120

acacctgagg tgacatgcgt ggtggtggac gtgtctcacg aggaccccga ggtgaagttc 180

aactggtacg tggacggcgt ggaggtgcac aacgccaaga caaagcctag agaggagcag 240

tacaactcta cataccgcgt ggtgtccgtg ctcacagtgc tgcaccagga ctggctgaac 300

ggcaaggagt acaagtgcaa ggtgtccaac aaggccctcc ccgcccctat tgaaaaaacc 360

attagcaagg ccaagggcca gccacgcgag cctcaggtgt acacactgcc tccttctagg 420

gacgagctga ccaagaacca ggtgtccctc acatgcctcg tgaagggctt ctaccctagc 480

gacattgccg tggagtggga gtctaacggc cagcctgaga acaactacaa gacaacacca 540

cctgtgctcg actctgacgg ctctttcttc ctgtactcta agctcacagt ggacaagagt 600

agatggcagc agggcaacgt gttctcttgc tctgtgatgc acgaggccct gcacaaccac 660

tacacccaga agtctctgtc tctgtctcca ggcaag 696

<210> 10

<211> 696

<212> DNA

<213> Chile person

<220>

<221> human IgG1-Fc region of TGF-beta receptor II

<222> (1)..(696)

<400> 10

gagcctaagt cttgtgacaa gacacacaca tgccctccct gtccagcccc tgagctgctg 60

ggcggcccta gcgtgtttct gttcccccct aagcctaaag atacactgat gattagcaga 120

acaccagagg tgacctgtgt ggtggtggat gtgagccacg aggaccctga ggtgaagttt 180

aactggtatg tggatggagt ggaagtgcac aatgctaaga ccaagcctag agaggagcag 240

tataatagca catatagagt ggtgtctgtg ctgaccgtgc tgcaccagga ctggctgaat 300

ggcaaagagt ataaatgtaa ggtgagcaac aaggccctgc ccgcccccat tgagaaaacc 360

atctctaagg ctaagggaca gccaagagag cctcaggtgt atacactgcc tccttctaga 420

gatgagctga ccaaaaacca ggttagcctg acatgcctgg tgaaaggctt ctatccaagc 480

gatatcgctg tggaatggga gagcaatggc cagcctgaga ataattataa aaccacccca 540

ccagtgctgg atagcgatgg cagctttttc ctgtattcta agctgacagt ggataaatct 600

aggtggcagc agggcaatgt gtttagctgc tctgtgatgc acgaggccct gcacaatcac 660

tatacacaga agagcctgag cctgagccct ggcaag 696

<210> 11

<211> 51

<212> DNA

<213> Chile person

<220>

<221> CD33 Signal peptide of PD-1

<222> (1)..(51)

<400> 11

atgccactgc tcctcctgct gccactgctc tgggccggcg ccctcgctat g 51

<210> 12

<211> 51

<212> DNA

<213> Chile person

<220>

<221> CD33 Signal peptide of TGF-beta receptor II

<222> (1)..(51)

<400> 12

atgcctctgc tgctgctgct gcctctgctg tgggccggcg ccctggccat g 51

<210> 13

<211> 1197

<212> DNA

<213> artificial sequence

<220>

<223> CD33 sp-PD-1-Fc

<400> 13

atgccactgc tcctcctgct gccactgctc tgggccggcg ccctcgctat gggctggttc 60

ctcgactctc ccgacagacc ttggaaccca cctacattca gccccgccct cctcgtggtg 120

accgagggcg acaacgccac cttcacatgc agcttcagca acacatccga gtctttcgtg 180

ctgaactggt atcggatgtc tccatctaac cagaccgaca agctcgccgc cttccccgag 240

gaccgctcgc agcctggcca ggattgcaga ttcagagtga cccagctccc aaacggcagg 300

gacttccaca tgagcgtggt gcgcgcccgg agaaacgact ctggcacata cctgtgcggc 360

gccattagcc tcgcccctaa ggcccagatt aaggagtccc tgcgggccga gctgagagtg 420

acagagcgga gagccgaggt gcctaccgcc cacccatctc catctcctag acccgccggc 480

cagttccaga cactcgtgga gcctaagtct tgcgacaaga cccacacatg cccaccttgc 540

cccgcccctg agctgctggg cggcccatcc gtgttcctgt tcccaccaaa gccaaaggac 600

acactcatga ttagcaggac acctgaggtg acatgcgtgg tggtggacgt gtctcacgag 660

gaccccgagg tgaagttcaa ctggtacgtg gacggcgtgg aggtgcacaa cgccaagaca 720

aagcctagag aggagcagta caactctaca taccgcgtgg tgtccgtgct cacagtgctg 780

caccaggact ggctgaacgg caaggagtac aagtgcaagg tgtccaacaa ggccctcccc 840

gcccctattg aaaaaaccat tagcaaggcc aagggccagc cacgcgagcc tcaggtgtac 900

acactgcctc cttctaggga cgagctgacc aagaaccagg tgtccctcac atgcctcgtg 960

aagggcttct accctagcga cattgccgtg gagtgggagt ctaacggcca gcctgagaac 1020

aactacaaga caacaccacc tgtgctcgac tctgacggct ctttcttcct gtactctaag 1080

ctcacagtgg acaagagtag atggcagcag ggcaacgtgt tctcttgctc tgtgatgcac 1140

gaggccctgc acaaccacta cacccagaag tctctgtctc tgtctccagg caagtga 1197

<210> 14

<211> 1182

<212> DNA

<213> artificial sequence

<220>

<223> CD33 sp-TGF-beta receptor II-Fc

<400> 14

atgcctctgc tgctgctgct gcctctgctg tgggccggcg ccctggccat gacaattcct 60

cctcacgtgc agaagagcgt gaataatgat atgattgtga ccgataataa cggcgccgtg 120

aaatttcccc agctgtgtaa gttttgtgat gtgagattca gcacctgtga taatcagaag 180

tcttgcatgt ctaactgttc catcacatcc atctgtgaga agccacagga ggtgtgcgtg 240

gccgtgtgga gaaagaatga tgagaatatc acacttgaga ccgtgtgcca cgacccaaaa 300

ctgccatacc acgattttat tctggaggat gccgcctctc ctaagtgcat tatgaaggag 360

aagaagaagc caggcgagac cttctttatg tgtagctgtt cctctgatga gtgtaatgat 420

aatattatct tcagcgaaga gtataacacc agtaatcctg acctgctgct tgtgattttc 480

caggagccta agtcttgtga caagacacac acatgccctc cctgtccagc ccctgagctg 540

ctgggcggcc ctagcgtgtt tctgttcccc cctaagccta aagatacact gatgattagc 600

agaacaccag aggtgacctg tgtggtggtg gatgtgagcc acgaggaccc tgaggtgaag 660

tttaactggt atgtggatgg agtggaagtg cacaatgcta agaccaagcc tagagaggag 720

cagtataata gcacatatag agtggtgtct gtgctgaccg tgctgcacca ggactggctg 780

aatggcaaag agtataaatg taaggtgagc aacaaggccc tgcccgcccc cattgagaaa 840

accatctcta aggctaaggg acagccaaga gagcctcagg tgtatacact gcctccttct 900

agagatgagc tgaccaaaaa ccaggttagc ctgacatgcc tggtgaaagg cttctatcca 960

agcgatatcg ctgtggaatg ggagagcaat ggccagcctg agaataatta taaaaccacc 1020

ccaccagtgc tggatagcga tggcagcttt ttcctgtatt ctaagctgac agtggataaa 1080

tctaggtggc agcagggcaa tgtgtttagc tgctctgtga tgcacgaggc cctgcacaat 1140

cactatacac agaagagcct gagcctgagc cctggcaagt ga 1182

<210> 15

<211> 42

<212> DNA

<213> vaccinia Virus

<220>

<221> pSE/L promoter

<222> (1)..(42)

<400> 15

aaaaattgaa attttatttt ttttttttgg aatataaata ag 42

<210> 16

<211> 10

<212> DNA

<213> vaccinia Virus

<220>

<221> T5NT stop codon

<222> (1)..(7)

<220>

<221> misc_feature

<222> (6)..(6)

<223> n may be a, t, g or c.

<220>

<221> misc_feature

<222> (8)..(10)

<223> n does not exist.

<400> 16

tttttntnnn 10

<210> 17

<211> 4392

<212> DNA

<213> artificial sequence

<220>

<223> WR-GO-001

<400> 17

atggagaatg ttcctaatgt atactttaat cctgtgttta tagagcccac gtttaaacat 60

tctttattaa gtgtttataa acacagatta atagttttat ttgaagtatt cgttgtattc 120

attctaatat atgtattttt tagatctgaa ttaaatatgt tcttcatgcc taaacgaaaa 180

atacccgatc ctattgatag attacgacgt gctaatctag cgtgtgaaga cgataaatta 240

atgatctatg gattaccatg gatgacaact caaacatctg cgttatcaat aaatagtaaa 300

ccgatagtgt ataaagattg tgcaaagctt ttgcgatcaa taaatggatc acaaccagta 360

tctcttaacg atgttcttcg cagatgatga ttcatttttt aagtatttgg ctagtcaaga 420

tgatgaatct tcattatctg atatattgca aatcactcaa tatctagact ttctgttatt 480

attattgatc caatcaaaaa ataaattaga agccgtgggt cattgttatg aatctctttc 540

agaggaatac agacaattga caaaattcac agactttcaa gattttaaaa aactgtttaa 600

caaggtccct attgttacag atggaagggt caaacttaat aaaggatatt tgttcgactt 660

tgtgattagt ttgatgcgat tcaaaaaaga atcctctcta gctaccaccg caatagatcc 720

tgttagatac atagatcctc gtcgcaatat cgcattttct aacgtgatgg atatattaaa 780

gtcgaataaa gtgaacaata attaattctt tattgtcaag gcctagaaaa acctcacttg 840

cctggagaca gagacagaga cttctgggtg tagtggttgt gcagggcctc gtgcatcaca 900

gagcaagaga acacgttgcc ctgctgccat ctactcttgt ccactgtgag cttagagtac 960

aggaagaaag agccgtcaga gtcgagcaca ggtggtgttg tcttgtagtt gttctcaggc 1020

tggccgttag actcccactc cacggcaatg tcgctagggt agaagccctt cacgaggcat 1080

gtgagggaca cctggttctt ggtcagctcg tccctagaag gaggcagtgt gtacacctga 1140

ggctcgcgtg gctggccctt ggccttgcta atggtttttt caataggggc ggggagggcc 1200

ttgttggaca ccttgcactt gtactccttg ccgttcagcc agtcctggtg cagcactgtg 1260

agcacggaca ccacgcggta tgtagagttg tactgctcct ctctaggctt tgtcttggcg 1320

ttgtgcacct ccacgccgtc cacgtaccag ttgaacttca cctcggggtc ctcgtgagac 1380

acgtccacca ccacgcatgt cacctcaggt gtcctgctaa tcatgagtgt gtcctttggc 1440

tttggtggga acaggaacac ggatgggccg cccagcagct caggggcggg gcaaggtggg 1500

catgtgtggg tcttgtcgca agacttaggc tccacgagtg tctggaactg gccggcgggt 1560

ctaggagatg gagatgggtg ggcggtaggc acctcggctc tccgctctgt cactctcagc 1620

tcggcccgca gggactcctt aatctgggcc ttaggggcga ggctaatggc gccgcacagg 1680

tatgtgccag agtcgtttct ccgggcgcgc accacgctca tgtggaagtc cctgccgttt 1740

gggagctggg tcactctgaa tctgcaatcc tggccaggct gcgagcggtc ctcggggaag 1800

gcggcgagct tgtcggtctg gttagatgga gacatccgat accagttcag cacgaaagac 1860

tcggatgtgt tgctgaagct gcatgtgaag gtggcgttgt cgccctcggt caccacgagg 1920

agggcggggc tgaatgtagg tgggttccaa ggtctgtcgg gagagtcgag gaaccagccc 1980

atagcgaggg cgccggccca gagcagtggc agcaggagga gcagtggcat ggcttattta 2040

tattccaaaa aaaaaaaata aaatttcaat ttttactagt aaaaattgaa attttatttt 2100

ttttttttgg aatataaata aggatgcctc tgctgctgct gctgcctctg ctgtgggccg 2160

gcgccctggc catgacaatt cctcctcacg tgcagaagag cgtgaataat gatatgattg 2220

tgaccgataa taacggcgcc gtgaaatttc cccagctgtg taagttttgt gatgtgagat 2280

tcagcacctg tgataatcag aagtcttgca tgtctaactg ttccatcaca tccatctgtg 2340

agaagccaca ggaggtgtgc gtggccgtgt ggagaaagaa tgatgagaat atcacacttg 2400

agaccgtgtg ccacgaccca aaactgccat accacgattt tattctggag gatgccgcct 2460

ctcctaagtg cattatgaag gagaagaaga agccaggcga gaccttcttt atgtgtagct 2520

gttcctctga tgagtgtaat gataatatta tcttcagcga agagtataac accagtaatc 2580

ctgacctgct gcttgtgatt ttccaggagc ctaagtcttg tgacaagaca cacacatgcc 2640

ctccctgtcc agcccctgag ctgctgggcg gccctagcgt gtttctgttc ccccctaagc 2700

ctaaagatac actgatgatt agcagaacac cagaggtgac ctgtgtggtg gtggatgtga 2760

gccacgagga ccctgaggtg aagtttaact ggtatgtgga tggagtggaa gtgcacaatg 2820

ctaagaccaa gcctagagag gagcagtata atagcacata tagagtggtg tctgtgctga 2880

ccgtgctgca ccaggactgg ctgaatggca aagagtataa atgtaaggtg agcaacaagg 2940

ccctgcccgc ccccattgag aaaaccatct ctaaggctaa gggacagcca agagagcctc 3000

aggtgtatac actgcctcct tctagagatg agctgaccaa aaaccaggtt agcctgacat 3060

gcctggtgaa aggcttctat ccaagcgata tcgctgtgga atgggagagc aatggccagc 3120

ctgagaataa ttataaaacc accccaccag tgctggatag cgatggcagc tttttcctgt 3180

attctaagct gacagtggat aaatctaggt ggcagcaggg caatgtgttt agctgctctg 3240

tgatgcacga ggccctgcac aatcactata cacagaagag cctgagcctg agccctggca 3300

agtgactttt tatgagctcg aattctatta tattttttat ctaaaaaact aaaaataaac 3360

attgattaaa ttttaatata atacttaaaa atggatgttg tgtcgttaga taaaccgttt 3420

atgtattttg aggaaattga taatgagtta gattacgaac cagaaagtgc aaatgaggtc 3480

gcaaaaaaac tgccgtatca aggacagtta aaactattac taggagaatt attttttctt 3540

agtaagttac agcgacacgg tatattagat ggtgccaccg tagtgtatat aggatctgct 3600

cccggtacac atatacgtta tttgagagat catttctata atttaggagt gatcatcaaa 3660

tggatgctaa ttgacggccg ccatcatgat cctattttaa atggattgcg tgatgtgact 3720

ctagtgactc ggttcgttga tgaggaatat ctacgatcca tcaaaaaaca actgcatcct 3780

tctaagatta ttttaatttc tgatgtgaga tccaaacgag gaggaaatga acctagtacg 3840

gcggatttac taagtaatta cgctctacaa aatgtcatga ttagtatttt aaaccccgtg 3900

gcgtctagtc ttaaatggag atgcccgttt ccagatcaat ggatcaagga cttttatatc 3960

ccacacggta ataaaatgtt acaacctttt gctccttcat attcagctga aatgagatta 4020

ttaagtattt ataccggtga gaacatgaga ctgactcgag ttaccaaatc agacgctgta 4080

aattatgaaa aaaagatgta ctaccttaat aagatcgtcc gtaacaaagt agttgttaac 4140

tttgattatc ctaatcagga atatgactat tttcacatgt actttatgct gaggaccgtg 4200

tactgcaata aaacatttcc tactactaaa gcaaaggtac tatttctaca acaatctata 4260

tttcgtttct taaatattcc aacaacatca actgaaaaag ttagtcatga accaatacaa 4320

cgtaaaatat ctagcaaaaa ttctatgtct aaaaacagaa atagcaagag atccgtacgc 4380

agtaataaat ag 4392

<210> 18

<211> 3199

<212> DNA

<213> artificial sequence

<220>

<223> WR-GO-002

<400> 18

atggagaatg ttcctaatgt atactttaat cctgtgttta tagagcccac gtttaaacat 60

tctttattaa gtgtttataa acacagatta atagttttat ttgaagtatt cgttgtattc 120

attctaatat atgtattttt tagatctgaa ttaaatatgt tcttcatgcc taaacgaaaa 180

atacccgatc ctattgatag attacgacgt gctaatctag cgtgtgaaga cgataaatta 240

atgatctatg gattaccatg gatgacaact caaacatctg cgttatcaat aaatagtaaa 300

ccgatagtgt ataaagattg tgcaaagctt ttgcgatcaa taaatggatc acaaccagta 360

tctcttaacg atgttcttcg cagatgatga ttcatttttt aagtatttgg ctagtcaaga 420

tgatgaatct tcattatctg atatattgca aatcactcaa tatctagact ttctgttatt 480

attattgatc caatcaaaaa ataaattaga agccgtgggt cattgttatg aatctctttc 540

agaggaatac agacaattga caaaattcac agactttcaa gattttaaaa aactgtttaa 600

caaggtccct attgttacag atggaagggt caaacttaat aaaggatatt tgttcgactt 660

tgtgattagt ttgatgcgat tcaaaaaaga atcctctcta gctaccaccg caatagatcc 720

tgttagatac atagatcctc gtcgcaatat cgcattttct aacgtgatgg atatattaaa 780

gtcgaataaa gtgaacaata attaattctt tattgtcaag gcctagaaaa acctcgaggc 840

ttatttatat tccaaaaaaa aaaaataaaa tttcaatttt tactagtaaa aattgaaatt 900

ttattttttt tttttggaat ataaataagg atgcctctgc tgctgctgct gcctctgctg 960

tgggccggcg ccctggccat gacaattcct cctcacgtgc agaagagcgt gaataatgat 1020

atgattgtga ccgataataa cggcgccgtg aaatttcccc agctgtgtaa gttttgtgat 1080

gtgagattca gcacctgtga taatcagaag tcttgcatgt ctaactgttc catcacatcc 1140

atctgtgaga agccacagga ggtgtgcgtg gccgtgtgga gaaagaatga tgagaatatc 1200

acacttgaga ccgtgtgcca cgacccaaaa ctgccatacc acgattttat tctggaggat 1260

gccgcctctc ctaagtgcat tatgaaggag aagaagaagc caggcgagac cttctttatg 1320

tgtagctgtt cctctgatga gtgtaatgat aatattatct tcagcgaaga gtataacacc 1380

agtaatcctg acctgctgct tgtgattttc caggagccta agtcttgtga caagacacac 1440

acatgccctc cctgtccagc ccctgagctg ctgggcggcc ctagcgtgtt tctgttcccc 1500

cctaagccta aagatacact gatgattagc agaacaccag aggtgacctg tgtggtggtg 1560

gatgtgagcc acgaggaccc tgaggtgaag tttaactggt atgtggatgg agtggaagtg 1620

cacaatgcta agaccaagcc tagagaggag cagtataata gcacatatag agtggtgtct 1680

gtgctgaccg tgctgcacca ggactggctg aatggcaaag agtataaatg taaggtgagc 1740

aacaaggccc tgcccgcccc cattgagaaa accatctcta aggctaaggg acagccaaga 1800

gagcctcagg tgtatacact gcctccttct agagatgagc tgaccaaaaa ccaggttagc 1860

ctgacatgcc tggtgaaagg cttctatcca agcgatatcg ctgtggaatg ggagagcaat 1920

ggccagcctg agaataatta taaaaccacc ccaccagtgc tggatagcga tggcagcttt 1980

ttcctgtatt ctaagctgac agtggataaa tctaggtggc agcagggcaa tgtgtttagc 2040

tgctctgtga tgcacgaggc cctgcacaat cactatacac agaagagcct gagcctgagc 2100

cctggcaagt gactttttat gagctcgaat tctattatat tttttatcta aaaaactaaa 2160

aataaacatt gattaaattt taatataata cttaaaaatg gatgttgtgt cgttagataa 2220

accgtttatg tattttgagg aaattgataa tgagttagat tacgaaccag aaagtgcaaa 2280

tgaggtcgca aaaaaactgc cgtatcaagg acagttaaaa ctattactag gagaattatt 2340

ttttcttagt aagttacagc gacacggtat attagatggt gccaccgtag tgtatatagg 2400

atctgctccc ggtacacata tacgttattt gagagatcat ttctataatt taggagtgat 2460

catcaaatgg atgctaattg acggccgcca tcatgatcct attttaaatg gattgcgtga 2520

tgtgactcta gtgactcggt tcgttgatga ggaatatcta cgatccatca aaaaacaact 2580

gcatccttct aagattattt taatttctga tgtgagatcc aaacgaggag gaaatgaacc 2640

tagtacggcg gatttactaa gtaattacgc tctacaaaat gtcatgatta gtattttaaa 2700

ccccgtggcg tctagtctta aatggagatg cccgtttcca gatcaatgga tcaaggactt 2760

ttatatccca cacggtaata aaatgttaca accttttgct ccttcatatt cagctgaaat 2820

gagattatta agtatttata ccggtgagaa catgagactg actcgagtta ccaaatcaga 2880

cgctgtaaat tatgaaaaaa agatgtacta ccttaataag atcgtccgta acaaagtagt 2940

tgttaacttt gattatccta atcaggaata tgactatttt cacatgtact ttatgctgag 3000

gaccgtgtac tgcaataaaa catttcctac tactaaagca aaggtactat ttctacaaca 3060

atctatattt cgtttcttaa atattccaac aacatcaact gaaaaagtta gtcatgaacc 3120

aatacaacgt aaaatatcta gcaaaaattc tatgtctaaa aacagaaata gcaagagatc 3180

cgtacgcagt aataaatag 3199

<210> 19

<211> 3214

<212> DNA

<213> artificial sequence

<220>

<223> WR-GO-003

<400> 19

atggagaatg ttcctaatgt atactttaat cctgtgttta tagagcccac gtttaaacat 60

tctttattaa gtgtttataa acacagatta atagttttat ttgaagtatt cgttgtattc 120

attctaatat atgtattttt tagatctgaa ttaaatatgt tcttcatgcc taaacgaaaa 180

atacccgatc ctattgatag attacgacgt gctaatctag cgtgtgaaga cgataaatta 240

atgatctatg gattaccatg gatgacaact caaacatctg cgttatcaat aaatagtaaa 300

ccgatagtgt ataaagattg tgcaaagctt ttgcgatcaa taaatggatc acaaccagta 360

tctcttaacg atgttcttcg cagatgatga ttcatttttt aagtatttgg ctagtcaaga 420

tgatgaatct tcattatctg atatattgca aatcactcaa tatctagact ttctgttatt 480

attattgatc caatcaaaaa ataaattaga agccgtgggt cattgttatg aatctctttc 540

agaggaatac agacaattga caaaattcac agactttcaa gattttaaaa aactgtttaa 600

caaggtccct attgttacag atggaagggt caaacttaat aaaggatatt tgttcgactt 660

tgtgattagt ttgatgcgat tcaaaaaaga atcctctcta gctaccaccg caatagatcc 720

tgttagatac atagatcctc gtcgcaatat cgcattttct aacgtgatgg atatattaaa 780

gtcgaataaa gtgaacaata attaattctt tattgtcaag gcctagaaaa acctcacttg 840

cctggagaca gagacagaga cttctgggtg tagtggttgt gcagggcctc gtgcatcaca 900

gagcaagaga acacgttgcc ctgctgccat ctactcttgt ccactgtgag cttagagtac 960

aggaagaaag agccgtcaga gtcgagcaca ggtggtgttg tcttgtagtt gttctcaggc 1020

tggccgttag actcccactc cacggcaatg tcgctagggt agaagccctt cacgaggcat 1080

gtgagggaca cctggttctt ggtcagctcg tccctagaag gaggcagtgt gtacacctga 1140

ggctcgcgtg gctggccctt ggccttgcta atggtttttt caataggggc ggggagggcc 1200

ttgttggaca ccttgcactt gtactccttg ccgttcagcc agtcctggtg cagcactgtg 1260

agcacggaca ccacgcggta tgtagagttg tactgctcct ctctaggctt tgtcttggcg 1320

ttgtgcacct ccacgccgtc cacgtaccag ttgaacttca cctcggggtc ctcgtgagac 1380

acgtccacca ccacgcatgt cacctcaggt gtcctgctaa tcatgagtgt gtcctttggc 1440

tttggtggga acaggaacac ggatgggccg cccagcagct caggggcggg gcaaggtggg 1500

catgtgtggg tcttgtcgca agacttaggc tccacgagtg tctggaactg gccggcgggt 1560

ctaggagatg gagatgggtg ggcggtaggc acctcggctc tccgctctgt cactctcagc 1620

tcggcccgca gggactcctt aatctgggcc ttaggggcga ggctaatggc gccgcacagg 1680

tatgtgccag agtcgtttct ccgggcgcgc accacgctca tgtggaagtc cctgccgttt 1740

gggagctggg tcactctgaa tctgcaatcc tggccaggct gcgagcggtc ctcggggaag 1800

gcggcgagct tgtcggtctg gttagatgga gacatccgat accagttcag cacgaaagac 1860

tcggatgtgt tgctgaagct gcatgtgaag gtggcgttgt cgccctcggt caccacgagg 1920

agggcggggc tgaatgtagg tgggttccaa ggtctgtcgg gagagtcgag gaaccagccc 1980

atagcgaggg cgccggccca gagcagtggc agcaggagga gcagtggcat ggcttattta 2040

tattccaaaa aaaaaaaata aaatttcaat ttttactagt aaaaattgaa attttatttt 2100

ttttttttgg aatataaata aggtcgactt tttatgagct cgaattctat tatatttttt 2160

atctaaaaaa ctaaaaataa acattgatta aattttaata taatacttaa aaatggatgt 2220

tgtgtcgtta gataaaccgt ttatgtattt tgaggaaatt gataatgagt tagattacga 2280

accagaaagt gcaaatgagg tcgcaaaaaa actgccgtat caaggacagt taaaactatt 2340

actaggagaa ttattttttc ttagtaagtt acagcgacac ggtatattag atggtgccac 2400

cgtagtgtat ataggatctg ctcccggtac acatatacgt tatttgagag atcatttcta 2460

taatttagga gtgatcatca aatggatgct aattgacggc cgccatcatg atcctatttt 2520

aaatggattg cgtgatgtga ctctagtgac tcggttcgtt gatgaggaat atctacgatc 2580

catcaaaaaa caactgcatc cttctaagat tattttaatt tctgatgtga gatccaaacg 2640

aggaggaaat gaacctagta cggcggattt actaagtaat tacgctctac aaaatgtcat 2700

gattagtatt ttaaaccccg tggcgtctag tcttaaatgg agatgcccgt ttccagatca 2760

atggatcaag gacttttata tcccacacgg taataaaatg ttacaacctt ttgctccttc 2820

atattcagct gaaatgagat tattaagtat ttataccggt gagaacatga gactgactcg 2880

agttaccaaa tcagacgctg taaattatga aaaaaagatg tactacctta ataagatcgt 2940

ccgtaacaaa gtagttgtta actttgatta tcctaatcag gaatatgact attttcacat 3000

gtactttatg ctgaggaccg tgtactgcaa taaaacattt cctactacta aagcaaaggt 3060

actatttcta caacaatcta tatttcgttt cttaaatatt ccaacaacat caactgaaaa 3120

agttagtcat gaaccaatac aacgtaaaat atctagcaaa aattctatgt ctaaaaacag 3180

aaatagcaag agatccgtac gcagtaataa atag 3214

<210> 20

<211> 233

<212> PRT

<213> artificial sequence

<220>

<223> anti-PD-1 antibody-LC

<400> 20

Met Ala Trp Ser Pro Leu Phe Leu Thr Leu Ile Thr His Cys Ala Gly

1 5 10 15

Ser Trp Ala Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu

20 25 30

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val

35 40 45

Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg

50 55 60

Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn

100 105 110

Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr

115 120 125

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

130 135 140

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

145 150 155 160

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

165 170 175

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

180 185 190

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

195 200 205

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

210 215 220

Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230

<210> 21

<211> 459

<212> PRT

<213> artificial sequence

<220>

<223> anti-PD-1 antibody-HC

<400> 21

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe

35 40 45

Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val

115 120 125

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

130 135 140

Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu

145 150 155 160

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

165 170 175

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

180 185 190

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

195 200 205

Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr

210 215 220

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

355 360 365

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

450 455

<210> 22

<211> 5338

<212> DNA

<213> artificial sequence

<220>

<223> WR-GO-004

<400> 22

atggagaatg ttcctaatgt atactttaat cctgtgttta tagagcccac gtttaaacat 60

tctttattaa gtgtttataa acacagatta atagttttat ttgaagtatt cgttgtattc 120

attctaatat atgtattttt tagatctgaa ttaaatatgt tcttcatgcc taaacgaaaa 180

atacccgatc ctattgatag attacgacgt gctaatctag cgtgtgaaga cgataaatta 240

atgatctatg gattaccatg gatgacaact caaacatctg cgttatcaat aaatagtaaa 300

ccgatagtgt ataaagattg tgcaaagctt ttgcgatcaa taaatggatc acaaccagta 360

tctcttaacg atgttcttcg cagatgatga ttcatttttt aagtatttgg ctagtcaaga 420

tgatgaatct tcattatctg atatattgca aatcactcaa tatctagact ttctgttatt 480

attattgatc caatcaaaaa ataaattaga agccgtgggt cattgttatg aatctctttc 540

agaggaatac agacaattga caaaattcac agactttcaa gattttaaaa aactgtttaa 600

caaggtccct attgttacag atggaagggt caaacttaat aaaggatatt tgttcgactt 660

tgtgattagt ttgatgcgat tcaaaaaaga atcctctcta gctaccaccg caatagatcc 720

tgttagatac atagatcctc gtcgcaatat cgcattttct aacgtgatgg atatattaaa 780

gtcgaataaa gtgaacaata attaattctt tattgtcaag gcctagaaaa accttacttc 840

cctaggctga gactcagtga tttttgggtg taatggttgt gcaacgcttc gtgcatcacg 900

gagcaggaaa agacattacc ttcctgccaa cggcttttat caacggtcag gcgcgagtac 960

agaaagaagc tgccgtcgct gtcaagcacg ggtggagtag tcttatagtt attctctggt 1020

tggccattac tctcccactc cacggctata tcgctagggt aaaaaccctt aaccagacag 1080

gtcagactta cctggttttt agtcatttct tcctgtgagg gtggtagggt gtagacctga 1140

ggttcccgag gctgtccctt ggctttgcta attgtctttt cgatggaaga gggcaatcct 1200

ttgtttgaca ccttgcactt atattccttg ccattcagcc aatcctggtg caacactgtc 1260

agcacgctga cgacccgata ggttgaattg aattgttcct cgcggggttt cgtctttgcg 1320

ttatgcactt caacgccgtc tacgtaccag ttgaattgca cttcgggatc ctcttgagag 1380

acatcaacca cgacacaagt cacttccggg gtccggctaa tcattagggt gtccttaggt 1440

tttggtggga acagaaacac ggacggtcca cccaagaact cgggggcagg acacggaggg 1500

caggggggtc catacttgga ttcgacccgc ttatctactt tagtattaga gggcttgtgg 1560

tcaacgttac aggtataagt tttcgtgcct agactagatg aaggaacagt gaccacggaa 1620

gatagtgaat agaggcccga gctctgcagc acggcaggga atgtgtgcac tccagaggtc 1680

agggcgccgc tgttccagga cacagtcacc ggttctggaa agtagtcctt cacgagacat 1740

cctagcgcag ctgtgctttc gcttgtgctc ctactgcagg gggcgagcgg gaacactgag 1800

ggtcccttgg tgcttgccga acttacggtc accaatgtgc cctgtcccca gtaatcgtcg 1860

tttgtggcgc agtagtatac ggctgtgtcc tcagcccgca gtgaattcat ctgtaggaac 1920

aatgtatttt tagagttgtc cctactaatg gtgaaccgtc ccttcacaga gtcggcgtaa 1980

taacgcttgg agccgtcata ccagataact gccacccact ccaagccctt gcctggcgct 2040

tgccgtaccc agtgcatccc gctgttagaa aaggtgatgc cggaagcctt acagtccaat 2100

cttagtgatc tgcctggctg gacaactccg ccgcctgact caaccagctg tacttggctg 2160

tgggcgcctg ttgccgcggc aaccaagaac aggattctcc aggtccaatc catctcgagc 2220

ttatttatat tccaaaaaaa aaaaataaaa tttcaatttt taaaaaattg aaattttatt 2280

tttttttttt ggaatataaa taaggtcgac atggcatggt ccccattatt tctgaccttg 2340

attactcact gcgccggctc ttgggctgaa atcgtactca cgcagtcccc tgctactctg 2400

agtctctcac caggagaacg cgctaccctt tcttgccgtg cgtcacagtc agtatcgtcc 2460

tatctggctt ggtatcagca aaaaccaggt caggcccccc gattattgat ttatgatgca 2520

tctaaccggg ctacagggat tcctgccaga tttagcggta gcgggagtgg aactgacttc 2580

actctaacca ttagctccct tgagccagag gatttcgccg tctactactg tcagcagtct 2640

tccaactggc ctcgtacttt cggacaggga acaaaggtgg aaatcaaacg taccgtggct 2700

gcacccagcg tgttcatttt tccaccaagc gacgagcagc tcaagagcgg aaccgcatcc 2760

gtagtatgtc tcctcaataa cttctaccca cgagaagcca aagtgcagtg gaaggtggat 2820

aatgccttgc aatccggaaa cagccaagaa agcgtgaccg aacaggattc aaaagacagc 2880

acctattctc tgtccagcac attgacactg agtaaagctg attatgagaa gcacaaggtc 2940

tacgcgtgtg aggttacaca tcaaggattg tcttcaccag tcaccaagag tttcaataga 3000

ggagagtgct gagaattctt tttattaaaa attgaaattt tatttttttt ttttggaata 3060

taaataagga tgcctctgct gctgctgctg cctctgctgt gggccggcgc cctggccatg 3120

acaattcctc ctcacgtgca gaagagcgtg aataatgata tgattgtgac cgataataac 3180

ggcgccgtga aatttcccca gctgtgtaag ttttgtgatg tgagattcag cacctgtgat 3240

aatcagaagt cttgcatgtc taactgttcc atcacatcca tctgtgagaa gccacaggag 3300

gtgtgcgtgg ccgtgtggag aaagaatgat gagaatatca cacttgagac cgtgtgccac 3360

gacccaaaac tgccatacca cgattttatt ctggaggatg ccgcctctcc taagtgcatt 3420

atgaaggaga agaagaagcc aggcgagacc ttctttatgt gtagctgttc ctctgatgag 3480

tgtaatgata atattatctt cagcgaagag tataacacca gtaatcctga cctgctgctt 3540

gtgattttcc aggagcctaa gtcttgtgac aagacacaca catgccctcc ctgtccagcc 3600

cctgagctgc tgggcggccc tagcgtgttt ctgttccccc ctaagcctaa agatacactg 3660

atgattagca gaacaccaga ggtgacctgt gtggtggtgg atgtgagcca cgaggaccct 3720

gaggtgaagt ttaactggta tgtggatgga gtggaagtgc acaatgctaa gaccaagcct 3780

agagaggagc agtataatag cacatataga gtggtgtctg tgctgaccgt gctgcaccag 3840

gactggctga atggcaaaga gtataaatgt aaggtgagca acaaggccct gcccgccccc 3900

attgagaaaa ccatctctaa ggctaaggga cagccaagag agcctcaggt gtatacactg 3960

cctccttcta gagatgagct gaccaaaaac caggttagcc tgacatgcct ggtgaaaggc 4020

ttctatccaa gcgatatcgc tgtggaatgg gagagcaatg gccagcctga gaataattat 4080

aaaaccaccc caccagtgct ggatagcgat ggcagctttt tcctgtattc taagctgaca 4140

gtggataaat ctaggtggca gcagggcaat gtgtttagct gctctgtgat gcacgaggcc 4200

ctgcacaatc actatacaca gaagagcctg agcctgagcc ctggcaagtg actttttatg 4260

agctcgaatt ctattatatt ttttatctaa aaaactaaaa ataaacattg attaaatttt 4320

aatataatac ttaaaaatgg atgttgtgtc gttagataaa ccgtttatgt attttgagga 4380

aattgataat gagttagatt acgaaccaga aagtgcaaat gaggtcgcaa aaaaactgcc 4440

gtatcaagga cagttaaaac tattactagg agaattattt tttcttagta agttacagcg 4500

acacggtata ttagatggtg ccaccgtagt gtatatagga tctgctcccg gtacacatat 4560

acgttatttg agagatcatt tctataattt aggagtgatc atcaaatgga tgctaattga 4620

cggccgccat catgatccta ttttaaatgg attgcgtgat gtgactctag tgactcggtt 4680

cgttgatgag gaatatctac gatccatcaa aaaacaactg catccttcta agattatttt 4740

aatttctgat gtgagatcca aacgaggagg aaatgaacct agtacggcgg atttactaag 4800

taattacgct ctacaaaatg tcatgattag tattttaaac cccgtggcgt ctagtcttaa 4860

atggagatgc ccgtttccag atcaatggat caaggacttt tatatcccac acggtaataa 4920

aatgttacaa ccttttgctc cttcatattc agctgaaatg agattattaa gtatttatac 4980

cggtgagaac atgagactga ctcgagttac caaatcagac gctgtaaatt atgaaaaaaa 5040

gatgtactac cttaataaga tcgtccgtaa caaagtagtt gttaactttg attatcctaa 5100

tcaggaatat gactattttc acatgtactt tatgctgagg accgtgtact gcaataaaac 5160

atttcctact actaaagcaa aggtactatt tctacaacaa tctatatttc gtttcttaaa 5220

tattccaaca acatcaactg aaaaagttag tcatgaacca atacaacgta aaatatctag 5280

caaaaattct atgtctaaaa acagaaatag caagagatcc gtacgcagta ataaatag 5338

<210> 23

<211> 5

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-HCDR 1

<222> (1)..(5)

<400> 23

Asn Ser Gly Met His

1 5

<210> 24

<211> 17

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-HCDR 2

<222> (1)..(17)

<400> 24

Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 25

<211> 4

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-HCDR 3

<222> (1)..(4)

<400> 25

Asn Asp Asp Tyr

1

<210> 26

<211> 11

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-LCDR 1

<222> (1)..(11)

<400> 26

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 27

<211> 7

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-LCDR 2

<222> (1)..(7)

<400> 27

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 28

<211> 9

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-LCDR 3

<222> (1)..(9)

<400> 28

Gln Gln Ser Ser Asn Trp Pro Arg Thr

1 5

<210> 29

<211> 113

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-VH

<222> (1)..(113)

<400> 29

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser

<210> 30

<211> 110

<212> PRT

<213> Chile person

<220>

<221> anti-PD-1 antibody-VL

<222> (1)..(110)

<400> 30

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

<210> 31

<211> 15

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-HCDR 1

<222> (1)..(15)

<400> 31

aacagcggga tgcac 15

<210> 32

<211> 51

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-HCDR 2

<222> (1)..(51)

<400> 32

gttatctggt atgacggctc caagcgttat tacgccgact ctgtgaaggg a 51

<210> 33

<211> 12

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-HCDR 3

<222> (1)..(12)

<400> 33

aacgacgatt ac 12

<210> 34

<211> 33

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-LCDR 1

<222> (1)..(33)

<400> 34

cgtgcgtcac agtcagtatc gtcctatctg gct 33

<210> 35

<211> 21

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-LCDR 2

<222> (1)..(21)

<400> 35

gatgcatcta accgggctac a 21

<210> 36

<211> 27

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-LCDR 3

<222> (1)..(27)

<400> 36

cagcagtctt ccaactggcc tcgtact 27

<210> 37

<211> 339

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-VH

<222> (1)..(339)

<400> 37

caagtacagc tggttgagtc aggcggcgga gttgtccagc caggcagatc actaagattg 60

gactgtaagg cttccggcat caccttttct aacagcggga tgcactgggt acggcaagcg 120

ccaggcaagg gcttggagtg ggtggcagtt atctggtatg acggctccaa gcgttattac 180

gccgactctg tgaagggacg gttcaccatt agtagggaca actctaaaaa tacattgttc 240

ctacagatga attcactgcg ggctgaggac acagccgtat actactgcgc cacaaacgac 300

gattactggg gacagggcac attggtgacc gtaagttcg 339

<210> 38

<211> 330

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-VL

<222> (1)..(330)

<400> 38

gaaatcgtac tcacgcagtc ccctgctact ctgagtctct caccaggaga acgcgctacc 60

ctttcttgcc gtgcgtcaca gtcagtatcg tcctatctgg cttggtatca gcaaaaacca 120

ggtcaggccc cccgattatt gatttatgat gcatctaacc gggctacagg gattcctgcc 180

agatttagcg gtagcgggag tggaactgac ttcactctaa ccattagctc ccttgagcca 240

gaggatttcg ccgtctacta ctgtcagcag tcttccaact ggcctcgtac tttcggacag 300

ggaacaaagg tggaaatcaa acgtaccgtg 330

<210> 39

<211> 1379

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-HC

<222> (1)..(983)

<400> 39

atggattgga cctggagaat cctgttcttg gttgccgcgg caacaggcgc ccacagccaa 60

gtacagctgg ttgagtcagg cggcggagtt gtccagccag gcagatcact aagattggac 120

tgtaaggctt ccggcatcac cttttctaac agcgggatgc actgggtacg gcaagcgcca 180

ggcaagggct tggagtgggt ggcagttatc tggtatgacg gctccaagcg ttattacgcc 240

gactctgtga agggacggtt caccattagt agggacaact ctaaaaatac attgttccta 300

cagatgaatt cactgcgggc tgaggacaca gccgtatact actgcgccac aaacgacgat 360

tactggggac agggcacatt ggtgaccgta agttcggcaa gcaccaaggg accctcagtg 420

ttcccgctcg ccccctgcag taggagcaca agcgaaagca cagctgcgct aggatgtctc 480

gtgaaggact actttccaga accggtgact gtgtcctgga acagcggcgc cctgacctct 540

ggagtgcaca cattccctgc cgtgctgcag agctcgggcc tctattcact atcttccgtg 600

gtcactgttc cttcatctag tctaggcacg aaaacttata cctgtaacgt tgaccacaag 660

ccctctaata ctaaagtaga taagcgggtc gaatccaagt atggaccccc ctgccctccg 720

tgtcctgccc ccgagttctt gggtggaccg tccgtgtttc tgttcccacc aaaacctaag 780

gacaccctaa tgattagccg gaccccggaa gtgacttgtg tcgtggttga tgtctctcaa 840

gaggatcccg aagtgcaatt caactggtac gtagacggcg ttgaagtgca taacgcaaag 900

acgaaacccc gcgaggaaca attcaattca acctatcggg tcgtcagcgt gctgacagtg 960

ttgcaccagg attggctgaa tggcaaggaa tataagtgca aggtgtcaaa caaaggattg 1020

ccctcttcca tcgaaaagac aattagcaaa gccaagggac agcctcggga acctcaggtc 1080

tacaccctac caccctcaca ggaagaaatg actaaaaacc aggtaagtct gacctgtctg 1140

gttaagggtt tttaccctag cgatatagcc gtggagtggg agagtaatgg ccaaccagag 1200

aataactata agactactcc acccgtgctt gacagcgacg gcagcttctt tctgtactcg 1260

cgcctgaccg ttgataaaag ccgttggcag gaaggtaatg tcttttcctg ctccgtgatg 1320

cacgaagcgt tgcacaacca ttacacccaa aaatcactga gtctcagcct agggaagta 1379

<210> 40

<211> 702

<212> DNA

<213> Chile person

<220>

<221> anti-PD-1 antibody-LC

<222> (1)..(315)

<400> 40

atggcatggt ccccattatt tctgaccttg attactcact gcgccggctc ttgggctgaa 60

atcgtactca cgcagtcccc tgctactctg agtctctcac caggagaacg cgctaccctt 120

tcttgccgtg cgtcacagtc agtatcgtcc tatctggctt ggtatcagca aaaaccaggt 180

caggcccccc gattattgat ttatgatgca tctaaccggg ctacagggat tcctgccaga 240

tttagcggta gcgggagtgg aactgacttc actctaacca ttagctccct tgagccagag 300

gatttcgccg tctactactg tcagcagtct tccaactggc ctcgtacttt cggacaggga 360

acaaaggtgg aaatcaaacg taccgtggct gcacccagcg tgttcatttt tccaccaagc 420

gacgagcagc tcaagagcgg aaccgcatcc gtagtatgtc tcctcaataa cttctaccca 480

cgagaagcca aagtgcagtg gaaggtggat aatgccttgc aatccggaaa cagccaagaa 540

agcgtgaccg aacaggattc aaaagacagc acctattctc tgtccagcac attgacactg 600

agtaaagctg attatgagaa gcacaaggtc tacgcgtgtg aggttacaca tcaaggattg 660

tcttcaccag tcaccaagag tttcaataga ggagagtgct ga 702

Claims

1. A modified oncolytic virus comprising a viral genome having a first heterologous polynucleotide encoding a first molecule capable of inhibiting an interaction between PD-1 and PD-L1 and a second heterologous polynucleotide encoding a second molecule capable of inhibiting TGF- β signaling.

2. The modified oncolytic virus of claim 1, wherein the oncolytic virus is selected from the group consisting of vaccinia virus, adenovirus, reovirus, herpes simplex virus, semliki forest virus, venezuelan equine encephalitis virus, parvovirus, chicken anemia virus, measles virus, coxsackie virus, vesicular stomatitis virus, saint valley virus, maraba virus, newcastle disease virus, and myxoma virus.

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

4. The modified oncolytic virus of claim 1, wherein the modified oncolytic virus is attenuated and capable of replication in tumor cells.

5. The modified oncolytic virus of claim 1, wherein the viral genome comprises at least one deletion or disruption such that the virus is capable of selective replication in tumor cells.

6. The modified oncolytic virus of claim 5, wherein the deletion or disruption is in an Open Reading Frame (ORF) encoding at least a portion of an enzyme that is both critical for replication of the virus and preferentially expressed in tumor cells over non-tumor cells.

7. The modified oncolytic virus of claim 6, wherein the enzyme is a kinase.

8. The modified oncolytic virus of claim 7, wherein the enzyme is thymidine kinase.

9. The modified oncolytic virus of claim 2, wherein the oncolytic virus is derived from a Western Reserve strain.

10. The modified oncolytic virus of claim 4, wherein the first heterologous polynucleotide and the second heterologous polynucleotide are inserted in place of the deletion.

11. The modified oncolytic virus of claim 1, wherein the first heterologous polynucleotide and the second heterologous polynucleotide are configured such that they are expressed in the same or different phases of the replication cycle of the modified oncolytic virus.

12. The modified oncolytic virus of claim 1, wherein the first molecule is a first fusion protein and the second molecule is a second fusion protein.

13. The modified oncolytic virus of claim 12, wherein the first heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: a first promoter-a polynucleotide encoding the first fusion protein-a first stop codon, and the second heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: second promoter-polynucleotide encoding the second fusion protein-second stop codon.

14. The modified oncolytic virus of claim 13, wherein the first fusion protein expressed from the first heterologous polynucleotide and the second fusion protein expressed from the second heterologous polynucleotide are expressed as separate proteins.

15. The modified oncolytic virus of claim 13, wherein the first heterologous polynucleotide is immediately upstream or immediately downstream of the second heterologous polynucleotide.

16. The modified oncolytic virus of claim 13, wherein the first promoter is capable of driving expression of the first fusion protein and the second promoter is capable of driving expression of the second fusion protein, wherein the first and second promoters are in a head-to-head orientation.

17. The modified oncolytic virus of claim 13, wherein the first promoter and the second promoter are the same or different.

18. The modified oncolytic virus of claim 17, wherein the first promoter and the second promoter are both early and late promoters.

19. The modified oncolytic virus of claim 18, wherein the early and late promoters are pses/ls.

20. The modified oncolytic virus of claim 13, wherein the first stop codon and the second stop codon are the same or different.

21. The modified oncolytic virus of claim 12, wherein the first fusion protein comprises a PD-1 extracellular domain (PD-1 ECD).

22. The modified oncolytic virus of claim 21, wherein the first fusion protein further comprises a first immunoglobulin Fc region.

23. The modified oncolytic virus of claim 22, wherein the first immunoglobulin Fc region is a first human IgG1 Fc region.

24. The modified oncolytic virus of claim 23, wherein the PD-1ECD is operably linked to the first immunoglobulin Fc region at the C-terminus of the PD-1 ECD.

25. The modified oncolytic virus of claim 21, wherein the first fusion protein further comprises a signal peptide.

26. The modified oncolytic virus of claim 25, wherein the signal peptide is operably linked to the PD-1ECD at the C-terminus of the signal peptide.

27. The modified oncolytic virus of claim 25, wherein the signal peptide is a CD33 signal peptide.

28. The modified oncolytic virus of claim 21, wherein the PD-1ECD comprises the amino acid sequence of SEQ ID NO:1 or a homologous sequence thereof having at least 80% sequence identity.

29. The modified oncolytic virus of claim 28, wherein the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID No. 7 or a homologous sequence thereof having at least 80% sequence identity.

30. The modified oncolytic virus of claim 23, wherein the first human IgG1 Fc region comprises the amino acid sequence of SEQ ID No. 3 or a homologous sequence thereof having at least 80% sequence identity.

31. The modified oncolytic virus of claim 30, wherein the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO 9 or a homologous sequence thereof having at least 80% sequence identity.

32. The modified oncolytic virus of claim 27, wherein the CD33 signal peptide comprises the amino acid sequence of SEQ ID No. 4 or a homologous sequence thereof having at least 80% sequence identity.

33. The modified oncolytic virus of claim 32, wherein the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID No. 11 or a homologous sequence thereof having at least 80% sequence identity.

34. The modified oncolytic virus of claim 12, wherein the first fusion protein comprises the amino acid sequence of SEQ ID No. 5 or a homologous sequence thereof having at least 80% sequence identity.

35. The modified oncolytic virus of claim 34, wherein the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID No. 13 or a homologous sequence thereof having at least 80% sequence identity.

36. The modified oncolytic virus of claim 1, wherein the first molecule is an anti-PD-1 antibody and the second molecule is a second fusion protein.

37. The modified oncolytic virus of claim 36, wherein the anti-PD-1 antibody comprises:

HCDR1 having the amino acid sequence of SEQ ID NO. 23 or a homologous sequence having at least 80% sequence identity,

HCDR2 having the amino acid sequence of SEQ ID NO. 24 or a homologous sequence having at least 80% sequence identity,

HCDR3 having the amino acid sequence of SEQ ID NO. 25 or a homologous sequence having at least 80% sequence identity,

LCDR1 having the amino acid sequence of SEQ ID NO. 26 or a homologous sequence having at least 80% sequence identity,

LCDR2 having the amino acid sequence of SEQ ID NO 27 or a homologous sequence thereof having at least 80% sequence identity, and

LCDR3 having the amino acid sequence of SEQ ID NO. 28 or a homologous sequence thereof having at least 80% sequence identity.

38. The modified oncolytic virus of claim 37, wherein

The HCDR1 is encoded by the nucleic acid sequence of SEQ ID NO. 31 or a homologous sequence thereof having at least 80% sequence identity,

the HCDR2 is encoded by the nucleic acid sequence of SEQ ID NO. 32 or a homologous sequence thereof having at least 80% sequence identity,

the HCDR3 is encoded by the nucleic acid sequence of SEQ ID NO. 33 or a homologous sequence thereof having at least 80% sequence identity,

the LCDR1 is encoded by the nucleic acid sequence of SEQ ID NO. 34 or a homologous sequence thereof having at least 80% sequence identity,

the LCDR2 is encoded by the nucleic acid sequence of SEQ ID NO. 35 or a homologous sequence thereof having at least 80% sequence identity, an

The LCDR3 is encoded by the nucleic acid sequence of SEQ ID NO. 36 or a homologous sequence thereof having at least 80% sequence identity.

39. The modified oncolytic virus of claim 36, wherein the anti-PD-1 antibody comprises a heavy chain variable region having the amino acid sequence of SEQ ID No. 29 or a homologous sequence thereof having at least 80% sequence identity and a light chain variable region having the amino acid sequence of SEQ ID No. 30 or a homologous sequence thereof having at least 80% sequence identity.

40. The modified oncolytic virus of claim 39, wherein the anti-PD-1 antibody heavy chain variable region is encoded by the nucleic acid sequence of SEQ ID NO:37 or a homologous sequence thereof having at least 80% sequence identity and the anti-PD-1 antibody light chain variable region is encoded by the nucleic acid sequence of SEQ ID NO:38 or a homologous sequence thereof having at least 80% sequence identity.

41. The modified oncolytic virus of claim 36, wherein the anti-PD-1 antibody comprises a full length heavy chain having the amino acid sequence of SEQ ID No. 21 or a homologous sequence thereof having at least 80% sequence identity and a full length light chain having the amino acid sequence of SEQ ID No. 20 or a homologous sequence thereof having at least 80% sequence identity.

42. The modified oncolytic virus of claim 41, wherein the anti-PD-1 antibody full length heavy chain is encoded by the nucleic acid sequence of SEQ ID No. 39 or a homologous sequence thereof having at least 80% sequence identity and the anti-PD-1 antibody full length light chain is encoded by the nucleic acid sequence of SEQ ID No. 40 or a homologous sequence thereof having at least 80% sequence identity.

43. The modified oncolytic virus of claim 36, wherein the first heterologous polynucleotide further comprises a third heterologous polynucleotide and a fourth heterologous polynucleotide, wherein the third heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: third promoter-polynucleotide encoding the heavy chain of the anti-PD-1 antibody-third stop codon, and wherein the fourth heterologous polynucleotide comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: fourth promoter-polynucleotide encoding the anti-PD-1 antibody light chain-fourth stop codon; and the second heterologous polynucleotide further comprises the following in-frame elements in the 5 'to 3' orientation of the sense strand: second promoter-polynucleotide encoding the second fusion protein-second stop codon.

44. The modified oncolytic virus of claim 43, wherein the third heterologous polynucleotide is immediately upstream or immediately downstream of the fourth heterologous polynucleotide.

45. The modified oncolytic virus of claim 44, wherein the third promoter is capable of driving expression of the anti-PD-1 antibody heavy chain and the fourth promoter is capable of driving expression of the anti-PD-1 antibody light chain, wherein the third promoter and the fourth promoter are in a head-to-head orientation.

46. The modified oncolytic virus of claim 36, wherein the first heterologous polynucleotide is immediately upstream or immediately downstream of the second heterologous polynucleotide.

47. The modified oncolytic virus of claim 46, wherein the anti-PD-1 antibody expressed from the first heterologous polynucleotide and the second fusion protein expressed from the second heterologous polynucleotide are expressed as separate proteins.

48. The modified oncolytic virus of claim 42, wherein the second promoter, the third promoter, and the fourth promoter are the same or different.

49. The modified oncolytic virus of claim 47, wherein the second promoter, the third promoter, and the fourth promoter are all early and late promoters.

50. The modified oncolytic virus of claim 48, wherein the early and late promoters are pSE/L.

51. The modified oncolytic virus of claim 42, wherein the second stop codon, the third stop codon, and the fourth stop codon are the same or different.

52. The modified oncolytic virus of claim 12 or 36, wherein the second fusion protein comprises a TGF- β receptor II extracellular domain (TGFBRII ECD).

53. The modified oncolytic virus of claim 52, wherein the second fusion protein further comprises a second immunoglobulin Fc region.

54. The modified oncolytic virus of claim 53, wherein the immunoglobulin Fc region is a second human IgG1Fc region.

55. The modified oncolytic virus of claim 54, wherein the TGFBRII ECD is operably linked to the second immunoglobulin Fc region at the C-terminus of the TGFBRIIECD.

56. The modified oncolytic virus of claim 52, wherein the second fusion protein further comprises a signal peptide.

57. The modified oncolytic virus of claim 56, wherein said signal peptide is operably linked to said TGFBRII ECD at the C-terminus of said signal peptide.

58. The modified oncolytic virus of claim 56, wherein the signal peptide is a CD33 signal peptide.

59. A modified oncolytic virus of claim 52, wherein said TGFBRII ECD comprises the amino acid sequence of SEQ ID NO. 2 or a homologous sequence thereof having at least 80% sequence identity.

60. The modified oncolytic virus of claim 59, wherein the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO. 8 or a homologous sequence thereof having at least 80% sequence identity.

61. The modified oncolytic virus of claim 54, wherein the second human IgG1 Fc region comprises the amino acid sequence of SEQ ID No. 3 or a homologous sequence thereof having at least 80% sequence identity.

62. The modified oncolytic virus of claim 61, wherein the amino acid sequence is encoded by the nucleic acid sequence of SEQ ID NO. 10 or a homologous sequence thereof having at least 80% sequence identity.

63. The modified oncolytic virus of claim 58, wherein the CD33 signal peptide comprises the amino acid sequence of SEQ ID No. 4 or a homologous sequence thereof having at least 80% sequence identity.

64. The modified oncolytic virus of claim 63, wherein the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID No. 12 or a homologous sequence thereof having at least 80% sequence identity.

65. The modified oncolytic virus of claim 12 or 36, wherein said second fusion protein comprises the amino acid sequence of SEQ ID No. 6 or a homologous sequence thereof having at least 80% sequence identity.

66. The modified oncolytic virus of claim 65, wherein the amino acid sequence is encoded by a nucleic acid sequence comprising SEQ ID No. 14 or a homologous sequence thereof having at least 80% sequence identity.

67. The modified oncolytic virus of claim 12, wherein the first fusion protein and the second fusion protein are capable of forming dimers.

68. The modified oncolytic virus of claim 67, wherein the dimer is formed by association of the first immunoglobulin Fc region with the second immunoglobulin Fc region.

69. The modified oncolytic virus of claim 1, having the nucleic acid sequence of SEQ ID No. 17 or SEQ ID No. 22.

70. A pharmaceutical composition comprising the modified oncolytic virus of any one of claims 1-69 and a pharmaceutically acceptable carrier.

71. A method of treating a tumor, the method comprising administering to a subject an effective amount of the modified oncolytic virus of any one of claims 1-69 or the pharmaceutical composition of claim 70.

72. The method of claim 71, wherein the subject is a human.

73. The method of claim 71, wherein the tumor is a solid tumor.

74. The method of claim 71, wherein the tumor is melanoma, non-small cell lung cancer, renal cell carcinoma, hodgkin's lymphoma, head and neck squamous cell carcinoma, bladder cancer, colorectal cancer, triple negative breast cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, colon cancer, pharyngeal squamous cell carcinoma, or ovarian teratoma.

75. The method of claim 71, wherein the route of administration is topical.

76. The method of claim 75, wherein the route of administration is intratumoral injection.