TW201825511A - Oncolytic virus expressing immune checkpoint modulators - Google Patents

Oncolytic virus expressing immune checkpoint modulators Download PDF

Info

Publication number
TW201825511A
TW201825511A TW106130884A TW106130884A TW201825511A TW 201825511 A TW201825511 A TW 201825511A TW 106130884 A TW106130884 A TW 106130884A TW 106130884 A TW106130884 A TW 106130884A TW 201825511 A TW201825511 A TW 201825511A
Authority
TW
Taiwan
Prior art keywords
nucleic acid
oncolytic
immune checkpoint
scfv
acid encoding
Prior art date
Application number
TW106130884A
Other languages
Chinese (zh)
Inventor
宋曉彤
Original Assignee
美商艾斯合顧問有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 美商艾斯合顧問有限公司 filed Critical 美商艾斯合顧問有限公司
Publication of TW201825511A publication Critical patent/TW201825511A/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Virology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present application provides oncolytic vaccinia virus encoding an immune checkpoint modulator, such as immune checkpoint inhibitor, wherein the expression of the immune checkpoint modulator is driven by a late promoter. The present application also provides oncolytic virus encoding an immune checkpoint modulator, and a bispecific molecule comprising antigen-binding domains specifically recognizing a tumor antigen and a cell surface molecule on an effector cell, such as CD3 on T lymphocytes. In some embodiments, the oncolytic virus further encodes a cytokine. Methods of treating cancer using one or more of the compositions are encompassed in the disclosure.

Description

表現免疫檢查點調節子的溶瘤病毒Oncolytic viruses expressing immune checkpoint regulators

本發明係關於溶瘤病毒、抗PD-1抗體及其使用方法。The present invention relates to oncolytic viruses, anti-PD-1 antibodies and methods of use.

溶瘤病毒(OV)由於能夠感染腫瘤細胞，在腫瘤細胞中複製及溶解腫瘤細胞且經連續數輪複製來擴散穿過腫瘤細胞而成為頗具前景的癌症治療劑(Russell等人, Nat Biotechnol. 2012, 30:658-670；Kelly及Russell, Mol Ther. 2007, 15:651-659)。其主要作用模式係溶解腫瘤細胞，由此可以誘導針對標靶轉移性疾病之抗原特異性T細胞反應，即使該等OV係局部遞送的。OV已在臨床前模型及臨床試驗中進行測試，但僅很少地觀察到完全臨床反應，突顯出對OV療法進一步改進的需求。許多OV與擴散穿過腫瘤之病毒有限及抗腫瘤T細胞反應之次佳活化有關(Heo等人, Nat Med. 2013, 19:329-336；Breitbach等人, Nature. 2011, 477:99-102；Kim等人, Mol Ther. 2006, 14:361-370；Hwang等人, Mol Ther. 2011, 19:1913-1922；Lun等人, Mol Ther. 2010, 18:1927-1936；Heo等人, Mol Ther. 2011, 19:1170-1179；Senzer等人, J Clin Oncol. 2009, 27:5763-5771；Adair等人, Sci Transl Med. 2012, 4:138ra77)。 越來越多的證據顯示，T細胞免疫療法能夠控制腫瘤生長並延長癌症患者之存活期。然而，可能歸因於腫瘤細胞之各種免疫逃避機制的限制，腫瘤特異性T細胞反應很難達成並保持(Shafer-Weaver等人, Adv Exp Med Biol. 2007, 601:357-368；Shafer-Weaver等人, J Immunol. 2009, 183:4848-4852)。免疫檢查點分子係在體內某些免疫細胞上表現之蛋白質，該等免疫細胞需要進行活化或抑制以起始免疫反應，例如攻擊異常細胞，諸如腫瘤細胞。「免疫逃避」可以包括腫瘤細胞之若干活動，諸如共刺激分子(諸如刺激性免疫檢查點分子)表現之下調，及抑制分子(諸如抑制性免疫檢查點分子)表現之上調。阻斷該等抑制性免疫檢查點分子在癌症治療之臨床前及臨床測試中顯示出極具前景的結果。不過，在一些情況下存在一些不想要的副作用。舉例而言，阻斷該等抑制性免疫檢查點分子(受體或配體)可能破壞免疫動態平衡及自身耐受性，從而引起自身免疫性/自發炎副作用(Corsello, S.等人, J Clin Endocrinol Metab (2013) 98(4):1361-7510)。免疫檢查點調節子不能在腫瘤部位有效地積累亦可能導致全身不良作用。 雙特異性接合分子，諸如包含T細胞表面分子結合結構域及腫瘤抗原結合結構域之分子，提供了一種將T細胞接合至腫瘤細胞的方式且顯示出一些臨床成果，諸如殺滅患有非霍奇金氏淋巴瘤(non-Hodgkin's lymphomas)及前驅B細胞急性淋巴母細胞白血病之患者體內的腫瘤細胞(Bargou等人, Science. 2008, 321:974-977；Topp等人, J Clin Oncol. 2011, 29:2493-2498；Nagorsen等人, Leuk Lymphoma.2009, 50:886-891)。然而，雙特異性接合分子，諸如雙特異性T細胞接合子，具有較短半衰期，需要連續輸注(Hammond等人, Cancer Res. 2007, 67:3927-3935；Lutterbuese等人, Proc Natl Acad Sci USA. 2010, 107:12605-12610；Friedrich等人, Mol Cancer Ther. 2012, 11:2664-2673；Choi等人, Proc Natl Acad Sci USA. 2013, 110:270-275)。另外，雙特異性T細胞接合子不能在腫瘤部位有效地積累亦導致全身不良作用，諸如在中樞神經系統內之不良作用。 本文所提及之所有出版物、專利、專利申請案及公開之專利申請案的揭示內容特此以全文引用的方式併入本文中。Oncolytic virus (OV) can infect tumor cells, replicate and lyse tumor cells in tumor cells and spread through tumor cells through successive rounds of replication to become a promising cancer therapeutic agent (Russell et al., Nat Biotechnol. 2012 , 30: 658-670; Kelly and Russell, Mol Ther. 2007, 15: 651-659). Its main mode of action is to lyse tumor cells, which can induce antigen-specific T cell responses against target metastatic disease, even if these OVs are delivered locally. OV has been tested in pre-clinical models and clinical trials, but only a full clinical response has been observed, highlighting the need for further improvement of OV therapy. Many OVs are associated with the limited virus spreading through tumors and the second best activation of anti-tumor T cell responses (Heo et al., Nat Med. 2013, 19: 329-336; Breitbach et al., Nature. 2011, 477: 99-102 ; Kim et al., Mol Ther. 2006, 14: 361-370; Hwang et al., Mol Ther. 2011, 19: 1913-1922; Lun et al., Mol Ther. 2010, 18: 1927-1936; Heo et al., Mol Ther. 2011, 19: 1170-1179; Senzer et al., J Clin Oncol. 2009, 27: 5763-5771; Adair et al., Sci Transl Med. 2012, 4: 138ra77). There is increasing evidence that T cell immunotherapy can control tumor growth and prolong the survival of cancer patients. However, it may be due to the limitation of various immune escape mechanisms of tumor cells, and tumor-specific T cell responses are difficult to achieve and maintain (Shafer-Weaver et al., Adv Exp Med Biol. 2007, 601: 357-368; Shafer-Weaver Et al., J Immunol. 2009, 183: 4848-4852). Immune checkpoint molecules are proteins that appear on certain immune cells in the body. These immune cells need to be activated or suppressed to initiate an immune response, such as attacking abnormal cells, such as tumor cells. "Immune evasion" can include several activities of tumor cells, such as the down-regulation of costimulatory molecules (such as stimulatory immune checkpoint molecules) and the up-regulation of inhibitory molecules (such as suppressive immune checkpoint molecules). Blocking these inhibitory immune checkpoint molecules has shown promising results in pre-clinical and clinical tests for cancer treatment. However, there are some undesirable side effects in some cases. For example, blocking these inhibitory immune checkpoint molecules (receptors or ligands) may disrupt the immune homeostasis and self-tolerance, causing autoimmune / spontaneous side effects (Corsello, S. et al., J Clin Endocrinol Metab (2013) 98 (4): 1361-7510). Failure to effectively accumulate immune checkpoint regulators at the tumor site may also cause systemic adverse effects. Bispecific conjugating molecules, such as molecules containing a T cell surface molecular binding domain and a tumor antigen binding domain, provide a way to join T cells to tumor cells and show some clinical results, such as killing non-Ho Tumor cells in patients with non-Hodgkin's lymphomas and precursor B-cell acute lymphoblastic leukemia (Bargou et al., Science. 2008, 321: 974-977; Topp et al., J Clin Oncol. 2011 , 29: 2493-2498; Nagorsen et al., Leuk Lymphoma. 2009, 50: 886-891). However, bispecific ligation molecules, such as bispecific T cell zygotes, have a short half-life and require continuous infusion (Hammond et al., Cancer Res. 2007, 67: 3927-3935; Lutterbuese et al., Proc Natl Acad Sci USA 2010, 107: 12605-12610; Friedrich et al., Mol Cancer Ther. 2012, 11: 2664-2673; Choi et al., Proc Natl Acad Sci USA. 2013, 110: 270-275). In addition, the inability of bispecific T cell zygote to accumulate effectively at the tumor site also leads to systemic adverse effects, such as those in the central nervous system. The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

本申請案之一態樣提供一種溶瘤牛痘病毒(VV)，其包含編碼免疫檢查點調節子之核酸，其中該核酸係可操作地連接至晚期啟動子。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，該晚期啟動子係F17R。 在根據上述溶瘤VV中之任一種的一些實施例中，該溶瘤VV係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉牛痘病毒(Modified Vaccinia Ankara，MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，該溶瘤VV包含胸苷激酶(TK)基因及痘瘡生長因子(VGF)基因之雙重缺失。 在根據上述溶瘤VV中之任一種的一些實施例中，該免疫檢查點調節子係刺激性免疫檢查點分子之活化劑。在一些實施例中，該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。在一些實施例中，該免疫檢查點調節子係結合至免疫檢查點分子之配體。 在根據上述溶瘤VV中之任一種的一些實施例中，該免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，該免疫檢查點調節子係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係特異性識別免疫檢查點分子之抗體，諸如抗PD-1抗體。在一些實施例中，該特異性識別PD-1之抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，該特異性識別PD-1之抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO:10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，免疫檢查點調節子係結合至免疫檢查點分子，諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4之配體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段的融合物。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子係結合到至少兩個不同的抑制性免疫檢查點分子之配體。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。 在根據上述溶瘤VV中之任一種的一些實施例中，溶瘤VV進一步包含編碼細胞介素，諸如GM CSF之第二核酸。 本申請案之另一態樣提供一種溶瘤病毒(OV)，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，該OV係選自由以下組成之群：牛痘病毒(VV)、塞內加谷病毒(Seneca Valley virus，SVV)、腺病毒、單純疱疹病毒1(HSV1)、單純疱疹病毒2(HSV2)、黏液瘤病毒、呼腸孤病毒、脊髓灰白質炎病毒、水泡性口炎病毒(VSV)、麻疹病毒(MV)、慢病毒、反轉錄病毒、麻疹病毒(morbillivirus)、流感病毒、辛德比病毒(Sindbis virus)及新城疫病毒(Newcastle disease virus，NDV)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該溶瘤VV係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉牛痘病毒(MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，該溶瘤VV包含胸苷激酶(TK)基因及痘瘡生長因子(VGF)基因之雙重缺失。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，免疫檢查點調節子係刺激性免疫檢查點分子之活化劑。在一些實施例中，該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。在一些實施例中，該免疫檢查點調節子係結合至免疫檢查點分子之配體。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，該免疫檢查點調節子係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係特異性識別免疫檢查點分子之抗體，諸如抗PD-1抗體。在一些實施例中，該特異性識別PD-1之抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，該特異性識別PD-1之抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO:10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，免疫檢查點調節子係結合至免疫檢查點分子，諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4之配體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段的融合物。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，腫瘤抗原係選自由以下組成之群：EpCAM、FAP、EphA2、HER2、GD2、EGFR、VEGFR2及磷脂醯肌醇蛋白聚糖-3(GPC3)。在一些實施例中，腫瘤抗原係EpCAM、FAP、EGFR或GPC3。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，效應細胞係選自由以下組成之群：T淋巴細胞、B淋巴細胞、自然殺手(NK)細胞、樹突狀細胞(DC)、巨噬細胞、單核細胞、嗜中性球及NKT細胞。在一些實施例中，效應細胞係T淋巴細胞，諸如細胞毒性T淋巴細胞。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，細胞表面分子係選自由以下組成之群：CD3、CD4、CD5、CD8、CD16、CD28、CD40、CD64、CD89、CD134、CD137、NKp46及NKG2D。在一些實施例中，細胞表面分子係CD3。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，第一抗原結合結構域及/或第二抗原結合結構域係單鏈可變片段(scFv)。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，第一抗原結合結構域與第二抗原結合結構域係藉由連接子連接。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之C末端。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，編碼免疫檢查點調節子之第一核酸係可操作地連接至晚期啟動子。在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，編碼雙特異性分子之第二核酸係可操作地連接至晚期啟動子。在一些實施例中，驅動免疫檢查點調節子及/或雙特異性分子表現之晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，該晚期啟動子係F17R。 在根據上述編碼免疫檢查點調節子及雙特異性分子之OV中之任一種的一些實施例中，溶瘤病毒進一步包含編碼細胞介素，諸如GM-CSF之第三核酸。 進一步提供一種醫藥組合物，其包含上述編碼免疫檢查點調節子之溶瘤VV或編碼免疫檢查點調節子及雙特異性分子之溶瘤病毒中之任一種，及醫藥學上可接受之載劑。在一些實施例中，提供一種醫藥組合物，其包括：包含編碼免疫檢查點調節子(諸如上述免疫檢查點調節子中之任一種)之第一核酸的第一OV；及包含編碼雙特異性接合分子(諸如上述雙特異性分子中之任一種)之第二核酸的第二OV，該雙特異性接合分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域；以及醫藥學上可接受之載劑。在一些實施例中，提供一種醫藥組合物，其包括：包含編碼免疫檢查點調節子(諸如上述免疫檢查點調節子中之任一種)之第一核酸的第一OV，及包含編碼細胞介素(諸如上述細胞介素中之任一種)之第二核酸的第二OV，以及醫藥學上可接受之載劑。在一些實施例中，提供一種醫藥組合物，其包括：包含編碼免疫檢查點調節子(諸如上述免疫檢查點調節子中之任一種)之第一核酸的第一OV；包含編碼雙特異性接合分子(諸如上述雙特異性分子中之任一種)之第二核酸的第二OV，該雙特異性接合分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域；及包含編碼細胞介素(諸如上述細胞介素中之任一種)之第三核酸的第三OV；以及醫藥學上可接受之載劑。 本申請案之另一態樣提供一種治療個體癌症之方法，其包含向個體投與有效量之上述醫藥組合物。在一些實施例中，提供一種治療個體癌症之方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子(諸如上述免疫檢查點調節子中之任一種)之第一核酸的第一OV，及醫藥學上可接受之第一載劑；以及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼雙特異性分子(諸如上述雙特異性分子中之任一種)之第二核酸的第二OV，及醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域。在一些實施例中，提供一種治療個體癌症之方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子(諸如上述免疫檢查點調節子中之任一種)之第一核酸的第一OV，及醫藥學上可接受之第一載劑；以及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼細胞介素(諸如上述細胞介素中之任一種)之第二核酸的第二OV，及醫藥學上可接受之第二載劑。在一些實施例中，提供一種治療個體癌症之方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子(諸如上述免疫檢查點調節子中之任一種)之第一核酸的第一OV，及醫藥學上可接受之第一載劑；有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼雙特異性分子(諸如上述雙特異性分子中之任一種)之第二核酸的第二OV，及醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域；以及有效量之第三醫藥組合物，該第三醫藥組合物包括包含編碼細胞介素(諸如上述細胞介素中之任一種)之第三核酸的第三OV，及醫藥學上可接受之第三載劑。 在一些實施例中，有效量係約105 至約1013 pfu。在一些實施例中，有效量係約109 pfu。在一些實施例中，該醫藥組合物係全身性投與，諸如靜脈內投與。在一些實施例中，該醫藥組合物係局部投與，諸如腫瘤內投與。在一些實施例中，該癌症係實體腫瘤，諸如結腸直腸癌、肝癌或乳癌。在一些實施例中，上述治療癌症之方法進一步包含向該個體投與另外的癌症療法，諸如手術、放射、化學療法、免疫療法、激素療法或其組合。在一些實施例中，個體係人類。One aspect of the present application provides an oncolytic vaccinia virus (VV) comprising a nucleic acid encoding an immune checkpoint regulator, wherein the nucleic acid is operably linked to a late promoter. In some embodiments, the late promoter sub-cluster system consisting of selected from the group consisting of:. F17R, I2L late promoter, L4R late promoter, P 7 5k early / late promoter, P EL early / late promoter, P 11k The late promoter, P SEL synthesizes the early / late promoter, and P SL synthesizes the late promoter. In some embodiments, the late promoter line is F17R. In some embodiments according to any of the above oncolytic VVs, the oncolytic VV is selected from the group consisting of: Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Vaccinia Ankara , MVA), Lister, King, IHD, Evans, USSR and Western Reserve (WR). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV includes a double deletion of the thymidine kinase (TK) gene and the acne growth factor (VGF) gene. In some embodiments according to any of the above oncolytic VVs, the immune checkpoint regulator is an activator of stimulatory immune checkpoint molecules. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is a ligand that binds to the immune checkpoint molecule. In some embodiments according to any of the above oncolytic VVs, the immune checkpoint regulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint regulator sub-line PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA , B7-H4, CD160, 2B4 or CD73 inhibitors. In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the antibody that specifically recognizes PD-1 comprises: a heavy chain variable region (VH), which includes (1) HVR-H1 containing the amino acid sequence SEQ ID NO: 1; (2) containing Amino acid sequence HVR-H2 of SEQ ID NO: 2; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 3; and light chain variable region (VL), including (1) amine-containing HVR-L1 of amino acid sequence SEQ ID NO: 4; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 5; and (3) HVR-L3 containing amino acid sequence SEQ ID NO: 6 In some embodiments, the antibody that specifically recognizes PD-1 comprises: a heavy chain variable region (VH), which includes (1) HVR-H1 containing the amino acid sequence SEQ ID NO: 7; (2) containing Amino acid sequence HVR-H2 of SEQ ID NO: 8; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 9; and light chain variable region (VL), including (1) amine-containing HVR-L1 of amino acid sequence SEQ ID NO: 10; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 11; and (3) HVR-L3 containing amino acid sequence SEQ ID NO: 12. In some embodiments, the immune checkpoint regulator is bound to an immune checkpoint molecule, such as a ligand of PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator is bound to a ligand of at least two different inhibitory immune checkpoint molecules. In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments according to any of the above oncolytic VVs, the oncolytic VV further comprises a second nucleic acid encoding a cytokine, such as GM CSF. Another aspect of the present application provides an oncolytic virus (OV) including a first nucleic acid encoding an immune checkpoint regulator and a second nucleic acid encoding a bispecific molecule, the bispecific molecule including specific recognition The first antigen binding domain of the tumor antigen and the second antigen binding domain that specifically recognizes the cell surface molecule on the effector cell. In some embodiments according to any of the above-mentioned OV encoding immune checkpoint regulators and bispecific molecules, the OV is selected from the group consisting of: vaccinia virus (VV), Seneca Valley virus (Seneca Valley virus) virus, SVV), adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, morbillivirus, influenza virus, Sindbis virus and Newcastle disease virus (NDV). In some embodiments, the OV is an oncolytic VV. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Ankara Vaccinia Virus (MVA), Lister, King, IHD, Evans, USSR and Western Reserve (WR). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV includes a double deletion of the thymidine kinase (TK) gene and the acne growth factor (VGF) gene. In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the immune checkpoint regulator is an activator of stimulatory immune checkpoint molecules. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is a ligand that binds to the immune checkpoint molecule. In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the immune checkpoint regulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint regulator sub-line PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA , B7-H4, CD160, 2B4 or CD73 inhibitors. In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the antibody that specifically recognizes PD-1 comprises: a heavy chain variable region (VH), which includes (1) HVR-H1 containing the amino acid sequence SEQ ID NO: 1; (2) containing Amino acid sequence HVR-H2 of SEQ ID NO: 2; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 3; and light chain variable region (VL), including (1) amine-containing HVR-L1 of amino acid sequence SEQ ID NO: 4; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 5; and (3) HVR-L3 containing amino acid sequence SEQ ID NO: 6 In some embodiments, the antibody that specifically recognizes PD-1 comprises: a heavy chain variable region (VH), which includes (1) HVR-H1 containing the amino acid sequence SEQ ID NO: 7; (2) containing Amino acid sequence HVR-H2 of SEQ ID NO: 8; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 9; and light chain variable region (VL), including (1) amine-containing HVR-L1 of amino acid sequence SEQ ID NO: 10; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 11; and (3) HVR-L3 containing amino acid sequence SEQ ID NO: 12. In some embodiments, the immune checkpoint regulator is bound to an immune checkpoint molecule, such as a ligand of PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2 and Phospholipid inositol-3 (GPC3). In some embodiments, the tumor antigens are EpCAM, FAP, EGFR or GPC3. In some embodiments according to any of the above OV encoding immune checkpoint regulators and bispecific molecules, the effector cell line is selected from the group consisting of: T lymphocytes, B lymphocytes, natural killer (NK) cells , Dendritic cells (DC), macrophages, monocytes, neutrophils and NKT cells. In some embodiments, the effector cell line T lymphocytes, such as cytotoxic T lymphocytes. In some embodiments according to any of the above OVs encoding immune checkpoint regulators and bispecific molecules, the cell surface molecules are selected from the group consisting of: CD3, CD4, CD5, CD8, CD16, CD28, CD40 , CD64, CD89, CD134, CD137, NKp46 and NKG2D. In some embodiments, the cell surface molecule line is CD3. In some embodiments according to any of the above OV encoding immune checkpoint regulators and bispecific molecules, the first antigen binding domain and / or the second antigen binding domain is a single chain variable fragment (scFv) . In some embodiments according to any of the above-mentioned OVs encoding immune checkpoint regulators and bispecific molecules, the first antigen binding domain and the second antigen binding domain are connected by a linker. In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the first antigen-binding domain is at the N-terminus of the second antigen-binding domain. In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the first antigen-binding domain is at the C-terminus of the second antigen-binding domain. In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the first nucleic acid encoding the immune checkpoint regulator is operably linked to the late promoter. In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the second nucleic acid encoding the bispecific molecule is operably linked to the late promoter. In some embodiments, the immune checkpoint regulator drive late promoter and / or expression of the bispecific molecule selected from the group consisting of a promoter system consisting of the group:. F17R, I2L late promoter, L4R late promoter, P 7 5k early / Late promoter, P EL early / late promoter, P 11k late promoter, P SEL synthetic early / late promoter, and P SL synthetic late promoter. In some embodiments, the late promoter line is F17R. In some embodiments according to any of the OVs encoding immune checkpoint regulators and bispecific molecules described above, the oncolytic virus further comprises a third nucleic acid encoding a cytokine, such as GM-CSF. Further provided is a pharmaceutical composition comprising any one of the oncolytic VV encoding the immune checkpoint regulator or the oncolytic virus encoding the immune checkpoint regulator and the bispecific molecule, and a pharmaceutically acceptable carrier . In some embodiments, a pharmaceutical composition is provided, comprising: a first OV comprising a first nucleic acid encoding an immune checkpoint regulator (such as any of the above immune checkpoint regulators); and comprising a coding bispecific The second OV of the second nucleic acid of the conjugating molecule (such as any of the above-mentioned bispecific molecules), the bispecific conjugating molecule comprising a first antigen binding domain that specifically recognizes a tumor antigen and a specific recognition on an effector cell The second antigen binding domain of the cell surface molecule; and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising: a first OV comprising a first nucleic acid encoding an immune checkpoint regulator (such as any of the above immune checkpoint regulators), and comprising a cytokine encoding The second OV of the second nucleic acid (such as any of the above-mentioned cytokines), and a pharmaceutically acceptable carrier. In some embodiments, there is provided a pharmaceutical composition comprising: a first OV comprising a first nucleic acid encoding an immune checkpoint regulator (such as any of the above immune checkpoint regulators); comprising a coding bispecific junction The second OV of the second nucleic acid of the molecule (such as any of the above-mentioned bispecific molecules), the bispecific conjugating molecule comprises a first antigen binding domain that specifically recognizes a tumor antigen and specifically recognizes cells on effector cells The second antigen-binding domain of the surface molecule; and a third OV containing a third nucleic acid encoding a cytokine (such as any of the above cytokines); and a pharmaceutically acceptable carrier. Another aspect of the present application provides a method of treating cancer in an individual, which comprises administering an effective amount of the above-mentioned pharmaceutical composition to the individual. In some embodiments, there is provided a method of treating cancer in an individual, comprising administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising an immune checkpoint regulator (such as the immune checkpoint described above) Any one of the modulators) the first OV of the first nucleic acid, and a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition including A second OV of a second nucleic acid of a molecule (such as any of the above-mentioned bispecific molecules), and a pharmaceutically acceptable second carrier, the bispecific molecule includes a first antigen binding that specifically recognizes a tumor antigen Domain and a second antigen binding domain that specifically recognizes cell surface molecules on effector cells. In some embodiments, there is provided a method of treating cancer in an individual, comprising administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising an immune checkpoint regulator (such as the immune checkpoint described above) Any one of the modulators) the first OV of the first nucleic acid, and a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition comprising The second OV of the second nucleic acid (such as any of the above-mentioned cytokines), and a pharmaceutically acceptable second carrier. In some embodiments, there is provided a method of treating cancer in an individual, comprising administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising an immune checkpoint regulator (such as the immune checkpoint described above) Any one of the modulators) the first OV of the first nucleic acid, and a pharmaceutically acceptable first carrier; an effective amount of a second pharmaceutical composition, the second pharmaceutical composition comprising a coding bispecific molecule The second OV of the second nucleic acid (such as any of the above bispecific molecules), and a pharmaceutically acceptable second carrier, the bispecific molecule includes a first antigen binding structure that specifically recognizes a tumor antigen Domain and a second antigen-binding domain that specifically recognizes cell surface molecules on effector cells; and an effective amount of a third pharmaceutical composition, which includes an encoding cytokine (such as The third OV of the third nucleic acid of any one), and a pharmaceutically acceptable third carrier. In some embodiments, the effective amount based of about 105 to about 10 13 pfu. In some embodiments, the effective amount is about 109 pfu. In some embodiments, the pharmaceutical composition is administered systemically, such as intravenously. In some embodiments, the pharmaceutical composition is administered locally, such as intratumorally. In some embodiments, the cancer is a solid tumor, such as colorectal cancer, liver cancer, or breast cancer. In some embodiments, the above method of treating cancer further comprises administering to the individual additional cancer therapy, such as surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof. In some embodiments, each system is human.

相關申請案 本申請案要求2016年9月9日提交的美國臨時專利申請案第62/385,930號及美國臨時專利申請案第62/385,933號之優先權，其內容以全文引用之方式併入本文中。 序列表以ASCII正文檔案提交 以下以ASCII正文檔案提交之內容係以全文引用的方式併入本文中：電腦可讀形式(CRF)之序列表(檔案名稱：768312000241SEQLIST.txt，記錄日期：2017年9月5日，大小：28 KB)。 本發明提供一種在晚期啟動子控制下編碼免疫檢查點調節子(諸如免疫檢查點抑制劑)的溶瘤牛痘病毒，及編碼免疫檢查點調節子(諸如免疫檢查點抑制劑)及雙特異性接合分子(在下文中又稱「雙特異性分子」、「接合分子」或「接合子」)之溶瘤病毒(諸如溶瘤VV)作為新策略以1)促進腫瘤部位處之T細胞活化，2)有效地溶解感染溶瘤病毒或未感染溶瘤病毒(旁觀者殺滅)之腫瘤細胞，及/或3)使全身不良作用減到最少以實現較高抗腫瘤活性，尤其是針對實體腫瘤。本發明部分基於發現溶瘤病毒(諸如溶瘤VV)、在腫瘤部位表現之免疫檢查點調節子及/或雙特異性分子可以提供協同作用。另外，使用僅在病毒感染腫瘤細胞之後活化的晚期啟動子(諸如晚期牛痘病毒啟動子F17R)驅動免疫檢查點調節子及/或雙特異性分子之表現可以避免全身毒性且允許腫瘤部位限制性遞送。 因此，本發明之一態樣提供一種溶瘤牛痘病毒，其包含編碼免疫檢查點調節子之核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。 本發明之另一態樣提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼免疫檢查點調節子(諸如免疫檢查點抑制劑)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域。 亦提供組合物(諸如醫藥組合物)，及使用該等溶瘤病毒(諸如溶瘤VV)治療癌症(諸如實體癌症)之方法。 本發明亦提供新穎抗PD-1抗體及其使用方法。 I. 定義 除非明確指示相反，否則本發明之實踐將採用在此項技術之技能範圍內的病毒學、免疫學、微生物學、分子生物學及重組型DNA技術之習知方法，其中有許多出於說明之目的而描述於下文中。該等技術在文獻中有完整解釋。參見例如，Current Protocols in Molecular Biology或Current Protocols in Immunology, John Wiley & Sons, New York, N.Y.(2009)；Ausubel等人, Short Protocols in Molecular Biology, 第3版, Wiley & Sons, 1995；Sambrook and Russell, Molecular Cloning: A Laboratory Manual (第3版, 2001)；Maniatis等人, Molecular Cloning: A Laboratory Manual (1982)；DNA Cloning: A Practical Approach, 第I及II卷 (D. Glover編)；Oligonucleotide Synthesis (N. Gait編, 1984)；Nucleic Acid Hybridization (B. Hames及S. Higgins編, 1985)；Transcription and Translation (B. Hames及S. Higgins編, 1984)；Animal Cell Culture (R. Freshney編, 1986)；Perbal, A Practical Guide to Molecular Cloning (1984)及其他類似參考文獻。 如本文所使用，「治療(treatment/treating)」係用於獲得有益或所希望之結果，包括臨床結果的一種方法。出於本發明之目的，有益或所希望之臨床結果包括(但不限於)以下一或多種：減輕由疾病引起之一或多種症狀、降低疾病程度、使疾病穩定(例如防止或延遲疾病之惡化)、預防或延遲疾病之擴散(例如轉移)、預防或延遲疾病之復發、延遲或減慢疾病之進展、改善疾病狀態、使疾病緩解(部分或總體緩解)、減小治療疾病所需之一或多種其他藥物之劑量、延遲疾病之進展、增加生活品質及/或延長存活期。「治療」亦涵蓋癌症病理結果之減輕。本發明之方法涵蓋該等治療態樣中之任一種或多種。 術語「預防(prevent)」及類似詞，諸如「預防(prevented/preventing)」等指示用於預防、抑制或降低疾病或病況，例如癌症復發之可能性的一種方法。其亦指延遲疾病或病況之復發，或延遲疾病或病況之症狀之復發。如本文所使用，「預防」及類似詞亦包括在疾病或病況復發之前，降低疾病或病況之強度、影響、症狀及/或負荷。 如本文所使用，「延遲」癌症發展意謂延緩、阻礙、減緩、阻滯、穩定及/或推遲該疾病之發展。此延遲可以取決於所治療之疾病及/或個體的病史而具有不同時間長度。「延遲」癌症發展之方法係當與不使用該方法相比較時，在給定時間範圍內降低疾病發展之機率及/或在給定時間範圍內減輕疾病程度之方法。該等比較通常係基於使用統計顯著數目之個體進行之臨床研究。癌症發展可以使用標準方法偵測，包括(但不限於)電腦化斷層掃描(CAT掃描)、磁共振成像(MRI)、腹部超音波、凝血測試、動脈攝影術或活組織檢查。發展亦可指可能最初不可偵測之癌症進展且包括發生、復發及發作。 本文所使用的術語「有效量」係指足以治療指定病症、病況或疾病，諸如改善、緩和、減輕及/或延遲其一或多種症狀的一種藥劑或藥劑組合之量。就癌症而言，有效量包含足以使腫瘤縮小及/或降低腫瘤生長速率(由此抑制腫瘤生長)或者預防或延遲其他不想要的細胞增殖之量。在一些實施例中，有效量係足以延遲發展的量。在一些實施例中，有效量係足以預防或延遲復發的量。有效量可以分一或多次投藥投與。藥物或組合物之有效量可以：(i)減少癌細胞之數量；(ii)減小腫瘤大小；(iii)在一定程度上抑制、阻滯、減緩且較佳停止周圍器官中之癌細胞浸潤；(iv)抑制(亦即，在一定程度上減緩且較佳停止)腫瘤轉移；(v)抑制腫瘤生長；(vi)預防或延遲腫瘤之發生及/或復發；及/或(vii)在一定程度上緩解一或多種癌症相關症狀。 如本文所使用，「個體」或「受試者」係指哺乳動物，包括(但不限於)人類、牛科動物、馬、貓科動物、犬科動物、嚙齒動物或靈長類動物。在一些實施例中，個體係人類。 術語「雙特異性T細胞接合子」或「BiTE」在在本文中可互換使用，以指具有多抗原決定基特異性且一種特異性係針對T細胞表面分子之抗體或其片段。 結合抗體所使用之術語「多特異性」係指具有多抗原決定基特異性(亦即，能夠特異性結合至一個生物分子上之兩個、三個或三個以上不同抗原決定基或能夠特異性結合至兩個、三個或三個以上不同生物分子上之抗原決定基)的抗體。 結合抗體所使用的術語「雙特異性」係指能夠特異性結合至一個生物分子上之兩個不同抗原決定基，或能夠特異性結合至兩個不同生物分子上之抗原決定基的抗體。除非另外指明，否則所列的雙特異性抗體結合抗原之次序係任意次序。也就是說，例如，術語「抗CD3/EpCAM」、「抗EpCAM/CD3」、「EpCAM×CD3」、「CD3×EpCAM」、「CD3-EpCAM」及「EpCAM-CD3」可互換使用，以指特異性結合至CD3及EpCAM兩者的雙特異性抗體。 如本文所使用，術語「免疫檢查點抑制劑」係指完全或部分地減少、抑制或干擾可以抑制T細胞活化及功能之一或多個抑制性免疫檢查點分子的分子。 如本文所使用，術語「刺激性免疫檢查點分子之活化劑」係指刺激、活化刺激性免疫檢查點分子介導之免疫反應，或增加該免疫反應之強度的分子。 「分離之」核酸係指已與其天然環境之組分分離之核酸分子。分離之核酸包括通常含有核酸分子之細胞中所含的核酸分子，但該核酸分子存在於染色體外或在不同於其天然染色體位置之染色體位置處。 如本文所使用，術語「載體」係指這樣一種核酸分子，其能夠傳播其所連接之另一核酸分子。該術語包括呈自我複製核酸結構之載體以及併入引入其之宿主細胞之基因組中的載體。某些載體能夠引導與其可操作地連接之核酸的表現。此類載體在本文中稱為「表現載體」。 如本文所使用，術語「轉染」或「轉型」或「轉導」係指將外源性核酸轉移至或引入宿主細胞中的一種方法。「轉染」或「轉化」或「轉導」細胞為已經外源性核酸轉染、轉化或轉導之細胞。該細胞包括原代受試者細胞及其後代。 術語「宿主細胞」、「宿主細胞株」及「宿主細胞培養物」可互換使用且指引入了外源性核酸之細胞，包括此類細胞之後代。宿主細胞包括「轉型體」及「轉型細胞」，其包括原代轉型細胞及由其得到的後代，不考慮繼代次數。後代之核酸含量與親代細胞可能不完全相同，而是可能含有突變。本文包括針對初始轉型細胞篩選或選擇的具有相同功能或生物活性之突變型後代。 「輔助治療(Adjuvant setting)」係指這樣一種臨床情況，其中個體具有癌症史，且一般(但未必)對療法，包括(但不限於)手術(例如手術切除)、放射療法及化學療法有反應。然而，由於有癌症史，故認為此等個體有發展該疾病之風險。在「輔助治療」中之治療或投藥係指後續治療模式。風險程度(例如當認為在輔助治療中之個體「高危」或「低危」時)取決於若干因素，最通常取決於在首次治療時疾病之程度。 「新輔助治療」係指在初始療法/確定性療法之前進行該方法的一種臨床情況。 應瞭解，本文所描述的本發明之實施例包括「由實施例組成」及/或「基本上由實施例組成」。 本文中提及「約」包括(且描述)針對該值或參數本身之變化。舉例而言，提及「約X」之描述包括有關「X」之描述。 如本文所使用，提及「不為」一值或參數一般意謂且描述「除一值或參數外」。舉例而言，方法不用於治療X型癌症意謂該方法用於治療除X型外的類型之癌症。 本文所使用之術語「約X-Y」具有與「約X至約Y」相同之含義。 除非上下文另外明確指示，否則如在本文及所附申請專利範圍中所使用，單數形式「一個(種)(a/an)」及「該(the)」包括複數個(種)指示物。 II. 表現免疫檢查點調節子之溶瘤病毒在晚期啟動子下表現免疫檢查點調節子的溶瘤牛痘病毒 本發明提供一種溶瘤牛痘病毒(VV)，其包含編碼免疫檢查點調節子之核酸，其中該核酸係可操作地連接至晚期啟動子。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，該晚期啟動子係F17R。在一些實施例中，該溶瘤VV係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉牛痘病毒(MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，該溶瘤VV包含胸苷激酶(TK)基因及牛痘病毒生長因子(VGF)基因之雙重缺失。在一些實施例中，免疫檢查點調節子係刺激性免疫檢查點分子之活化劑(諸如CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑)。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係特異性識別免疫檢查點分子之抗體，諸如抗PD-1抗體。在一些實施例中，免疫檢查點調節子係結合至免疫檢查點分子，諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4之配體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之第二核酸。 晚期溶瘤牛痘病毒啟動子包括(但不限於)F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，該晚期啟動子係F17R。 因此，在一些實施例中，提供一種溶瘤VV，其包含編碼免疫檢查點調節子之核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係刺激性免疫檢查點分子之活化劑(諸如CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑)。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係特異性識別免疫檢查點分子之抗體，諸如抗PD-1抗體。在一些實施例中，免疫檢查點調節子係結合至免疫檢查點分子，諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4之配體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 免疫檢查點調節子可以為刺激性免疫檢查點分子之活化劑，包括(但不限於)CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑。 因此，在一些實施例中，提供一種溶瘤VV，其包含編碼刺激性免疫檢查點分子之活化劑(諸如CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑)的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼刺激性免疫檢查點分子之活化劑(諸如CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑)的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。在一些實施例中，該免疫檢查點調節子係結合至免疫檢查點分子之配體。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 免疫檢查點調節子可以為免疫檢查點抑制劑，包括(但不限於)PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。 因此，在一些實施例中，提供一種溶瘤VV，其包含編碼免疫檢查點抑制劑之核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼免疫檢查點抑制劑之核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，該免疫檢查點抑制劑係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。在一些實施例中，免疫檢查點抑制劑係PD-1之抑制劑。在一些實施例中，該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係結合至免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)之配體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼特異性識別抑制性免疫檢查點分子之抗體的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼特異性識別抑制性免疫檢查點分子之抗體的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，抑制性免疫檢查點分子係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73。在一些實施例中，抑制性免疫檢查點分子係PD-1。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼PD-1之抑制劑的核酸，其中該核酸可操作地連接至F17R晚期啟動子。在一些實施例中，該PD-1之抑制劑係特異性識別PD-1之抗體。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)，其中該PD-1之抑制劑係特異性識別PD-1之抗體。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至F17R晚期啟動子，其中該PD-1之抑制劑係特異性識別PD-1之抗體。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸可操作地連接至F17R晚期啟動子，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至F17R晚期啟動子，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸可操作地連接至晚期啟動子(諸如F17R)，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸可操作地連接至F17R晚期啟動子，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1之抑制劑的核酸，其中該核酸係可操作地連接至F17R晚期啟動子，其中該PD-1之抑制劑係特異性識別PD-1之抗體，該抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼結合至PD-L1及/或PD-L2之配體的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼結合至PD-L1及/或PD-L2之配體的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，該結合至PD-L1及/或PD-L2之配體包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)，且其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至F17R晚期啟動子，且其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼結合至HHLA2之配體的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼結合至HHLA2之配體的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，該結合至HHLA2之配體包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)，且其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至F17R晚期啟動子，且其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼結合至CD47及CXCR4之配體的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼結合至CD47及CXCR4之配體的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，該結合至CD47及CXCR4之配體包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至F17R晚期啟動子。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 在一些實施例中，提供一種溶瘤VV，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)，且其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，提供一種溶瘤VV，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的核酸，其中該核酸係可操作地連接至F17R晚期啟動子，且其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之核酸。 表現免疫檢查點調節子及雙特異性接合分子之溶瘤病毒 本發明亦提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼免疫檢查點調節子之第一核酸及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該溶瘤病毒係選自由以下組成之群：牛痘病毒(VV)、塞內加谷病毒(SVV)、腺病毒、單純疱疹病毒1(HSV1)、單純疱疹病毒2(HSV2)、黏液瘤病毒、呼腸孤病毒、脊髓灰白質炎病毒、水泡性口炎病毒(VSV)、麻疹病毒(MV)、慢病毒、反轉錄病毒、麻疹病毒、流感病毒、辛德比病毒及新城疫病毒(NDV)。在一些實施例中，溶瘤病毒係溶瘤VV。在一些實施例中，該溶瘤VV係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉牛痘病毒(MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤病毒包含TK基因及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係刺激性免疫檢查點分子之活化劑(諸如CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑)。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，該免疫檢查點調節子係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，該免疫檢查點調節子係結合至免疫檢查點分子之配體。在一些實施例中，免疫檢查點調節子係PD-L1/PD-L2、HHLA-2、CD47或CXCR4之配體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，腫瘤抗原係選自由以下組成之群：EpCAM、FAP、EphA2、HER2、GD2、EGFR、VEGFR2及磷脂醯肌醇蛋白聚糖-3(GPC3)。在一些實施例中，腫瘤抗原EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係選自由以下組成之群：T淋巴細胞、B淋巴細胞、自然殺手(NK)細胞、樹突狀細胞(DC)、巨噬細胞、單核細胞、嗜中性球及NKT細胞。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係選自由以下組成之群：CD3、CD4、CD5、CD8、CD16、CD28、CD40、CD64、CD89、CD134、CD137、NKp46及NKG2D。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及/或第二抗原結合結構域係單鏈可變片段(scFv)。在一些實施例中，第一與第二抗原結合結構域係藉由連接子連接。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之C末端。在一些實施例中，編碼免疫檢查點調節子之第一核酸係可操作地連接至晚期啟動子。在一些實施例中，編碼雙特異性分子之第二核酸係可操作地連接至晚期啟動子。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，該晚期啟動子係F17R。在一些實施例中，溶瘤病毒進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 本文所描述的表現免疫檢查點調節子及雙特異性接合分子之溶瘤病毒可以：1)促進腫瘤部位處T細胞之活化；2)有效地溶解感染或未感染溶瘤病毒之腫瘤細胞(旁觀者殺滅)；3)藉由在腫瘤內選擇性遞送並保持免疫檢查點調節子，來使免疫檢查點分子之非腫瘤限制性阻斷引起的全身自身免疫/自發炎副作用減到最少；4)藉由在腫瘤內選擇性遞送並保持雙特異性接合分子來使全身不良事件減到最少；及/或5)增強在T細胞存在下雙特異性接合分子介導之腫瘤溶解活性。 在一些實施例中，提供一種溶瘤VV，其包含編碼免疫檢查點調節子之第一核酸及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，溶瘤VV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 免疫檢查點調節子可以為刺激性免疫檢查點分子之活化劑，包括(但不限於)CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑。 因此，在一些實施例中，提供一種OV，其包含編碼刺激性免疫檢查點分子活化劑(諸如CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑)的第一核酸及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，該免疫檢查點調節子係特異性識別刺激性免疫檢查點分子之抗體。在一些實施例中，該免疫檢查點調節子係結合至刺激性免疫檢查點分子之配體。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 免疫檢查點調節子可以為免疫檢查點抑制劑，包括(但不限於)PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。 因此，在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點抑制劑係PD-1之抑制劑。在一些實施例中，免疫檢查點抑制劑係抗PD-1抗體。在一些實施例中，免疫檢查點抑制劑包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，PD-1之抑制劑係抗PD-1抗體。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒， 其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域第一抗原結合結構域諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO:10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼結合至PD-L1及/或PD-L2之配體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該結合至PD-L1及/或PD-L2之配體包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼結合至HHLA2之配體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該結合至HHLA2之配體包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼結合至CD47及CXCR4之配體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該結合至CD47及CXCR4之配體包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 腫瘤抗原可以為腫瘤相關抗原(TAA)或腫瘤特異性抗原(TSA)。在一些實施例中，TAA或TSA係在實體腫瘤之細胞上表現。腫瘤抗原包括(但不限於)EpCAM、FAP、EphA2、HER2、GD2、EGFR、VEGFR2及磷脂醯肌醇蛋白聚糖-3。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。 效應細胞包括(但不限於)T淋巴細胞、B淋巴細胞、自然殺手(NK)細胞、樹突狀細胞(DC)、巨噬細胞、單核細胞、嗜中性球、NKT細胞或類似細胞。在一些實施例中，效應細胞係T淋巴細胞。在一些實施例中，效應細胞係細胞毒性T淋巴細胞。 在效應細胞上之細胞表面分子包括(但不限於)CD3、CD4、CD5、CD8、CD16、CD28、CD40、CD64、CD89、CD134、CD137、NKp46、NKG2D或類似物。在一些實施例中，細胞表面分子係CD3。 因此，在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點抑制劑係特異性識別抑制性免疫檢查點分子之抗體。在一些實施例中，免疫檢查點抑制劑係PD-1之抑制劑。在一些實施例中，免疫檢查點抑制劑係抗PD-1抗體。在一些實施例中，免疫檢查點抑制劑係結合至抑制性免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)的配體。在一些實施例中，免疫檢查點抑制劑包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點抑制劑係特異性識別抑制性免疫檢查點分子之抗體。在一些實施例中，免疫檢查點抑制劑係PD-1之抑制劑。在一些實施例中，免疫檢查點抑制劑係抗PD-1抗體。在一些實施例中，免疫檢查點抑制劑係結合至抑制性免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)的配體。在一些實施例中，免疫檢查點抑制劑包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點抑制劑係特異性識別抑制性免疫檢查點分子之抗體。在一些實施例中，免疫檢查點抑制劑係PD-1之抑制劑。在一些實施例中，免疫檢查點抑制劑係抗PD-1抗體。在一些實施例中，免疫檢查點抑制劑係結合至抑制性免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)的配體。在一些實施例中，免疫檢查點抑制劑包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點抑制劑係特異性識別抑制性免疫檢查點分子之抗體。在一些實施例中，免疫檢查點抑制劑係PD-1之抑制劑。在一些實施例中，免疫檢查點抑制劑係抗PD-1抗體。在一些實施例中，免疫檢查點抑制劑係結合至抑制性免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)的配體。在一些實施例中，免疫檢查點抑制劑包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點抑制劑(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點抑制劑係特異性識別抑制性免疫檢查點分子之抗體。在一些實施例中，免疫檢查點抑制劑係PD-1之抑制劑。在一些實施例中，免疫檢查點抑制劑係抗PD-1抗體。在一些實施例中，免疫檢查點抑制劑係結合至抑制性免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)的配體。在一些實施例中，免疫檢查點抑制劑包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點抑制劑包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，PD-1之抑制劑係抗PD-1抗體。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，PD-1之抑制劑係抗PD-1抗體。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，PD-1之抑制劑係抗PD-1抗體。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，PD-1之抑制劑係抗PD-1抗體。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，PD-1之抑制劑係抗PD-1抗體。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包含(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3的第二抗原結合結構域(諸如scFv)，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 本文所描述之雙特異性分子可以具有任何形式。在一些實施例中，第一抗原結合結構域係scFv。在一些實施例中，第二抗原結合結構域係scFv。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一與第二抗原結合結構域係藉由連接子連接。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之C末端。 因此，在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該第一scFv係在該第二scFv之N末端。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，第一scFv及第二scFv係藉由連接子連接。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1之抑制劑之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv，及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二scFv，其中該第一scFv係在該第二scFv之N末端。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，PD-1之抑制劑係抗PD-1抗體。在一些實施例中，第一scFv及第二scFv係藉由連接子連接。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包含：重鏈可變區(VH)，其包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，其包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。在一些實施例中，抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，提供一種溶瘤病毒，其包含編碼特異性識別PD-1之抗體的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別T淋巴細胞上之CD3之第二scFv，其中該抗PD-1抗體包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中第一scFv係在第二scFv之N末端。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，提供一種溶瘤病毒，其包含編碼PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，提供一種溶瘤病毒，其包含編碼TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(例如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EpCAM之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別FAP之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別EGFR之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別GPC3之第一scFv及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二scFv，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，提供一種溶瘤病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)之融合物的第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一scFv及特異性識別T淋巴細胞上之CD3的第二scFv，其中該SIRPα細胞外結構域包含胺基酸序列SEQ ID NO: 29，且該CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該OV係WR株VV。在一些實施例中，該OV包含TK及VGF基因之雙重缺失。在一些實施例中，第一scFv係在第二scFv之N末端。在一些實施例中，第一及/或第二核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。 本文所描述的編碼雙特異性分子、免疫檢查點調節子及/或細胞介素之核酸可以可操作地連接至啟動子。在一些實施例中，編碼雙特異性分子、免疫檢查點調節子及細胞介素之核酸中的至少兩個係可操作地連接至相同啟動子。在一些實施例中，編碼雙特異性分子、免疫檢查點調節子及細胞介素之核酸全部可操作地連接至相同啟動子。在一些實施例中，編碼雙特異性分子、免疫檢查點調節子及細胞介素之核酸全部可操作地連接至不同啟動子。在一些實施例中，該啟動子係晚期啟動子。在一些實施例中，該啟動子係牛痘病毒啟動子。在一些實施例中，該啟動子係晚期VV啟動子。在一些實施例中，該啟動子係F17R。 溶瘤病毒 溶瘤病毒能夠在分裂細胞(例如癌細胞)中選擇性複製，同時使非分裂細胞(例如正常細胞)不受損害。當受感染之分裂細胞因溶解而破壞時，其釋放新的感染性病毒粒子感染周圍的分裂細胞。癌細胞為許多病毒之理想宿主，因為其等具有失活的抗病毒干擾素路徑或具有突變之腫瘤抑制基因，使病毒複製能夠不受阻礙地進行 (Chernajovsky等人, 2006, British Med. J. 332: 170-2)。 例示性溶瘤病毒包括(但不限於)牛痘病毒(VV)、塞內加谷病毒(SVV)、腺病毒(AdV)、單純疱疹病毒(HSV，諸如HSV1及HSV2)、呼腸孤病毒、黏液瘤病毒(MYXV)、脊髓灰白質炎病毒、水泡性口炎病毒(VSV)、麻疹病毒(MV)、慢病毒、反轉錄病毒、麻疹病毒、流感病毒、辛德比病毒、新城疫病毒(NDV)或類似病毒(參見例如，Kirn等人, Nat. Med. 7:781 (2001)；Coffey等人, Science 282:1332 (1998)；Lorence等人, Cancer Res. 54:6017 (1994)；及Peng等人, Blood 98:2002 (2001))。在一些實施例中，本文所描述之溶瘤病毒係溶瘤牛痘病毒(VV)。 溶瘤病毒可以利用DNA或RNA作為其遺傳物質。溶瘤DNA病毒可以具有衣殼對稱性，即呈二十面體或複合物。二十面體溶瘤DNA病毒可以為裸病毒或包含包膜。溶瘤DNA病毒科包括腺病毒科(例如腺病毒，基因組大小為36-38kb)、疱疹病毒科(例如HSV1，基因組大小為120-200 kb)及痘病毒科(例如牛痘病毒及黏液瘤病毒，基因組大小為130-280 kb)。溶瘤RNA病毒包括具有二十面體或螺旋形衣殼對稱性之RNA病毒。二十面體溶瘤病毒係不含包膜之裸病毒且包括呼腸孤病毒科(例如呼腸孤病毒，具有22-27 kb之基因組)及小RNA病毒科(例如脊髓灰白質炎病毒，基因組大小為7.2-8.4 kb)。螺旋形溶瘤RNA病毒具有包膜且包括彈狀病毒科(例如VSV，基因組大小為13-16 kb)及副黏病毒科(例如MV及NDV，基因組大小為16-20 kb)。 在一些實施例中，溶瘤病毒係牛痘病毒(VV)。在一些實施例中，VV可以為Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉牛痘病毒(MVA)、Lister、King、IHD、Evans、USSR或Western Reserve(WR)株。在一些實施例中，VV係WR株。 在一些實施例中，藉由改變一或多個病毒基因來修飾本發明之溶瘤病毒(諸如溶瘤VV)。該等修飾較佳使得合成缺陷性蛋白質，該缺陷性蛋白質無法確保在正常條件下未修飾基因產生之該蛋白質之活性(或合成缺乏)。修飾涵蓋病毒基因或其調控元件內一或多個核苷酸(相鄰或不相鄰)之缺失、突變及/或取代。修飾可以藉由熟習此項技術者已知之多種方式，使用習知重組技術進行。例示性修飾揭示於文獻中且改變DNA代謝、宿主毒力、IFN路徑(參見例如，Guse等人, 2011, Expert Opinion Biol. Ther. 11(5): 595-608)及類似特性所涉及之病毒基因的修飾尤佳。 在一些實施例中，溶瘤病毒(諸如溶瘤VV)包含胸苷激酶(TK)基因之失活突變，由此產生陰性TK表型。在一些實施例中，VV之TK基因缺失。TK酶涉及脫氧核糖核苷酸之合成。TK係正常細胞中病毒複製所需的，因為該等細胞一般具有低濃度核苷酸，但其在含有高核苷酸濃度之分裂細胞中並非必需的。因此，TK缺失明顯限制休眠細胞中之病毒複製，使有效病毒複製僅在活躍分裂之細胞(例如癌細胞)中發生。 替代地或組合地，亦可實行其他策略以進一步增加病毒的腫瘤特異性。適合修飾之代表性實例包括破壞病毒基因組中之VGF編碼基因。VGF(表示VV生長因子)係在細胞感染之後早期表現的一種分泌蛋白質且其功能看來對於病毒在正常細胞中擴散很重要。VGF缺失之牛痘病毒的複製在休眠(非癌症)細胞中大幅減少。在一些實施例中，本發明之VV不表現功能性痘瘡生長因子(VGF)。在一些實施例中，本發明之溶瘤病毒係缺乏TK及VGF兩種活性之牛痘病毒。經顯示，TK及VGF缺失之影響具有協同性。 因此，在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤VV包含TK基因及VGF基因之雙重缺失。 在一些實施例中，提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之核酸，進一步包含編碼雙特異性分子之核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，溶瘤病毒係溶瘤VV。在一些實施例中，溶瘤病毒係WR株。在一些實施例中，溶瘤病毒包含TK基因及VGF基因之雙重缺失。 據設想，本發明亦關於一種溶瘤病毒載體，其包含本發明中所描述之核酸分子。如本文所使用，術語「病毒載體」係根據其在此項技術中公認之含義使用。該術語係指包括至少一個病毒來源之元件且可以包裝於病毒載體粒子中的核酸載體構築體。病毒載體粒子可以用於在活體外或在活體內將DNA、RNA或其他核酸轉移至細胞中的目的。溶瘤病毒載體包括(但不限於)牛痘病毒(VV)載體、塞內加谷病毒(SVV)載體、腺病毒(AdV)載體、單純疱疹病毒載體(例如HSV1載體)、呼腸孤病毒載體、黏液瘤病毒(MYXV)載體、脊髓灰白質炎病毒載體、水泡性口炎病毒(VSV)載體、麻疹病毒(MV)載體、慢病毒載體、反轉錄病毒載體、麻疹病毒載體、流感病毒載體、辛德比病毒載體及新城疫病毒(NDV)載體。 在一些實施例中，溶瘤病毒載體係溶瘤VV載體。因此，本發明係關於一種溶瘤VV載體，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之核酸，其中該核酸係可操作地連接至晚期啟動子(諸如F17R)。亦提供一種溶瘤病毒載體(諸如溶瘤VV載體)，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之核酸，進一步包含編碼雙特異性分子之核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)的第二抗原結合結構域(諸如scFv)。 本申請案亦提供一種免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種)。該等免疫檢查點調節子可以併入溶瘤病毒，諸如溶瘤VV中，或者可以分離形式提供。 免疫檢查點調節子 免疫檢查點係免疫系統中增大(刺激性分子)或減小信號(抑制性分子)之分子。免疫檢查點蛋白質調控並維持自身耐受性以及生理免疫反應之持續時間及幅度。刺激性檢查點分子包括(但不限於)屬於腫瘤壞死因子(TNF)受體超家族的CD27、CD40、OX40、GITR及CD137，以及屬於B7-CD28超家族之CD28及ICOS。抑制性檢查點分子包括(但不限於)計劃性死亡蛋白1(PD-1)、細胞毒性T淋巴細胞相關蛋白質4(CTLA-4)、淋巴細胞活化基因-3(LAG-3)、T細胞免疫球蛋白結構域及黏蛋白結構域3(TIM-3、HAVCR2)、T細胞活化之V結構域Ig抑制因子(VISTA、B7-H5)、B7-H3、B7-H4(VTCN1)、HHLA2(B7-H7)、B及T淋巴細胞衰減因子(BTLA)、吲哚胺2,3-雙加氧酶(IDO)、殺手細胞免疫球蛋白樣受體(KIR)、腺苷A2A受體(A2AR)、Ig及ITIM結構域蛋白T細胞免疫受體(TIGIT)、2B4(CD244)及其配體。多種檢查點蛋白質已得到廣泛研究，諸如CTLA-4與其配體CD80(B7-1)及CD86，以及PD-1與其配體PD-L1(B7-H1、CD274)及PD-L2(B7-DC、CD273)(參見例如，Pardoll, Nature Reviews Cancer 12: 252-264 (2012))。 免疫檢查點調節子可以為免疫檢查點抑制劑(抑制性免疫檢查點分子之抑制劑)或刺激性免疫檢查點分子之活化劑。免疫檢查點抑制劑(抑制性免疫檢查點分子之抑制劑)在本發明中備受關注，諸如PD-1(CD279)、PD-L1(B7-H1、CD274)、PD-L2(B7-DC、CD273)、LAG-3、TIM-3(HAVCR2)、BTLA、CTLA-4、TIGIT、VISTA(B7-H5)、B7-H4(VTCN1)、CD160(BY55)、HHLA2(B7-H7)、CXCR4、2B4(CD244)、CD73、B7-1(CD80)、B7-H3(CD276)、KIR或IDO之抑制劑。 在一些實施例中，免疫檢查點調節子係刺激性免疫檢查點分子之活化劑。在一些實施例中，刺激性免疫檢查點分子之活化劑係刺激性免疫檢查點分子之天然或經工程改造之配體，包括例如OX40之配體(例如OX40L)、CD28之配體(例如CD80、CD86)、ICOS之配體(例如B7RP1)、4-1BB之配體(例如4-1BBL、Ultra 4-1BBL)、CD27之配體(例如CD70)、CD40之配體(例如CD40L)及TCR之配體(例如I類或II類MHC分子，IMCgp100)。在一些實施例中，刺激性免疫檢查點分子之活化劑係分泌之蛋白質。在一些實施例中，刺激性免疫檢查點分子之活化劑係抗體(諸如促效性抗體)，諸如抗CD28、抗OX40、抗ICOS、抗GITR、抗4-1BB、抗CD27、抗CD40、抗CD3及抗HVEM。 在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，免疫檢查點抑制劑靶向T細胞。在一些實施例中，免疫檢查點抑制劑靶向腫瘤細胞。舉例而言，在一些情況下，腫瘤細胞當連接至特定T細胞受體時，可以切斷活化之T細胞。不過，免疫檢查點抑制劑可以防止腫瘤細胞連接至T細胞，由此使T細胞保持活化(參見例如，Howard West, JAMA Oncol. 1(1):115 (2015))。在一些實施例中，免疫檢查點抑制劑係抑制性免疫檢查點分子之天然或經工程改造之配體，包括例如CTLA-4之配體(例如B7.1、B7.2)、TIM-3之配體(例如半乳糖凝集素-9)、A2A受體之配體(例如腺苷、瑞加德松(Regadenoson))、LAG-3之配體(例如I類MHC或II類MHC分子)、BTLA之配體(例如HVEM、B7-H4)、KIR之配體(例如I類MHC或II類MHC分子)、PD-1之配體(例如PD-L1、PD-L2)、IDO之配體(例如NKTR-218、因多莫得(Indoximod)、NLG919)、HHLA2之配體(例如TMIGD2)、CXCR4之配體(例如CXCL12)及CD47之配體(例如SIRP-α受體)。在一些實施例中，免疫檢查點抑制劑係分泌的。在一些實施例中，免疫檢查點抑制劑係靶向抑制性免疫檢查點蛋白質之抗體(諸如拮抗性抗體)，包括(但不限於)抗CTLA-4、抗TIM-3、抗LAG-3、抗KIR、抗PD-1、抗PD-L1、抗CD73、抗B7-H3、抗CD47、抗BTLA、抗VISTA、抗A2AR、抗B7-1、抗B7-H4、抗CD52、抗IL-10、抗IL-35及抗TGF-β。在一些實施例中，免疫檢查點抑制劑係選自由以下組成之群之抑制性檢查點分子的抑制劑：PD-1、PD-L1、LAG-3、TIM-3、HHLA2、CD47、CXCR4、CD160、CD73、BLTA、B7-H4、TIGIT及VISTA。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係特異性識別PD-1之抗體。在一些實施例中，該免疫檢查點調節子係結合至免疫檢查點分子之配體。在一些實施例中，該免疫檢查點調節子抑制劑係結合至PD-L1及/或PD-L2之配體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域與免疫球蛋白Fc片段的融合物。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，免疫檢查點調節子係結合至HHLA2之配體。在一些實施例中，免疫檢查點調節子係TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子係結合到至少兩個不同的抑制性免疫檢查點分子之配體(例如雙特異性配體)，諸如結合至CD47及CXCR4兩者之配體。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。 PD-1 PD-1係調控T細胞活化及耐受性之B7/CD28共刺激分子家族之一部分，且因此拮抗性抗PD-1抗體可以用於克服耐受性。已確定PD-1為B7-4之受體。B7-4在結合至免疫細胞上之抑制性受體時可以抑制免疫細胞活化。PD-1/PD-L1路徑接合引起T細胞效應功能、細胞介素分泌及增殖之抑制。(Turnis等人, OncoImmunology 1(7):1172-1174, 2012)。較高的PD-1含量與耗竭或長期刺激之T細胞有關。另外，增加PD-1表現與癌症患者之存活期縮短相關。用於下調PD-1、B7-4以及免疫細胞中B7-4與PD-1抑制信號之間之相互作用的試劑可以增強免疫反應。 PD-L1/PD-L2 PD-L1(計劃性細胞死亡配體1)又稱為分化簇274(CD274)或B7同源物1(B7-H1)。PD-L1用作PD-1之配體，在諸如妊娠、組織同種異體移植(tissue allographs)、自身免疫疾病及其他疾病狀態(諸如肝炎及癌症)之特定事件期間抑制免疫系統方面起主要作用。PD-1受體/PD-L1配體複合物之形成將傳輸抑制性信號，由此減少淋巴結處CD8+ T細胞之增殖。 PD-L2(計劃性細胞死亡1配體2)又稱為B7-DC。PD-L2用作PD-1之配體。在某些情況下，PD-L2及其抑制劑可以分別用作PD-L1及其抑制劑之替代物。 TMIGD2 HHLA2在乳癌、肺癌、甲狀腺癌、黑素瘤、胰臟癌、卵巢癌、肝癌、膀胱癌、結腸癌、前列腺癌、腎癌、食道癌及血液惡性病白血病及淋巴瘤之許多人類癌症中廣泛表現。HHLA2路徑代表腫瘤微環境內之一種新穎免疫抑制機制且為人類癌症療法的引人注目之標靶。已鑑別出TMIGD2為HHLA2之受體。阻斷HHLA2/TMIGD2可以作為癌症免疫療法的一種有效策略。 CD47 CD47係腫瘤細胞所採用的抗吞噬配體，其藉由將抑制信號傳輸通過其受體信號調控蛋白質α(SIRPα)來削弱抗體效應功能。干擾CD47-SIRPα相互作用可以增強抗腫瘤免疫反應。 CXCR4 趨化細胞素CXCL12及其受體CXCR4在包括卵巢癌在內之人類癌症中廣泛表現，在人類癌症中，其在腫瘤細胞增殖、侵襲及血管生成層面上與疾病進展有關。由腫瘤組織及周圍基質產生之CXCL12刺激VEGF介導之血管生成及自骨髓之內皮祖細胞之募集。另外，經顯示，CXCL12在腫瘤部位募集抑制性CD11b+Gr1+骨髓細胞及pDC，且誘導腫瘤內T調控細胞(Treg)定位，由此阻止破壞腫瘤之免疫機制。因此，調節CXCL12/CXCR4軸可以影響腫瘤發病機制之多個方面，包括免疫調節異常。若干CXCR4拮抗劑已在臨床前模型中展示抗腫瘤功效且已在早期臨床試驗中進行評價。 在一些實施例中，本發明之溶瘤病毒(諸如溶瘤VV)可以包含編碼本文所描述之免疫檢查點分子之任何抗體或抗原結合片段的核酸。舉例而言，本發明之溶瘤病毒(諸如溶瘤VV)可以包含編碼上述抗PD-1抗體之scFv形式的核酸。在一些實施例中，本發明之溶瘤病毒(諸如溶瘤VV)可以包含編碼包含本文所描述之任何抗體片段或全長抗體之任何其他功能變異體或衍生物之融合蛋白的核酸。舉例而言，本發明之溶瘤病毒(諸如溶瘤VV)可以包含編碼上述抗PD-1抗體之抗原結合結構域與IgG4片段之融合物的核酸。 本文中涵蓋之免疫檢查點調節子係蛋白質或肽。在一些實施例中，免疫檢查點調節子包含單一多肽鏈。在一些實施例中，免疫檢查點調節子包含多於一個(諸如2、3、4或更多個中之任一個)多肽鏈。免疫檢查點調節子之多肽鏈可具有任何長度，諸如至少約10、20、50、100、200、300、500或更多個中之任一種氨基酸長度。在多鏈免疫檢查點調節子之情況下，編碼該等多肽鏈之核酸序列可以可操作地連接至相同啟動子或不同啟動子。 在一些實施例中，免疫檢查點調節子係分泌蛋白質。在一些實施例中，免疫檢查點調節子係抗體。天然抗體，諸如單株抗體係與特定抗原免疫反應之免疫球蛋白分子。在一些實施例中，該抗體係促效性抗體。在一些實施例中，該抗體係拮抗性抗體。在一些實施例中，抗體係單株抗體。在一些實施例中，抗體係全長抗體。在一些實施例中，抗體係選自由以下組成之群的抗原結合片段：VH 、VL 、VNAR 、VH H、Fab、Fab'、F(ab')2 、Fv、微型抗體、scFv、sc(Fv)2 、三鏈抗體、四鏈抗體、scFv-scFv(諸如BiTE® )、微型抗體、scFv-Fc、三功能抗體及全長抗體之其他抗原結合子序列或其經工程改造之組合。在一些實施例中，抗體係人類抗體、人類化抗體或嵌合抗體。在一些實施例中，抗體係單價抗體。在一些實施例中，抗體係多價抗體，諸如二價抗體或四價抗體。在一些實施例中，抗體係雙特異性抗體。在一些實施例中，抗體係多特異性抗體。在一些實施例中，抗體係單結構域抗體(sdAb)。在一些實施例中，抗體係僅含重鏈之抗體，諸如駱駝抗體或其衍生物。在一些實施例中，抗體係單鏈抗體。在一些實施例中，抗體係scFv。在一些實施例中，抗體係包含抗體片段(諸如含Fc之融合蛋白，例如PD-1細胞外結構域-Fc融合蛋白)或全長抗體之任何其他功能變異體或衍生物的融合蛋白。 在一些實施例中，免疫檢查點調節子係包含重鏈及輕鏈之抗體。在一些實施例中，重鏈包含VH 結構域。在一些實施例中，重鏈進一步包含一或多個恆定結構域，諸如CH 1、CH 2、CH 3或其任何組合。在一些實施例中，輕鏈包含VL 結構域。在一些實施例中，輕鏈進一步包含恆定結構域，諸如CL 。在一些實施例中，重鏈與輕鏈經由複數個二硫鍵彼此連接。在一些實施例中，抗體包含Fc，諸如人類IgG1、IgG2、IgG3或IgG4之Fc片段。在一些實施例中，抗體不包含Fc片段。在一些實施例中，免疫檢查點調節子係Fab。在一些實施例中，免疫檢查點調節子係全長抗PD-1抗體。 溶瘤病毒(諸如溶瘤VV)可以表現任何數量(諸如1、2、3、4、5、6或更多個中之任一個)之免疫檢查點調節子。在一些實施例中，溶瘤病毒包含編碼單一免疫檢查點調節子之核酸。在一些實施例中，溶瘤病毒包含編碼至少兩個免疫檢查點調節子之一或多個核酸。在一些實施例中，編碼至少兩個免疫檢查點調節子之核酸可操作地連接至相同啟動子。在一些實施例中，編碼至少兩個免疫檢查點調節子之核酸可操作地連接至不同啟動子。在一些實施例中，編碼本發明之免疫檢查點調節子及雙特異性分子之核酸可操作地連接至相同啟動子。在一些實施例中，編碼本發明之免疫檢查點調節子及雙特異性分子之核酸可操作地連接至不同啟動子。 多鏈免疫調節抗體之重鏈多肽及輕鏈多肽係由單一核酸或由兩個核酸在本發明之溶瘤病毒(諸如溶瘤VV)中共表現。在一些實施例中，重鏈多肽與輕鏈多肽係以等莫耳比表現。在一些實施例中，重鏈多肽與輕鏈多肽係大致以下任一比率表現：10:1、8:1、6:1、5:1、4:1、3:1、2:1、3:2、4:3、5:4、1:1、4:5、3:4、2:3、1:2、1:3、1:4、1:5、1:6、1:8或1:10。在一些實施例中，重鏈多肽與輕鏈多肽係以以下任一比率表現：約1:10至約1:5、約1:5至約1:3、約1:4至約1:2、約1:2至約1:1、約1:1至約2:1、約2:1至約4:1、約3:1至約5:1、約5:1至約10:1、約1:2至約2:1、約1:3至約3:1、約1:5至約5:1或約1:10至約10:1。重鏈多肽與輕鏈多肽之間的最佳表現比率可以有助於抗體摺疊及組裝過程。參見例如，Schlatter S等人, Biotechnol Prog.21(1): 122-33 (2005)。 多鏈免疫調節抗體中重鏈多肽與輕鏈多肽之間之各種表現比率可以藉由操縱異源核酸及/或編碼重鏈及輕鏈之核酸之複本數量，及/或誘導序列及/或連接至該編碼重鏈及輕鏈之核酸之啟動子的強度來達成。在一些實施例中，編碼重鏈之核酸與編碼輕鏈之核酸係可操作地連接至相同啟動子。在一些實施例中，編碼重鏈之核酸與編碼輕鏈之核酸係可操作地連接至不同啟動子。在一些實施例中，編碼重鏈之核酸的啟動子與編碼輕鏈之核酸的啟動子可以同時經誘導。在一些實施例中，編碼重鏈之核酸的啟動子與編碼輕鏈之核酸的啟動子可以依序誘導。在一些實施例中，編碼重鏈之核酸的啟動子係在誘導編碼輕鏈之核酸的啟動子之前誘導。在一些實施例中，編碼重鏈之核酸的啟動子係在誘導編碼輕鏈之核酸的啟動子之後誘導。在一些實施例中，編碼重鏈之核酸的啟動子與編碼輕鏈之核酸的啟動子具有大致以下任一強度比率：10:1、8:1、6:1、5:1、4:1、3:1、2:1、3:2、4:3、5:4、1:1、4:5、3:4、2:3、1:2、1:3、1:4、1:5、1:6、1:8或1:10。在一些實施例中，編碼重鏈之核酸的啟動子與編碼輕鏈之核酸的啟動子具有以下任一強度比率：約1:10至約1:5、約1:5至約1:3、約1:4至約1:2、約1:2至約1:1、約1:1至約2:1、約2:1至約4:1、約3:1至約5:1、約5:1至約10:1、約1:2至約2:1、約1:3至約3:1、約1:5至約5:1或約1:10至約10:1。 在一些實施例中，免疫檢查點調節子係包含抗體片段或全長抗體之任何其他功能變異體或衍生物的融合蛋白。在一些實施例中，免疫檢查點調節子係含Fc之融合蛋白。在一些實施例中，免疫檢查點調節子包含本文所描述之抗體之抗原結合結構域(諸如包含CDRs之片段)與Fc片段之融合物。舉例而言，本發明之溶瘤病毒可以包含編碼抗PD-1抗體之抗原結合結構域與IgG4片段之融合物的核酸。在一些實施例中，免疫檢查點調節子係本文所描述之抑制性免疫檢查點分子之配體的細胞外結構域(諸如PD-1細胞外結構域)與Fc片段的融合物。在一些實施例中，Fc片段可以為人類IgG1、IgG2、IgG3或IgG4之Fc片段。在一些實施例中，該Fc片段係IgG4 Fc。 在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-Fc融合蛋白。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，PD-1細胞外結構域係由包含SEQ ID NO:26之核酸序列編碼。 在一些實施例中，免疫檢查點調節子係TMIGD2細胞外結構域-Fc融合蛋白。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，TMIGD2細胞外結構域-Fc融合蛋白進一步包括含胺基酸序列SEQ ID NO:28之信號肽。 在一些實施例中，免疫檢查點調節子特異性結合兩個不同的免疫檢查點分子，諸如CD47及CXCR4。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段之融合物。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段藉由連接子，諸如IgG1鉸鏈連接至SIRPα細胞外結構域。在一些實施例中，CXCL12片段係藉由包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。在一些實施例中，SIRPα-CXCL12-Fc融合蛋白進一步包含包含胺基酸序列SEQ ID NO: 32之信號肽。在一些實施例中，SIRPα-CXCL12-Fc融合蛋白具有(N'至C')信號肽-SIRPα細胞外結構域-連接子-CXCL12-Fc片段之結構。 細胞介素 如本文所使用，術語「細胞介素(cytokine/cytokines)」係指影響免疫系統之細胞的通用生物分子種類。該定義意圖包括(但不限於)局部作用或可以在血液中循環之該等生物分子，且其當在本發明中使用時用於調控或調節個體對癌症之免疫反應。用於實踐本發明之例示性細胞介素包括(但不限於)干擾素(諸如IFN-α、IFN-β、IFN-γ)、所有白細胞介素(例如IL-1至IL-29，尤其是IL-1、IL-2、IL-6、IL-7、IL-10、IL-12、IL-15、IL-18、IL-23、IL-24及IL-27)、腫瘤壞死因子(例如TNF-α及TNF-β)、紅血球生成素(EPO)、MIP3a、ICAM、巨噬細胞群落刺激因子(M-CSF)、粒細胞群落刺激因子(G-CSF)及顆粒球-巨噬細胞群落刺激因子(GM-CSF)。GM-CSF係由巨噬細胞、T細胞、肥大細胞、NK細胞、內皮細胞及纖維母細胞分泌的充當細胞介素之單體糖蛋白。GM-CSF誘導多種免疫活性細胞群之活化、增殖及分化，由此促進體液及細胞介導之免疫的產生(Warren及Weiner, 2000)。在一些實施例中，細胞介素係GM-CSF。 在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一核酸，其中該編碼免疫檢查點調節子之第一核酸係可操作地連接至晚期啟動子(諸如F17R)，進一步包含編碼細胞介素(諸如GM-CSF)之第二核酸。在一些實施例中，編碼免疫檢查點調節子及細胞介素之核酸係可操作地連接至同一晚期啟動子(諸如F17R)。在一些實施例中，編碼免疫檢查點調節子及細胞介素之核酸係可操作地連接至不同啟動子。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑(諸如特異性識別免疫檢查點分子之抗體)。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係結合至免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)之配體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-Fc融合蛋白。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。 在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼抗PD-1抗體或其抗原結合片段之第一核酸，其中該編碼抗PD-1抗體或其抗原結合片段之第一核酸係可操作地連接至晚期啟動子(諸如F17R)，進一步包含編碼GM-CSF之第二核酸。 在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼PD-1細胞外結構域-Fc融合蛋白之第一核酸，其中該編碼PD-1細胞外結構域-Fc融合蛋白之第一核酸係可操作地連接至晚期啟動子(諸如F17R)，進一步包含編碼GM-CSF之第二核酸。在一些實施例中，該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。 在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼TMIGD2細胞外結構域-Fc融合蛋白之第一核酸，其中該編碼TMIGD2細胞外結構域-Fc融合蛋白之第一核酸係可操作地連接至晚期啟動子(諸如F17R)，進一步包含編碼GM-CSF之第二核酸。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。 在一些實施例中，提供一種溶瘤牛痘病毒，其包含編碼SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白之第一核酸，其中該編碼SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白之第一核酸係可操作地連接至晚期啟動子(諸如F17R)，進一步包含編碼GM-CSF之第二核酸。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。 在一些實施例中，提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)，該溶瘤病毒進一步包含編碼細胞介素(諸如GM-CSF)之第三異源核酸。在一些實施例中，編碼免疫檢查點調節子、雙特異性接合分子及細胞介素之核酸中的至少兩個係可操作地連接至相同啟動子。在一些實施例中，編碼免疫檢查點調節子、雙特異性接合分子及細胞介素之核酸全部可操作地連接至相同啟動子。在一些實施例中，編碼免疫檢查點調節子、雙特異性接合分子及細胞介素之核酸全部可操作地連接至不同啟動子。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑(諸如特異性識別免疫檢查點分子之抗體)。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係結合至免疫檢查點分子(諸如PD-L1/PD-L2、HHLA-2、CD47或CXCR4)之配體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-Fc融合蛋白。在一些實施例中，該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。在一些實施例中，該Fc片段係IgG4 Fc。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。 在一些實施例中，提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼抗PD-1抗體或其抗原結合片段之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)，該溶瘤病毒進一步包含編碼GM-CSF之第三核酸。 在一些實施例中，提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼PD-1細胞外結構域-Fc融合蛋白之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)，該溶瘤病毒進一步包含編碼GM-CSF之第三核酸。在一些實施例中，該PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。 在一些實施例中，提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼TMIGD2細胞外結構域-Fc融合蛋白之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)，該溶瘤病毒進一步包含編碼GM-CSF之第三核酸。在一些實施例中，TMIGD2細胞外結構域包含胺基酸序列SEQ ID NO:27。 在一些實施例中，提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)，該溶瘤病毒進一步包含編碼GM-CSF之第三核酸。在一些實施例中，SIRPα細胞外結構域包含胺基酸序列SEQ ID NO:29。在一些實施例中，CXCL12片段包含胺基酸序列SEQ ID NO: 30。在一些實施例中，CXCL12片段係藉由連接子，諸如IgG1鉸鏈，或包含胺基酸序列SEQ ID NO: 31之連接子連接至SIRPα細胞外結構域。 調控序列 熟習此項技術者顯而易知，可以向本發明中所包含之OV核酸分子中添加調控序列。此類調控序列(控制元件)係技術人員已知的且可以包括啟動子、用於轉錄(例如聚腺苷酸轉錄終止序列)、mRNA運輸(例如核定位信號序列)、加工(例如剪接信號)、穩定性(例如內含子及非編碼5'及3'序列)、轉譯(例如起始子Met、三聯前導序列、IRES核糖體結合位點、信號肽等)之適當起始、調控及/或終止之另外的元件，及用於將插入序列引入病毒載體中之插入位點。在一些實施例中，調控序列係啟動子、轉錄強化子及/或可以採用允許適當表現本發明之雙特異性分子、免疫檢查點調節子及細胞介素的序列。 術語「調控序列」係指影響所連接之編碼序列之表現所需的DNA序列。此類控制序列之性質取決於宿主生物體而不同。在原核生物中，控制序列一般包括啟動子、核糖體結合位點及終止子。在真核生物中，控制序列一般包括啟動子、終止子且在一些情況下包括強化子、反式活化蛋白或轉錄因子。術語「控制序列」在最低限度下意欲包括表現需要存在之所有組分且亦可包括另外的有利組分。 術語「可操作地連接」係指所描述之組分處於准許其以其預期方式作用的關係中的併接。「可操作地連接」至編碼序列的控制序列係以使得該編碼序列在與該等控制序列相容之條件下表現的方式連接。在控制序列係啟動子之情況下，熟習此項技術者顯而易知較佳使用雙股核酸。 如本文所使用，「啟動子」、啟動子區或啟動子元件或調控區或調控元件係指這樣一種DNA或RNA區段，其控制其以操作方式連接之DNA或RNA之轉錄。啟動子區包括RNA聚合酶識別、結合及轉錄起始所涉及之特定序列。此外，啟動子包括調節RNA聚合酶之識別、結合及轉錄起始活性(亦即，結合一或多個轉錄因子)的序列。該等序列可以為順式作用的或可以對反式作用因子起反應。取決於調控之性質，啟動子可以為組成性或調控性的。調控性啟動子可以為誘導性或環境反應性的(例如對諸如pH、厭氧條件、滲壓劑、溫度、光或細胞密度之提示起反應)。許多此類啟動子序列係此項技術中已知的。參見例如，美國專利第4,980,285號、第5,631,150號、第5,707,928號、第5,759,828號、第5,888,783號、第5,919,670號，以及Sambrook等人, Molecular Cloning: A Laboratory Manual, 第2版, Cold Spring Harbor Press (1989)。 據設想，本發明亦關於一種溶瘤病毒載體(諸如溶瘤VV載體)，其包含本發明所描述之核酸分子。如本文所使用，術語「病毒載體」係根據其在此項技術中公認之含義使用。該術語係指包括至少一個病毒來源之元件且可以包裝於病毒載體粒子中的核酸載體構築體。病毒載體粒子可以用於在活體外或在活體內將DNA、RNA或其他核酸轉移至細胞中的目的。在某一實施例中，溶瘤病毒載體係VV載體。VV可以為Wyeth或Western Reserve(WR)株。VV可以在其基因組中具有缺失或在一或多個基因中具有突變。牛痘病毒之胸苷激酶基因可能已缺失。牛痘病毒可以在編碼牛痘病毒生長因子之基因中具有突變。在一些實施例中，溶瘤病毒載體係慢病毒載體。慢病毒載體係可商購的，包括例如自Clontech(Mountain View, Calif.)或GeneCopoeia(Rockville, Md.)購得。 「表現載體」係可用以轉型所選宿主且使編碼序列在該所選宿主中表現的構築體。表現載體可以例如為選殖載體、二元載體或整合載體。表現包含核酸分子之轉錄，較佳轉錄成可轉譯之mRNA。確保在真核細胞中表現之調控元件係熟習此項技術者熟知的。在真核細胞之情況下，其通常包含確保轉錄起始之啟動子且視情況包含確保轉錄物之轉錄終止及穩定化的聚腺苷酸信號。允許在真核宿主細胞中表現之調控元件之實例係在酵母中之AOX1或GAL1啟動子，或在哺乳動物及其他動物細胞中之CMV、SV40、RSV啟動子(勞斯肉瘤病毒)、CMV強化子、SV40強化子或血球蛋白內含子。 除負責轉錄起始之元件之外，此類調控元件亦可在聚核苷酸之下游包含轉錄終止信號，諸如SV40-聚腺苷酸位點或tk-聚腺苷酸位點。另外，取決於所用表現系統，能夠將多肽引導至細胞區室或將其分泌至培養基中的前導序列可以添加至所述核酸序列之編碼序列中且為此項技術中熟知的。前導序列，且較佳能夠引導轉譯之蛋白質之分泌的前導序列或其一部分係在適當階段與轉譯、起始及終止序列一起組裝於周質間隙或細胞外介質中。視情況，異源核酸序列可以編碼包括賦予所需特徵，例如所表現之重組產物之穩定化或簡化純化之N末端標識肽的融合蛋白。適合表現載體係此項技術中已知的，諸如Okayama-Berg cDNA表現載體pcDV1(Pharmacia)、pEF-Neo、pCDM8、pRc/CMV、pcDNA1、pcDNA3(Invitrogen)、pEF-DHFR及pEF-ADA(Raum等人, Cancer Immunol Immunother (2001) 50(3), 141-150)或pSPORT1 (GIBCO BRL)。 熟習此項技術者應瞭解，調控序列之選擇可以取決於諸如核酸分子本身、插入其之病毒、宿主細胞或受試者、所需表現量等因素。啟動子具有特別重要的意義。在本發明之情形中，其可以組成性引導核酸分子在許多類型宿主細胞或某些特定宿主細胞(例如腫瘤特異性調控序列)中表現或響應於特定事件或外源因素(例如溫度、營養添加劑、激素等)或根據病毒週期之階段(例如晚期或早期)進行調控。為了使病毒製造最佳且避開所表現之多肽之潛在毒性，亦可使用在該製造步驟期間響應於特定事件或外源因素而受到阻遏的啟動子。 在一些實施例中，表現控制序列係載體中能夠轉型或轉染真核宿主細胞的真核啟動子系統，但亦可使用原核宿主之控制序列。一旦載體併入適當宿主中，該宿主即維持在適於高水準表現核苷酸序列之條件下，且按需要，隨後可以收集及純化本發明之多肽。 啟動子可以為天然或異源的(相對於本文所描述之溶瘤病毒)。可以使用任何適合的啟動子，包括合成及天然存在之啟動子以及經修飾之啟動子。如本文所使用，天然啟動子係病毒內源性啟動子且其核苷酸序列及其在病毒基因組中之位置相較於野生型病毒係未修飾的。合成啟動子係異源性啟動子，其核苷酸序列在自然界中未發現。合成啟動子可以為具有合成序列或衍生自天然啟動子之序列或其部分的核酸分子。合成啟動子亦可為由衍生自不同天然啟動子之不同元件構成的雜合啟動子。有關例示性牛痘病毒合成啟動子，參見例如US9005602。 病毒啟動子可以包括(但不限於)VV啟動子、痘病毒啟動子、腺病毒晚期啟動子、牛痘ATI啟動子或T7啟動子。啟動子可以為牛痘病毒啟動子、合成啟動子、在至少感染早期期間引導轉錄之啟動子、在至少感染中期期間引導轉錄之啟動子、在感染早期/晚期期間引導轉錄之啟動子或在至少感染晚期期間引導轉錄之啟動子。 啟動子可以大致分類為組成性啟動子或調控性啟動子，諸如誘導性啟動子。 適於在哺乳動物細胞中組成性表現的啟動子包括(但不限於)巨細胞病毒(CMV)即刻早期啟動子(US 5,168,062)、RSV啟動子、腺病毒主要晚期啟動子、磷酸甘油酸激酶(PGK)啟動子(Adra等人, 1987, Gene 60: 65-74)、單純疱疹病毒(HSV)-l之胸苷激酶(TK)啟動子及T7聚合酶啟動子(WO98/10088)。牛痘病毒啟動子特別適合於在溶瘤痘病毒中表現。代表性實例包括(不限於)痘瘡7.5K、H5R、11K7.5(Erbs等人, 2008, Cancer Gene Ther. 15(1): 18-28)、TK、p28、pll、pB2R、pA35R及K1L啟動子，以及合成啟動子，諸如Chakrabarti等人(1997, Biotechniques 23: 1094-7；Hammond等人, 1997, J. Virol Methods 66: 135-8；以及Kumar及Boyle, 1990, Virology 179: 151-8)中所述之該等啟動子，以及早期/晚期嵌合啟動子。適於溶瘤麻疹病毒之啟動子包括(不限於)引導麻疹轉錄單元之表現的任何啟動子(Brandler及Tangy, 2008, CIMID 31: 271)。 誘導性啟動子屬於調控性啟動子類別。誘導性啟動子可以在一或多種條件下誘導，諸如物理條件、宿主細胞之微環境或宿主細胞之生理狀態、誘導劑(亦即，誘導試劑)或其組合。 適於表現之啟動子可以在活體外(例如在適合培養之細胞株中)或在活體內(例如在適合動物模型中或在受試者體內)進行測試。當編碼的免疫檢查點調節子包含抗體且尤其是mAb時，適合表現該免疫檢查點調節子之較重組分的啟動子之實例包含CMV、SV及牛痘病毒pH5R、F17R及pllK7.5啟動子；適合表現該免疫檢查點調節子之較輕組分的啟動子之實例包含PGK、β-肌動蛋白及牛痘病毒p7.5K、F17R及pA35R啟動子。 啟動子可以用較強或較弱啟動子替代，其中替代引起病毒減毒之變化。如本文所使用，用較強啟動子替代啟動子係指自基因組移除啟動子且將其用使轉錄起始水準相對於所替代之啟動子有所增加的啟動子替代。通常，較強啟動子結合聚合酶複合物之能力相對於所替代之啟動子有所提高。因此，可操作地連接至較強啟動子之開放閱讀框架具有較高基因表現水準。類似地，用較弱啟動子替代啟動子係指自基因組移除啟動子且將其用使轉錄起始水準相對於所替代之啟動子有所降低的啟動子替代。通常，較弱啟動子結合聚合酶複合物之能力相對於所替代之啟動子有所減弱。因此，可操作地連接至較弱啟動子之開放閱讀框架具有較低基因表現水準。病毒可以展現由於使用較強啟動子與較弱啟動子而引起之特徵差異，諸如減毒。舉例而言，在牛痘病毒中，合成早期/晚期啟動子及晚期啟動子係相對較強之啟動子，而痘瘡合成早期啟動子、P7.5k早期/晚期啟動子、P7.5k早期啟動子及P28晚期啟動子係相對較弱之啟動子(參見例如，Chakrabarti等人(1997)BioTechniques 23 (6) 1094-1097)。 在一些實施例中，啟動子係牛痘病毒啟動子。用於本發明中之例示性牛痘病毒啟動子可以包括(但不限於)P7.5k 、P11k 、PSE 、PSEL 、PSL 、H5R、TK、P28、C11R、G8R、F17R、I3L、I8R、A1L、A2L、A3L、H1L、H3L、H5L、H6R、H8R、D1R、D4R、D5R、D9R、D11L、D12L、D13L、M1L、N2L、P4b或K1啟動子。 在一些實施例中，該啟動子係牛痘病毒天然啟動子。如本文所使用，天然啟動子係病毒內源性啟動子且其核苷酸序列及其在病毒基因組中之位置相較於野生型病毒係未修飾的。在一些實施例中，啟動子係牛痘病毒合成啟動子(參見例如US9005602)。合成啟動子係異源性啟動子，其核苷酸序列在自然界中未發現。合成啟動子可以為具有合成序列或衍生自天然啟動子之序列或其部分的核酸分子。合成啟動子亦可為由衍生自不同天然啟動子之不同元件構成的雜合啟動子。 例示性痘瘡早期、中期及晚期啟動子包括例如痘瘡P7 . 5k 早期/晚期啟動子、痘瘡PEL 早期/晚期啟動子、痘瘡P13 早期啟動子、痘瘡P11k 晚期啟動子及本文中別處所列的痘瘡啟動子。例示性合成啟動子包括例如PSE 合成早期啟動子、PSEL 合成早期/晚期啟動子、PSL 合成晚期啟動子、本文中別處所列的痘瘡合成啟動子(Patel等人,Proc. Natl. Acad. Sci. USA 85: 9431-9435 (1988)；Davison及Moss,J Mol Biol 210: 749-769 (1989)；Davison等人,Nucleic Acids Res. 18: 4285-4286 (1990)；Chakrabarti等人,BioTechniques 23: 1094-1097 (1997))。可以在相同病毒或兩種不同病毒中使用不同啟動子之組合表現不同基因產物。 在一些實施例中，啟動子在至少感染晚期期間引導轉錄(諸如F17R啟動子)。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。 在一些實施例中，採用在至少感染晚期期間引導轉錄之啟動子(諸如F17R啟動子)。晚期牛痘病毒啟動子F17R僅在VV感染腫瘤細胞之後活化，因此藉由使用F17R啟動子將進一步增強來自VV之轉殖基因之腫瘤選擇性表現。本發明之免疫檢查點調節子、雙特異性接合分子及/或細胞介素(諸如GM-CSF)之晚期表現亦將在T細胞活化及T細胞介導之腫瘤溶解之前允許充分病毒複製。 因此，在本發明之一些實施例中提供一種溶瘤牛痘病毒，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之核酸，其中該編碼免疫檢查點調節子之核酸係可操作地連接至晚期啟動子。在一些實施例中，該晚期啟動子係F17R。在一些實施例中，表現免疫檢查點調節子之溶瘤牛痘病毒進一步包含編碼細胞介素之第二核酸。在一些實施例中，編碼細胞介素之核酸亦可操作地連接至啟動子。在一些實施例中，編碼細胞介素之核酸係連接至晚期啟動子。在一些實施例中，編碼細胞介素之核酸係連接至F17R。在一些實施例中，細胞介素係GM-CSF。 在一些實施例中，提供一種溶瘤病毒(諸如溶瘤VV)，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)，其中該編碼免疫檢查點調節子之核酸及/或編碼雙特異性分子之核酸係可操作地連接至啟動子。在一些實施例中，該啟動子係溶瘤牛痘病毒啟動子。在一些實施例中，該啟動子係晚期啟動子。在一些實施例中，該啟動子係F17R啟動子。在一些實施例中，編碼免疫檢查點調節子之核酸及編碼雙特異性分子之核酸係可操作地連接一個啟動子。在一些實施例中，編碼免疫檢查點調節子之核酸及編碼雙特異性分子之核酸係可操作地連接兩個相同的啟動子。在一些實施例中，編碼免疫檢查點調節子之核酸及編碼雙特異性分子之核酸係可操作地連接至不同啟動子。在一些實施例中，編碼免疫檢查點調節子之核酸及編碼雙特異性分子之核酸係在病毒感染之相同或類似階段期間經引導轉錄。在一些實施例中，編碼免疫檢查點調節子之核酸及編碼雙特異性分子之核酸係在病毒感染之不同階段期間經引導轉錄。在一些實施例中，編碼免疫檢查點調節子之核酸及編碼雙特異性分子之核酸係可操作地連接至相同或類似強度之啟動子。在一些實施例中，編碼免疫檢查點調節子之核酸及編碼雙特異性分子之核酸係可操作地連接至不同強度之啟動子。在一些實施例中，編碼雙特異性分子之核酸係可操作地連接至比可操作地連接至編碼免疫檢查點調節子之核酸之啟動子強的啟動子。在一些實施例中，編碼雙特異性分子之核酸係可操作地連接至比可操作地連接至編碼免疫檢查點調節子之核酸之啟動子弱的啟動子。 另外的調控元件可以包括轉錄以及轉譯強化子。有利地，上文所描述的本發明之溶瘤病毒載體包含可選擇及/或可評分之標記物。可用於選擇經轉型細胞之可選擇標記物基因係熟習此項技術者熟知的且包含作為選擇之基礎的抗代謝物抗性，例如：dhfr，其賦予對甲胺喋呤(methotrexate)之抗性(Reiss, Plant Physiol. (Life-Sci. Adv.) 13 (1994), 143-149)； npt，其賦予對胺基糖苷類新黴素(新黴素)、卡那黴素(kanamycin)及巴龍黴素(paromycin)之抗性(Herrera-Estrella, EMBO J. 2 (1983), 987-995)及hygro，其賦予對潮黴素(hygromycin)之抗性(Marsh, Gene 32 (1984), 481-485)。已描述另外的可選擇基因，即trpB，其使得細胞能利用吲哚代替色胺酸；hisD，其使得細胞能利用組胺醇代替組胺酸(Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047)；甘露糖-6-磷酸異構酶，其使得細胞能利用甘露糖(WO 94/20627)及ODC(鳥胺酸脫羧酶)，其賦予對鳥胺酸脫羧酶抑制劑2-(二氟甲基)-DL-鳥胺酸DFMO之抗性(McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory編)或來自土麯黴(Aspergillus terreus)之脫胺酶賦予對殺稻瘟菌素S(Blasticidin S)之抗性(Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338)。 有用的可評分標記物亦係熟習此項技術者已知的且係可商購的。有利地，該標記物係編碼螢光素酶(Giacomin, Pl. Sci. 116(1996), 59-72；Scikantha, J. Bact. 178(1996), 121)、綠色螢光蛋白(Gerdes, FEBS Lett. 389(1996), 44-47)、YFP或β-葡糖苷酸酶(Jefferson, EMBO J. 6(1987), 3901-3907)之基因。可評分標記物特別適用於簡單且快速地篩選含有所述載體之細胞、組織及生物體。 適當時，可以有利地包括另外的調控元件以促進插入本發明溶瘤病毒之病毒基因組中的至少一個基因(亦即，雙特異性接合分子、免疫檢查點調節子及/或細胞介素)之表現、運輸及生物活性。舉例而言，可以包括信號肽(或前導序列)以促進受感染細胞之分泌。信號肽通常插入蛋白質之N末端緊接在Met起始子之後。信號肽之選擇範圍較寬且係熟習此項技術者可獲得的。亦可設想添加跨膜結構域以促進經編碼蛋白質錨定於受感染細胞之適合膜(例如質膜)中。跨膜結構域通常插入該蛋白質之C末端，剛好在終止密碼子之前或緊密鄰近終止密碼子。眾多的跨膜結構域可用於此項技術中(參見例如WO99/03885)。 在一些實施例中，本文所描述之免疫檢查點調節子進一步包含在免疫檢查點調節子N末端融合之信號肽。在一些實施例中，免疫檢查點調節子包含在VH結構域之N末端融合之信號肽，諸如SEQ ID NO: 21。在一些實施例中，免疫檢查點調節子包含在VL結構域之N末端融合之信號肽，諸如SEQ ID NO: 23。在一些實施例中，信號肽可以由核酸SEQ ID NO: 22或SEQ ID NO:24編碼。在一些實施例中，信號肽包含胺基酸序列SEQ ID NO:28或SEQ ID NO: 32。 作為額外實例，亦可以添加肽標籤(通常為能夠由可用抗血清或化合物識別的短肽序列)用於後續經編碼基因產物之表現、運輸或純化。在本發明之情形下可以使用多種標籤肽，包括(但不限於)PK標籤、FLAG八肽、MYC標籤、HIS標籤(通常為4至10個組胺酸殘基之鏈段)及e標籤(US 6,686,152)。當採用若干標籤時，該等標籤肽可以獨立地安置於蛋白質之N末端，或替代地安置於其C末端，或替代地安置於內部或該等位置中之任一個處。標籤肽可以藉由免疫偵測分析法，使用抗標籤抗體偵測。作為另一實例，可以改變糖基化以便增加經編碼基因產物之生物活性(例如增加)。此類修飾可以例如藉由使糖基化位點內之一或多個殘基突變來實現。改變糖基化模式可以增加抗體之ADCC能力及/或其對其標靶之親和力。 所描述的引入宿主中之核酸分子或載體可以整合至宿主基因組中或其可以保持在染色體外。 宿主可以為任何真核細胞或原核細胞。特定言之，設想所述宿主可以為哺乳動物細胞。宿主細胞包括(但不限於)CV-1、BS-C-1、HuTK-143B、BHK-21、CEF、CHO細胞、COS細胞、骨髓瘤細胞株如SP2/0或NS/0細胞。 雙特異性接合分子 在一些實施例中，本發明之表現免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之溶瘤病毒(諸如溶瘤VV)進一步包含編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。 本發明揭示一種溶瘤病毒(諸如溶瘤VV)，其包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一核酸，進一步包含編碼接合分子之第二核酸，該接合分子包含特異性識別一或多種腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，接合分子係雙特異性分子。在一些實施例中，細胞表面分子係T淋巴細胞表面標記物(諸如CD3)，因此接合分子係T細胞接合子(TE)。在一些實施例中，本發明揭示一種溶瘤病毒(諸如溶瘤VV)，其表現免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)，進一步表現雙特異性T細胞接合子(BiTE)。 一旦該接合分子之第二抗原結合結構域結合至效應細胞，該第二抗原結合結構域即可活化效應細胞。在一些實施例中，當接合分子之第二抗原結合結構域結合至免疫細胞上之細胞表面分子，且第一抗原結合結構域結合至腫瘤細胞抗原時，免疫細胞殺滅腫瘤細胞。 腫瘤抗原 在一些實施例中，接合分子之第一抗原結合結構域特異性識別的抗原係腫瘤相關抗原(TAA)或腫瘤特異性抗原(TSA)。在某一實施例中，TAA或TSA係在癌細胞上表現。在一些實施例中，TAA或TSA係在血液癌細胞上表現。在一些實施例中，TAA或TSA係在實體腫瘤之細胞上表現。作為非限制性實例，某些形式之實體腫瘤癌症包括神經膠母細胞瘤、非小細胞肺癌、除非小細胞肺癌外之肺癌、乳癌、卵巢癌、前列腺癌、胰臟癌、肝癌、結腸直腸癌、胃癌、脾臟癌、皮膚癌(諸如黑素瘤)、除神經膠母細胞瘤外之腦癌、腎癌、甲狀腺癌、頭頸腫瘤、膀胱癌、食道癌或類似癌症。在一些實施例中，TAA或TSA係以下一或多種，例如接合子上之scFv對以下一或多種具有特異性：EphA2、HER2、GD2、GPC3、5T4、8H9、αv β6 整合素、B7-H3、B7-H6、CAIX、CA9、CD19、CD20、CD22、κ輕鏈、CD30、CD33、CD38、CD44、CD44v6、CD44v7/8、CD70、CD123、CD138、CD171、CEA、CSPG4、EGFR、EGFRvIII、EGP2、EGP40、EPCAM、ERBB3、ERBB4、ErbB3/4、FAP、FAR、FBP、胎兒AchR、葉酸受體a、GD2、GD3、HLA-AI MAGE A1、HLA-A2、IL11Ra、IL13Ra2、KDR、λ(Lambda)、Lewis-Y、MCSP、間皮素、Muc1、Muc16、NCAM、NKG2D配體、NY-ESO-1、PRAME、PSCA、PSC1、PSMA、ROR1、SURVIVIN、TAG72、TEM1、TEM8、VEGFR2、癌胚抗原、HMW-MAA、VEGF受體，且其他例示性抗原係存在於腫瘤細胞外基質中之抗原，諸如纖維結合蛋白、肌腱蛋白之癌胚變異體或腫瘤壞死區。 在一些實施例中，接合分子之第一抗原結合結構域特異性識別之抗原係選自由以下組成之群：EpCAM、FAP、EphA2、HER2、GD2、EGFR、VEGFR2及GPC3。在一些實施例中，腫瘤抗原係EpCAM、FAP、EGFR或GPC3。 在一些實施例中，接合分子之第一抗原結合結構域特異性識別之抗原係上皮細胞黏附分子(EpCAM、CD326)，又稱為17-1A、ESA、AUA1、EGP40等，其係由314個胺基酸構成的一種40 kDa跨膜醣蛋白。EpCAM在各種類型之上皮細胞及大多數類型之人類惡性病中特異性表現。舉例而言，EpCAM在結腸癌、肺癌、前列腺癌、肝癌、胰臟癌、乳癌及卵巢癌中大量表現。 在一些實施例中，接合分子之第一抗原結合結構域特異性識別之抗原係纖維母細胞活化蛋白(FAP)。纖維母細胞係分泌富含膠原蛋白及其他大分子之細胞外基質的結締組織細胞。腫瘤基質(亦即，負載腫瘤之組織)中之纖維母細胞合成FAP，一種充當絲胺酸蛋白酶之II型跨膜蛋白。FAP在與結腸癌、乳癌及肺癌有關的超過90%基質纖維母細胞中選擇性過度表現。FAP亦在一些腫瘤細胞，諸如人類惡性神經膠質瘤細胞株U87及鼠類路易斯肺癌(lewis lung cancer)細胞株LL2中表現(Kraman等人,Science 330: 827-830 (2010))。據報導，FAP之過度表現使得促進腫瘤生長且增加轉移可能，而用抗FAP抗體治療抑制腫瘤生長。 在一些實施例中，接合分子之第一抗原結合結構域特異性識別之抗原係表皮生長因子受體(EGFR)。EGFR係由包括EGFR(ErbB-1)、Her2/neu(ErbB-2)、Her3(ErbB-3)及Her4(ErbB-4)的密切相關之受體構成之ErbB家族之成員。EGFR之活化引起受體酪胺酸激酶活化及一系列下游信號傳導事件，該等信號傳導事件介導細胞增殖、活動性、黏附、侵襲及對化學療法之抗性以及細胞凋亡抑制，該等過程對於癌細胞不斷增殖及存活至關重要。EGFR之表現與包括胃、結腸、膀胱、乳房、前列腺、子宮內膜、腎及腦(例如神經膠質瘤)之多種腫瘤類型之不良預後相關。 EphA2稱為EPH受體A2(艾普瑞林型(ephrin type)-A受體2；EPHA2；ARCC2；CTPA；CTPP1；或ECK)，其係人體中由蛋白質酪胺酸激酶家族之艾普瑞林受體亞家族中的EPHA2基因所編碼之蛋白質。此亞家族中之受體一般包括單一激酶結構域及包含富含Cys結構域及2個纖維結合蛋白III型重複序列之胞外區；本發明之抗體之實施例可以靶向該等結構域中之任一個。艾普瑞林受體由於其各別細胞外結構域序列之相似性以及其結合艾普瑞林-A配體及艾普瑞林-B配體之親和力而分成兩組，且EphA2編碼結合艾普瑞林-A配體之蛋白質。例示性人類EphA2核酸序列係在GenBank®登錄號NM_004431中，且例示性人類EphA2多肽序列係在GenBank®登錄號NP_004422中，該兩個序列均整體併入本文中。 HER2稱為人類表皮生長因子受體2(Neu、ErbB-2、CD340或pi 85)，其係人體中由表皮生長因子受體(EFR/ErbB)家族中之ERBB2基因編碼的一種蛋白質。HER2含有細胞外配體結合結構域、跨膜結構域及可以與多種信號傳導分子相互作用之細胞內結構域。 GD2係在神經外胚層來源之腫瘤，包括人類神經母細胞瘤及黑素瘤上表現的雙唾液酸神經節苷脂，且在正常組織上，主要在人類小腦及周圍神經上之表現高度受限。GD2存在且集中於細胞表面上，且腦醯胺部分之兩條烴鏈包埋於質膜中且寡醣位於細胞外表面上，其中其存在細胞外分子或鄰近細胞表面之識別點。 在一些實施例中，接合分子之第一抗原結合結構域特異性識別之抗原係磷脂醯肌醇蛋白聚糖-3(GPC3)。磷脂醯肌醇蛋白聚糖-3(GPC3)係在贅生性肝細胞中以較高頻率再表現之癌胚抗原。GPC3基因編碼70 kDa之前驅核心蛋白，可以經弗林蛋白酶(furin)裂解以產生40 kDa之胺基(N)末端蛋白質及30 kDa之膜結合羧基(C)末端蛋白質。該C末端藉由糖基化磷脂醯肌醇(GPI)錨連接至細胞膜。 血管內皮生長因子受體2(VEGFR2、KDR3)係一種VEGF受體，其係血管生成之最有效之特異性陽性調控因子之一。VEGFR2在腫瘤相關內皮細胞中大量表現且促進腫瘤生長、侵襲及轉移(Dias等人,J Clin Invest. 106(4):511-521, 2000；Santos等人,Blood 103(10):3883-3889, 2004；St. Croix等人,Science 289:1197-1202, 2000)。此外，VEGFR2亦在若干腫瘤細胞，包括：B細胞淋巴瘤及白血病、多發性骨髓瘤、尿道上皮膀胱癌、乳癌及肺癌等之表面上表現(El-Obeid等人,Leuk Res. 28(2): 133-137, 2004；Kumar等人,Leukemia 17(10):2025-2031, 2003；Gakiopoulou-Givalou等人,Histopathology 43(3):272-279, 2003；Kranz等人,Int J Cancer 84(3):293-298, 1999；Decaussin等人,J Pathol. 188(4):369-377, 1999)。相對於正常血管內皮細胞，在腫瘤細胞上相對較高之表現量表明，VEGFR2係適合的腫瘤療法標靶。 因此，在一些實施例中，雙特異性分子包含特異性識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。 在一些實施例中，雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。 在一些實施例中，雙特異性分子包含特異性識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。 在一些實施例中，雙特異性分子包含特異性識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。 效應細胞表面分子 例示性效應細胞包括(但不限於)T淋巴細胞、B淋巴細胞、自然殺手(NK)細胞、樹突狀細胞(DC)、巨噬細胞、單核細胞、嗜中性球、NKT細胞或類似細胞。在一些實施例中，效應細胞係T淋巴細胞。在一些實施例中，效應細胞係細胞毒性T淋巴細胞。在一些實施例中，效應細胞係同種異體的。在一些實施例中，效應細胞係自體的。 本發明之效應細胞上的細胞表面分子係在特定細胞類型或有限數量之細胞類型之外部細胞壁或質膜上發現的分子。細胞表面分子之實例包括(但不限於)膜蛋白質，諸如受體、轉運蛋白、離子通道、質子泵及G蛋白偶聯受體；細胞外基質分子，諸如黏附分子(例如整合素、鈣黏素、選擇素或NCAMS)；參見例如美國專利第7,556,928號，其以全文引用之方式併入本文中。效應細胞上之細胞表面分子包括(但不限於)CD3、CD4、CD5、CD8、CD16、CD27、CD28、CD40、CD64、CD89、CD134、CD137、CD278、NKp46、NKp30、NKG2D及不變TCR。 接合分子之細胞表面分子結合結構域可以活化免疫細胞。熟練技術人員認識到，免疫細胞具有不同細胞表面分子。舉例而言，CD3係T細胞上之細胞表面分子，而CD16、NKG2D或NKp30係NK細胞上之細胞表面分子，且CD3或不變TCR係NKT細胞上之細胞表面分子。因此，活化T細胞之接合分子可以具有與活化NK細胞之接合分子不同的細胞表面分子結合結構域。在一些實施例中，例如在免疫細胞係T細胞之一些實施例中，活化分子係以下一或多種：CD3，例如CD3γ、CD3δ或CD3ε；或CD27、CD28、CD40、CD134、CD137及CD278。在其他一些實施例中，例如在免疫細胞係NK細胞之一些實施例中，細胞表面分子係CD16、NKG2D或NKp30，或在免疫細胞係NKT細胞之一些實施例中，細胞表面分子係CD3或不變TCR。 CD3包含三條不同的多肽鏈(ε、δ及γ鏈)，其係由T細胞表現之抗原。該三條CD3多肽鏈與T細胞受體(TCR)及ζ鏈締合形成TCR複合物，該TCR複合物具有活化T細胞中之信號傳導級聯之功能。當前，許多治療策略靶向TCR信號轉導以使用抗人類CD3單株抗體治療疾病。CD3特異性抗體OKT3係首個經批准用於人類治療用途之單株抗體，且在臨床上用作免疫調節劑用於治療同種異體移植排斥反應。 在一些實施例中，第二抗原結合結構域特異性結合至T淋巴細胞上之CD3。在一些實施例中，人類CD3特異性結構域之VH及VL區係來源於CD3特異性抗體，諸如X35-3、VIT3、BMA030 (BW264/56)、CLB-T3/3、CRIS7、YTH12.5、Fl 11-409、CLB-T3.4.2、TR-66、WT32、SPv-T3b、11D8、XIII-141、XIII-46、XIII-87、12F6、T3/RW2-8C8、T3/RW2-4B6、OKT3D、M-T301、SMC2、WT31、F101.01或布林莫單抗(Blinatumomab)(Blincyto® ，即CD19-CD3雙特異性抗體)。該等CD3特異性抗體為此項技術中熟知且尤其描述於Tunnacliffe等人, Int Immunol. 1(5):546-50 (1989)中。在一些實施例中，VH及VL區係來源於能夠特異性識別人類CD3-ε鏈或人類CD3-ζ鏈之抗體/抗體衍生物及類似物。在一些實施例中，人類CD3特異性結構域之VH及VL區係來源於布林莫單抗(Blincyto®，即CD19-CD3雙特異性抗體)。在一些實施例中，第二抗原結合結構域特異性結合至人類CD3-ε鏈或人類CD3-ζ鏈內之抗原決定基。 熟練技術人員應認識到，TCR複合物係具有可變TCR α及β鏈及三個二聚合信號傳導模組CD3δ/ε、CD3γ/ε及CD3ζ/ζ或ζ/η之八聚體複合物。儘管在一些情況下，本文所描述之接合分子以一個scFv靶向CD3ε，但本發明中亦涵蓋以一個特異性scFv靶向其他CD3分子，尤其CD3ζ，或TCR α及β鏈。在一些實施例中，本發明中涵蓋不作為TCR複合物之一部分的靶向分子(例如CD27、CD28、CD40、CD134、CD137及CD278)。 因此，在一些實施例中，雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3之第二抗原結合結構域(諸如scFv)。 在一些實施例中，雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別NK細胞上之CD16之第二抗原結合結構域(諸如scFv)。 在一些實施例中，雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別NK細胞上之NKG2D之第二抗原結合結構域(諸如scFv)。 在一些實施例中，雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別NK細胞上之NKp30之第二抗原結合結構域(諸如scFv)。 接合分子形式 本文所描述之接合分子可以呈此項技術中已知之任何形式。此類接合分子一般包含腫瘤抗原結合結構域及效應細胞表面分子結合結構域。接合分子之抗原結合結構域可以經設計以便結合至標靶細胞上存在之一或多種腫瘤抗原，而接合分子之效應細胞表面分子結合結構域可以經設計以便結合至效應細胞，例如T淋巴細胞上存在之一或多個細胞表面分子。一旦接合分子之效應細胞表面分子結合結構域結合效應細胞，效應細胞表面分子結合結構域即可活化效應細胞。在一些實施例中，當接合子之效應細胞表面分子結合結構域結合至效應細胞上之細胞表面分子，且腫瘤抗原結合結構域結合至腫瘤細胞上之腫瘤抗原時，效應細胞殺滅腫瘤細胞。 在一些實施例中，接合分子係由兩部分組成的(例如包含可視情況藉由連接子接合的腫瘤抗原結合結構域及效應細胞表面分子結合結構域)，或可以係由三部分或多個部分組成的(例如包含一或多個腫瘤抗原結合結構域及/或一或多個效應細胞表面分子結合結構域，或其他結構域，包括一或多個共刺激結構域及/或一或多個二聚合、三聚合或多聚合結構域)。 在一些實施例中，接合子係雙特異性的(例如包含可視情況藉由連接子接合的腫瘤抗原結合結構域及效應細胞表面分子結合結構域，其中腫瘤抗原與細胞表面分子不同)。接合分子亦可為多特異性的(例如包含可視情況藉由連接子接合的一個腫瘤抗原結合結構域及兩個效應細胞表面分子結合結構域，其中該腫瘤抗原與該兩個細胞表面分子皆不同)。 接合分子可以呈此項技術中已知之任何形式(參見例如，Weidle等人, Cancer Genomics Proteomics, 10(1):1-18, 2013；Geering and Fussenegger, Trends Biotechnol., 33(2):65-79, 2015；Stamova等人, Antibodies, 1(2):172-198, 2012)。接合子可以為包含Fc區之「IgG源性分子」之形式種類。舉例而言，接合分子可以呈(但不限於)以下形式：共用LC(輕鏈)、DAF(雙重作用Fab，其包含具有雙重特異性之進化之Fv)、互換Mab、IgG-dsscFv2(二硫化物穩定之scFv2)、DVD(雙重可變結構域)、IgG-dsFv(二硫化物穩定之Fv)、經加工IgG-dsFv、IgG-scFab(單鏈Fab)、scFab-dsscFv或Fv2-Fc。可以使用孔中節(Knobs-into-holes)技術在例如共用LC、互換Mab、IgG-dsF、IgG-scFab或Fv2-Fc中進行不同H鏈之異二聚合。接合子亦可為「低Fc雙特異性」形式種類，該種類通常包括含經由連接子融合在一起的具有不同特異性之Fab的個別scFv。舉例而言，接合子可以呈(但不限於)以下形式：Fab-scFv2、Fab-scFv、scFv-scFv scFv(諸如BiTE® )、雙功能抗體、scBsDb(單鏈雙特異性雙功能抗體)、DART(雙重親和力再靶向分子)、TandAb(四價串聯抗體)、scBsTaFv(單鏈雙特異性串聯可變結構域)、DNL-F(ab)3 (對接鎖定三價Fab)、scFv-HSA-scFv(scFv-人類血清白蛋白-scFv)或bssdAb(雙特異性單結構域抗體)。二價或三價Fab-Fv或Fab-Fv2形式係藉由VH-CH1及/或L鏈與scFv融合而產生。scFv-scFv分子(諸如BiTE® )係藉由融合具有不同特異性之scFv而產生。連接肽長度可以經調節以使得VH與VL正確地配對，諸如在雙功能抗體、DART及TandAb中。該等分子可以進一步藉由鏈間二硫鍵(例如在DART中，或在包含scFv之抗體之VH與VL之間)穩定。接合分子亦可呈抗體模擬物之形式種類，該等抗體模擬物係包含使人聯想抗體之抗原結合結構域的經工程改造之小蛋白質(Geering及ussenegger, Trends Biotechnol., 33(2):65-79, 2015)。該等分子係來源於現有的人類支架蛋白質且包含單一多肽。呈抗體模擬物形式種類之例示性接合子可以為經設計錨蛋白重複蛋白(DARPin；包含藉由N末端及C末端帽結構域側接的3-5個完全合成之錨蛋白重複序列)、親合力多聚體(avimer；包含多個A結構域之高親和力蛋白質，每個結構域對標靶具有較低親和力)或抗運載蛋白(基於脂質運載蛋白之支架，具有四個可進入環，各自之序列可以隨機化)。根據本發明採用之接合分子可以為前述接合子形式中之任一種的化學修飾之衍生物，或其可以包含配體、肽或其組合。根據本發明採用之接合分子可以使用此項技術中已知之習知技術進一步修飾，例如藉由使用單獨或組合形式的胺基酸缺失、插入、取代、添加及/或重組，及/或此項技術中已知之任何其他修飾(例如轉譯後修飾及化學修飾，諸如糖基化及磷酸化)。有關此類修飾之化學/生物化學或分子生物學方法係此項技術中已知的且尤其描述於實驗室手冊中(參見Sambrook等人; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 第2版1989及第3版2001；Gerhardt等人; Methods for General and Molecular Bacteriology; ASM Press, 1994；Lefkovits; Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press, 1997；Golemis; Protein-Protein Interactions: A Molecular Cloning Manual; Cold Spring Harbor Laboratory Press, 2002)。 在一些實施例中，本發明之雙特異性接合分子係雙特異性單鏈Fv(scFv)。scFv一般含有藉由連接肽連接之VH及VL結構域。分泌型接合子係由來自細胞之信號肽(允許分泌)隨後藉由連接肽(Lx、Ly、Lz)連接之2個scFv構成。連接子之長度及序列可以足以確保第一及第二結構域各自可以彼此獨立地保持其不同的結合特異性。雙特異性單鏈分子為此項技術中已知且描述於如下文獻中：WO 99/54440；Mack, J. Immunol. (1997), 158, 3965-3970；Mack, PNAS, (1995), 92, 7021-7025；Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197；Loffler, Blood, (2000), 95, 6, 2098-2103；及Bruhl, J. Immunol., (2001), 166, 2420-2426。 在一些實施例中，本發明之例示性分子形式提供一種溶瘤病毒(諸如溶瘤VV)，其包括編碼含信號肽隨後兩個scFv之多肽的核酸，其中該第一scFv特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)，且第二scFv特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)。scFv各自包含一個VH 及一個VL 區。雙特異性scFv可以為串聯雙-scFv或雙功能抗體。雙特異性scFv可以佈置成不同形式：VH α-Lx-VL α-Ly-VH β-Lz-VL β、VL α-Lx-VH α-Ly-VH β-Lz-VL β、VL α-Lx-VH α-Ly-VL β-Lz-VH β、VH α-Lx-VL α-Ly-VL β-Lz-VH β、VH α-Lx-VL β-Ly-VH β-Lz-VL α、VL α-Lx-VL β-Ly-VH β-Lz-VH α、VH α-Lx-VH β-Ly-VL β-Lz-VL α、VL α-Lx-VH β-Ly-VL β-Lz-VH α、VH β-Lx-VL α-Ly-VH α-Lz-VL β、VL β-Lx-VL α-Ly-VH α-Lz-VH β、VH β-Lx-VH α-Ly-VL α-Lz-VL β、VL β-Lx-VH α-Ly-VL α-Lz-VH β。因此，具有以上可能之佈置的雙特異性scFv係該雙特異性單鏈接合分子之特定實施例。連接子Lx、Ly及Lz可以相同或不同。 連接子可以為任何長度之肽連接子。在一些實施例中，抗原結合結構域(諸如scFv)之VH與VL之間的肽連接子係1個胺基酸至20個胺基酸長、2個胺基酸至19個胺基酸長、3個胺基酸至18個胺基酸長、4個胺基酸至17個胺基酸長、5個胺基酸至17個胺基酸長、6個胺基酸至17個胺基酸長、7個胺基酸至18個胺基酸長、8個胺基酸至17個胺基酸長、9個胺基酸至17個胺基酸長、10個胺基酸至17個胺基酸長、11個胺基酸至16個胺基酸長、12個胺基酸至17個胺基酸長、13個胺基酸至16個胺基酸長、14個胺基酸至16個胺基酸長或14個胺基酸至15個胺基酸長。在一些實施例中，抗原結合結構域(諸如scFv)之VH與VL之間的肽連接子係1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20個中之任一種胺基酸長度。在一些實施例中，抗原結合結構域(諸如scFv)之VH與VL之間的肽連接子係14或15個胺基酸長。在一些實施例中，第一與第二抗原結合結構域(諸如scFv)之間之肽連接子係1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20個中之任一種胺基酸長度。在一些實施例中，第一與第二抗原結合結構域(諸如scFv)之間之肽連接子係5個胺基酸長。 此類肽連接子之基本技術特徵在於，該肽連接子不包含任何聚合活性。包含不存在二級結構促進作用之肽連接子之特徵係此項技術中已知的且描述於例如Dall'Acqua等人(Biochem. (1998) 37, 9266-9273)、Cheadle等人(Mol Immunol (1992) 29, 21-30)以及Raag及Whitlow (FASEB (1995) 9(1), 73-80)中。在「肽連接子」之情形下，尤佳胺基酸為Gly。此外，不促進任何二級結構之肽連接子係較佳的。結構域彼此之連接可以藉由例如基因工程改造提供。製備融合及操作性連接之雙特異性單鏈構築體及在哺乳動物細胞或細菌中表現其之方法係此項技術中熟知的(例如 WO 99/54440；Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. 1989及1994，或Sambrook等人, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001)。 肽連接子可以為蛋白酶，尤其基質金屬蛋白酶(MMP)不可裂解之穩定連接子。 連接子亦可為可撓性連接子。例示性可撓性連接子包括甘胺酸聚合物(G)n 、甘胺酸-絲胺酸聚合物(包括例如(GS)n 、(GSGGS)n 及(GGGS)n ，其中n係至少一之整數)、甘胺酸-丙胺酸聚合物、丙胺酸-絲胺酸聚合物及此項技術中已知之其他可撓性連接子。甘胺酸及甘胺酸-絲胺酸聚合物係相對非結構化的，且因此能夠充當各組分之間之中性繫鏈。甘胺酸甚至比丙胺酸更易進入φ-ψ間隙，且所受限制比具有較長側鏈之殘基要小得多(參見Scheraga, Rev. Computational Chem. 11 173-142 (1992))。普通熟練技術人員應認識到，雙特異性抗體分子之設計可以包括具有完全或部分可撓性之連接子，由此該連接子可以包括可撓性連接子部分以及引起較低可撓性結構之一或多個部分，由此提供所需之雙特異性抗體分子結構。 在一些實施例中，第一抗原結合結構域(諸如scFv)之VH及VL結構域係藉由長度足以使各結構域以允許結合至腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之方式摺疊的連接子連接在一起。關於此實施例，此類連接子可以包含例如胺基酸序列GGGGSGGGGSGGGGS (SEQ ID NO: 33)，或GGGGSGGGGSGGSA (SEQ ID NO: 34)。在一些實施例中，第二抗原結合結構域(諸如scFv)之VH結構域及VL結構域係藉由長度足以使各結構域以允許結合至效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之方式摺疊的連接子連接在一起。關於此實施例，此類連接子可以包含例如胺基酸序列GGGGSGGGGSGGGGS (SEQ ID NO: 33)。在一些實施例中，第一與第二抗原結合結構域(諸如scFv)係藉由長度足以使各結構域以允許結合至腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)及效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)兩者之方式摺疊的連接子連接在一起。關於此實施例，此類連接子可以包含例如胺基酸序列GGGGS (SEQ ID NO: 35)。 在一些實施例中，接合分子另外包含一或多個其他結構域，例如細胞介素、共刺激結構域、抑制T細胞活化之負調控分子之結構域，或其組合中之一或多種。在一些實施例中，細胞介素係IL-15、IL-2及/或IL-7。在一些實施例中，共刺激結構域係CD27、CD80、CD83、CD86、CD134或CD137。在一些實施例中，抑制T細胞活化之負調控分子之結構域係PD-1、PD-L1、CTLA4或B7-H4。 因此，在一些實施例中，本文所描述之接合分子包含識別EpCAM之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3之第二抗原結合結構域(諸如scFv)。在一些實施例中，第一抗原結合結構域係scFv。在一些實施例中，第二抗原結合結構域係scFv。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一與第二抗原結合結構域係藉由連接子連接。在一些實施例中，第一抗原結合結構域係在雙特異性分子之N末端。在一些實施例中，第一抗原結合結構域係在雙特異性分子之C末端。 在一些實施例中，接合分子包含識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3之第二抗原結合結構域(諸如scFv)。在一些實施例中，第一抗原結合結構域係scFv。在一些實施例中，第二抗原結合結構域係scFv。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一與第二抗原結合結構域係藉由連接子連接。在一些實施例中，第一抗原結合結構域係在雙特異性分子之N末端。在一些實施例中，第一抗原結合結構域係在雙特異性分子之C末端。 在一些實施例中，接合分子包含識別EGFR之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3之第二抗原結合結構域(諸如scFv)。在一些實施例中，第一抗原結合結構域係scFv。在一些實施例中，第二抗原結合結構域係scFv。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一與第二抗原結合結構域係藉由連接子連接。在一些實施例中，第一抗原結合結構域係在雙特異性分子之N末端。在一些實施例中，第一抗原結合結構域係在雙特異性分子之C末端。 在一些實施例中，接合分子包含識別GPC3之第一抗原結合結構域(諸如scFv)及特異性識別T淋巴細胞上之CD3之第二抗原結合結構域(諸如scFv)。在一些實施例中，第一抗原結合結構域係scFv。在一些實施例中，第二抗原結合結構域係scFv。在一些實施例中，第一及第二抗原結合結構域均為scFv。在一些實施例中，第一與第二抗原結合結構域係藉由連接子連接。在一些實施例中，第一抗原結合結構域係在雙特異性分子之N末端。在一些實施例中，第一抗原結合結構域係在雙特異性分子之C末端。 III. 治療癌症之方法 醫藥組合物 本申請案進一步提供包含有效量的本文所描述之溶瘤牛痘病毒中之任一種及視情況之醫藥學上可接受之載劑的醫藥組合物，該溶瘤牛痘病毒包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之核酸，其中該編碼免疫檢查點調節子之核酸係可操作地連接至晚期啟動子(諸如F17R)。本申請案亦提供包含有效量之溶瘤病毒(諸如溶瘤VV)中之任一種及視情況之醫藥學上可接受之載劑的醫藥組合物，該溶瘤病毒包含編碼免疫檢查點調節子(諸如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一核酸，及編碼本文所描述之雙特異性分子之第二核酸。 在一些實施例中，本申請案亦提供一種醫藥組合物，其包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種)之第一核酸的第一OV(例如溶瘤VV)；及包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；以及視情況醫藥學上可接受之載劑。在一些實施例中，提供一種醫藥組合物，其包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種)之第一核酸的第一OV(例如溶瘤VV)；及包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第二核酸的第二OV(例如溶瘤VV)；以及視情況醫藥學上可接受之載劑。在一些實施例中，提供一種醫藥組合物，其包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種)之第一核酸的第一OV(例如溶瘤VV)；包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；及包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三核酸的第三OV(例如溶瘤VV)；以及視情況醫藥學上可接受之載劑。在一些實施例中，該溶瘤病毒係選自由以下組成之群：牛痘病毒(VV)、塞內加谷病毒(SVV)、腺病毒、單純疱疹病毒1(HSV1)、單純疱疹病毒2(HSV2)、黏液瘤病毒、呼腸孤病毒、脊髓灰白質炎病毒、水泡性口炎病毒(VSV)、麻疹病毒(MV)、慢病毒、反轉錄病毒、麻疹病毒、流感病毒、辛德比病毒及新城疫病毒(NDV)。在一些實施例中，該OV係溶瘤VV。在一些實施例中，該溶瘤VV係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉牛痘病毒(MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。在一些實施例中，該溶瘤VV係WR株。在一些實施例中，溶瘤病毒包含TK基因及VGF基因之雙重缺失。在一些實施例中，免疫檢查點調節子係刺激性免疫檢查點分子之活化劑(諸如CD27、CD28、CD40、CD122、CD137、OX40、GITR或ICOS之活化劑)。在一些實施例中，免疫檢查點調節子係免疫檢查點抑制劑。在一些實施例中，該免疫檢查點調節子係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。在一些實施例中，免疫檢查點調節子係PD-1之抑制劑。在一些實施例中，該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，該免疫檢查點調節子係結合至免疫檢查點分子之配體。在一些實施例中，免疫檢查點調節子係PD-L1/PD-L2、HHLA-2、CD47或CXCR4之配體。在一些實施例中，免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，腫瘤抗原係選自由以下組成之群：EpCAM、FAP、EphA2、HER2、GD2、EGFR、VEGFR2及磷脂醯肌醇蛋白聚糖-3(GPC3)。在一些實施例中，腫瘤抗原係EpCAM。在一些實施例中，腫瘤抗原係FAP。在一些實施例中，腫瘤抗原係EGFR。在一些實施例中，腫瘤抗原係GPC3。在一些實施例中，效應細胞係選自由以下組成之群：T淋巴細胞、B淋巴細胞、自然殺手(NK)細胞、樹突狀細胞(DC)、巨噬細胞、單核細胞、嗜中性球及NKT細胞。在一些實施例中，效應細胞係T淋巴細胞(諸如細胞毒性T淋巴細胞)。在一些實施例中，細胞表面分子係選自由以下組成之群：CD3、CD4、CD5、CD8、CD16、CD28、CD40、CD64、CD89、CD134、CD137、NKp46及NKG2D。在一些實施例中，細胞表面分子係CD3。在一些實施例中，第一及/或第二抗原結合結構域係單鏈可變片段(scFv)。在一些實施例中，第一與第二抗原結合結構域係藉由連接子連接。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之N末端。在一些實施例中，第一抗原結合結構域係在第二抗原結合結構域之C末端。在一些實施例中，編碼免疫檢查點調節子之第一核酸係可操作地連接至晚期啟動子。在一些實施例中，編碼雙特異性分子之第二核酸係可操作地連接至晚期啟動子。在一些實施例中，該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。在一些實施例中，該晚期啟動子係F17R。 如本文所使用，「載劑」包括在所使用之劑量及濃度下對暴露於其之細胞或哺乳動物無毒的醫藥學上可接受之載劑、賦形劑或穩定劑。通常生理學上可接受之載劑係水性pH緩衝溶液。適合醫藥學載劑之實例係此項技術中熟知的且包括磷酸鹽緩衝生理食鹽水溶液、水、乳液(諸如油/水乳液)、各種類型之潤濕劑、無菌溶液等。可接受之載劑、賦形劑或穩定劑在所用劑量及濃度下對接受者無毒。 包含此類載劑之醫藥組合物可以藉由熟知之習知方法調配。溶劑或稀釋劑較佳為等滲、低滲或較弱高滲性的且具有相對較低之離子強度。代表性實例包括無菌水、生理食鹽水(例如氯化鈉)、林格氏溶液(Ringer's solution)、葡萄糖、海藻糖或蔗糖溶液、漢克氏溶液(Hank's solution)及其他生理上平衡之水性鹽溶液(參見例如，最新版本的Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams&Wilkins)。 本發明之醫藥組合物可以局部(諸如腫瘤內)或全身投與。投與一般為非經腸投與，例如靜脈內投與；DNA亦可直接投與標靶部位，例如藉由基因槍遞送至內部或外部標靶部位，或藉由導管投與動脈中之部位。在一些實施例中，該醫藥組合物係皮下投與。在一些實施例中，該醫藥組合物係靜脈內投與。用於非經腸投藥之製劑包括無菌水性或非水性溶液、懸浮液及乳液。非水性溶劑之實例係丙二醇、聚乙二醇、植物油(諸如橄欖油)，及可注射有機酯(諸如油酸乙酯)。水性載劑包括水、醇/水性溶液、乳液或懸浮液，包括生理食鹽水及緩衝介質。非經腸媒劑包括氯化鈉溶液、林格氏右旋糖(Ringer's dextrose)、右旋糖及氯化鈉、乳酸化林格氏液或不揮發性油。靜脈內媒劑包括流體及營養補充劑、電解質補充劑(諸如基於林格氏右旋糖之電解質補充劑)及類似物。亦可存在防腐劑及其他添加劑，諸如抗菌劑、抗氧化劑、螯合劑及惰性氣體及類似物。此外，本發明之醫藥組合物可包含蛋白質載劑，較佳為人類來源之蛋白質載劑，如例如血清白蛋白或免疫球蛋白。此項技術中可利用呈冷凍、液體形式或凍乾形式之各種病毒調配物(例如WO98/02522、WO01/66137、WO03/053463、WO2007/056847及WO2008/114021等)。可以藉由涉及真空乾燥及冷凍乾燥之方法獲得固體(例如乾燥粉末或凍乾)組合物(參見例如WO2014/053571)。據設想，除免疫檢查點調節子(及/或雙特異性接合分子、細胞介素)或其編碼核酸分子或載體(如本發明中所描述)外，取決於該醫藥組合物之預定用途，本發明之醫藥組合物亦可包含其他生物活性劑。 在一些實施例中，本文所描述之醫藥組合物經適當地緩衝以用於人類用途。適合緩衝液包括(不限於)能夠維持生理或略呈鹼性之pH(例如約pH 7至約pH 9)的磷酸鹽緩衝液(例如PBS)、碳酸氫鹽緩衝液及/或Tris緩衝液。在一些實施例中，亦可藉由添加適合張力調節劑，諸如甘油使該醫藥組合物與血液等滲。 在一些實施例中，醫藥組合物係包含在單次用小瓶，諸如單次用密封小瓶中。在一些實施例中，該醫藥組合物係包含在多次用小瓶中。在一些實施例中，該醫藥組合物係作為整體包含在容器中。在一些實施例中，該醫藥組合物經低溫保藏。 溶瘤病毒之治療用途 本申請案之一態樣係關於治療癌症之方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包含溶瘤牛痘病毒及視情況醫藥學上可接受之載劑，該溶瘤牛痘病毒包含編碼免疫檢查點調節子(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)的核酸，其中該編碼免疫檢查點調節子之核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，該個體係癌症患者或者易患癌症或懷疑患有癌症之患者。本發明包括本文所涵蓋及/或由本文所涵蓋之方法產生的編碼免疫檢查點調節子之核酸序列、編碼免疫檢查點調節子之載體(諸如溶瘤病毒載體)。 本申請案之另一態樣係關於治療癌症之方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑，該溶瘤病毒包含編碼免疫檢查點調節子(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，該個體係癌症患者或者易患癌症或懷疑患有癌症之患者。本發明包括本文所涵蓋及/或由本文所涵蓋之方法產生的編碼免疫檢查點調節子之核酸序列、編碼免疫檢查點調節子之載體(諸如溶瘤病毒載體)。 本發明部分涵蓋病毒、蛋白質構築體(諸如雙特異性分子或免疫檢查點調節子)、核酸分子及/或載體(諸如溶瘤病毒載體)，其可以單獨投與或以與另一療法之任何組合且在至少一些態樣中與醫藥學上可接受之載劑或賦形劑一起投與。在一些實施例中，在投與病毒或蛋白質構築體之前，可以將其與此項技術中熟知的適合醫藥學載劑及賦形劑組合。根據本發明製備之組合物可以用於治療或延遲癌症之發作或惡化。 在一些實施例中，提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與有效量之醫藥組合物及視情況醫藥學上可接受之載劑，該醫藥組合物包括含編碼免疫檢查點調節子(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之核酸的溶瘤牛痘病毒，其中該編碼免疫檢查點調節子之核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，該治療癌症之方法具有以下生物活性中之一或多種：(1)殺滅癌細胞(包括旁觀者殺滅)；(2)抑制癌細胞增殖；(3)誘導腫瘤中之免疫反應；(4)減小腫瘤大小；(5)減輕患有癌症之個體之一或多種症狀；(6)抑制腫瘤轉移；(7)延長存活期；(8)延長癌症發展之時間；(9)預防、抑制癌症復發或降低癌症復發之可能性；(10)誘導周圍T細胞之再分佈；及(11)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔。在一些實施例中，由本文所描述之醫藥組合物介導的殺滅癌細胞之方法可以達成至少約40%、50%、60%、70%、80%、90%、95%或更高百分比中之任一者之腫瘤細胞死亡率。在一些實施例中，由本文所描述之醫藥組合物介導的殺滅癌細胞之方法可以達成至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或更高百分比中之任一者之旁觀者腫瘤細胞(未感染溶瘤VV之腫瘤細胞)死亡率。在一些實施例中，由本文所描述之醫藥組合物介導的減小腫瘤大小之方法可以使腫瘤大小減小至少約10%(包括例如至少約20%、30%、40%、60%、70%、80%、90%或100%中之任一者)。在一些實施例中，由本文所描述之醫藥組合物介導的抑制腫瘤轉移之方法可以抑制至少約10%(包括例如至少約20%、30%、40%、60%、70%、80%、90%或100%中之任一者)之轉移。在一些實施例中，由本文所描述之醫藥組合物介導的延長個體(諸如人類)之存活期的方法可以使個體之存活期延長至少1、2、3、4、5、6、7、8、9、10、11、12、18或24個月中之任一者。在一些實施例中，由本文所描述之醫藥組合物介導的延長癌症進展時間之方法可以使癌症進展時間延長至少1、2、3、4、5、6、7、8、9、10、11或12週中之任一者。 在一些實施例中，提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑，該溶瘤病毒包含編碼免疫檢查點調節子(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，該治療癌症之方法具有以下生物活性中之一或多種：(1)殺滅癌細胞(包括旁觀者殺滅)；(2)抑制癌細胞增殖；(3)誘導周圍T細胞之再分佈；(4)誘導腫瘤中之免疫反應；(5)減小腫瘤大小；(6)減輕患有癌症之個體之一或多種症狀；(7)抑制腫瘤轉移；(8)延長存活期；(9)延長癌症進展之時間；(10)預防、抑制癌症復發或降低癌症復發之可能性；(11)在腫瘤中誘導基質破壞或殺滅腫瘤基質細胞；(12)促進溶瘤病毒擴散通過腫瘤；(13)促進腫瘤中之T細胞浸潤；及(14)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔。在一些實施例中，由本文所描述之醫藥組合物介導的殺滅癌細胞之方法可以達成至少約40%、50%、60%、70%、80%、90%、95%或更高百分比中之任一者之腫瘤細胞死亡率。在一些實施例中，由本文所描述之醫藥組合物介導的殺滅癌細胞之方法可以達成至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或更高百分比中之任一者之旁觀者腫瘤細胞(未感染溶瘤VV之腫瘤細胞)死亡率。在一些實施例中，由本文所描述之醫藥組合物介導的減小腫瘤大小之方法可以使腫瘤大小減小至少約10%(包括例如至少約20%、30%、40%、60%、70%、80%、90%或100%中之任一者)。在一些實施例中，由本文所描述之醫藥組合物介導的抑制腫瘤轉移之方法可以抑制至少約10%(包括例如至少約20%、30%、40%、60%、70%、80%、90%或100%中之任一者)之轉移。在一些實施例中，由本文所描述之醫藥組合物介導的延長個體(諸如人類)之存活期的方法可以使個體之存活期延長至少1、2、3、4、5、6、7、8、9、10、11、12、18或24個月中之任一者。在一些實施例中，由本文所描述之醫藥組合物介導的延長癌症發展時間之方法可以使癌症發展時間延長至少1、2、3、4、5、6、7、8、9、10、11或12週中之任一者。 在一些實施例中，亦提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域與IgG4 Fc之融合物、TMIGD2細胞外結構域與IgG4 Fc之融合物，或SIRPα細胞外結構域及CXCL12片段與IgG4 Fc之融合物)之第一核酸的第一OV(例如溶瘤VV)；及包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；以及視情況醫藥學上可接受之載劑。在一些實施例中，亦提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域與IgG4 Fc之融合物、TMIGD2細胞外結構域與IgG4 Fc之融合物，或SIRPα細胞外結構域及CXCL12片段與IgG4 Fc之融合物)之第一核酸的第一OV(例如溶瘤VV)；及包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第二核酸的第二OV；以及視情況醫藥學上可接受之載劑。在一些實施例中，亦提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域與IgG4 Fc之融合物、TMIGD2細胞外結構域與IgG4 Fc之融合物，或SIRPα細胞外結構域及CXCL12片段與IgG4 Fc之融合物)之第一核酸的第一OV(例如溶瘤VV)；包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；及包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三核酸的第三OV(例如溶瘤VV)；以及視情況醫藥學上可接受之載劑。在一些實施例中，亦提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包含第一OV(例如溶瘤VV)及視情況醫藥學上可接受之載劑，該第一OV包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域與IgG4 Fc之融合物、TMIGD2細胞外結構域與IgG4 Fc之融合物，或SIRPα細胞外結構域及CXCL12片段與IgG4 Fc之融合物)之第一核酸；及有效量之第二醫藥組合物，該第二醫藥組合物包含第二OV(例如溶瘤VV)及視情況醫藥學上可接受之第二載劑，該第二OV包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包含第一OV(例如溶瘤VV)及視情況醫藥學上可接受之第一載劑，該第一OV包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域與IgG4 Fc之融合物、TMIGD2細胞外結構域與IgG4 Fc之融合物，或SIRPα細胞外結構域及CXCL12片段與IgG4 Fc之融合物)之第一核酸；及有效量之第二醫藥組合物，該第二醫藥組合物包含第二OV(例如溶瘤VV)及視情況醫藥學上可接受之第二載劑，該第二OV包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第二核酸。在一些實施例中，提供一種治療個體(諸如人類)之癌症之方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包含第一OV(例如溶瘤VV)及視情況醫藥學上可接受之第一載劑，該第一OV包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域與IgG4 Fc之融合物、TMIGD2細胞外結構域與IgG4 Fc之融合物，或SIRPα細胞外結構域及CXCL12片段與IgG4 Fc之融合物)之第一核酸；有效量之第二醫藥組合物，該第二醫藥組合物包含第二OV(例如溶瘤VV)及視情況醫藥學上可接受之載劑，該第二OV包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；以及有效量之第三醫藥組合物，該第三醫藥組合物包含第三OV(例如溶瘤VV)及視情況醫藥學上可接受之第三載劑，該第三OV包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三核酸。在一些實施例中，該治療癌症之方法具有以下生物活性中之一或多種：(1)殺滅癌細胞(包括旁觀者殺滅)；(2)抑制癌細胞增殖；(3)誘導周圍T細胞之再分佈；(4)誘導腫瘤中之免疫反應；(5)減小腫瘤大小；(6)減輕患有癌症之個體之一或多種症狀；(7)抑制腫瘤轉移；(8)延長存活期；(9)延長癌症進展之時間；(10)預防、抑制癌症復發或降低癌症復發之可能性；(11)在腫瘤中誘導基質破壞或殺滅腫瘤基質細胞(例如當表現FAP-CD3 T細胞接合子時)；(12)促進溶瘤病毒擴散通過腫瘤(例如當表現FAP-CD3 T細胞接合子時)；(13)促進腫瘤中之T細胞浸潤(例如當表現FAP-CD3 T細胞接合子時)；及(14)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔。在一些實施例中，由本文所描述之醫藥組合物介導的殺滅癌細胞之方法可以達成至少約40%、50%、60%、70%、80%、90%、95%或更高百分比中之任一者之腫瘤細胞死亡率。在一些實施例中，由本文所描述之醫藥組合物介導的殺滅癌細胞之方法可以達成至少約10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或更高百分比中之任一者之旁觀者腫瘤細胞(未感染OV之腫瘤細胞)死亡率。在一些實施例中，由本文所描述之醫藥組合物介導的減小腫瘤大小之方法可以使腫瘤大小減小至少約10%(包括例如至少約20%、30%、40%、60%、70%、80%、90%或100%中之任一者)。在一些實施例中，由本文所描述之醫藥組合物介導的抑制腫瘤轉移之方法可以抑制至少約10%(包括例如至少約20%、30%、40%、60%、70%、80%、90%或100%中之任一者)之轉移。在一些實施例中，由本文所描述之醫藥組合物介導的延長個體(諸如人類)之存活期的方法可以使個體之存活期延長至少1、2、3、4、5、6、7、8、9、10、11、12、18或24個月中之任一者。在一些實施例中，由本文所描述之醫藥組合物介導的延長癌症進展時間之方法可以使癌症進展時間延長至少1、2、3、4、5、6、7、8、9、10、11或12週中之任一者。 編碼免疫檢查點調節子之溶瘤牛痘病毒之功效，及表現本文所描述之免疫檢查點抑制劑及雙特異性接合分子之溶瘤病毒(諸如溶瘤VV)之功效可能因腫瘤基質作為病毒擴散及T細胞之物理屏障而受到限制。臨床前研究顯示，共靶向腫瘤細胞及腫瘤基質顯著增強免疫療法之抗腫瘤活性。在一些實施例中，本發明之溶瘤病毒(諸如溶瘤VV)包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原FAP之第一抗原結合結構域(例如scFv)及特異性識別效應細胞上之細胞表面分子(例如T淋巴細胞上之CD3)的第二抗原結合結構域(例如scFv)。在一些實施例中，共表現免疫檢查點調節子及雙特異性分子之溶瘤病毒係溶瘤VV。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-Fc融合蛋白。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。 因此，在一些實施例中，提供一種在個體(諸如人類)之腫瘤中誘導基質破壞或殺滅腫瘤基質細胞之方法，其包含向個體投與有效量的包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑之醫藥組合物，該溶瘤病毒包含編碼免疫檢查點調節子(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。在一些實施例中，提供一種在個體(諸如人類)之腫瘤中誘導基質破壞或殺滅腫瘤基質細胞的方法，其包含向個體投與：第一醫藥組合物，該第一醫藥組合物包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一OV，及視情況醫藥學上可接受之第一載劑；及第二醫藥組合物，該第二醫藥組合物包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之OV，及視情況醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)(及視情況第三醫藥組合物，其包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種)之第三OV，及視情況醫藥學上可接受之第三載劑)。在一些實施例中，提供一種在個體(諸如人類)之腫瘤中誘導基質破壞或殺滅腫瘤基質細胞的方法，其包含向個體投與醫藥組合物，該醫藥組合物包括：編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一OV；及編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二OV，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；及/或編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV；以及視情況醫藥學上可接受之載劑。在一些實施例中，該溶瘤病毒係牛痘病毒。在一些實施例中，該方法進一步在T細胞存在下誘導旁觀者基質破壞或殺滅旁觀者腫瘤基質細胞(未感染本文所描述之溶瘤病毒之細胞)。在一些實施例中，該方法進一步包含向個體投與有效量之另一醫藥組合物，該醫藥組合物包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑，該溶瘤病毒包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之核酸，該雙特異性分子包含特異性識別不為FAP之腫瘤抗原(例如EpCAM、EGFR或GPC3)之第一抗原結合結構域(例如scFv)及特異性識別效應細胞上之細胞表面分子(例如T淋巴細胞上之CD3)之第二抗原結合結構域(例如scFv)。在一些實施例中，該另一醫藥組合物中之溶瘤病毒係牛痘病毒。在一些實施例中，帶FAP-T細胞接合子之溶瘤病毒(FAP-TEA-OV)增強非FAP-TEA-OV，諸如EpCAM-TEA-OV(在下文中亦稱為EpCAM-CD3-OV)、EGFR-TEA-OV(在下文中亦稱為EGFR-CD3-OV)或GPC3-TEA-OV(在下文中亦稱為GPC3-CD3-OV)之抗腫瘤活性。在一些實施例中，由共表現FAP-TE(在下文中亦稱為FAP-CD3)之溶瘤病毒表現的免疫檢查點調節子進一步增強FAP-TE及/或非FAP-TE之抗腫瘤作用。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-IgG4 Fc。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。 在一些實施例中，亦提供一種在個體(諸如人類)中促進溶瘤病毒擴散通過腫瘤之方法，其包含向個體投與有效量的包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑之醫藥組合物，該溶瘤病毒包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。在一些實施例中，提供一種在個體(諸如人類)中促進溶瘤病毒擴散通過腫瘤之方法，其包含向個體投與：第一醫藥組合物，該第一醫藥組合物包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一OV，及視情況醫藥學上可接受之第一載劑；及第二醫藥組合物，該第二醫藥組合物包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之OV，及視情況醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)(及視情況第三醫藥組合物，其包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種)之第三OV，及視情況醫藥學上可接受之第三載劑)。在一些實施例中，提供一種在個體(諸如人類)中促進溶瘤病毒擴散通過腫瘤之方法，其包含向個體投與醫藥組合物，該醫藥組合物包括：編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一OV；及編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二OV，該雙特異性分子包含特異性識別FAP之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；及/或編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV；以及視情況醫藥學上可接受之載劑。在一些實施例中，該溶瘤病毒係牛痘病毒。在一些實施例中，該方法進一步包含向個體投與有效量之另一醫藥組合物，該醫藥組合物包含溶瘤病毒及視情況醫藥學上可接受之載劑，該溶瘤病毒包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之核酸，該雙特異性分子包含特異性識別不為FAP之腫瘤抗原(例如EpCAM、EGFR或GPC3)之第一抗原結合結構域(例如scFv)及特異性識別效應細胞上之細胞表面分子(例如T淋巴細胞上之CD3)之第二抗原結合結構域(例如scFv)。在一些實施例中，該另一醫藥組合物中之溶瘤病毒係牛痘病毒。在一些實施例中，帶有FAP-T細胞接合子之OV(FAP-TEA-OV)增進非FAP-TEA-OV，諸如EpCAM-TEA-OV、EGFR-TEA-OV或GPC3-TEA-OV之擴散。在一些實施例中，由共表現FAP-TE之溶瘤病毒表現的免疫檢查點調節子進一步增強FAP-TE及/或非FAP-TE之作用。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-IgG4 Fc。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。 在一些實施例中，提供一種增加個體(諸如人類)中之T細胞腫瘤浸潤之方法，其包含向個體投與有效量的包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑之醫藥組合物，該溶瘤病毒包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，OV進一步包含編碼細胞介素(諸如GM-CSF)之第三核酸。在一些實施例中，提供一種增加個體(諸如人類)中之T細胞腫瘤浸潤之方法，其包含向個體投與：第一醫藥組合物，該第一醫藥組合物包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一OV，及視情況醫藥學上可接受之第一載劑；及第二醫藥組合物，該第二醫藥組合物包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之OV，及視情況醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)(及視情況第三醫藥組合物，其包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種)之第三OV，及視情況醫藥學上可接受之第三載劑)。在一些實施例中，提供一種增加個體(諸如人類)中之T細胞腫瘤浸潤之方法，其包含向個體投與醫藥組合物，該醫藥組合物包括：編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如免疫檢查點抑制劑，例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白，或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之第一OV；及編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二OV，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；及/或編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV；以及視情況醫藥學上可接受之載劑。在一些實施例中，該溶瘤病毒係牛痘病毒。在一些實施例中，該方法在T細胞存在下誘導旁觀者腫瘤細胞(未感染本文所描述之溶瘤病毒之細胞)中之T細胞免疫反應。在一些實施例中，該方法進一步包含向個體投與有效量之另一醫藥組合物，該醫藥組合物包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑，該溶瘤病毒包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之核酸，該雙特異性分子包含特異性識別腫瘤抗原FAP之第一抗原結合結構域(例如scFv)及特異性識別效應細胞上之細胞表面分子(例如T淋巴細胞上之CD3)之第二抗原結合結構域(例如scFv)。在一些實施例中，該另一醫藥組合物中之溶瘤病毒係牛痘病毒。在一些實施例中，該另一醫藥組合物之FAP-TEA-VV(在下文中亦稱為FAP-CD3-VV)增進感染本文所描述之溶瘤病毒之腫瘤細胞及/或未感染本文所描述之溶瘤病毒之旁觀者腫瘤細胞中的T細胞浸潤。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-IgG4 Fc。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。 本文所描述之方法適於治療多種癌症，包括實體癌症及液體癌症兩種。該等方法適用於所有分期之癌症，包括早期癌症、非轉移癌、原發癌、晚期癌症、局部晚期癌症、轉移癌或緩解期癌症。本文所描述之方法可以在輔助治療或新輔助治療(亦即，該方法可以在初始療法/確定性療法之前進行)中用作第一療法、第二療法、第三療法或利用此項技術中已知的其他類型癌症療法之組合療法，該等其他類型癌症療法諸如化學療法、手術、激素療法、放射、基因療法、免疫療法(諸如T細胞療法)、骨髓移植、幹細胞移植、靶向療法、超低溫療法、超音波療法、光動力療法、射頻消融或類似療法。在一些實施例中，該方法用於治療預先經過治療之個體。在一些實施例中，該癌症係難以用先前療法治療的。在一些實施例中，該方法用於治療先前未經過治療之個體。 可以用本發明之方法治療的癌症之實例包括(但不限於)皮腺癌、AIDS相關癌症(例如AIDS相關淋巴瘤)、肛門癌、闌尾癌、星形細胞瘤(例如小腦及大腦星形細胞瘤)、基底細胞癌、膽管癌(例如肝外膽管癌)、膀胱癌、骨癌(骨肉瘤及惡性纖維組織細胞瘤)、腦腫瘤(例如神經膠質瘤、腦幹神經膠質瘤、小腦或大腦星形細胞瘤(例如毛細胞型星形細胞瘤、彌漫性星形細胞瘤、多形性(惡性)星形細胞瘤)、惡性神經膠質瘤、室管膜瘤、少突神經膠質瘤、腦膜瘤、顱咽管瘤、血管母細胞瘤、神經管母細胞瘤、幕上原始神經外胚層腫瘤、視覺路徑及下丘腦神經膠質瘤，及神經膠母細胞瘤)、乳癌、支氣管腺瘤/類癌、類癌(例如胃腸類癌)、原發灶不明癌、中樞神經系統淋巴瘤、子宮頸癌、結腸癌、結腸直腸癌、慢性骨髓增生性病症、子宮內膜癌(例如子宮癌)、室管膜瘤、食道癌、尤文氏肉瘤家族腫瘤(Ewing's family of tumor)、眼癌(例如眼內黑素瘤及視網膜母細胞瘤)、膽囊癌、胃(gastric/stomach)癌、胃腸類癌、胃腸基質腫瘤(GIST)、生殖細胞腫瘤(例如顱外、性腺外、卵巢腫瘤)、妊娠期滋養細胞腫瘤、頭頸癌、肝細胞(肝)癌(例如肝癌瘤及肝細胞腫瘤)、下咽癌、胰島細胞癌瘤(內分泌胰臟癌)、喉癌、喉癌、白血病、唇及口腔癌、口腔癌、肝癌、肺癌(例如小細胞肺癌、非小細胞肺癌、肺腺癌及肺鱗狀細胞癌)、淋巴贅瘤(例如淋巴瘤)、神經管母細胞瘤、黑素瘤、間皮瘤、轉移性鱗狀細胞頸癌、口腔癌、多發性內分泌瘤症候群、骨髓發育不良症候群、骨髓發育不良/骨髓增生性疾病、鼻腔及鼻竇癌、鼻咽癌、神經母細胞瘤、口咽癌、卵巢癌(例如卵巢上皮癌、卵巢生殖細胞腫瘤、卵巢低惡性腫瘤)、胰臟癌、副甲狀腺癌、陰莖癌、腹膜癌、咽癌、嗜鉻細胞瘤、松果體母細胞瘤及幕上原始神經外胚層腫瘤、垂體腫瘤、胸膜肺母細胞瘤、淋巴瘤、原發性中樞神經系統淋巴瘤(小神經膠質細胞瘤)、直腸癌、腎癌、腎盂及輸尿管癌(移行細胞癌)、橫紋肌肉瘤、唾液腺癌、皮膚癌(例如非黑素瘤(例如鱗狀細胞癌)、黑素瘤及梅克爾細胞癌(Merkel cell carcinoma))、小腸癌、鱗狀細胞癌、睪丸癌、咽喉癌、胸腺瘤及胸腺癌、甲狀腺癌、尿道癌、陰道癌、外陰癌、威爾姆氏腫瘤(Wilms' tumor)，及移植後淋巴增生性病症(PTLD)、與母斑病有關之異常血管增殖、水腫(諸如與腦腫瘤有關之水腫)及梅格斯氏症候群(Meigs' syndrome)。投與本文所描述之醫藥組合物可用於所有分期及類型之癌症，包括微小殘留病、早期實體腫瘤、晚期實體腫瘤及/或轉移性實體腫瘤。 在一些實施例中，該方法適於治療具有異常PD-1或PD-L1/PD-L2表現活性及/或信號傳導之癌症，包括(作為非限制性實例)血液癌症及/或實體腫瘤。可使用本發明抗體抑制生長之一些癌症包括通常對免疫療法起反應之癌症。適於治療之癌症之非限制性實例包括黑素瘤(例如轉移性惡性黑素瘤)、腎癌(例如透明細胞癌瘤)、前列腺癌(例如激素難治性前列腺腺癌)、乳癌、結腸癌及肺癌(例如非小細胞肺癌)。另外，本發明包括可使用本發明抗體抑制生長之難治性或復發性惡性病。可使用本發明抗體治療之其他癌症的實例包括骨癌；胰臟癌；皮膚癌；頭頸癌；皮膚或眼內惡性黑素瘤；子宮癌；卵巢癌；直腸癌；肛門區癌；胃癌；睾丸癌；子宮癌；輸卵管癌；子宮內膜癌；子宮頸癌；陰道癌；外陰癌；霍奇金氏病(Hodgkin's Disease)；非霍奇金氏淋巴瘤(non-Hodgkin's lymphoma)；食道癌；小腸癌；內分泌系統癌症；甲狀腺癌；副甲狀腺癌；腎上腺癌；軟組織肉瘤；尿道癌；陰莖癌；慢性或急性白血病，包括急性骨髓白血病、慢性骨髓白血病、急性淋巴母細胞性白血病、慢性淋巴細胞性白血病；兒童實體腫瘤；淋巴細胞性淋巴瘤；膀胱癌；腎癌或尿道癌；腎盂癌；中樞神經系統(CNS)贅瘤；原發性CNS淋巴瘤；腫瘤血管生成；脊軸腫瘤；腦幹神經膠質瘤；垂體腺瘤；卡波西氏肉瘤(Kaposi's sarcoma)；表皮樣癌；鱗狀細胞癌；T細胞淋巴瘤；環境誘發性癌症，包括由石棉誘發之癌症；及該等癌症之組合。本發明亦可用於治療轉移性癌症，尤其是表現PD-L1之轉移性癌症(Iwai等人 (2005)Int. Immunol. 17:133-144)。 因此，在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常PD-1或PD-L1/PD-L2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量的包含溶瘤牛痘病毒及視情況醫藥學上可接受之載劑之醫藥組合物，該溶瘤牛痘病毒包含編碼免疫檢查點調節子(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之核酸，其中該編碼免疫檢查點調節子之核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-Fc融合蛋白。在一些實施例中，該方法具有以下生物活性中之一或多種：(1)殺滅癌細胞；(2)抑制癌細胞增殖；(3)誘導腫瘤中之免疫反應；(4)減小腫瘤大小；(5)減輕患有癌症之個體之一或多種症狀；(6)抑制腫瘤轉移；(7)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔；(8)延長存活期；及/或(9)延長癌症進展之時間。在一些實施例中，該免疫療法反應性實體腫瘤係乳癌、結腸癌或肝癌。 在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常PD-1或PD-L1/PD-L2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量的包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑之醫藥組合物，該溶瘤病毒包含：編碼免疫檢查點調節子(諸如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸；及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常PD-1或PD-L1/PD-L2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸的第一OV(例如溶瘤VV)；及包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；以及視情況醫藥學上可接受之載劑。在一些實施例中，該醫藥組合物進一步包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV。在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常PD-1或PD-L1/PD-L2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸的第一OV(例如溶瘤VV)，及視情況醫藥學上可接受之第一載劑；及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，及視情況醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，該方法進一步包含向個體投與有效量之第三醫藥組合物，該第三醫藥組合物包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV，及視情況醫藥學上可接受之第三載劑。在一些實施例中，免疫檢查點調節子係抗PD-1抗體。在一些實施例中，免疫檢查點調節子係PD-1細胞外結構域-Fc融合蛋白。在一些實施例中，該方法具有以下生物活性中之一或多種：(1)殺滅癌細胞；(2)抑制癌細胞增殖；(3)誘導腫瘤中之免疫反應；(4)減小腫瘤大小；(5)減輕患有癌症之個體之一或多種症狀；(6)抑制腫瘤轉移；(7)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔；(8)延長存活期；(9)延長癌症進展之時間；(10)增加T細胞腫瘤浸潤；及/或(11)增強所提及之作用中之任一種。舉例而言，PD-1細胞外結構域-Fc融合蛋白之共表現可以增強在共表現之雙特異性接合分子存在下活化T細胞之腫瘤細胞殺滅作用，增加T細胞之細胞介素產生，或消除或降低由腫瘤細胞表現之抑制性免疫檢查點分子引起的腫瘤逃避作用。在一些實施例中，該免疫療法反應性實體腫瘤係乳癌、結腸癌或肝癌。 在一些實施例中，該方法適於治療具有異常HHLA2及/或TMIGD2表現、活性及/或信號傳導之癌症，包括(但不限於)乳癌、肺癌、甲狀腺癌、黑素瘤、胰臟癌、卵巢癌、肝癌、膀胱癌、結腸癌、前列腺癌、腎癌、食道癌以及血液惡性病白血病及淋巴瘤。 因此，在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常HHLA2及/或TMIGD2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量的包含溶瘤牛痘病毒及視情況醫藥學上可接受之載劑之醫藥組合物，該溶瘤牛痘病毒包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之核酸，其中該編碼免疫檢查點調節子之核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，該方法具有以下生物活性中之一或多種：(1)殺滅癌細胞；(2)抑制癌細胞增殖；(3)誘導腫瘤中之免疫反應；(4)減小腫瘤大小；(5)減輕患有癌症之個體之一或多種症狀；(6)抑制腫瘤轉移；(7)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔；(8)延長存活期；及/或(9)延長癌症進展之時間。在一些實施例中，該免疫療法反應性實體腫瘤係乳癌、結腸癌或肝癌。 在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常HHLA2及/或TMIGD2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量的包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑的醫藥組合物，該溶瘤病毒包含：編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸；及編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常HHLA2及/或TMIGD2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸的第一OV(例如溶瘤VV)；及包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；以及視情況醫藥學上可接受之載劑。在一些實施例中，該醫藥組合物進一步包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV。在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常HHLA2及/或TMIGD2表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸的第一OV(例如溶瘤VV)，及視情況醫藥學上可接受之第一載劑；及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，及視情況醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，該方法進一步包含向個體投與有效量之第三醫藥組合物，該第三醫藥組合物包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV，及視情況醫藥學上可接受之第三載劑。在一些實施例中，免疫檢查點調節子包含TMIGD2細胞外結構域與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，該方法具有以下一或多種生物活性：(1)殺滅癌細胞；(2)抑制癌細胞增殖；(3)誘導腫瘤中之免疫反應；(4)減小腫瘤大小；(5)減輕患有癌症之個體之一或多種症狀；(6)抑制腫瘤轉移；(7)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔；(8)延長存活期；(9)延長癌症進展之時間；(10)增加T細胞腫瘤浸潤；及/或(11)增強所提及之作用中之任一種。舉例而言，TMIGD2細胞外結構域-Fc融合蛋白之共表現可以增強活化之T細胞在共表現之雙特異性接合分子存在下殺滅腫瘤細胞之作用，提高T細胞之細胞介素產生，或者消除或降低由腫瘤細胞表現之抑制性免疫檢查點分子引起的腫瘤逃避作用。在一些實施例中，該免疫療法反應性實體腫瘤係乳癌、結腸癌或肝癌。 在一些實施例中，該方法適於治療具有異常CD47/SIRPα表現、活性及/或信號傳導，及/或異常CXCL12/CXCR4表現、活性及/或信號傳導之癌症，包括(但不限於)卵巢癌、黑素瘤、前列腺癌、卵巢癌、多發性骨髓瘤、乳癌、肺癌、肝癌。 因此，在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常CD47/SIRPα及/或CXCL12/CXCR4表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量的包含溶瘤牛痘病毒及視情況醫藥學上可接受之載劑之醫藥組合物，該溶瘤牛痘病毒包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之核酸，其中該編碼免疫檢查點調節子之核酸係可操作地連接至晚期啟動子(諸如F17R)。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，該方法具有以下生物活性中之一或多種：(1)殺滅癌細胞；(2)抑制癌細胞增殖；(3)誘導腫瘤中之免疫反應；(4)減小腫瘤大小；(5)減輕患有癌症之個體之一或多種症狀；(6)抑制腫瘤轉移；(7)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔；(8)延長存活期；及/或(9)延長癌症進展之時間。在一些實施例中，該免疫療法反應性實體腫瘤係乳癌或肝癌。 在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常CD47/SIRPα及/或CXCL12/CXCR4表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量的包含溶瘤病毒(諸如溶瘤VV)及視情況醫藥學上可接受之載劑的醫藥組合物，該溶瘤病毒包含：編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸；及編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常CD47/SIRPα及/或CXCL12/CXCR4表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包括：包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸的第一OV(例如溶瘤VV)；及包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)；以及視情況醫藥學上可接受之載劑。在一些實施例中，該醫藥組合物進一步包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)之第三OV。在一些實施例中，提供一種治療個體之免疫療法反應性實體腫瘤(諸如癌瘤或腺癌，諸如具有異常CD47/SIRPα及/或CXCL12/CXCR4表現、活性及/或信號傳導之癌症)的方法，其包含向個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子(諸如本文所描述之免疫檢查點調節子中之任一種，例如PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑)之第一核酸的第一OV(例如溶瘤VV)，及視情況醫藥學上可接受之第一載劑；及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼雙特異性分子(諸如本文所描述之雙特異性分子中之任一種)之第二核酸的第二OV(例如溶瘤VV)，及視情況醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)。在一些實施例中，該方法進一步包含向個體投與有效量之第三醫藥組合物，該第三醫藥組合物包含編碼細胞介素(諸如本文所描述之細胞介素中之任一種，例如GM-CSF)及視情況醫藥學上可接受之第三載劑。在一些實施例中，免疫檢查點調節子包含SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段(諸如IgG4 Fc)的融合物。在一些實施例中，該方法具有以下生物活性中之一或多種：(1)殺滅癌細胞；(2)抑制癌細胞增殖；(3)誘導腫瘤中之免疫反應；(4)減小腫瘤大小；(5)減輕患有癌症之個體之一或多種症狀；(6)抑制腫瘤轉移；(7)降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔；(8)延長存活期；(9)延長癌症進展之時間；(10)增加T細胞腫瘤浸潤；及/或(11)增強所提及之作用中之任一種。舉例而言，SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白之共表現可以增強在共表現之雙特異性接合分子存在下活化T細胞之腫瘤細胞殺滅作用，增加T細胞之細胞介素產生，或消除或降低由腫瘤細胞表現之抑制性免疫檢查點分子引起的腫瘤逃避作用。在一些實施例中，該免疫療法反應性實體腫瘤係乳癌或肝癌。 在一些實施例中，癌症係例如EpCAM陽性、FAP陽性、EGFR陽性或GPC3陽性的。在一些實施例中，該癌症對於本文所列腫瘤相關抗原或腫瘤特異性抗原中之任一種呈陽性，例如展示在其細胞表面上。 在一些實施例中，本文所描述之方法適於治療在癌細胞之表面上過度表現EpCAM之癌症，諸如EpCAM陽性實體癌症。舉例而言，個體(諸如人類)中癌細胞表現之EpCAM可以至少約為正常細胞之超過2、5、10、20、50、100、200、500、1000倍或更高倍數中之任一者。EpCAM陽性實體癌症可以為癌瘤或腺癌。EpCAM陽性實體癌症包括(但不限於)小腸癌、結腸直腸癌、肺癌、子宮頸癌、肝癌、胃癌、胰臟癌、皮膚癌(諸如黑素瘤)、腎癌、膀胱癌、甲狀腺癌、前列腺癌、卵巢癌、乳癌、膽管癌及頭頸癌。 在一些實施例中，本文所描述之方法適於治療在腫瘤細胞或腫瘤基質纖維母細胞上過度表現FAP之癌症，諸如FAP陽性實體癌症。舉例而言，個體(諸如人類)中腫瘤基質纖維母細胞表現之FAP至少約為正常纖維母細胞之超過2、5、10、20、50、100、200、500、1000倍或更高倍數中之任一者。FAP陽性實體癌症可以為癌瘤或腺癌。在一些實施例中，FAP陽性實體癌症係選自由以下組成之群：結腸直腸癌、乳癌、腦癌、肺癌及皮膚癌(諸如黑素瘤)。 在一些實施例中，本文所描述之方法適於治療在癌細胞之表面上過度表現EGFR之癌症，諸如EGFR陽性實體癌症。舉例而言，個體(諸如人類)中癌細胞表現之EGFR至少約為正常細胞之超過2、5、10、20、50、100、200、500、1000倍或更高倍數中之任一者。EGFR陽性實體癌症可以為癌瘤或腺癌。EGFR介導之癌症包括(但不限於)神經膠母細胞瘤、頭頸癌、胰臟癌、肺癌、神經系統癌症、胃腸癌、前列腺癌、卵巢癌、乳癌、腎癌、視網膜癌、皮膚癌、肝癌、泌尿生殖器癌症及膀胱癌。 在一些實施例中，本文所描述之方法適於治療在癌細胞之表面上過度表現GPC3之癌症，諸如GPC3陽性實體癌症。舉例而言，個體(諸如人類)中癌細胞表現之GPC3可以至少約為正常細胞之超過2、5、10、20、50、100、200、500、1000倍或更高倍數中之任一者。GPC3陽性實體癌症可以例如為鱗狀細胞癌或肝細胞癌(HCC)。 在一些實施例中，本文所描述之方法適於治療結腸直腸癌，諸如腺癌，胃腸類癌、胃腸基質腫瘤、平滑肌肉瘤、黑素瘤或鱗狀細胞癌。在一些實施例中，本文所描述之方法適於治療肝癌，諸如肝細胞癌瘤、纖維板層變化型肝細胞癌或混合型肝細胞膽管癌。在一些實施例中，本文所描述之方法適於治療乳癌，諸如早期乳癌、非轉移性乳癌、晚期乳癌、IV期乳癌、局部晚期乳癌、轉移性乳癌、緩解型乳癌、輔助治療中之乳癌或新輔助治療中之乳癌。在一些實施例中，乳癌係纖維腺瘤或乳管內乳頭瘤。在一些實施例中，乳癌係HER2陽性或HER2陰性的。在一些具體實例中，乳癌係三陰性乳癌。 有效量的本文所描述之醫藥組合物中所採用的病毒(諸如VV)之數量將取決於多種情況，諸如引入之目的、所治療癌症之特定類型及分期、所使用之方案(例如投藥次數及途徑)、病毒之穩定性、所編碼之雙特異性分子及免疫檢查點調節子之活性，及類似情況。本文所描述之醫藥組合物之劑量方案將由主治醫師及臨床因素決定。如醫療技術中所熟知，對於任何一位患者，劑量取決於許多因素，包括患者之體格、體表面積、年齡、欲投與之特定化合物、性別、投藥時間及途徑、一般健康狀況及同時投與之其他藥物。在一些實施例中，本文所描述之醫藥組合物之有效量低於誘發毒理學作用(亦即，超過臨床上可接受之毒性水準之作用)之量或係當將醫藥組合物投與個體時可以控制或耐受潛在副作用之量。 在一些實施例中，有效量的本文所描述之醫藥組合物中之溶瘤病毒(諸如溶瘤VV)係約105 至約1013 pfu，包括例如以下任一種：約105 至約1012 pfu、約106 至約1013 pfu、約106 至約1012 pfu、約107 至約1013 pfu、約107 至約1012 pfu、約107 至約1011 pfu、約107 至約1010 pfu、約107 至約109 pfu、約108 至約1013 pfu、約108 至約1012 pfu、約108 至約1011 pfu、約108 至約1010 pfu、約108 至約109 pfu、約109 至約1013 pfu、約109 至約1012 pfu、約109 至約1011 pfu、約109 至約1010 pfu、約1010 至約1013 pfu、約1011 至約1013 pfu，或約1012 至約1013 pfu。在一些實施例中，有效量的本文所描述之醫藥組合物中之OV(諸如溶瘤VV)係約1013 pfu、約1012 pfu、約1011 pfu、約1010 pfu、約109 pfu、約108 pfu、約107 pfu、約106 pfu或約105 pfu。在一些實施例中，有效量的本文所描述之醫藥組合物中之OV(諸如溶瘤VV)係約105 至約1013 pfu。在一些實施例中，有效量的本文所描述之醫藥組合物中之OV(諸如溶瘤VV)係約107 至約109 pfu。在一些實施例中，有效量的本文所描述之醫藥組合物中之OV(諸如溶瘤VV)係約109 pfu。 在一些實施例中，該醫藥組合物係一次性投與(例如快速注射)。在一些實施例中，該醫藥組合物係多次投與(諸如2、3、4、5、6或更多次中之任一者)。若多次投藥，則其可以藉由相同或不同途徑進行且可在相同部位或在替代部位進行。該醫藥組合物可以每週兩次、每週3次、每週4次、每週5次、每天一次、每天一次不間斷、每週一次、每週一次不間斷、每2週一次、每3週一次、每月一次、每2個月一次、每3個月一次、每4個月一次、每5個月一次、每6個月一次、每7個月一次、每8個月一次、每9個月一次、每10個月一次、每11個月一次或每年一次投與。投藥之間之時間間隔可以為約24小時至48小時、2天至3天、3天至5天、5天至1週、1週至2週、2週至1個月、1個月至2個月、2個月至3個月、3個月至6個月或6個月至一年中之任一種。時間間隔亦可為不規律的(例如遵循腫瘤進展)。在一些實施例中，給藥時程不存在間斷。在一些實施例中，包含本文所描述之溶瘤病毒之醫藥組合物可以在約105 pfu至約1013 pfu範圍內(諸如約107 pfu至約109 pfu，或約109 pfu)之劑量投與一次或若干次(例如2、3、4、5、6、7、或8次等)。每次投藥之間之時間間隔可以在約1天至約8週、約2天至約6週、約3天至約4週、約1週至約3週，或每兩週間變化。在一些實施例中，包含本文所描述之溶瘤病毒(諸如溶瘤VV)之醫藥組合物係以約1或2週之時間間隔靜脈內或腫瘤內投與在約105 pfu至約1013 pfu範圍內(諸如約107 pfu至約109 pfu，或約109 pfu)之劑量2至5次(例如3次)。熟習醫學技術者可藉由監測患者之疾病病徵並相應地調整治療而容易地確定針對特定患者之最佳劑量及治療方案。 本文所描述之醫藥組合物可以適於多種投與模式，包括例如全身或局部投與。在一些實施例中，該醫藥組合物係非經腸、經皮(在真皮中)、管內、動脈內(在動脈中)、肌肉內(在肌肉中)、鞘內或靜脈內投與。在一些實施例中，該醫藥組合物係皮下(在皮膚下)投與。在一些實施例中，該醫藥組合物係靜脈內投與。在一些實施例中，本文所描述之醫藥組合物係經由輸注或注射投與個體。在一些實施例中，該醫藥組合物係直接注射至腫瘤部位(腫瘤內，即直接注射至腫瘤中或其附近)。包含編碼本文所描述之雙特異性分子之溶瘤病毒載體及醫藥學上可接受之載劑的醫藥組合物亦涵蓋在本發明中。溶瘤病毒載體可以例如藉由基因槍遞送至內部或外部標靶位點或藉由導管遞送至動脈中之部位，來直接投與至標靶位點。投藥可以使用習知注射器及針(例如Quadrafuse注射針)或此項技術中可利用的能夠促進或改善受試者中活性劑之遞送的任何化合物或裝置。 在一些實施例中，待治療之個體係哺乳動物。哺乳動物之實例包括(但不限於)人類、猴、大鼠、小鼠、倉鼠、天竺鼠、狗、貓、兔、豬、綿羊、山羊、馬、牛及類似哺乳動物。在一些實施例中，個體係人類。 IV. 抗體 本申請案之各種態樣利用了抗體。本申請案提供新穎抗體、抗體片段或其抗原結合片段。該等抗體可以獨立使用，或可以併入本文所描述之OV(諸如溶瘤VV)或溶瘤病毒載體(諸如溶瘤VV載體)中之任一種中。 術語「抗體」係以最廣義使用且涵蓋各種抗體結構，包括(但不限於)單株抗體、多株抗體、多特異性抗體(例如雙特異性抗體)及抗體片段，只要其展現所需抗原結合活性即可。 基礎4鏈抗體單元係由兩條相同輕(L)鏈及兩條相同重(H)鏈構成的雜四聚糖蛋白。IgM抗體由5個基礎雜四聚體單元以及稱為J鏈之另外的多肽組成，且含有10個抗原結合位點，而IgA抗體包含2至5個基礎4鏈單元，該等單元可以聚合以與J鏈組合形成多價群集體。在IgG之情況下，該4鏈單元一般係約150,000道爾頓。各L鏈經一個共價二硫鍵連接至H鏈，而取決於H鏈同型，兩條H鏈經一或多個二硫鍵彼此連接。H鏈及L鏈各自亦具有規律間隔之鏈內二硫橋。各H鏈在N末端具有可變結構域(VH )，對於α及γ鏈中每一個隨後具有三個恆定結構域(CH )且對於μ及ε同型具有四個CH 結構域。各L鏈在N端具有可變結構域(VL )，隨後在其另一端具有恆定結構域。VL 與VH 對準，且CL 與重鏈(CH 1)之第一恆定結構域對準。咸信特定胺基酸殘基在輕鏈與重鏈可變結構域之間形成界面。VH 及VL 對一起形成單一抗原結合位點。有關不同類別抗體之結構及特性，參見例如，Basic and Clinical Immunology , 第8版, Daniel P. Sties, Abba I. Terr及Tristram G. Parsolw (編), Appleton & Lange, Norwalk, Conn, 1994, 第71頁及第6章。來自任何脊椎動物物種之L鏈可基於其恆定結構域之胺基酸序列而指定為稱為κ及λ之兩種明顯不同類型之一。取決於免疫球蛋白之重鏈恆定結構域(CH )之胺基酸序列，免疫球蛋白可指定為不同類別或同型。存在5種類別之免疫球蛋白：IgA、IgD、IgE、IgG及IgM，其分別具有指定為α、δ、ε、γ及μ之重鏈。基於CH 序列及功能之相對較小差異，γ及α類別進一步分成亞類，例如人類表現以下亞類：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1及IgA2。 「人類抗體」係具有對應於由人類產生及/或已使用如本文所揭示之任一種人類抗體製備技術所製備之抗體之胺基酸序列的抗體。此人類抗體定義明確地排除包含非人類抗原結合殘基之人類化抗體。人類抗體可以使用此項技術中已知之各種技術，包括噬菌體展示文庫製造。Hoogenboom及Winter,J. Mol. Biol., 227:381 (1991)；Marks等人,J. Mol. Biol., 222:581 (1991)。另外，可用於製備人類單株抗體之方法描述於Cole等人,Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, 第77頁(1985)；Boerner等人,J. Immunol., 147(1):86-95 (1991)中。亦參見van Dijk及van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001)。人類抗體可以藉由以下方式製備：將抗原投與經修飾以響應於抗原攻擊產生此類抗體但內源性基因座已失能之轉殖基因動物，例如經免疫之xenomice(關於XENOMOUSE™技術，參見例如美國專利第6,075,181號及第6,150,584號)。關於經由人類B細胞融合瘤技術產生之人類抗體，亦參見例如Li等人,Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006)。 如本文中所使用，術語「重組人類抗體」意欲包括藉由重組手段製備、表現、產生或分離之所有人類抗體，諸如自諸如NS0或CHO細胞之宿主細胞或自關於人類免疫球蛋白基因轉殖基因之動物(例如，小鼠)分離的抗體，或使用轉染至宿主細胞中之重組表現載體表現的抗體。此類重組人類抗體具有呈重新佈置形式之可變區及恆定區。根據本發明之重組人類抗體已經歷活體內體細胞超突變。因此，重組抗體VH及VL區之胺基酸序列係來源於且關於人類生殖系VH及VL序列，但可能並非在活體內天然存在於人類抗體生殖系譜系內的序列。 非人類(例如鼠類)抗體之「人類化」形式係含有來源於非人類免疫球蛋白之最小序列之嵌合抗體。在一個實施例中，人類化抗體係這樣一種人類免疫球蛋白(接受者抗體)，其中來自接受者之HVR(在下文中定義)之殘基經來自具有所需特異性、親和力及/或能力之非人類物種(供體抗體)，諸如小鼠、大鼠、兔或非人類靈長類動物之HVR的殘基置換。在一些情況下，人類免疫球蛋白之構架(「FR」)殘基經相應非人類殘基置換。此外，人類化抗體可包含在接受者抗體或供體抗體中未發現之殘基。可進行此等修飾以進一步優化抗體效能，諸如結合親和力。一般而言，人類化抗體將包含至少一個且通常兩個可變結構域之大體上全部，其中所有或大體上所有高變環對應於非人類免疫球蛋白序列之高變環，且所有或大體上所有FR區係人類免疫球蛋白序列之FR區，但該等FR區可包括一或多個改良抗體效能(諸如結合親和力、異構化、免疫原性等)之個別FR殘基取代。FR中此等胺基酸取代之數量通常在H鏈中不超過6個且在L鏈中不超過3個。人類化抗體視情況亦將包含免疫球蛋白恆定區(Fc)，通常人類免疫球蛋白恆定區之至少一部分。有關更多細節，參見例如，Jones等人,Nature 321:522-525 (1986)；Riechmann等人,Nature 332:323-329 (1988)；及Presta,Curr. Op. Struct. Biol. 2:593-596 (1992)。亦參見例如，Vaswani及Hamilton,Ann. Allergy, Asthma &Immunol. 1:105-115 (1998)；Harris,Biochem. Soc. Transactions 23:1035-1038 (1995)；Hurle及Gross,Curr. Op. Biotech. 5:428-433 (1994)；以及美國專利第6,982,321號及第7,087,409號。 術語「嵌合」抗體係指通常利用重組DNA技術製備之抗體，其中重鏈及/或輕鏈之一部分係來源於特定來源或物種，而重鏈及/或輕鏈之其餘部分係來源於不同來源或物種。嵌合抗體可以包含鼠類可變區及人類恆定區。「嵌合抗體」亦可為恆定區已自原始抗體修飾或改變以產生根據本發明之特性，尤其是C1q結合及/或Fc受體(FcR)結合的抗體。此類嵌合抗體又稱為「類別轉換抗體」。嵌合抗體係包含編碼免疫球蛋白可變區之DNA區段及編碼免疫球蛋白恆定區之DNA區段的免疫球蛋白基因之表現產物。用於產生嵌合抗體之方法涉及此項技術中熟知的習知重組DNA及基因轉染技術。參見例如Morrison, S.L.等人, Proc. Natl. Acad. Sci. USA 81 (1984) 6851- 6855；美國專利第5,202,238號及第5,204,244號。 如本文所使用，術語「單株抗體」係指一種自大體上同質之抗體群獲得之抗體，亦即，構成該群體之個別抗體除可能天然存在之突變及/或可能少量存在之轉譯後修飾(例如，異構化、醯胺化)之外其餘相同。單株抗體具有高度特異性，針對單一抗原位點。與通常包括針對不同決定子(抗原決定基)之不同抗體的多株抗體製劑相對，各單株抗體係針對抗原上之單一決定子。除特異性之外，單株抗體之有利之處亦在於，其係藉由融合瘤培養物合成，未受其他免疫球蛋白污染。修飾語「單株」指示抗體之特徵為自大體上同質之抗體群獲得，且不應理解為需要藉由任何特定方法產生該抗體。舉例而言，根據本發明使用之單株抗體可以藉由多種技術製備，包括例如融合瘤方法(例如Kohler及Milstein.,Nature, 256:495-97 (1975)；Hongo等人,Hybridoma, 14 (3): 253-260 (1995)；Harlow等人,Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 第2版, 1988)；Hammerling等人,Monoclonal Antibodies and T -Cell Hybridomas 563-681 (Elsevier, N.Y., 1981))、重組DNA方法(參見例如，美國專利第4,816,567號)、噬菌體展示技術 (參見例如，Clackson等人,Nature, 352: 624-628 (1991)；Marks等人,J. Mol. Biol. 222: 581-597 (1992)；Sidhu等人,J. Mol. Biol. 338(2): 299-310 (2004)；Lee等人,J. Mol. Biol. 340(5): 1073-1093 (2004)；Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004)；及Lee等人,J. Immunol. Methods 284(1-2): 119-132 (2004)，及用於在具有部分或全部人類免疫球蛋白基因座或編碼人類免疫球蛋白序列之基因的動物中產生人類或人類樣抗體的技術(參見例如，WO 1998/24893；WO 1996/34096；WO 1996/33735；WO 1991/10741；Jakobovits等人,Proc. Natl. Acad. Sci. USA 90: 2551 (1993)；Jakobovits等人,Nature 362: 255-258 (1993)；Bruggemann等人,Year in Immunol. 7:33 (1993)；美國專利第5,545,807號、第5,545,806號、第5,569,825號、第5,625,126號、第5,633,425號及第5,661,016號；Marks等人,Bio/Technology 10: 779-783 (1992)；Lonberg等人,Nature 368: 856-859 (1994)；Morrison,Nature 368: 812-813 (1994)；Fishwild等人,Nature Biotechnol. 14: 845-851 (1996)；Neuberger,Nature Biotechnol. 14: 826 (1996)；以及Lonberg及Huszar,Intern. Rev. Immunol. 13: 65-93 (1995)。 術語「全長抗體」、「完整抗體」或「全抗體」可互換使用以指呈大體上完整形式之抗體，與抗體片段相對。具體而言，全長4鏈抗體包括具有重鏈及輕鏈之抗體，包括Fc區在內。恆定結構域可以為天然序列恆定結構域(例如人類天然序列恆定結構域)或其胺基酸序列變異體。在一些情況下，完整抗體可以具有一或多種效應功能。 「抗體片段」包含完整抗體之一部分，較佳完整抗體之抗原結合區及/或可變區。抗體片段之實例包括Fab、Fab'、F(ab')2 及Fv片段；雙功能抗體；線性抗體(參見美國專利第5,641,870號，實例2；Zapata等人,Protein Eng. 8(10): 1057-1062 [1995])；單鏈抗體分子及由抗體片段形成之多特異性抗體。木瓜蛋白酶消化抗體產生兩個相同的抗原結合片段，稱作「Fab」片段；及殘餘「Fc」片段，該名稱反映易於結晶之能力。Fab片段由完整L鏈以及H鏈之可變區結構域(VH )及一條重鏈之第一恆定結構域(CH 1)組成。各Fab片段就抗原結合而言係單價的，亦即，其具有單一抗原結合位點。胃蛋白酶處理抗體產生單一較大的F(ab')2 片段，該片段大致對應於經二硫鍵連接的具有不同抗原結合活性之兩個Fab片段且仍能夠交聯抗原。Fab'片段與Fab片段之不同之處在於，Fab'片段在CH 1結構域之羧基末端具有若干另外的殘基，包括一或多個來自抗體鉸鏈區之半胱胺酸。Fab'-SH在本文中係針對恆定結構域之半胱胺酸殘基攜帶游離硫醇基的Fab'之名稱。F(ab')2 抗體片段最初係以在其間具有鉸鏈半胱胺酸之Fab'片段對形式產生。抗體片段之其他化學偶合亦係已知的。 術語「恆定結構域」係指具有比含有抗原結合位點之免疫球蛋白其他部分，即可變結構域保守之胺基酸序列的免疫球蛋白分子部分。恆定結構域含有重鏈之CH 1、CH 2及CH 3結構域(統稱為CH)及輕鏈之CHL (或CL)結構域。 來自任何哺乳動物物種之抗體(免疫球蛋白)的「輕鏈」可以基於其恆定結構域之胺基酸序列而指定為稱為κ及λ的兩種明顯不同之類型之一。 術語「雙功能抗體」係指具有兩個抗原結合位點之抗體片段，該等片段包含連接至同一多肽鏈(VH-VL)中之輕鏈可變結構域(VL)的重鏈可變結構域(VH)。藉由使用過短以致同一鏈上之兩個結構域之間不能配對的連接子，迫使該等結構域與另一條鏈之互補結構域配對，且產生兩個抗原結合位點。雙功能抗體可以為二價或雙特異性的。雙功能抗體更充分地描述於例如EP 404,097；WO 1993/01161；Hudson等人, Nat. Med. 9:129-134 (2003)；及Hollinger等人, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)中。三功能抗體及四功能抗體亦描述於Hudson等人, Nat. Med. 9:129-134 (2003)中。 「Fc」片段包含藉由二硫鍵保持在一起的兩個H鏈之羧基末端部分。抗體之效應功能係由Fc區中之序列決定，該區亦為由在某些類型細胞上所發現之Fc受體(FcR)識別的部分。 「Fv」係含有完整抗原識別及抗原結合位點之最小抗體片段。此片段由緊密、非共價締合之一個重鏈可變區結構域與一個輕鏈可變區結構域之二聚體組成。由此等兩個結構域之摺疊產生六個高變環(來自H鏈及L鏈各3個環)，其提供用於抗原結合之胺基酸殘基且賦予抗體抗原結合特異性。然而，即使單一可變結構域(或僅包含對抗原具有特異性之三個HVR的一半Fv)能夠識別及結合抗原，但其親和力低於完整結合位點。 「單鏈Fv」亦簡稱為「sFv」或「scFv」，其為包含連接至單一多肽鏈中之VH 及VL 抗體結構域的抗體片段。在一些實施例中，scFv多肽在VH 與VL 結構域之間進一步包含多肽連接子，其使scFv能夠形成抗原結合所需之結構。scFv係此項技術中已知的，參見例如Pluckthun,The Pharmacology of Monoclonal Antibodies , 第113卷, Rosenburg及Moore編, Springer-Verlag, New York, 第269-315頁(1994)。 抗體之「可變區」或「可變結構域」係指抗體之重鏈或輕鏈之胺基末端結構域。重鏈及輕鏈之可變結構域可分別稱為「VH 」及「VL 」。該等結構域一般為抗體變化最大之部分(相對於同種類之其他抗體)且含有抗原結合位點。 術語「可變」係指可變結構域某些區段之序列在抗體間廣泛不同的事實。V結構域介導抗原結合且確定特定抗體對其特定抗原之特異性。然而，可變性在整個可變結構域範圍內並非均勻分佈。相反，其集中在輕鏈可變結構域及重鏈可變結構域中的三個稱作高變區(HVR)之區段中。可變結構域之保守性較高的部分稱為構架區(FR)。天然重鏈及輕鏈之可變結構域各包含四個FR區，該等FR區由三個HVR連接，大體上呈β-片層構形，該等HVR形成連接β-片層結構之環且在一些情況下形成β-片層結構之一部分。各鏈中之HVR藉由FR區保持在一起且與來自其他鏈之HVR緊密靠近，由此促進抗體抗原結合位點之形成(參見Kabat等人,Sequences of Immunological Interest , 第五版, National Institute of Health, Bethesda, Md. (1991))。恆定結構域不直接參與抗體與抗原之結合，但展現各種效應功能，諸如抗體依賴性細胞毒性中抗體的參與。 當在本文中使用時，術語「高變區」、「HVR」或「HV」係指抗體可變結構域中序列具有高變性及/或形成結構確定之環的區域。一般而言，4鏈抗體包含六個HVR；三個在VH (H1、H2、H3)中，且三個在VL (L1、L2、L3)中。在天然4鏈抗體中，H3及L3展示該六個HVR之大部分多樣性，且咸信尤其是H3在賦予抗體精細特異性方面起到獨特作用。參見例如，Xu等人,Immunity 13:37-45 (2000)；Johnson及Wu,Methods in Molecular Biology 248:1-25 (Lo編, Human Press, Totowa, N.J., 2003)。實際上，僅由重鏈組成的天然存在之駱駝抗體在無輕鏈存在下亦具有功能且穩定。參見例如，Hamers-Casterman等人,Nature 363:446-448 (1993)；Sheriff等人,Nature Struct. Biol. 3:733-736 (1996)。 已使用多種HVR描述且涵蓋於本文中。Kabat「互補決定區」(或「CDR」)係基於序列可變性且最常用(Kabat等人,Sequences of Proteins of Immunological Interest, 第5版, Public Health Service, National Institutes of Health, Bethesda, Md. (1991))。而Chothia提及結構環之位置(Chothia及Lesk,J. Mol. Biol. 196:901-917 (1987))。AbM HVR表示Kabat HVR與Chothia結構環之間的折中，且由Oxford Molecular之AbM抗體模擬軟體使用。「接觸」HVR係基於可用複雜晶體結構之分析。來自該等HVR中每一個的殘基描述於下表1中。 表1. HVR描述。 HVR可包含如下「擴展HVR」：VL 中之24-36或24-34 (L1)、46-56或50-56 (L2)及89-97或89-96 (L3)，及VH 中之26-35 (H1)、50-65或49-65 (H2)及93-102、94-102或95-102 (H3)。可變結構域殘基係根據Kabat等人(同上文)中關於此等定義中之每一者編號。 表述「如Kabat中之可變結構域殘基編號」或「如Kabat中之胺基酸位置編號」及其變化形式係指Kabat等人(同上文)中用於抗體編譯之重鏈可變結構域或輕鏈可變結構域之編號系統。使用此編號系統，實際線性胺基酸序列可含有對應於可變結構域之FR或HVR之縮短或插入的較少或另外的胺基酸。舉例而言，重鏈可變結構域可以包括在H2之殘基52之後的單一胺基酸插入(根據Kabat之殘基52a)及在重鏈FR殘基82之後插入的殘基(例如根據Kabat之殘基82a、82b及82c等)。對於既定抗體，可藉由將抗體序列之同源區與「標準」Kabat編號序列比對來確定殘基之Kabat編號。 「構架」或「FR」殘基係除如本文所定義之HVR殘基外的可變結構域殘基。可變結構域之FR一般由四個FR結構域組成：FR1、FR2、FR3及FR4。因此，HVR及FR序列一般按以下順序出現在VH (或VL)中：FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。 除非本文另外指明，否則免疫球蛋白重鏈中殘基之編號係如Kabat等人(同上文)中之EU索引之編號。「Kabat中之EU索引」係指人類IgG1 EU抗體之殘基編號。 如本文中所使用，術語「結合」「特異性結合至」、「特異性識別」或「對......具有特異性」係指在異質分子(包括生物分子)群存在下決定標靶之存在的可量測且可再現之相互作用，諸如標靶與抗體之間的結合。舉例而言，結合至，或特異性識別，或特異性結合至標靶(其可以為抗原決定基)之抗體係結合此標靶之親和力、親合力、容易性及/或持續時間強於結合至其他標靶的抗體。在一些實施例中，如例如藉由放射免疫分析(RIA)所量測，抗體與不相關標靶之結合程度比抗體與該標靶之結合低約10%。在一些實施例中，抗體特異性結合至蛋白質上之抗原決定基，該抗原決定基在來自不同物種之蛋白質間保守。在另一個實施例中，特異性結合可以包括排他性結合，但並非必需。 如本文所使用，關於肽、多肽或抗體序列之「胺基酸序列一致性百分比(%)」及「同源性」定義為在比對序列且必要時引入空位以達成最大序列一致性百分比之後，且不考慮任何保守取代為序列一致性之一部分，候選序列中與特定肽或多肽序列中之胺基酸殘基一致的胺基酸殘基之百分比。出於測定胺基酸序列一致性百分比之目的進行的比對可以藉由在此項技術之技能範圍內的各種方式實現，例如使用公開可得的電腦軟體，諸如BLAST、BLAST-2、ALIGN或MEGALIGN™(DNASTAR)軟體。熟習此項技術者可確定用於量測比對之適當參數，包括用於在所比較序列之全長內達成最大比對所需的任何算法。 在一些實施例中，提供一種特異性結合至PD-1之抗體、抗體片段或抗原結合結構域(在下文中又稱為「抗PD-1抗體」、「抗PD-1抗體片段」、「PD-1結合片段」或「PD-1結合結構域」)。兩種例示性抗PD-1抗體或其抗原結合片段係1H7e3及4F11C3。 在一些實施例中，抗PD-1抗體或其抗原結合片段(諸如全長抗體或scFv)包括含來自SEQ ID NO: 13之一個、兩個或三個HVR之重鏈可變區(VH)，及/或含來自SEQ ID NO: 14之一個、兩個或三個HVR之輕鏈可變區(VL)。在一些實施例中，抗PD-1抗體或其抗原結合片段(諸如全長抗體或scFv)包括含來自SEQ ID NO: 13之三個HVR之重鏈可變區(VH)，及/或含來自SEQ ID NO: 14之三個HVR之輕鏈可變區(VL)。 在一些實施例中，抗PD-1抗體包括抗原結合結構域(諸如scFv)，其包含：重鏈可變區(VH)，該重鏈可變區包括含胺基酸序列SEQ ID NO: 1之HVR-H1；含胺基酸序列SEQ ID NO: 2之HVR-H2；及含胺基酸序列SEQ ID NO: 3之HVR-H3；及/或輕鏈可變區(VL)，該輕鏈可變區包括含胺基酸序列SEQ ID NO: 4之HVR-L1；含胺基酸序列SEQ ID NO: 5之HVR-L2；及含胺基酸序列SEQ ID NO: 6之HVR-L3。 在一些實施例中，抗PD-1抗體包含抗原結合結構域(諸如scFv)，其包括：包含與胺基酸序列SEQ ID NO: 13至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%一致之胺基酸序列的重鏈可變區(VH)；及/或包含與胺基酸序列SEQ ID NO: 14至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%一致之胺基酸序列的輕鏈可變區(VL)。在一些實施例中，抗PD-1抗體或其抗原結合片段(諸如scFv)包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。 在一些實施例中，抗PD-1抗體或其抗原結合片段(諸如全長抗體或scFv)包括含來自SEQ ID NO: 15之一個、兩個或三個HVR的重鏈可變區(VH)，及/或含來自SEQ ID NO: 16之一個、兩個或三個HVR的輕鏈可變區(VL)。在一些實施例中，抗PD-1抗體或其抗原結合片段(諸如全長抗體或scFv)包括含來自SEQ ID NO: 15之三個HVR之重鏈可變區(VH)，及/或含來自SEQ ID NO: 16之三個HVR之輕鏈可變區(VL)。 在一些實施例中，抗PD-1抗體包括抗原結合結構域(諸如scFv)，其包含：重鏈可變區(VH)，該重鏈可變區包括含胺基酸序列SEQ ID NO: 7之HVR-H1；含胺基酸序列SEQ ID NO: 8之HVR-H2；及含胺基酸序列SEQ ID NO: 9之HVR-H3；及/或輕鏈可變區(VL)，該輕鏈可變區包括含胺基酸序列SEQ ID NO: 10之HVR-L1；含胺基酸序列SEQ ID NO: 11之HVR-L2；及含胺基酸序列SEQ ID NO: 12之HVR-L3。 在一些實施例中，抗PD-1抗體包含抗原結合結構域(諸如scFv)，其包括：包含與胺基酸序列SEQ ID NO: 15至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%一致之胺基酸序列的重鏈可變區(VH)；及/或包含與胺基酸序列SEQ ID NO: 16至少約85%、至少約86%、至少約87%、至少約88%、至少約89%、至少約90%、至少約91%、至少約92%、至少約93%、至少約94%、至少約95%、至少約96%、至少約97%、至少約98%或至少約99%一致之胺基酸序列的輕鏈可變區(VL)。在一些實施例中，抗PD-1抗體或其抗原結合片段(諸如scFv)包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。 在一些實施例中，抗PD-1抗體係全長抗體。在一些實施例中，全長抗PD-1抗體包含來自免疫球蛋白，諸如IgA、IgD、IgE、IgG及IgM之Fc序列。在一些實施例中，全長抗PD-1抗體包含IgG，諸如IgG1、IgG2、IgG3或IgG4中之任一者的Fc序列。在一些實施例中，全長抗PD-1抗體包含人類免疫球蛋白之Fc序列。在一些實施例中，全長抗PD-1抗體包含已改變或以其他方式變化以使其具有增強之抗體依賴性細胞毒性(ADCC)或補體依賴性細胞毒性(CDC)效應功能的Fc序列。 亦提供一種與本文所描述之抗PD-1抗體中之任一者競爭結合PD-1的分離之抗體或其抗原結合片段。在一些實施例中，提供一種與本文所描述之抗PD-1抗體中之任一者結合至相同抗原決定基的分離之抗體或其抗原結合片段。用於篩選具有所需特異性之抗體的方法包括(但不限於)酶聯結免疫吸附劑分析法(ELISA)及此項技術內已知的其他免疫介導之技術。 熟習此項技術者應認識到，無需過度實驗，可藉由確定一種單株抗體是否阻止本發明之單株抗體(例如具有含胺基酸序列SEQ ID NO: 13之可變重鏈及/或含胺基酸序列SEQ ID NO: 14之可變輕鏈的抗PD-1抗體，或具有含胺基酸序列SEQ ID NO: 15之可變重鏈及/或含胺基酸序列SEQ ID NO: 16之可變輕鏈的抗PD-1抗體)結合至PD-1來確定該單株抗體是否具有與本發明抗體相同的特異性。若如由本發明之單株抗體結合減少所示，測試單株抗體與本發明之單株抗體競爭，則該兩種單株抗體結合至同一抗原決定基或緊密相關之抗原決定基。 用於確定一種單株抗體是否具有本發明單株抗體之特異性的替代方法係將本發明之單株抗體與可溶性PD-1蛋白質(與該單株抗體通常具有反應性之蛋白質)預培育，且接著添加測試單株抗體以確定該測試單株抗體結合PD-1之能力是否受到抑制。若測試單株抗體受到抑制，則極有可能其具有與本發明之單株抗體相同或在功能上等效的抗原決定基特異性。 在一些實施例中，抗PD-1抗體係單株抗體，諸如單價抗體。在一些實施例中，抗PD-1抗體係全長抗體。在一些實施例中，抗PD-1抗原結合片段係呈Fab、Fab'、F(ab')2 、單鏈Fv(scFv)、Fv片段、雙功能抗體或線性抗體形式。 在一些實施例中，抗PD-1抗體係結合至PD-1且亦結合一或多個其他標靶並視情況抑制其功能的多特異性抗體。多特異性抗體係對兩種或兩種以上不同抗原具有結合特異性的單株、較佳人類或人類化抗體(例如對至少兩種抗原具有結合特異性之雙特異性抗體)。在一些實施例中，抗PD-1抗體係雙特異性分子，其中該雙特異性分子進一步包含特異性識別本文所描述之另一抑制性免疫檢查點分子(例如PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73)之第二抗原結合片段。在一些實施例中，抗PD-1抗體係雙特異性分子，其中該雙特異性分子進一步包含特異性識別及活化本文所描述之刺激性免疫檢查點分子(例如OX40L、CD80、CD86、B7RP1、4-1BBL、Ultra 4-1BBL、CD70、CD40L、或I類或II類MHC分子、IMCgp100)之第二抗原結合片段。 在一些實施例中，多特異性抗PD-1分子係例如雙功能抗體(Db)、單鏈雙功能抗體(scDb)、串聯scDb(Tandab)、線性二聚scDb(LD-scDb)、環形二聚scDb(CD-scDb)、二-雙功能抗體、串聯scFv、串聯二scFv (例如雙特異性T細胞接合子)、串聯三scFv、三功能抗體、雙特異性Fab2 、二微型抗體、四功能抗體、scFv-Fc-scFv融合物、雙重親和力再靶向(DART)抗體、雙可變結構域(DVD)抗體、IgG-scFab、scFab-ds-scFv、Fv2-Fc、IgG-scFv融合物、對接鎖定(DNL)抗體、孔中節(knob-into-hole；KiH)抗體(藉由KiH技術製備之雙特異性IgG)、DuoBody(藉由Duobody技術製備之雙特異性IgG)、異多聚體抗體或異結合物抗體。在一些實施例中，多特異性抗PD-1分子係串聯scFv(例如串聯二scFv，諸如雙特異性T細胞接合子)。 進一步提供包含上述抗PD-1抗體或其抗原結合片段中之任一者的融合蛋白、結合物或分離之細胞。 在一些實施例中，該抗PD-1抗體或其抗原結合片段係結合至治療劑(例如細胞毒性劑、放射性同位素及化學治療劑)或藉由成像在患者樣品中或在活體內偵測PD-1之標記(例如放射性同位素、螢光染料及酶)。在一些實施例中，抗PD-1抗體或其抗原結合片段係結合至毒素。 本文所描述之抗PD-1抗體或抗原結合片段可以用於多種治療及診斷方法中。進一步提供治療個體之癌症的方法，其包含向該個體投與有效量的上述抗PD-1抗體或其抗原結合片段或其醫藥組合物。舉例而言，抗PD-1抗體(或其抗原結合片段)可以單獨使用或與其他藥劑組合使用以治療以異常PD-L1或PD-1表現為特徵之疾病，或對免疫療法起反應之癌症，包括(但不限於)黑素瘤(例如轉移性惡性黑色素瘤)、腎癌(例如透明細胞癌瘤)、前列腺癌(例如激素難治性前列腺腺癌)、乳癌、結腸癌、肝癌及肺癌(例如非小細胞肺癌)。本發明之抗PD-1抗體或抗原結合片段亦可用於治療轉移性癌症，尤其是表現PD-L1之轉移性癌症(Iwai等人 (2005)Int. Immunol. 17:133-144)。本文所提供之抗體亦可用於偵測患者或患者樣品中之PD-1蛋白質。 進一步提供編碼抗PD-1抗體或其抗原結合片段的分離之核酸、包含該編碼抗PD-1抗體或其抗原結合片段之核酸的OV(諸如溶瘤VV)、表現該抗PD-1抗體或其抗原結合片段的分離之細胞、包含該抗PD-1抗體或其抗原結合片段中之任一者的醫藥組合物、編碼其之OV(諸如溶瘤VV)、表現其之宿主細胞、使用此類醫藥組合物治療個體癌症之方法。在一些實施例中，該醫藥組合物係經靜脈內投與待治療的個體。在一些實施例中，該醫藥組合物係經腫瘤內投與待治療的個體。在一些實施例中，待治療之個體係人類。 單株抗體 本發明之單株抗體之篩選亦可例如藉由量測PD-1介導之信號傳導，且確定測試單株抗體是否能夠調節、阻斷、抑制、減少、拮抗、中和或以其他方式干擾PD-1介導之信號傳導來進行。該等分析法可以包括競爭性結合分析法。另外，該等分析法可以量測生物讀出。 可以使用此項技術中已知之各種程序製造針對PD-1或針對其衍生物、片段、類似物、同源物或直系同源物之單株抗體。參見例如，Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY，以引用之方式併入本文中。完全人類抗體係輕鏈及重鏈(包括HVR)之完整序列源自人類基因之抗體分子。此類抗體在本文中稱為「人類抗體」或「完全人類抗體」。人類單株抗體係例如使用以下提供之實例中所描述的程序製備。人類單株抗體亦可藉由使用三源融合瘤技術；人類B細胞融合瘤技術(參見Kozbor等人, 1983 Immunol Today 4: 72)；及EBV融合瘤技術以產生人類單株抗體(參見Cole等人, 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 第77-96頁)來製備。人類單株抗體可以經利用且可以藉由使用人類融合瘤(參見Cote等人, 1983. Proc Natl Acad Sci USA 80: 2026-2030)或藉由在活體外用埃-巴二氏病毒(Epstein Barr Virus)轉型人類B細胞(參見Cole等人, 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 第77-96頁)產生。 抗體係藉由熟知技術，諸如使用蛋白質A或蛋白質G之親和層析法純化，由此主要提供免疫血清之IgG溶離份。隨後或替代地，作為免疫球蛋白所搜尋之標靶的特定抗原或其抗原決定基可以固定在管柱上以藉由免疫親和層析法純化免疫特異性抗體。免疫球蛋白之純化論述於例如D.Wilkinson(The Scientist, The Scientist, Inc.出版, Philadelphia PA, 第14卷, 第8期 (2000年4月17日), 第25-28頁)中。 本發明之PD-1抗體係單株抗體。調節、阻斷、抑制、減少、拮抗、中和或以其他方式干擾PD-1介導之細胞信號傳導的單株抗體係例如藉由用膜結合及/或可溶性PD-1，諸如人類PD-1或其免疫原性片段、衍生物或變異體對動物免疫接種來產生。或者，用經含編碼PD-1之核酸分子之載體轉染的細胞對動物免疫接種，由此PD-1經表現且與經轉染細胞之表面相關聯。或者，藉由篩選含有結合至PD-1之抗體或抗原結合結構域序列的文庫來獲得抗體。此文庫係例如在噬菌體中以蛋白質或肽與噬菌體外殼蛋白之融合物形式製備，其係在組裝之噬菌體粒子之表面上表現且編碼DNA序列包含在該等噬菌體粒子內(亦即，「噬菌體展示文庫」)。接著，針對與PD-1之反應性篩選由骨髓瘤/B細胞融合物產生之融合瘤。 單株抗體係例如使用融合瘤方法，諸如Kohler及Milstein, Nature, 256:495(1975)所描述之方法製備。在融合瘤方法中，通常用免疫劑對小鼠、倉鼠或其他適當的宿主動物免疫接種以使淋巴細胞產生或能夠產生將特異性結合至免疫劑之抗體。或者，淋巴細胞可在活體外經免疫接種。 免疫劑通常將包括蛋白質抗原、其片段或其融合蛋白。一般而言，若需要人類來源之細胞，則使用末梢血液淋巴細胞(「PBL」)；或若需要非人類哺乳動物來源之細胞，則使用脾細胞或淋巴結細胞。接著使用適合融合劑(諸如聚乙二醇)使淋巴細胞與永生化細胞株融合以形成融合瘤細胞(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 第59-103頁)。永生化細胞株通常係經轉型之哺乳動物細胞，尤其是嚙齒動物、牛科動物及人類來源之骨髓瘤細胞。通常，採用大鼠或小鼠骨髓瘤細胞株。融合瘤細胞可以在較佳含有一或多種抑制不融合之永生化細胞生長或存活之物質的適合培養基中培養。舉例而言，若親本細胞缺乏酶次黃嘌呤-鳥嘌呤磷酸核糖基轉移酶(HGPRT或HPRT)，則用於融合瘤之培養基通常將包括次黃嘌呤、胺基蝶呤及胸苷(「HAT培養基」)，該等物質阻止缺乏HGPRT之細胞生長。 較佳永生化細胞株係高效融合，支持所選產抗體細胞穩定高水準表現抗體且對諸如HAT培養基之培養基敏感的永生化細胞株。更佳永生化細胞株係鼠類骨髓瘤細胞株，其可以獲自例如Salk Institute Cell Distribution Center(San Diego, California)及American Type Culture Collection(Manassas, Virginia)。已描述用於產生人類單株抗體之人類骨髓瘤及小鼠-人類雜骨髓瘤細胞株(參見Kozbor, J. Immunol., 133:3001 (1984)；Brodeur等人, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) 第51-63頁))。 接著可以分析培養融合瘤細胞之培養基中針對抗原之單株抗體之存在。較佳藉由免疫沈澱或藉由活體外結合分析法(諸如放射免疫分析(RIA)或酶聯結免疫吸附劑分析法(ELISA))測定由融合瘤細胞產生之單株抗體的結合特異性。該等技術及分析法係此項技術中已知的。單株抗體之結合親和力可例如藉由Munson及Pollard, Anal. Biochem., 107:220 (1980)之Scatchard分析測定。另外，在單株抗體之治療應用中，鑑別對標靶抗原具有高度特異性之抗體很重要。 在鑑別出所需融合瘤細胞之後，可藉由限制性稀釋程序次選殖該等純系且藉由標準方法使其生長(參見Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) 第59-103頁)。適用於此目的之培養基包括例如達爾伯克改良型伊格爾培養基(Dulbecco's Modified Eagle's Medium)及RPMI-1640培養基。或者，融合瘤細胞可以在活體內腹水形式於哺乳動物中生長。 次純系所分泌之單株抗體可藉由習知免疫球蛋白純化程序，諸如蛋白質A-瓊脂糖、羥磷灰石層析法、凝膠電泳、透析或親和層析法自培養基或腹水液分離或純化。 單株抗體亦可藉由重組DNA法製得，諸如美國專利第4,816,567號中描述之方法。編碼本發明之單株抗體的DNA可以使用習知程序(例如藉由使用能夠特異性結合至編碼抗體之重鏈及輕鏈之基因的寡核苷酸探針)容易地分離及測序。分離後，即可將DNA放入表現載體中，隨後將該等表現載體轉染至不另外產生免疫球蛋白蛋白質之宿主細胞，諸如中國倉鼠卵巢(COS)細胞、人胚腎(HEK)293細胞、猴COS細胞、PER.C6®、NS0細胞、SP2/0、YB2/0或骨髓瘤細胞中，以在重組宿主細胞中合成單株抗體。亦可例如藉由用人類重鏈及輕鏈恆定結構域之編碼序列取代同源鼠類序列 (參見美國專利第4,816,567號；Morrison, Nature 368, 812-13 (1994))或藉由將非免疫球蛋白多肽編碼序列之全部或部分共價接合至該免疫球蛋白編碼序列來修飾DNA。此類非免疫球蛋白多肽可以取代抗體之恆定結構域，或可以取代抗體之一個抗原組合位點的可變結構域以產生嵌合二價抗體。 人類抗體及抗體之人類化 本發明之單株抗體包括完全人類抗體或人類化抗體。該等抗體適於投與人類，而不會引起人類針對所投與之免疫球蛋白的免疫反應。 可以使用此項技術中已知之任何程序產生抗PD-1抗體。舉例而言，可以在小鼠中使用改良之多位點重複免疫(Repetitive Immunization Multiple Sites，RIMMS)之免疫策略且隨後產生融合瘤來鑑別抗PD-1抗體。在其他替代性方法中，例如使用噬菌體展示方法，使用僅含有人類序列之抗體來產生抗PD-1抗體。此類方法係此項技術中熟知的，例如見於WO92/01047及美國專利第6,521,404號，其以引用的方式併入本文中。在此方法中，使用天然或重組來源之PD-1或其片段篩選載有任意輕鏈及重鏈對之噬菌體的組合文庫。在另一方法中，可以藉由以下方法製造抗PD-1抗體，其中該程序之至少一個步驟包括用人類PD-1蛋白質對轉殖基因非人類動物免疫接種。在此方法中，此異種非人類動物之部分內源性重鏈及/或κ輕鏈基因座已經失能且不能進行響應於抗原而產生編碼免疫球蛋白之基因所需的重排。此外，至少一個人類重鏈基因座及至少一個人類輕鏈基因座已穩定轉染至動物中。因此，響應於所投與之抗原，該等人類基因座重排以提供編碼對該抗原具有免疫特異性之人類可變區的基因。因此，在免疫接種後，xenomouse產生分泌完全人類免疫球蛋白之B細胞。 此項技術中熟知多種用於產生異種非人類動物之技術。舉例而言，參見美國專利第6,075,181號及第6,150,584號，其以全文引用的方式併入本文。此通用策略係結合1994年公開之首個XenoMouse™品系產生來證實。參見Green等人, Nature Genetics 7:13-21 (1994)，其以全文引用的方式併入本文中。亦參見美國專利第6,162,963號、第6,150,584號、第6,114,598號、第6,075,181號及第5,939,598號；及日本專利第3 068 180 B2號、第3 068 506 B2號及第3 068 507 B2號；及歐洲專利第EP 0 463 151 B1號，以及國際專利申請案第WO 94/02602號、第WO 96/34096號、第WO 98/24893號、第WO 00/76310號及相關家族成員。 在一種替代方法中，利用了「微型基因座(minilocus)」方法，其中經由包括來自Ig基因座之片段(個體基因)來模擬外源性Ig基因座。因此，一或多個VH基因、一或多個DH 基因、一或多個JH 基因、μ恆定區及第二恆定區(較佳為γ恆定區)形成構築體以插入動物中。參見例如，美國專利第5,545,806號、第5,545,807號、第5,591,669號、第5,612,205號、第5,625,825號、第5,625,126號、第5,633,425號、第5,643,763號、第5,661,016號、第5,721,367號、第5,770,429號、第5,789,215號、第 5,789,650號、第5,814,318號、第5,877,397號、第5,874,299號、第6,023,010號及第6,255,458號；及歐洲專利第EP 0 546 073 B1號；及國際專利申請案第WO 92/03918號、第WO 92/22645號、第WO 92/22647號、第WO 92/22670號、第WO 93/12227號、第WO 94/00569號、第WO 94/25585號、第WO 96/14436號、第WO 97/13852號及第WO 98/24884號及相關家族成員。 亦已展示自小鼠產生人類抗體，其中經由微胞融合引入了較大染色體片段或完整染色體。參見歐洲專利申請案第773 288號及第843 961號。 人類抗小鼠抗體(HAMA)反應已引導該行業製備嵌合或其他人類化抗體。儘管嵌合抗體具有人類恆定區及免疫可變區，但預期將觀察到某些人類抗嵌合抗體(HACA)反應，特別是在長期或多劑量使用該抗體時。因此，為了減少或以其他方式緩解HAMA或HACA反應之問題及/或影響，需要提供針對PD-1之完全人類抗體。 亦使用適當文庫，經由人類化、嵌合及展示技術製造出具有降低之免疫原性的抗體。應瞭解，鼠類抗體或來自其他物種之抗體可以使用此項技術中熟知之技術人類化或靈長類化。參見例如，Winter及Harris Immunol Today 14:43 46 (1993)以及Wright等人, Crit, Reviews in Immunol. 12125-168 (1992)。所關注抗體可以藉由重組DNA技術，用相應人類序列替代CH1、CH2、CH3、鉸鏈結構域及/或構架結構域進行工程改造(參見WO 92102190以及美國專利第5,530,101號、第5,585,089號、第5,693,761號、第5,693,792號、第5,714,350號及第5,777,085號)。另外，使用Ig cDNA構築嵌合免疫球蛋白基因係此項技術中已知的(Liu等人, P.N.A.S. 84:3439 (1987)及J. Immunol. 139:3521 (1987))。自融合瘤或其他產生該抗體之細胞分離mRNA並用於製造cDNA。所關注cDNA可以使用特定引子，藉由聚合酶鏈反應擴增(美國專利第4,683,195號及第4,683,202號)。或者，製備文庫且篩選以分離出所關注序列。接著使編碼抗體可變區之DNA序列與人類恆定區序列融合。人類恆定區基因之序列可以見於Kabat等人(1991) Sequences of Proteins of immunological Interest, N.I.H.出版號91-3242。人類C區基因易於自已知純系獲得。同型之選擇將由所需效應功能導引，諸如補體結合或在抗體依賴性細胞毒性中之活性。較佳之同型係IgG1、IgG2、IgG3及IgG4。可以使用人類輕鏈恆定區κ或λ中之任一者。接著，利用習知方法表現嵌合人類化抗體。 抗體片段，諸如Fv、F(ab')2 及Fab可以藉由裂解完整蛋白質，例如藉由蛋白酶或化學裂解來製備。或者，設計截短之基因。舉例而言，編碼一部分F(ab')2 片段之嵌合基因將包括編碼H鏈CH1結構域及鉸鏈區之DNA序列，隨後為轉譯終止密碼子，由此得到截短之分子。 可以使用H區、L區及J區之共同序列設計用作引子之寡核苷酸以將有用的限制性位點引入J區中，隨後將V區區段連接至人類C區區段。可以藉由定點突變誘發，在人類序列中之類似位置放入限制性位點來修飾C區cDNA。 表現載體包括質體、反轉錄病毒、YAC、EBV源性游離基因體及類似物。適宜載體係編碼功能完整之人類CH或CL免疫球蛋白序列且具有經工程改造以使得可以容易地插入及表現任何VH或VL序列之適當限制性位點的載體。在此類載體中，剪接通常在插入之J區中的剪接供體位點與在人類C區之前的剪接受體位點之間，以及出現在人類CH外顯子內之剪接區處發生。聚腺苷酸化及轉錄終止在編碼區下游之天然染色體位點處發生。所得嵌合抗體可以接合至任何強啟動子，包括反轉錄病毒LTR，例如SV-40早期啟動子(Okayama等人, Mol. Cell. Bio. 3:280 (1983))、勞斯肉瘤病毒LTR(Gorman等人, P.N.A.S. 79:6777 (1982))及莫洛尼鼠類白血病(moloney murine leukemia)病毒LTR(Grosschedl等人, Cell 41:885 (1985))。另外，應瞭解，可以使用天然Ig啟動子及類似物。 另外，可以經由展示型技術，包括(但不限於)噬菌體展示、反轉錄病毒展示、核糖體展示及其他技術，使用此項技術中熟知之技術產生人類抗體或來自其他物種之抗體，且所得分子可以經歷另外的成熟，諸如親和力成熟，此類技術亦為此項技術中熟知的。Wright等人, Crit, Reviews in Immunol. 12125-168 (1992)；Hanes及Plückthun PNAS USA 94:4937-4942 (1997) (核糖體展示)；Parmley及Smith Gene 73:305-318 (1988) (噬菌體展示)；Scott, TIBS, 第17:241-245卷 (1992)；Cwirla等人, PNAS USA 87:6378-6382 (1990)；Russel等人, Nucl. Acids Research 21:1081-1085 (1993)；Hoganboom等人, Immunol. Reviews 130:43-68 (1992)；Chiswell及McCafferty TIBTECH; 10:80-8A (1992)；及美國專利第5,733,743號。若利用展示技術來產生非人類抗體，則此類抗體可以如上文所描述進行人類化。 使用該等技術，可以產生針對PD-1表現細胞、PD-1之可溶性形式、其抗原決定基或肽及其表現文庫之抗體(參見例如，美國專利第5,703,057號)，之後可以如上文所描述，針對本文所描述之活性篩選該等表現文庫。 本發明之抗PD-1抗體可以藉由含有編碼上述單鏈抗體之DNA區段的載體表現。 該等載體可以包括載體、脂質體、裸DNA、佐劑輔助之DNA、基因槍、導管等。載體包括具有靶向部分(例如針對細胞表面受體之配體)及核酸結合部分(例如聚離胺酸)之化學結合物，諸如WO 93/64701中所述；病毒載體(例如DNA或RNA病毒載體)；融合蛋白，諸如PCT/US95/02140(WO 95/22618)中所述，該等融合蛋白係含有標靶部分(例如對標靶細胞具有特異性之抗體)及核酸結合部分(例如魚精蛋白)的融合蛋白；質體；噬菌體等。該等載體可以為染色體、非染色體或合成的。 較佳載體包括病毒載體、融合蛋白及化學結合物。反轉錄病毒載體包括莫洛尼鼠類白血病病毒。DNA病毒載體較佳。該等載體包括痘載體，諸如正痘或禽痘載體；疱疹病毒載體，諸如單純疱疹I病毒(HSV)載體(參見Geller, A. I.等人, J. Neurochem, 64:487 (1995)；Lim, F.等人, DNA Cloning: Mammalian Systems, D. Glover編(Oxford Univ. Press, Oxford England) (1995)；Geller, A. I.等人, Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993)；Geller, A. I.等人, Proc Natl. Acad. Sci USA 87:1149 (1990))、腺病毒載體(參見LeGal LaSalle等人, Science, 259:988 (1993)；Davidson等人, Nat. Genet 3:219 (1993)；Yang等人, J. Virol. 69:2004 (1995))及腺相關病毒載體(參見Kaplitt, M. G.等人, Nat. Genet. 8:148 (1994))。 痘病毒載體將基因引入細胞之細胞質中。禽痘病毒載體僅引起核酸之短期表現。對於將核酸引入神經細胞中而言，腺病毒載體、腺相關病毒載體及單純疱疹病毒(HSV)載體較佳。腺病毒載體引起之表現時間(約2個月)比腺相關病毒(約4個月)短，腺相關病毒又比HSV載體短。牛痘病毒載體能夠在眾多不同類型細胞中倍增。所選特定載體將取決於標靶細胞及所治療之病況。引入可以藉由標準技術，例如感染、轉染、轉導或轉型進行。基因轉移模式之實例包括例如裸DNA、CaPO4 沈澱、DEAE葡聚糖、電穿孔、原生質體融合、脂質體轉染、細胞顯微注射及病毒載體。 可以採用載體靶向基本上任何所需標靶細胞。舉例而言，可以使用立體定位注射將載體(例如腺病毒、HSV)引導至所需位置。另外，可以藉由腦室內(icv)輸注，使用小型泵輸注系統，諸如SynchroMed輸注系統遞送粒子。亦已證實基於總體流動(稱為對流)之方法在將大分子遞送至腦部延伸區域方面有效且可用於將載體遞送至標靶細胞(參見Bobo等人, Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994)；Morrison等人, Am. J. Physiol. 266:292-305 (1994))。其他可以使用的方法包括導管、靜脈內、非經腸、腹膜內及皮下注射，以及口服或其他已知之投藥途徑。 可以使用該等載體表現大量抗體，該等抗體可以多種方式使用。例如，用以偵測樣品中PD-1之存在。亦可使用抗PD-1抗體嘗試結合至PD-1及破壞PD-1介導之信號傳導。 技術可以適合於製造對本發明之抗原蛋白質具有特異性的單鏈抗體(參見例如，美國專利第4,946,778號)。另外，方法可適合於構築Fab表現文庫(參見例如Huse等人, 1989 Science 246: 1275-1281)以便快速且有效地鑑別對一種蛋白質或其衍生物、片段、類似物或同源物具有所需特異性的單株Fab片段。可以藉由此項技術中已知之技術製造含有針對蛋白質抗原之個體基因型的抗體片段，包括(但不限於)：(i)藉由胃蛋白酶消化抗體分子產生的F(ab')2 片段；(ii)藉由還原F( ab ') 2 片段之二硫橋鍵產生的Fab片段；(iii)藉由用木瓜蛋白酶及還原劑處理抗體分子產生的Fab片段；及(iv) Fv 片段。 本發明亦包括Fv 、Fab、Fab'及F(ab')2 抗PD-1片段、單鏈抗PD-1抗體、單結構域抗體(例如奈米抗體或VHH)、多特異性(諸如雙特異性)抗PD-1抗體及異結合抗PD-1抗體。 用於製備雙特異性抗體之方法係此項技術中已知的。傳統上，雙特異性抗體之重組製造係基於共表現兩個免疫球蛋白重鏈/輕鏈對，其中該兩個重鏈具有不同特異性(Milstein及Cuello, Nature, 305:537-539 (1983))。由於免疫球蛋白重鏈及輕鏈之隨機分類，此等融合瘤(四源融合瘤)產生十種不同抗體分子之潛在混合物，其中僅一種具有適當的雙特異性結構。適當分子之純化通常係藉由親和層析步驟實現。類似程序揭示於1993年5月13日公開之WO 93/08829及Traunecker等人, EMBO J., 10:3655-3659 (1991)中。 具有所需結合特異性之抗體可變結構域(抗體-抗原組合位點)可與免疫球蛋白恆定結構域序列融合。較佳與包含鉸鏈、CH2及CH3區之至少一部分的免疫球蛋白重鏈恆定結構域進行融合。較佳使含有輕鏈結合所需位點之第一重鏈恆定區(CH1)存在於至少一種融合物中。將編碼免疫球蛋白重鏈融合物及必要時免疫球蛋白輕鏈之DNA插入獨立表現載體中，且共轉染至適合宿主生物體中。有關產生雙特異性抗體之其他細節，參見例如Suresh等人, Methods in Enzymology, 121:210 (1986)。 根據WO 96/27011中描述之另一種方法，抗體分子對之間的界面可經工程改造以使自重組細胞培養物回收之異二聚體之百分比最大。較佳界面包含抗體恆定結構域CH3區之至少一部分。在此方法中，來自第一抗體分子界面之一或多個小胺基酸側鏈經較大側鏈(例如酪胺酸或色胺酸)置換。在第二抗體分子的界面上，藉由用較小胺基酸側鏈(例如丙胺酸或蘇胺酸)置換較大胺基酸側鏈產生大小與較大側鏈相同或類似的補償性「空腔」。由此提供了使異二聚體產量增加超過其他非所需最終產物(諸如同二聚體)的機制。 雙特異性抗體可以全長抗體或抗體片段形式製備。自抗體片段產生雙特異性抗體之技術已描述於文獻中。舉例而言，雙特異性抗體可使用化學鍵聯製備。Brennan等人, Science 229:81 (1985)描述一種程序，其中將完整抗體蛋白水解裂解以產生F(ab')2 片段。該等片段在二硫醇錯合劑亞砷酸鈉存在下還原以使鄰近二硫醇穩定並阻止分子間二硫化物形成。接著將所產生之Fab'片段轉化為硫代硝基苯甲酸酯(TNB)衍生物。接著，藉由巰基乙胺還原使Fab'-TNB衍生物之一再轉化為Fab'-硫醇且與等莫耳量的其他Fab'-TNB衍生物混合以形成雙特異性抗體。所產生之雙特異性抗體可用作選擇性固定酶之試劑。 另外，可以自大腸桿菌直接回收Fab'片段且以化學方式偶合以形成雙特異性抗體。Shalaby等人, J. Exp. Med. 175:217-225 (1992)描述完全人類化雙特異性抗體F(ab')2 分子之製造。各Fab'片段獨立地自大腸桿菌分泌且經歷活體外定向化學偶合而形成雙特異性抗體。 亦已描述直接自重組細胞培養物製備及分離雙特異性抗體片段之各種技術。舉例而言，已使用白胺酸拉鏈產生雙特異性抗體。Kostelny等人, J. Immunol. 148(5):1547-1553 (1992)。來自Fos及Jun蛋白質的白胺酸拉鏈肽藉由基因融合而連接至兩種不同抗體的Fab'部分。抗體同二聚體在鉸鏈區還原而形成單體且接著再氧化而形成抗體異二聚體。此方法亦可用於產生抗體同二聚體。Hollinger等人,Proc. Natl. Acad. Sci. USA 90:6444-6448(1993)所描述的「雙功能抗體」技術提供了用於製備雙特異性抗體片段之替代機制。該等片段包含藉由連接子連接至輕鏈可變結構域(VL )的重鏈可變結構域(VH )，該連接子太短而不允許同一鏈上的兩個結構域之間配對。因此，迫使一個片段之VH 及VL 結構域與另一片段之互補VL 及VH 結構域配對，由此形成兩個抗原結合位點。亦已報導藉由使用單鏈Fv(scFv)二聚體製備雙特異性抗體片段的另一種策略。參見Gruber等人, J. Immunol. 152:5368 (1994)。 涵蓋具有兩價以上之抗體。舉例而言，可以製備三特異性抗體。參見例如，Tutt等人, J. Immunol. 147:60 (1991)。 例示性雙特異性抗體可以結合至兩個不同的抗原決定基，其中至少一個來源於本發明之蛋白質抗原中。或者，免疫球蛋白分子之抗-抗原臂可以與結合至白細胞上之觸發分子，諸如T細胞受體分子(例如CD2、CD3、CD28或B7)，或IgG之Fc受體(FcγR)，諸如FcγRI(CD64)、FcγRII(CD32)及FcγRIII(CD16)的臂組合，由此將細胞防禦機構集中於表現特定抗原之細胞。亦可使用雙特異性抗體將細胞毒性劑引導至表現特定抗原之細胞。此等抗體具有抗原結合臂及結合細胞毒性劑或放射性核種螯合劑(諸如EOTUBE、DPTA、DOTA或TETA)之臂。 異結合物抗體亦在本發明之範疇內。異結合物抗體係由兩個共價接合之抗體構成。舉例而言，已提出此類抗體可使免疫系統細胞靶向不想要的細胞(參見美國專利第4,676,980號)且用於治療HIV感染(參見WO 91/00360、O 92/200373、EP 03089)。預期該等抗體可在活體外使用合成蛋白質化學中之已知方法(包括涉及交聯劑之方法)製備。舉例而言，可使用二硫化物交換反應或藉由形成硫醚鍵來構築免疫毒素。適於此目的的試劑之實例包括亞胺基硫醇酯及4-巰基丁醯亞胺甲酯及揭示於例如美國專利第4,676,980號中之試劑。 可需要針對效應功能修飾本發明抗體以便增強例如抗體治療異常PD-1信號傳導相關疾病及病症之有效性。舉例而言，可將半胱胺酸殘基引入Fc區中，由此允許在此區域中形成鏈間二硫鍵。由此產生的同二聚抗體可以具有改良的內化能力及/或增加的補體介導之細胞殺滅作用及抗體依賴性細胞毒性(ADCC)(參見Caron等人, J. Exp Med., 176: 1191-1195 (1992)及Shopes, J. Immunol., 148: 2918-2922 (1992))。或者，抗體可經工程改造以具有雙Fc區且因此可具有增強之補體溶解及ADCC能力(參見Stevenson等人, Anti-Cancer Drug Design, 3: 219-230 (1989))。 本發明亦關於免疫結合物，其包含抗體與細胞毒性劑諸如毒素(例如細菌、真菌、植物或動物來源之酶活性毒素，或其片段)、放射性同位素(亦即，放射性結合物)或藉由成像偵測在患者樣品中或在活體內之標靶抗原(諸如PD-1)的標記(例如放射性同位素、螢光染料及酶)之結合物。 可使用之酶活性毒素及其片段包括白喉A鏈、白喉毒素之非結合活性片段、外毒素A鏈(來自綠膿桿菌(Pseudomonas aeruginosa))、篦麻毒素A鏈、相思子毒素A鏈、莫迪素A鏈(modeccin A chain)、α-帚麴菌素(alpha-sarcin)、油桐(Aleurites fordii)蛋白、康乃馨蛋白、美洲商陸(Phytolaca americana)蛋白(PAPI、PAPII及PAP-S)、苦瓜(momordica charantia)抑制劑、麻瘋樹毒蛋白(curcin)、巴豆毒素(crotin)、肥皂草(sapaonaria officinalis)抑制劑、白樹素(gelonin)、有絲分裂素(mitogellin)、侷限麴菌素(restrictocin)、酚黴素、伊諾黴素(enomycin)及黴菌毒素(tricothecene)。多種放射性核種可用於產生放射性結合之抗體。實例包括212 Bi、131 I、131 In、90 Y及186 Re。 抗體與細胞毒性劑之結合物係使用多種雙官能蛋白質偶合劑來製備，該等蛋白質偶合劑諸如N-琥珀醯亞胺基-3-(2-吡啶基二硫醇)丙酸酯(SPDP)、亞胺基硫雜環戊烷(IT)、醯亞胺酯之雙官能衍生物(諸如二亞胺代己二酸二甲酯鹽酸鹽)、活性酯(諸如辛二酸二琥珀醯亞胺酯)、醛(諸如戊二醛)、雙疊氮基化合物(諸如雙(對疊氮基苯甲醯基)己二胺)、雙重氮鎓衍生物(諸如雙-(對重氮鎓苯甲醯基)-乙二胺)、二異氰酸酯(諸如2,6-二異氰酸甲苯酯)及雙活性氟化合物(諸如1,5-二氟-2,4-二硝基苯)。舉例而言，蓖麻毒素免疫毒素可如Vitetta等人, Science 238: 1098 (1987)中所述製備。碳-14標記之1-異硫氰基苯甲基-3-甲基二伸乙三胺五乙酸(MX-DTPA)係用於將放射性核苷酸結合至抗體之例示性螯合劑。(參見WO94/11026)。 一般熟習此項技術者應認識到，可以將多種可能的部分偶合至所得本發明之抗體(參見例如，「Conjugate Vaccines」, Contributions to Microbiology and Immunology, J. M. Cruse及R. E. Lewis, Jr (編), Carger Press, New York, (1989)，其完整內容以引用之方式併入本文中)。 偶合可以藉由將結合兩個分子之任何化學反應實現，只要該抗體與另一部分保持其對應的活性即可。此連接可以包括許多化學機制，例如共價結合、親和力結合、插入、配位結合及錯合。不過，較佳結合係共價結合。共價結合可以藉由現有側鏈之直接縮合或藉由併入外部橋連分子實現。許多二價或多價連接劑可用於將蛋白質分子，諸如本發明之抗體偶合至其他分子。舉例而言，代表性偶合劑可以包括有機化合物，諸如硫酯、碳化二亞胺、琥珀醯亞胺酯、二異氰酸酯、戊二醛、重氮苯及六亞甲基二胺。預期此清單並非此項技術中已知之各種類別偶合劑的詳盡清單，而實際上為較常見之偶合劑的示例(參見Killen及Lindstrom, Jour. Immun. 133:1335-2549 (1984)；Jansen等人, Immunological Reviews 62:185-216 (1982)；及Vitetta等人, Science 238:1098 (1987))。 較佳連接子描述於文獻中(參見例如Ramakrishnan, S.等人, Cancer Res. 44:201-208 (1984)描述使用間順丁烯二醯亞胺基苯甲醯基-N-羥基琥珀醯亞胺酯(MBS))。亦參見美國專利第5,030,719號，描述使用鹵代乙醯基醯肼衍生物藉助於寡肽連接子偶合至抗體。尤佳連接子包括：(i)EDC (1-乙基-3-(3-二甲胺基-丙基)碳化二亞胺鹽酸鹽；(ii)SMPT(4-琥珀醯亞胺基氧基羰基-α-甲基-α-(2-吡啶基-二硫基)-甲苯(Pierce Chem.Co., 目錄號(21558G)；(iii)SPDP(琥珀醯亞胺基-6[3-(2-吡啶基二硫基)丙醯胺基]己酸酯(Pierce Chem.Co., 目錄號21651G)；(iv)磺基-LC-SPDP(磺基琥珀醯亞胺基6[3-(2-吡啶基二硫基)-丙醯胺]己酸酯(Pierce Chem. Co., 目錄號2165-G)；及(v)結合至EDC的磺基-NHS(N-羥基磺基-琥珀醯亞胺：Pierce Chem. Co., 目錄號24510)。 上述連接子含有具有不同屬性之組分，由此產生具有不同物理化學特性之結合物。舉例而言，烷基羧酸之磺基-NHS酯的穩定性大於芳族羧酸之磺基-NHS酯。含有NHS-酯之連接子溶解性低於磺基-NHS酯。另外，連接子SMPT含有位阻二硫鍵，且可以形成具有增加之穩定性的結合物。二硫化物鍵聯一般不如其他鍵聯穩定，因為該二硫化物鍵聯在活體外裂解，使得不易獲得結合物。特定言之，磺基-NHS可以增強碳化二亞胺偶合之穩定性。碳化二亞胺偶合(諸如EDC)當結合磺基-NHS使用時，形成的酯對水解之抗性大於單獨碳化二亞胺偶合反應。 本文所揭示之抗體亦可調配成免疫脂質體形式。含有抗體之脂質體係藉由此項技術中已知之方法製備，諸如Epstein等人, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985)；Hwang等人, Proc. Natl Acad. Sci. USA, 77: 4030 (1980)；及美國專利第4,485,045號及第4,544,545號中所述。具有延長之循環時間的脂質體揭示於美國專利第5,013,556號中。 特別有用之脂質體可藉由逆相蒸發法，用包含磷脂醯膽鹼、膽固醇及PEG衍生化之磷脂醯乙醇胺(PEG-PE)的脂質組合物產生。脂質體經具有確定孔徑之過濾器擠出以產生具有所需直徑之脂質體。 針對PD-1之抗體之用途 應瞭解，根據本發明之治療性實體之投與將與適合載劑、賦形劑及併入調配物中以提供經改良之轉移、遞送、耐受性及類似者的其他試劑一起投與。多種適當的調配物可以見於所有醫藥化學家已知之處方集中：Remington's Pharmaceutical Sciences (第15版, Mack Publishing Company, Easton, PA (1975))，特別是其中Blaug, Seymour之第87章。此等調配物包括例如散劑、糊劑、軟膏、凝膠劑、蠟、油、脂質、含有囊泡之脂質(陽離子型或陰離子型) (諸如Lipofectin™)、DNA結合物、無水吸收糊劑、水包油及油包水乳液、乳液卡波蠟(emulsions carbowax) (具有各種分子量之聚乙二醇)、半固體凝膠及含有卡波蠟之半固體混合物。前述混合物中之任一者可以適用於根據本發明之治療及療法中，條件為調配物中之活性成分未因該調配物而失活且該調配物係生理上相容的且可耐受投藥途徑。有關醫藥化學家熟知之調配物、賦形劑及載劑之其他資訊，亦參見Baldrick P. 「Pharmaceutical excipient development: the need for preclinical guidance.」 Regul. Toxicol Pharmacol. 32(2):210-8 (2000)；Wang W. 「Lyophilization and development of solid protein pharmaceuticals.」 Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN 「Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.」 J Pharm Sci. 89(8):967-78 (2000)；Powell等人, 「Compendium of excipients for parenteral formulations」 PDA J Pharm Sci Technol. 52:238-311 (1998)及其中之引述。 在一些實施例中，本發明之抗PD-1抗體可以用作治療劑。此類試劑一般將用於診斷、預後、監測、治療、緩解及/或預防受試者之與異常PD-1或PD-L1/PD-L2表現、活性及/或信號傳導有關的疾病或病變。治療方案係藉由使用標準方法鑑別患有與異常PD-1或PD-L1/PD-L2表現、活性及/或信號傳導相關之疾病或病症(或有患該疾病或病症之風險)，例如癌症或其他贅生性病症之受試者，例如人類患者來進行。將抗體製劑，較佳對其標靶抗原具有高特異性及高親和力之抗體製劑投與受試者，且一般將由於其與標靶結合而具有作用。該抗體之投與可以廢除或抑制或干擾標靶(例如PD-1)之表現、活性及/或信號傳導功能。該抗體之投與可以廢除或抑制或干擾標靶(例如PD-1)與其天然結合之內源性配體的結合。舉例而言，該抗體結合至標靶且調節、阻斷、抑制、降低、拮抗、中和或以其他方式干擾PD-1表現、活性及/或信號傳導。 與異常PD-1或PD-L1/PD-L2表現、活性及/或信號傳導有關之疾病或病症包括(作為非限制性實例)血液癌症及/或實體腫瘤。可使用本發明抗體抑制生長之癌症包括通常對免疫療法起反應之癌症。供治療之癌症之實例包括(但不限於)黑素瘤(例如轉移性惡性黑色素瘤)、腎癌(例如透明細胞癌瘤)、前列腺癌(例如激素難治性前列腺腺癌)、乳癌、結腸癌、肝癌及肺癌(例如非小細胞肺癌)。本發明亦可用於治療轉移性癌症，尤其是表現PD-L1之轉移性癌症(Iwai等人(2005) Int. Immunol. 17:133-144)。 視情況，針對PD-1之抗體可與以下免疫原性試劑組合：諸如癌細胞、經純化之腫瘤抗原(包括重組蛋白質、肽及碳水化合物分子)、細胞及經編碼免疫刺激性細胞介素之基因轉染的細胞(He等人(2004) J. Immunol. 173:4919-28)。可使用之腫瘤疫苗之非限制性實例包括黑素瘤抗原之肽，諸如gp100、MAGE抗原、Trp-2、MART1及/或酪胺酸酶之肽，或經轉染以表現細胞介素GM-CSF之腫瘤細胞。本發明之抗PD-1抗體亦可與本文所描述之雙特異性接合分子，諸如包含特異性識別腫瘤抗原(諸如EpCAM、FAP、EGFR或GPC3)之第一抗原結合結構域(諸如scFv)及特異性識別效應細胞上之細胞表面分子(諸如T淋巴細胞上之CD3)之第二抗原結合結構域(諸如scFv)的雙特異性分子組合。 與癌症及其他贅生性病症相關之症狀包括例如炎症、發熱、全身不適、發熱、疼痛(通常侷限於發炎區域)、食慾不振、體重減輕、水腫、頭痛、疲勞、皮疹、貧血、肌無力、肌肉疲勞，及腹部症狀，諸如腹痛、腹瀉或便秘。 本發明抗體之治療有效量一般係指達成治療目的所需之量。如上所指出，此可為抗體與其標靶抗原之間之結合相互作用，在某些情況下，該相互作用干擾標靶之功能。需要投與之量另外將取決於抗體對其特異性抗原之結合親和力，且亦將取決於所投與之抗體自投與其之自由體積其他受試者耗竭之速率。本發明之抗體或抗體片段之治療有效劑量的常見範圍可以為(作為非限制性實例)約0.1 mg/kg體重至約100 mg/kg體重。常用給藥頻率可以例如在每天兩次至一週一次或兩週一次之範圍內。 治療之有效性係結合用於診斷或治療特定炎性相關病症之任何已知方法測定。該炎性相關病症之一或多種症狀的緩解指示該抗體提供臨床益處。 在另一個實施例中，針對PD-1之抗體可以用於此項技術內已知與PD-1定位及/或定量相關之方法中(例如用於量測適當生理樣品內PD-1之含量、用於診斷方法中、用於使蛋白質成像及類似方法)。在給定實施例中，利用含有抗體源性抗原結合結構域的對PD-1具有特異性之抗體或其衍生物、片段、類似物或同源物作為藥理學活性化合物(在下文中稱為「治療劑」)。 在另一個實施例中，可以使用對PD-1具有特異性之抗體，藉由標準技術，諸如免疫親和層析法或免疫沈澱分離PD-1多肽。針對PD-1蛋白質(或其片段)之抗體可以在診斷上用於監測組織中之蛋白質含量作為臨床測試程序之一部分，例如以確定給定治療方案之功效。可以藉由將該抗體與可偵測物質偶合(亦即，以物理方式連接)來幫助偵測。可偵測物質之實例包括各種酶、輔基、螢光物質、發光物質、生物發光物質及放射性物質。適合酶之實例包括辣根過氧化酶、鹼性磷酸酶、β-半乳糖苷酶或乙醯膽鹼酯酶；適合輔基複合物之實例包括抗生蛋白鏈菌素/生物素及抗生物素蛋白/生物素；適合螢光物質之實例包括傘酮、螢光素、異硫氰酸螢光素、若丹明、二氯三嗪基胺螢光素、丹磺醯氯或藻紅素；發光物質之實例包括魯米諾(luminol)；生物發光物質之實例包括螢光素酶、螢光素及發光蛋白質；且適合放射性物質之實例包括125 I、131 I、35 S或3 H。 在一些實施例中，根據本發明之抗PD-1抗體可以用作偵測樣品中PD-1(或其蛋白質片段)之存在的試劑。在一些實施例中，該抗體含有可偵測標記。抗體係多株抗體，或更佳為單株抗體。使用完整抗體或其片段(例如scFv)。關於探針或抗體之術語「標記」意圖涵蓋藉由將可偵測物質偶合(亦即，以物理方式連接)至探針或抗體進行的探針或抗體直接標記，以及藉由與經直接標記之另一試劑反應進行的探針或抗體間接標記。間接標記之實例包括使用螢光標記之二次抗體偵測一次抗體，及用生物素對DNA探針末端標記以使其可用螢光標記之抗生蛋白鏈菌素偵測。術語「生物樣品」意欲包括自受試者分離的組織、細胞及生物流體，以及存在於受試者體內之組織、細胞及流體。因此，在術語「生物樣品」之用法內包括血液及血液之成分或組分，包括血清、血漿，或淋巴。亦即，可以使用本發明之偵測方法在活體外在生物樣品中以及在活體內偵測分析物mRNA、蛋白質或基因組DNA。舉例而言，用於偵測分析物mRNA之活體外技術包括Northern雜交及原位雜交。用於偵測分析物蛋白質之活體外技術包括酶聯結免疫吸附劑分析法(ELISA)、西方墨點法(Western blot)、免疫沈澱法及免疫螢光法。用於偵測分析物基因組DNA之活體外技術包括Southern雜交。用於進行免疫分析法之程序描述於例如「ELISA: Theory and Practice: Methods in Molecular Biology」, 第42卷, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995；「Immunoassay」, E. Diamandis及T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996；及「Practice and Theory of Enzyme Immunoassays」, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985。另外，用於偵測分析物蛋白質之活體內技術包括將經標記之抗分析物蛋白質抗體引入受試者體內。舉例而言，可以用放射性標記物對抗體標記，該放射性標記物在受試者體內之存在及位置可以藉由標準成像技術偵測。 在一些實施例中，個體係哺乳動物。哺乳動物之實例包括(但不限於)人類、猴、大鼠、小鼠、倉鼠、天竺鼠、狗、貓、兔、豬、綿羊、山羊、馬、牛及類似哺乳動物。在一些實施例中，個體係人類。 亦提供一種醫藥組合物，其包含有效量之本文所述之抗PD-1抗體、表現本文所述之抗PD-1抗體之宿主細胞或編碼本文所述之抗PD-1抗體之溶瘤病毒(諸如溶瘤VV)，及視情況醫藥學上可接受之載劑。 亦提供一種治療個體之癌症之方法，其包含向個體投與有效量之醫藥組合物，該醫藥組合物包含本文所述之抗PD-1抗體、表現本文所述之抗PD-1抗體之宿主細胞或編碼本文所述之抗PD-1抗體之溶瘤病毒(諸如溶瘤VV)，及視情況醫藥學上可接受之載劑。治療作用可以包括(但不限於)殺滅癌細胞、抑制癌細胞增殖、誘導周圍T細胞之再分佈、誘導腫瘤中之免疫反應、減小腫瘤大小、抑制腫瘤轉移、降低先前存在之腫瘤轉移(諸如淋巴結轉移)之發生率或負擔、延長個體之存活期、延長癌症進展之時間等。在一些實施例中，該醫藥組合物係經靜脈內或腫瘤內投與個體。在一些實施例中，個體係人類。 V. 製備方法 本文所描述的抗PD-1抗體(其抗原結合片段)、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白可以藉由此項技術中已知之蛋白質表現及純化方法中之任一種製備。 在一些實施例中，本申請案提供編碼以下任一種之多肽鏈中之一或多種的分離之核酸：本文所描述之抗PD-1抗體(或其抗原結合片段)、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白。 在一些實施例中，該分離之核酸包含第一核酸序列SEQ ID NO: 17及第二核酸序列SEQ ID NO: 18。該等分離之核酸可以為DNA或RNA。在一些實施例中，本文所描述之分離之核酸係可操作地連接至啟動子。在一些實施例中，該啟動子係晚期啟動子。在一些實施例中，該啟動子係VV啟動子。在一些實施例中，該啟動子係VV晚期啟動子。在一些實施例中，該啟動子係F17R。 在一些實施例中，該分離之核酸包含第一核酸序列SEQ ID NO: 19及第二核酸序列SEQ ID NO: 20。該等分離之核酸可以為DNA或RNA。在一些實施例中，本文所描述之分離之核酸係可操作地連接至啟動子。在一些實施例中，該啟動子係晚期啟動子。在一些實施例中，該啟動子係VV啟動子。在一些實施例中，該啟動子係VV晚期啟動子。在一些實施例中，該啟動子係F17R。 在一些實施例中，將分離之核酸插入載體，諸如表現載體、病毒載體(諸如溶瘤VV載體)或選殖載體中。為表現該等核酸，可以將載體引入宿主細胞中以允許在該宿主細胞內表現該等核酸。該等表現載體可以含有多種用於控制表現之元件，包括(但不限於)啟動子序列、轉錄起始序列、強化子序列、可選擇標記物及信號序列。適當時，該等元件可由一般熟習此項技術者選擇。舉例而言，啟動子序列可經選擇以促進聚核苷酸在載體中之轉錄。適合啟動子序列包括(但不限於)T7啟動子、T3啟動子、SP6啟動子、β-肌動蛋白啟動子、EF1a啟動子、CMV啟動子、SV40啟動子及牛痘病毒啟動子(諸如F17R)。強化子序列可經選擇以增進核酸之轉錄。可選擇標記物可經選擇以允許自不含該載體之宿主細胞中選出經插入而含有該載體之宿主細胞，例如可選擇標記物可以為賦予抗生素抗性之基因。信號序列可經選擇以允許將表現之多肽運輸至宿主細胞外。在一些實施例中，分離之核酸進一步包含編碼信號肽之核酸序列。 在一些實施例中，編碼免疫檢查點調節子(及/或雙特異性接合分子、細胞介素，諸如本文所描述者)的溶瘤病毒係牛痘病毒。牛痘病毒由於若干特徵而在癌症基因療法中引起關注。其對癌細胞具有天然的趨向性且藉由缺失部分病毒基因可以顯著增強選擇性。在一些實施例中，編碼免疫檢查點調節子(及/或雙特異性接合分子、細胞介素，諸如本文所描述者)的溶瘤病毒(諸如溶瘤VV)包含胸苷激酶(TK)基因及牛痘病毒生長因子(VGF)基因之雙重缺失(vvDD病毒株)。TK及VGF基因係病毒在正常細胞複製所需的，但在癌細胞中複製不需要。可以分別在賦予活性的TK或VGF區域中工程改造成TK或VGF缺失。舉例而言，此可以藉由將含有免疫檢查點調節子(及/或雙特異性接合分子、細胞介素，諸如本文所描述者)之pSEM-1穿梭質體重組於Western Reserve牛痘病毒(WR VV)之VSC20病毒株(缺失VGF之病毒株)的TK基因中來產生。VSC20病毒株可以藉由將處於p11啟動子控制下之lacZ基因插入病毒VGF基因之兩個複本中，由此使VGF失活來構築。構築的穿梭載體pSEM-1可以在T細胞活化之前允許充分病毒複製的啟動子，諸如F17R晚期啟動子之轉錄控制下表現免疫檢查點調節子(及/或雙特異性接合分子、細胞介素，諸如本文所描述者)。在一些實施例中，VV可以進一步表現標記物，諸如DsRed2、YFP、GFP或YFP-GFP，以允許病毒選擇。在一些實施例中，感染性監測標記物可以在與驅動免疫檢查點調節子(及/或本文所描述之雙特異性接合分子)表現相同之啟動子的轉錄控制下表現。在一些實施例中，病毒選擇標記物可以在不同啟動子，諸如Pse/I啟動子或P7.5啟動子之轉錄控制下表現。在一些實施例中，病毒選擇標記物可以相同取向側接loxP位點。為了構築編碼免疫檢查點調節子(及/或雙特異性接合分子，細胞介素，諸如本文所描述者)之重組病毒，可以將含有免疫檢查點調節子(及/或雙特異性接合分子、細胞介素，諸如本文所描述者)之穿梭載體pSEM-1轉染至人類143 TK細胞中。接著，可以用感染倍率(MOI)為0.1之病毒VSC20感染細胞。在3至5(例如五)輪蝕斑選擇及擴增並證實免疫檢查點調節子(及/或雙特異性接合分子、細胞介素，諸如本文所描述者)之表現之後，可選出一個純系進行擴增及純化。在一些實施例中，可以自重組病毒移除選擇標記物，例如YFP-GFP卡匣。舉例而言，在一些實施例中，可以使病毒在表現Cre重組酶之細胞質形式的U2OS細胞株(U2OS-Cre)上傳代。在3至5(例如五)輪蝕斑選擇及擴增以證實免疫檢查點調節子(及/或雙特異性接合分子、細胞介素，諸如本文所描述者)之表現之後，可選出一個可選擇標記物陰性(例如YFP-GFP陰性)純系進行擴增及純化。 熟習此項技術者應認識到，可以使用任何適合的方法在所關注基因中產生失活突變，包括突變誘發、聚合酶鏈反應、同源重組或熟習此項技術者已知之任何其他基因工程改造技術。突變可以涉及核苷酸序列、單一基因或數組基因之修飾。突變可以涉及單一核苷酸(諸如點突變，其涉及DNA序列內單一核苷酸鹼基之移除、添加或取代)或其可以涉及大量核苷酸之插入或缺失。突變可以作為諸如DNA複製保真度之誤差之事件的結果而自發地發生，或在暴露於化學或物理誘變劑之後誘導。突變亦可經由使用熟習此項技術者熟知之特定靶向方法定點進行。 獲得的本發明病毒可以藉由用於病毒複製之習知方法，例如用該病毒感染宿主細胞(諸如293細胞)進行複製。 據設想，出於其他目的，溶瘤病毒核酸分子可以含有例如硫酯鍵及/或核苷酸類似物。修飾可以用於使核酸分子針對細胞中之內切核酸酶及/或外切核酸酶穩定化。核酸分子可以藉由包含允許該核酸分子在細胞中轉錄之嵌合基因的適當溶瘤載體轉錄。就此而言，亦應理解，此類聚核苷酸可以用於「基因靶向」或「基因治療」方法。核酸分子亦可經標記。用於偵測核酸之方法係此項技術中熟知的，例如Southern及Northern墨點法、PCR或引子延伸。此實施例可以用於篩選方法中以驗證例如在基因療法方法期間成功引入上述核酸分子。 在一些實施例中，提供一種分離之宿主細胞，其包含編碼上述抗PD-1抗體(或其抗原結合片段)的分離之核酸，編碼上述PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白的分離之核酸。包含本文所描述之分離之核酸的宿主細胞可用於表現或選殖本文所描述的抗PD-1抗體(或其抗原結合片段)、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白。適合宿主細胞可以包括(但不限於)原核細胞、真菌細胞、酵母細胞，或高級真核細胞，諸如哺乳動物細胞。此項技術中充分確定抗體及抗原結合片段在諸如大腸桿菌之原核細胞中的表現。有關評述，參見例如Pluckthun, A. BioTechnology 9: 545-551 (1991)。熟習此項技術者亦可用在培養中於真核細胞中表現作為製造抗體或其抗原結合片段之選擇，參見近期評述，例如Ref, M. E. (1993) Curr. Opinion Biotech. 4: 573-576；Trill J. J.等人(1995) Curr. Opinion Biotech 6: 553-560。高級真核細胞，特別是來源於多細胞生物體之細胞可以用於表現糖基化多肽。適合的高級真核細胞包括(但不限於)無脊椎動物細胞及昆蟲細胞，以及脊椎動物細胞。 載體可以使用此項技術中已知之任何適合方法引入宿主細胞中，包括(但不限於)DEAE-葡聚糖介導之遞送、磷酸鈣沈澱法、陽離子性脂質介導之遞送、脂質體介導之轉染、電穿孔、微彈轟擊、受體介導之基因遞送，由聚離胺酸、組蛋白、殼聚糖及肽介導之遞送。用於轉染及轉型細胞以表現所關注載體之標準方法係此項技術中熟知的。在一些實施例中，宿主細胞包含編碼第一多肽之第一載體及編碼第二多肽之第二載體。在一些實施例中，宿主細胞包含單一載體，該載體包含編碼第一多肽及第二多肽的分離之核酸。 在一些實施例中，本申請案提供表現本文所描述的抗PD-1抗體(或其抗原結合片段)、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白及/或雙特異性接合分子中之任一種的方法，其包含培養含有載體的分離之宿主細胞並自細胞培養物回收該抗PD-1抗體(或其抗原結合片段)、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白，及/或雙特異性接合分子。該等分離之宿主細胞係在允許表現插入載體中之分離之核酸之條件下培養。用於表現聚核苷酸之適合條件可以包括(但不限於)適合培養基、培養基中適合的宿主細胞密度、所需養分之存在、補充因子之存在、適合溫度及濕度，以及不存在微生物污染物。一般熟習此項技術者可以選擇適於該表現目的之適合條件。 在一些實施例中，宿主細胞中表現之多肽可以形成二聚體且因此產生本文所描述之抗PD-1抗體(或其抗原結合片段)及/或雙特異性接合分子。在一些實施例中，宿主細胞中表現之多肽可以形成多肽複合物，該複合物係同二聚體。在宿主細胞表現第一聚核苷酸及第二聚核苷酸的一些實施例中，該第一聚核苷酸與該第二聚核苷酸可以形成多肽複合物，該複合物係異二聚體。 在一些實施例中，多肽複合物(諸如雙特異性接合分子或抗PD-1抗體或其抗原結合片段)可在宿主細胞內部形成。舉例而言，可以藉助於相關酶及/或輔因子在宿主細胞內部形成二聚體。在一些實施例中，可以自細胞分泌出多肽複合物。在一些實施例中，可以自宿主細胞分泌出第一多肽及第二多肽且在宿主細胞外部形成二聚體。 在一些實施例中，第一多肽及第二多肽可以分開表現且使其在適合條件下二聚合以形成雙特異性接合分子或抗PD-1抗體(或其抗原結合片段)。舉例而言，第一多肽及第二多肽可以在適合緩衝液中組合且使第一蛋白質單體與第二蛋白質單體經由適當相互作用，諸如疏水相互作用二聚合。在一些實施例中，第一多肽及第二多肽可以在含有能促進第一多肽與第二多肽二聚合之酶及/或輔因子的適合緩衝液中組合。在一些實施例中，第一多肽及第二多肽可以在適合媒劑中組合且使其在適合試劑及/或催化劑存在下彼此反應。 所表現之多肽及/或多肽複合物可以使用任何適合方法收集。多肽及/或多肽複合物可以在細胞內、周質空間中表現或在細胞外分泌至培養基中。若多肽及/或多肽複合物係在細胞內表現，則可以將含有多肽及/或多肽複合物之宿主細胞溶解且可以藉由離心或超過濾移除不想要之碎片，自溶解產物分離出多肽及/或多肽複合物。若多肽及/或多肽複合物係分泌至大腸桿菌之周質空間中，則可以在諸如乙酸鈉(pH 3.5)、EDTA及苯甲基磺醯基氟(PMSF)之試劑存在下解凍細胞糊狀物，保持約30分鐘，且可以藉由離心移除細胞碎片(Carter等人, BioTechnology 10:163-167 (1992))。若多肽及/或多肽複合物係分泌至培養基中，則可以使用可商購之蛋白質濃縮過濾器，例如Amincon或Millipore Pellicon超過濾裝置收集及濃縮細胞培養物之上清液。在收集及濃縮步驟中可以包括蛋白酶抑制劑及/或抗生素以抑制蛋白質降解及/或受污染微生物之生長。 所表現之多肽及/或多肽複合物可以藉由適合方法進一步純化，諸如(但不限於)親和層析法、羥磷灰石層析法、尺寸排阻層析法、凝膠電泳、透析、在離子交換管柱上進行之離子交換部分分離、乙醇沈澱、逆相HPLC、二氧化矽層析法、在肝素瓊脂糖上進行之層析法、在陰離子或陽離子交換樹脂(諸如聚天冬胺酸管柱)上進行之層析法、層析聚焦、SDS-PAGE及硫酸銨沈澱(有關評述，參見Bonner, P. L., Protein purification, 由Taylor & Francis出版, 2007；Janson, J. C.等人, Protein purification: principles, high resolution methods and applications, 由Wiley-VCH出版, 1998)。 在一些實施例中，多肽及/或多肽二聚體複合物可以藉由親和層析法純化。在一些實施例中，蛋白質A層析法或蛋白質A/G(蛋白質A與蛋白質G之融合蛋白)層析法可用於純化包含衍生自抗體CH2結構域及/或CH3結構域之組分的多肽及/或多肽複合物(Lindmark等人, J. Immunol. Meth. 62:1-13 (1983))；Zettlit, K. A., Antibody Engineering, 第V部分, 531-535, 2010)。在一些實施例中，蛋白質G層析法可用於純化包含IgG γ3重鏈之多肽及/或多肽複合物(Guss等人, EMBO J. 5:1567 1575 (1986))。在一些實施例中，蛋白質L層析法可用於純化包含κ輕鏈之多肽及/或多肽複合物(Sudhir, P., Antigen engineering protocols, 第26章, 由Humana Press出版, 1995；Nilson, B. H. K.等人, J. Biol. Chem., 267, 2234-2239 (1992))。親和配體所連接之基質最常為瓊脂糖，但其他基質亦為可用的。與用瓊脂糖可達成之情形相比，機械穩定性基質(諸如受控微孔玻璃或聚(苯乙烯二乙烯基)苯)允許較快流動速率及較短處理時間。在抗體包含CH3結構域的情況下，Bakerbond ABX樹脂(J.T. Baker, Phillipsburg, N.J.)可用於純化。 VI. 製品及套組 本文所描述之組合物中之任一種可以包含在套組中(例如表現免疫檢查點調節子(例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之溶瘤牛痘病毒、表現免疫檢查點調節子及雙特異性接合分子之溶瘤病毒、抗PD-1抗體組合物、表現抗PD-1抗體之宿主細胞或編碼抗PD-1抗體之溶瘤病毒)。在一非限制性實例中，一或多種病毒及/或用以產生或操作病毒之試劑可以包含在套組中。該套組之組分係提供於適合容器構件中。 該等套組之一些組分可包裝於水性介質中或以凍乾形式包裝。該等套組之容器構件一般將包括至少一個小瓶、試管、燒瓶、瓶、注射器或其他容器構件，在其中可以放入且較佳適當地等分組分。在套組中存在多於一種組分之情況下，該套組一般亦將含有第二、第三或其他另外的容器，其中可以分別放入另外的組分。各種組分組合亦可包含在小瓶中。該等套組通常亦包括用於容納供商業出售之封閉限制式組分的構件。此類容器可以包括保持有所需小瓶的射出模製或吹塑模製之塑料容器。 當該套組之組分係以一種及/多種液體溶液形式提供時，該液體溶液係水溶液，且無菌水溶液特別有用。在一些情況下，容器構件本身可為注射器、滴管及/或其他此類設備，調配物可自其施用於身體之受感染區域，注射至動物中及/或甚至施加至套組之其他組分及/或與套組之其他組分混合。 該套組之組分亦可以乾粉形式提供。當試劑及/或組分係以乾粉形式提供時，可藉由添加適合溶劑來使粉末復原。預想該溶劑亦可提供於另一容器構件中。該等套組亦可包含用於容納無菌、醫藥學上可接受之緩衝液及/或其他稀釋劑的第二容器構件。 在一些實施例中，用於療法中之病毒係提供於套組中，且在一些情況下病毒基本上係該套組之唯一組分。該套組可以包含改良所需病毒之試劑及材料。在具體實施例中，該等試劑及材料包括表現構築體、用於擴增所需序列之引子、限制性酶、用於納入病毒中之一或多種DNA、核苷酸、適合緩衝液或緩衝試劑、鹽、諸如此類，且在一些情況下，該等試劑包括編碼如本文中所描述之接合分子及/或其對應之調控元件的載體及/或DNA。 在一些實施例中，在該套組中存在一或多個適於自個體提取一或多種樣品之設備。該等設備可以為注射器、刮刀，諸如此類。 在一些實施例中，除病毒實施例外，該套組亦包括第二癌症療法，諸如化學療法、激素療法及/或免疫療法。套組可以針對個體之特定癌症定製且包含用於該個體之對應的第二癌症療法。 本文所描述之套組可以進一步包括自商業及使用者觀點看所需要之其他材料，包括其他緩衝劑、稀釋劑、過濾器、針、注射器及附有關於執行本文所描述之任何方法之說明的藥品說明書。 本申請案進一步提供包含呈適合包裝形式的本文所描述之組合物(諸如醫藥組合物)的製品。用於本文所描述之組合物(諸如表現免疫檢查點調節子(例如抗PD-1抗體、PD-1細胞外結構域-Fc融合蛋白、TMIGD2細胞外結構域-Fc融合蛋白或SIRPα細胞外結構域及CXCL12片段-Fc融合蛋白)之溶瘤牛痘病毒、表現免疫檢查點調節子及雙特異性接合分子之溶瘤病毒、抗PD-1抗體組合物、表現抗PD-1抗體之宿主細胞或編碼抗PD-1抗體之溶瘤病毒)的適合包裝係此項技術中已知的，且包括例如小瓶(諸如密封小瓶)、容器、安瓿、瓶、罐、可撓性包裝(例如密封聚酯薄膜(Mylar)或塑膠袋)及類似物。該等製品可進一步經滅菌及/或密封。 例示性實施例 實施例1. 一種包含編碼免疫檢查點調節子之核酸的溶瘤牛痘病毒，其中該核酸係可操作地連接至晚期啟動子。 實施例2. 如實施例1之溶瘤牛痘病毒，其中該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子及PSL 合成晚期啟動子。 實施例3. 如實施例2之溶瘤牛痘病毒，其中該晚期啟動子係F17R。 實施例4. 如實施例1至3中任一項之溶瘤牛痘病毒，其中該溶瘤牛痘病毒係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉痘苗病毒(MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。 實施例5. 如實施例4之溶瘤牛痘病毒，其中該溶瘤牛痘病毒係WR株。 實施例6. 如實施例5之溶瘤牛痘病毒，其中該溶瘤牛痘病毒包含胸苷激酶(TK)基因及痘瘡生長因子(VGF)基因之雙重缺失。 實施例7. 如實施例1至6中任一項之溶瘤牛痘病毒，其中該免疫檢查點調節子係刺激性免疫檢查點分子之活化劑。 實施例8. 如實施例1至6中任一項之溶瘤牛痘病毒，其中該免疫檢查點調節子係免疫檢查點抑制劑。 實施例9. 如實施例8之溶瘤牛痘病毒，其中該免疫檢查點抑制劑係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。 實施例10. 如實施例9之溶瘤牛痘病毒，其中該免疫檢查點抑制劑係PD-1之抑制劑。 實施例11. 如實施例1至10中任一項之溶瘤牛痘病毒，其中該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。 實施例12. 如實施例11之溶瘤牛痘病毒，其中該免疫檢查點調節子係抗PD-1抗體，其包含：重鏈可變區(VH)，該重鏈可變區包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及輕鏈可變區(VL)，該輕鏈可變區包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。 實施例13. 如實施例11之溶瘤牛痘病毒，其中該免疫檢查點調節子係抗PD-1抗體，其包含：重鏈可變區(VH)，該重鏈可變區包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及輕鏈可變區(VL)，該輕鏈可變區包括(1)含胺基酸序列SEQ ID NO:10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。 實施例14. 如實施例1至9中任一項之溶瘤牛痘病毒，其中該免疫檢查點調節子係結合至該免疫檢查點分子之配體。 實施例15. 如實施例14之溶瘤牛痘病毒，其中該免疫檢查點分子係PD-L1、PD-L2、HHLA-2、CD47或CXCR4。 實施例16. 如實施例15之溶瘤牛痘病毒，其中該免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段之融合物、TMIGD2細胞外結構域與免疫球蛋白Fc片段之融合物，或SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段之融合物。 實施例17. 如實施例16之溶瘤牛痘病毒，其中該Fc片段係IgG4 Fc。 實施例18. 如實施例1至17中任一項之溶瘤牛痘病毒，其進一步包含編碼細胞介素之第二核酸。 實施例19. 如實施例18之溶瘤牛痘病毒，其中該細胞介素係GM-CSF。 實施例20. 一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域。 實施例21. 如實施例20之溶瘤病毒，其中該溶瘤病毒係選自由以下組成之群：牛痘病毒(VV)、塞內加谷病毒(SVV)、腺病毒、單純疱疹病毒1(HSV1)、單純疱疹病毒2(HSV2)、黏液瘤病毒、呼腸孤病毒、脊髓灰白質炎病毒、水泡性口炎病毒(VSV)、麻疹病毒(MV)、慢病毒、反轉錄病毒、麻疹病毒、流感病毒、辛德比病毒及新城疫病毒(NDV)。 實施例22. 如實施例21之溶瘤病毒，其中該溶瘤病毒係溶瘤牛痘病毒。 實施例23. 如實施例22之溶瘤病毒，其中該溶瘤牛痘病毒係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉痘苗病毒(MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。 實施例24. 如實施例23之溶瘤病毒，其中該溶瘤牛痘病毒係WR株。 實施例25. 如實施例24之溶瘤病毒，其中該溶瘤牛痘病毒包含胸苷激酶(TK)基因及痘瘡生長因子(VGF)基因之雙重缺失。 實施例26. 如實施例20至25中任一項之溶瘤病毒，其中該免疫檢查點調節子係刺激性免疫檢查點分子之活化劑。 實施例27. 如實施例20至25中任一項之溶瘤病毒，其中該免疫檢查點調節子係免疫檢查點抑制劑。 實施例28. 如實施例27之溶瘤病毒，其中該免疫檢查點抑制劑係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。 實施例29. 如實施例28之溶瘤病毒，其中該免疫檢查點抑制劑係PD-1之抑制劑。 實施例30. 如實施例20至29中任一項之溶瘤病毒，其中該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。 實施例31. 如實施例30之溶瘤病毒，其中該免疫檢查點調節子係抗PD-1抗體，其包含：重鏈可變區(VH)，該重鏈可變區包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及輕鏈可變區(VL)，該輕鏈可變區包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。 實施例32. 如實施例30之溶瘤病毒，其中該免疫檢查點調節子係抗PD-1抗體，其包含：重鏈可變區(VH)，該重鏈可變區包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及輕鏈可變區(VL)，該輕鏈可變區包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。 實施例33. 如實施例20至28中任一項之溶瘤病毒，其中該免疫檢查點調節子係結合至該免疫檢查點分子之配體。 實施例34. 如實施例33之溶瘤病毒，其中該免疫檢查點分子係PD-L1、PD-L2、HHLA-2、CD47或CXCR4。 實施例35. 如實施例34之溶瘤病毒，其中該免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段之融合物、TMIGD2細胞外結構域與免疫球蛋白Fc片段之融合物，或SIRPα細胞外結構域及CXCL12片段與免疫球蛋白Fc片段之融合物。 實施例36. 如實施例35之溶瘤病毒，其中該Fc片段係IgG4 Fc。 實施例37. 如實施例20至36中任一項之溶瘤病毒，其中該腫瘤抗原係選自由以下組成之群：EpCAM、FAP、EphA2、HER2、GD2、EGFR、VEGFR2及磷脂醯肌醇蛋白聚糖-3(GPC3)。 實施例38. 如實施例37之溶瘤病毒，其中該腫瘤抗原係EpCAM。 實施例39. 如實施例37之溶瘤病毒，其中該腫瘤抗原係FAP。 實施例40. 如實施例37之溶瘤病毒，其中該腫瘤抗原係EGFR。 實施例41. 如實施例37之溶瘤病毒，其中該腫瘤抗原係GPC3。 實施例42. 如實施例20至41中任一項之溶瘤病毒，其中該效應細胞係選自由以下組成之群：T淋巴細胞、B淋巴細胞、自然殺手(NK)細胞、樹突狀細胞(DC)、巨噬細胞、單核細胞、嗜中性球及NKT細胞。 實施例43. 如實施例42之溶瘤病毒，其中該效應細胞係T淋巴細胞。 實施例44. 如實施例43之溶瘤病毒，其中該T淋巴細胞係細胞毒性T淋巴細胞。 實施例45. 如實施例20至44中任一項之溶瘤病毒，其中該細胞表面分子係選自由以下組成之群：CD3、CD4、CD5、CD8、CD16、CD28、CD40、CD64、CD89、CD134、CD137、NKp46及NKG2D。 實施例46. 如實施例45之溶瘤病毒，其中該細胞表面分子係CD3。 實施例47. 如實施例20至46中任一項之溶瘤病毒，其中該第一抗原結合結構域係單鏈可變片段(scFv)。 實施例48. 如實施例20至47中任一項之溶瘤病毒，其中該第二抗原結合結構域係scFv。 實施例49. 如實施例20至48中任一項之溶瘤病毒，其中該第一抗原結合結構域及該第二抗原結合結構域係藉由連接子連接。 實施例50. 如實施例20至49中任一項之溶瘤病毒，其中該第一抗原結合結構域係在該第二抗原結合結構域之N末端。 實施例51. 如實施例20至49中任一項之溶瘤病毒，其中該第一抗原結合結構域係在該第二抗原結合結構域之C末端。 實施例52. 如實施例20至51中任一項之溶瘤病毒，其中該編碼免疫檢查點調節子之第一核酸係可操作地連接至晚期啟動子。 實施例53. 如實施例20至52中任一項之溶瘤病毒，其中該編碼雙特異性分子之第二核酸係可操作地連接至晚期啟動子。 實施例54. 如實施例52或53之溶瘤病毒，其中驅動免疫檢查點調節子及/或雙特異性分子之表現的該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子及PSL 合成晚期啟動子。 實施例55. 如實施例54之溶瘤病毒，其中該晚期啟動子係F17R。 實施例56. 如實施例20至55中任一項之溶瘤病毒，其中該溶瘤病毒進一步包含編碼細胞介素之第三核酸。 實施例57. 如實施例56之溶瘤病毒，其中該細胞介素係GM-CSF。 實施例58. 一種醫藥組合物，其包含如實施例1至57中任一項之溶瘤病毒，及醫藥學上可接受之載劑。 實施例59. 一種治療個體之癌症的方法，其包含向個體投與有效量之如實施例58之醫藥組合物。 實施例60. 如實施例59之方法，其中該有效量係約105 至約1013 pfu。 實施例61. 如實施例60之方法，其中該有效量係約109 pfu。 實施例62. 如實施例59至61中任一項之方法，其中該醫藥組合物係全身性投與。 實施例63. 如實施例62之方法，其中該醫藥組合物係靜脈內投與。 實施例64. 如實施例59至61中任一項之方法，其中該醫藥組合物係局部投與。 實施例65. 如實施例64之方法，其中該醫藥組合物係腫瘤內投與。 實施例66. 如實施例59至65中任一項之方法，其中該癌症係實體腫瘤。 實施例67. 如實施例66之方法，其中該癌症係選自由結腸直腸癌、肝癌及乳癌組成之群。 實施例68. 如實施例59至67中任一項之方法，其進一步包含向該個體投與另外的癌症療法。 實施例69. 如實施例68之方法，其中該另外的癌症療法係手術、放射、化學療法、免疫療法、激素療法或其組合。 實施例70. 如實施例59至69中任一項之方法，其中該個體係人類。 實施例71. 一種抗PD-1抗體，其包含：重鏈可變區(VH)，該重鏈可變區包括(1)含胺基酸序列SEQ ID NO: 1之HVR-H1；(2)含胺基酸序列SEQ ID NO: 2之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 3之HVR-H3；及輕鏈可變區(VL)，該輕鏈可變區包括(1)含胺基酸序列SEQ ID NO: 4之HVR-L1；(2)含胺基酸序列SEQ ID NO: 5之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 6之HVR-L3。 實施例72. 如實施例71之抗PD-1抗體，其中該構築體之抗原結合結構域包括含胺基酸序列SEQ ID NO: 13之重鏈可變區(VH)，及/或含胺基酸序列SEQ ID NO: 14之輕鏈可變區(VL)。 實施例73. 一種抗PD-1抗體，其包含：重鏈可變區(VH)，該重鏈可變區包括(1)含胺基酸序列SEQ ID NO: 7之HVR-H1；(2)含胺基酸序列SEQ ID NO: 8之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 9之HVR-H3；及輕鏈可變區(VL)，該輕鏈可變區包括(1)含胺基酸序列SEQ ID NO: 10之HVR-L1；(2)含胺基酸序列SEQ ID NO: 11之HVR-L2；及(3)含胺基酸序列SEQ ID NO: 12之HVR-L3。 實施例74. 如實施例73之抗PD-1抗體，其中該構築體之抗原結合結構域包括含胺基酸序列SEQ ID NO: 15之重鏈可變區(VH)及/或含胺基酸序列SEQ ID NO: 16之輕鏈可變區(VL)。 實施例75. 如實施例71至74中任一項之構築體，其中該抗PD-1抗體係全長抗體。 實施例76. 如實施例71至74中任一項之構築體，其中該抗PD-1抗體係scFv。 實施例77. 一種分離之核酸，其編碼如實施例71至76中任一項之抗PD-1抗體。 實施例78. 如實施例77之分離之核酸，其包含第一核酸序列SEQ ID NO: 17及第二核酸序列SEQ ID NO: 18。 實施例79. 如實施例77之分離之核酸，其包含第一核酸序列SEQ ID NO: 19及第二核酸序列SEQ ID NO: 20。 實施例80. 如實施例77至79中任一項之分離之核酸，其中該分離之核酸係可操作地連接至啟動子。 實施例81. 如實施例80之分離之核酸，其中該啟動子係晚期啟動子。 實施例82. 如實施例81之分離之核酸，其中該啟動子係牛痘病毒晚期啟動子。 實施例83. 如實施例82之分離之核酸，其中牛痘病毒晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子及PSL 合成晚期啟動子。 實施例84. 如實施例83之分離之核酸，其中該牛痘病毒晚期啟動子係F17R。 實施例85. 一種分離之宿主細胞，其包含如實施例77至84中任一項之分離之核酸。 實施例86. 一種溶瘤病毒，其包含如實施例77至84中任一項之核酸。 實施例87. 一種醫藥組合物，其包含如實施例71至76中任一項之抗PD-1抗體、如實施例85之分離之宿主細胞或如實施例86之溶瘤病毒，及醫藥學上可接受之載劑。 實施例88. 一種治療個體之癌症的方法，其包含向該個體投與有效量之如實施例87之醫藥組合物。 實施例89. 如實施例88之方法，其中該醫藥組合物係經靜脈內或腫瘤內投與該個體。 實施例90. 如實施例88或89之方法，其中該個體係人類。 實施例91. 一種醫藥組合物，其包括：包含編碼免疫檢查點調節子之第一核酸的第一OV；包含編碼雙特異性分子之第二核酸的第二OV，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域；及醫藥學上可接受之載劑。 實施例92. 如實施例91之醫藥組合物，其包括：包含編碼如實施例20至36、52、54及55中任一項之免疫檢查點調節子之第一核酸的第一OV；包含編碼如實施例20、37至51及53至55中任一項之雙特異性分子之第二核酸的第二OV；及醫藥學上可接受之載劑。 實施例93. 一種醫藥組合物，其包括：包含編碼免疫檢查點調節子之第一核酸的第一OV、包含編碼細胞介素之第二核酸的第二OV及醫藥學上可接受之載劑。 實施例94. 如實施例93之醫藥組合物，其包括：包含編碼如實施例20至36、52、54及55中任一項之免疫檢查點調節子之第一核酸的第一OV；包含編碼如實施例56或57細胞介素之第二核酸的第二OV；及醫藥學上可接受之載劑。 實施例95. 一種醫藥組合物，其包括：包含編碼免疫檢查點調節子之第一核酸的第一OV；包含編碼雙特異性分子之第二核酸的第二OV，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域；包含編碼細胞介素之第三核酸的第三OV；及醫藥學上可接受之載劑。 實施例96. 如實施例95之醫藥組合物，其包括：包含編碼如實施例20至36、52、54及55中任一項之免疫檢查點調節子之第一核酸的第一OV；包含編碼如實施例20、37至51及53至55中任一項之雙特異性分子之第二核酸的第二OV；包含編碼如實施例56或57細胞介素之第三核酸的第三OV；及醫藥學上可接受之載劑。 實施例97. 一種治療個體之癌症的方法，其包含向該個體投與有效量之如實施例91至96中任一項之醫藥組合物。 實施例98. 一種治療個體之癌症的方法，其包含向該個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子之第一核酸的第一OV及醫藥學上可接受之第一載劑；及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼雙特異性分子之第二核酸的第二OV及醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域。 實施例99. 如實施例98之方法，其包含向該個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼如實施例20至36、52、54及55中任一項之免疫檢查點調節子之第一核酸的第一OV，及醫藥學上可接受之第一載劑；及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼如實施例20、37至51及53至55中任一項之雙特異性分子之第二核酸的第二OV，及醫藥學上可接受之第二載劑。 實施例100. 一種治療個體之癌症的方法，其包含向該個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子之第一核酸的第一OV，及醫藥學上可接受之第一載劑；及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼細胞介素之第二核酸的第二OV，及醫藥學上可接受之第二載劑。 實施例101. 如實施例100之方法，其包含向該個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼如實施例20至36、52、54及55中任一項之免疫檢查點調節子之第一核酸的第一OV，及醫藥學上可接受之第一載劑；及有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼如實施例56或57之細胞介素之第二核酸的第二OV，及醫藥學上可接受之第二載劑。 實施例102. 一種治療個體癌症之方法，其包含向該個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼免疫檢查點調節子之第一核酸的第一OV，及醫藥學上可接受之第一載劑；有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼雙特異性分子之第二核酸的第二OV，及醫藥學上可接受之第二載劑，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域；及有效量之第三醫藥組合物，該第三醫藥組合物包括包含編碼細胞介素之第三核酸的第三OV，及醫藥學上可接受之第三載劑。 實施例103. 如實施例102之方法，其包含向該個體投與：有效量之第一醫藥組合物，該第一醫藥組合物包括包含編碼如實施例20至36、52、54及55中任一項之免疫檢查點調節子之第一核酸的第一OV，及醫藥學上可接受之第一載劑；有效量之第二醫藥組合物，該第二醫藥組合物包括包含編碼如實施例20、37至51及53至55中任一項之雙特異性分子之第二核酸的第二OV，及醫藥學上可接受之第二載劑；及有效量之第三醫藥組合物，該第三醫藥組合物包括包含編碼如實施例56或57之細胞介素之第三核酸的第三OV，及醫藥學上可接受之第三載劑。 實例 以下實例僅意欲為本發明之示例，且因此不應視為以任何方式限制本發明。以下實例及詳細描述係藉助於說明而非作為限制提供。 在本發明中，使用表現重組PD1-IgG4-Fc融合物之溶瘤牛痘病毒(在下文中稱為「PD1-Ig-VV」)阻斷癌細胞上之PD1配體與T細胞上之PD1受體之間的共抑制相互作用，由此增強抗腫瘤免疫反應。產生PD1-IgG4-Fc重組蛋白(在下文中稱為「PD1-Ig」)，其中PD1細胞外結構域與免疫球蛋白G4之恆定(Fc)結構域融合，有效地產生PD1與其配體之間之相互作用的抑制劑。 實例1：PD1-Ig-VV、GPC3-CD3-VV、FAP-CD3-VV及PD1-Ig-FAP-CD3-VV之構築 產生溶瘤牛痘病毒(VV)構築體PD1-Ig-VV以表現具有PD-1細胞外結構域與免疫球蛋白G4(IgG4)之恆定(Fc)結構域之融合物的重組蛋白。PD-1細胞外結構域包含胺基酸序列SEQ ID NO:25。GPC3-CD3(在下文中亦稱為GPC3-TE(GPC3 T細胞接合子))係靶向肝細胞癌(HCC)腫瘤抗原磷脂醯肌醇蛋白聚糖-3(GPC3)及T細胞上之CD3的雙特異性分子。產生溶瘤VV構築體GPC3-CD3-VV(在下文中亦稱為GPC3-TEA-VV)以表現分泌性GPC3-scFv-人類CD3-scFv(GPC3-CD3)。FAP-CD3(在下文中亦稱為FAP-TE)係靶向癌症相關纖維母細胞上之纖維母細胞活化蛋白(FAP)抗原及T細胞上之CD3的雙特異性分子。產生溶瘤VV構築體FAP-CD3-VV(在下文中亦稱為FAP-TEA-VV)以表現分泌性FAP-scFv-人類CD3-scFv(FAP-CD3或FAP-TE)。產生溶瘤VV構築體PD1-Ig-FAP-CD3-VV(在下文中亦稱為帶PD1-Ig-FAP-TEA-VV(PD1-Ig-FAP-T細胞接合子之VV))以共表現分泌性PD1-IgG4-Fc及分泌性FAP-scFv-人類CD3-scFv(FAP-CD3)。藉由T2A自裂解序列連接編碼PD1-IgG4-Fc與FAP-CD3之核酸序列。抗FAP scFv包含：重鏈可變區(VH)，該重鏈可變區包括(1)含胺基酸序列SEQ ID NO:36之HVR-H1；(2)含胺基酸序列SEQ ID NO: 37之HVR-H2；及(3)含胺基酸序列SEQ ID NO: 38之HVR-H3；及輕鏈可變區(VL)，該輕鏈可變區包括(1)含胺基酸序列SEQ ID NO: 39之HVR-L1；(2)含胺基酸序列SEQ ID NO:40之HVR-L2；及(3)含胺基酸序列SEQ ID NO:41之HVR-L3。抗FAP scFv包括含胺基酸序列SEQ ID NO:42之VH及含胺基酸序列SEQ ID NO:43之VL。抗FAP scFv包含胺基酸序列SEQ ID NO:44。FAP-CD3雙特異性分子包含胺基酸序列SEQ ID NO:45。共表現之PD1-IgG4-Fc包括含胺基酸序列SEQ ID NO:25之PD-1細胞外結構域。 藉由將含有PD1-Ig、T細胞接合子(TE)或PD1-Ig及FAP-CD3之pSEM-1質體形式重組於WR牛痘病毒(WR VV)之VSC20病毒株之TK基因中來產生編碼分泌性PD1-IgG4-Fc(PD1-Ig-VV)、GPC3-CD3(GPC3-CD3-VV或GPC3-TEA-VV)、FAP-CD3(FAP-CD3-VV或FAP-TEA-VV)或共表現分泌性PD1-IgG4-Fc及FAP-CD3(PD1-Ig-FAP-CD3-VV或PD1-Ig-FAP-TEA-VV)之牛痘病毒(Western Reserve株)。首先，構築含有PD1-Ig、T細胞接合子或PD1-Ig及FAP-CD3兩者之穿梭載體pSEM-1 (圖1)。使插入之PD1-Ig、TE或PD1-Ig及FAP-CD3兩者在F17R晚期啟動子之轉錄控制下表現以允許在T細胞活化之前進行充分病毒複製。該等VV亦表現YFP-GFP標記物以允許病毒選擇。使YFP-GFP在P7.5啟動子之轉錄控制下表現，且loxP位點以相同取向側接可選擇標記物YFP-GFP(圖1)。為了構築編碼PD1-Ig、TE或PD1-Ig及FAP-CD3兩者之重組病毒，首先將穿梭載體pSEM-1轉染至人類143 TK細胞中。接著用病毒VSC20以0.1之感染倍率(MOI)感染細胞。在五輪蝕斑選擇及擴增以證實PD1-Ig、TE或PD1-Ig及FAP-CD3兩者之表現之後，選出一個純系進行擴增及純化。為了自重組病毒移除YFP-GFP卡匣，使病毒在表現Cre重組酶之細胞質形式的U2OS細胞株(U2OS-Cre)上傳代。在五輪蝕斑選擇及擴增以證實PD1-Ig、TE或PD1-Ig及FAP-CD3兩者之表現之後，選出一個YFP-GFP陰性純系進行擴增及純化。 實例2：表現編碼PD1-Ig之牛痘病毒及測試其與Huh7細胞上之PD-L1的結合 為了測試PD1-Ig-VV表現PD1-Ig，收集經PD1-Ig-VV(如實例1中所描述)或VV-GFP對照(編碼GFP之牛痘病毒)感染的人類骨肉瘤143 TK細胞並藉由西方墨點法分析上清液。偵測PD1-Ig為85-90 KDa(圖2A)。 為了測試VV表現之PD1-Ig與Huh7細胞上之PD-L1配體的結合，將Huh7細胞與來自感染VV-GFP或PD1-Ig-VV之人類143 TK細胞的上清液一起培育兩小時。針對APC-抗Fc對細胞進行FACS分析以偵測PD1-IgG4-Fc(有關FACS方案，參見下文)。當相較於與GFP一起培育之細胞時，與PD1-Ig上清液一起培育之Huh7細胞顯示Fc陽性細胞增加，表明PD1-Ig結合至Huh7細胞上之PD-L1(圖2B)。FACS 根據通用方案進行FACS。簡言之，收集細胞並用含有1% FBS之PBS(Sigma, St. Louis, MO；FACS緩衝液)洗滌一次，隨後添加抗體。接著在冰上於暗處培育細胞30分鐘，洗滌一次，並在冰上於暗處與螢光素結合之二次抗體(若適用)一起培育30分鐘，且隨後洗滌一次。隨後將洗滌過的細胞固定於0.5%三聚甲醛/FACS緩衝液中，隨後分析。對於每一樣品，使用FACSCalibur儀器(BD, Becton Dickinson, Mountain View, CA)，利用Cell Quest軟體(Becton Dickinson)或利用FCS Express軟體(De Novo Software, Los Angeles, CA)分析10,000個細胞。 實例3：PD1-Ig增強活化T細胞介導的GPC3-CD3依賴性Huh7-GFP細胞殺滅 為了測試PD1-Ig增強雙特異性T細胞接合子之抗腫瘤作用的能力，使PD1-Ig與GPC3-CD3，即靶向肝細胞癌(HCC)腫瘤抗原磷脂醯肌醇蛋白聚糖-3(GPC3)及T細胞上之CD3的雙特異性分子(GPC3-scFv-CD3-scFv；有關構築參見實例1)一起共表現。用空病毒(-GPC3-CD3)、表現GPC3-CD3之牛痘病毒(+GPC3-CD3)感染或用編碼GPC3-CD3之牛痘病毒及編碼PD1-Ig之牛痘病毒(GPC3-CD3+PD1-Ig)共感染Huh7-GFP細胞(圖3A)。在2.5% FBS培養基中用VV以MOI 1感染腫瘤細胞兩小時，隨後在完全培養基中培養。在37℃下培育未刺激之人類PBMC(有關方案參見下文)兩小時以移除黏附細胞且將非黏附性PBMC以效應子:標靶(E:T)比率為1:1(圖3A上圖)或5:1(圖3A下圖)添加至Huh7-GFP細胞培養物中。如實例2中所描述，利用FACS針對CD3-APC及Huh7-GFP分選細胞，並藉由GFP評估Huh7細胞活力。末梢血液單核細胞 ( PBMC ) 為產生PBMC群，根據貝勒醫學院機構審查委員會(Institutional Review Board of Baylor College Medicine)批准之方案，自健康供體獲得血液樣品。末梢血液經Ficoll梯度處理，且所得PBMC在補充有10%熱滅活FCS及2 mmol/L GLUTAMAX之洛斯維·帕克紀念研究所(Roswell Park Memorial Institute) 1640(Thermo Scientific HyClone, Waltham, MA；Lonza, Basel, Switzerland)中培養。 自圖3A可以看出，當與陰性對照細胞(-GPC3-CD3)相比較時，在兩種E:T比率下表現GPC3-CD3之存活Huh7-GFP癌細胞之數量減少。在表現PD1-Ig及GPC3-CD3兩者(GPC3-CD3+PD1-Ig)之Huh7細胞中GFP信號進一步減弱。此表明PD1-Ig增強在T細胞存在下GPC3-CD3介導之Huh7腫瘤細胞溶解，且該作用在更高E:T比率(比較圖3A下圖與上圖)下進一步增強。使用螢光顯微鏡檢查亦觀測到在T細胞存在下PD1-Ig誘導之腫瘤溶解作用增強以及GFP信號減弱(圖3B)。 為進一步說明PD1-Ig可以增強在T細胞存在下GPC3-CD3介導之腫瘤細胞殺滅作用，使用FACS作圖，以細胞凋亡標記物磷脂結合蛋白-V及碘化丙錠(PI)分析感染VV之Huh7細胞。簡言之，收集與PBMC在5:1之E:T下共培養的感染VV之Huh7細胞並將其與重組磷脂結合蛋白-V一起培育。在即將用流式細胞測量術分析之前，將PI添加至所有樣品中。分析細胞中之磷脂結合蛋白-V結合以及PI吸收，並指出呈磷脂結合蛋白-V陽性、PI陽性或雙重陽性之細胞的百分比(圖3C)。 感染對照病毒(-GPC3-CD3)之Huh7細胞僅具有8.94%的PI/磷脂結合蛋白-V雙重陽性細胞(圖3C左圖)，而表現GPC3-CD3之細胞具有24.1%的雙重陽性細胞(圖3C中圖)。用編碼PD1-Ig之VV及編碼GPC3-CD3之VV共感染使得磷脂結合蛋白-V/PI雙重陽性Huh7腫瘤細胞之百分比增大至44.2%(圖3C右圖)，指示腫瘤溶解增加。該等資料指示，在Huh7人類肝癌細胞中共表現PD1-Ig可以加強在T細胞存在下GPC3-CD3介導之腫瘤溶解作用。 實例4：PD1-Ig不響應於GPC3-CD3影響T細胞表型 為表徵PD1-Ig對T細胞之影響，將未刺激之人類PBMC(1×106 個/孔)單獨培養或與感染VV之Huh7細胞(0.4×106 個/孔)一起在24孔盤中培育兩天。接著收集細胞並用針對細胞表面標記物CD3及CD69(CD69係T細胞活化標記物)(圖4A)或CD45RA及CCR7(圖4B)之螢光素結合抗體染色，隨後進行FACS分析，如實例2中所描述。顯示出細胞群百分比。使用針對CD45RA及CCR7(用於區分CD4+與CD8+ T細胞之標記物)之抗體作為陰性對照，因為PD-1主要在CD4-/CD8-陰性T細胞上表現(圖4B)。將PBMC單獨培養(圖4A及4B，左圖)或與用空病毒(自左側起第二圖，頂部及底部，-GPC3-CD3)、用編碼GPC3-CD3之VV(自右側起第二圖，GPC3-CD3/培養基)感染，或用編碼GPC3-CD3之牛痘病毒及編碼PD1-Ig之牛痘病毒共感染(右圖，GPC3-CD3/PD1-Ig)之Huh7腫瘤細胞共培養。 當將PBMC與表現GPC3-CD3之Huh7細胞共培養時，CD3+/CD69+細胞顯著增加至57.9%(圖4A)。在與表現GPC3-CD3及PD1-Ig兩者之Huh7細胞共培養的PBMC中雙重陽性細胞之百分含量略微增加至59.7%(圖4A，右圖)。因此，FACS偵測CD69(一種淋巴活化抗原)顯示在與GPC3-CD3表現Huh7細胞一起培育之PBMC中T細胞活化之明顯增加；不過，在添加PD1-Ig之後，此數量並未顯著增加，表明PD1-Ig不影響T細胞表型。 實例5：PD1-Ig增進T細胞之細胞介素產生之GPC3-CD3依賴性增加 為了研究PD1-Ig之存在是否增加T細胞之細胞介素產生，將PBMC與僅表現GPC3-CD3或表現GPC3-CD3及PD1-Ig之Huh7細胞共培養。對於在Huh7細胞中共表現GPC3-CD3及PD1-Ig，用編碼GPC3- CD3之VV(GPC3-CD3-VV)及編碼PD1-Ig之VV(PD1-Ig-VV)共感染Huh7細胞。用VV以MOI 1感染Huh7細胞，如上文所描述添加PBMC，並在病毒感染後24小時至48小時，收集細胞培養物並使用酶聯結免疫吸附分析法(ELISA)分析促炎性細胞介素IFNγ、TNFα及IL-2之存在(分別為圖5A、5B及5C)。當相較於與感染對照病毒之Huh7一起培養的T細胞時，與GPC3-CD3表現Huh7細胞一起培養的T細胞產生較高量之IFNγ、TNFα及IL-2(分別為圖5A至5C，「培養基」相對於「-GPC3-CD3」)。當將T細胞與表現GPC3-CD3及PD1-Ig兩者之Huh7細胞一起培養時，IFNγ、TNFα及IL-2之釋放進一步增大(分別為圖5A至5C，「PD1-Ig」)。IFNγ、TNFα及IL-2之釋放量在GPC3-CD3及PD1-Ig存在下甚至大於在GPC3-CD3及抗PD-1抗體存在下(分別為圖5A至5C，「αPD1」；目錄號10377-mhT28-200，Sino Biological)或在GPC3-CD3及抗PD-L1抗體存在下(分別為圖5A至5C，「αPD-L1」；Clone29E.2A3, BXCell)之釋放量。此表明PD1-Ig可以加強GPC3-CD3誘導T細胞釋放細胞介素之能力。 實例6：PD1-Ig-VV在活體內抑制SK-Br-3乳癌腫瘤生長 在植入人類腫瘤細胞之小鼠異種移植模型中評價PD1-Ig-VV之活體內功效。為了建立乳癌之小鼠異種移植模型，將4×106 個SK-Br-3人類乳癌細胞皮下接種至NSG小鼠之右側腹中。隨後在第8天，將PBS、1×108 pfu之對照VV或PD1-Ig-VV注射至右側腹腫瘤中，並在第11天靜脈內植入2×107 個未活化之人類PBMC細胞(圖6A)。僅接受PBS/PBMC之小鼠充當對照。在腫瘤體積分析中，接受PBS/PBMC之小鼠到第21天產生約3600 mm3 之腫瘤體積(圖6B及圖6C左圖)。接受對照VV之小鼠在PBMC存在下腫瘤體積中等地減小(圖6B及圖6C中圖)。注射PD1-Ig-VV之小鼠在第21天在PBMC存在下顯示腫瘤體積顯著減小(圖6B及圖6C右圖)，表明PD1-Ig-VV可以在活體內抑制SK-Br-3乳癌腫瘤生長。 實例7：PD1-Ig-VV在活體內抑制HT-29結腸直腸腺癌腫瘤生長 為了將PD1-Ig-VV抑制腫瘤生長之能力擴展至另一腫瘤模型，採用HT-29結腸直腸癌異種移植模型。為了建立此模型，將4×106 個HT-29結腸直腸腺癌細胞皮下接種至NSG小鼠之右側腹中，隨後在第8天，將PBS、1×108 pfu之對照VV或PD1-Ig-VV注射至右側腹腫瘤中，接著在第11天靜脈內植入2×107 個未活化之人類PBMC細胞(圖7A)。在24天之療程中檢查腫瘤體積顯示，僅注射PBS或在PBMC存在下注射PBS之對照小鼠在第24天產生平均為約1000 mm3 之腫瘤(圖7B)。注射對照病毒GFP-VV且植入PBMC之小鼠的腫瘤體積略有減小(平均為約350 mm3 )。不過，注射PD1-Ig-VV且植入PBMC之小鼠顯示腫瘤生長之顯著抑制，且平均腫瘤大小接近0 mm3 。該等結果表明，PD1-Ig-VV可以在多種小鼠異種移植模型中在活體內強烈抑制腫瘤生長。 實例8：共表現PD1-Ig及FAP-CD3之牛痘病毒及其與U87細胞上之PD-L1及FAP之結合的測試 為了測試PD1-Ig-FAP-CD3-VV共表現PD1-Ig及FAP-CD3，收集經PD1-Ig-FAP-CD3-VV(如實例1中所描述)或VV-GFP對照(編碼GFP之VV)感染的人類骨肉瘤143 TK細胞並藉由西方墨點法分析上清液。 為了測試VV表現之PD1-Ig與U87(人類神經膠母細胞瘤)細胞上PD-L1配體之結合，將U87細胞與來自感染VV-GFP或PD1-Ig-FAP-CD3-VV之人類143 TK細胞的上清液一起培育兩小時。針對APC-抗Fc對細胞進行FACS分析以偵測PD1-IgG4-Fc(有關FACS方案參見實例2)。當相較於與GFP上清液一起培育之細胞時，預期與PD1-Ig上清液一起培育之U87細胞顯示Fc陽性細胞增加，表明PD1-Ig結合至U87細胞上之PD-L1。 為了測試VV表現之FAP CD3與FAP陽性U87細胞上FAP抗原之結合，將U87細胞與來自感染VV-GFP或PD1-Ig-FAP-CD3-VV之人類143 TK細胞的上清液一起培育兩小時。針對抗CD3 scFv抗體對細胞進行FACS分析以偵測FAP-CD3(FAP-TE)(有關FACS方案參見實例2)。當相較於與GFP上清液一起培育之細胞時，預期與FAP-CD3上清液一起培育之U87細胞顯示FAP-CD3陽性陽性細胞增加，表明FAP-CD3結合至U87細胞上之FAP。 實例9：PD1-Ig增強活化T細胞介導之FAP-CD3依賴性U87細胞殺滅 為了測試PD1-Ig增強雙特異性T細胞接合子之抗腫瘤作用的能力，用空病毒、僅編碼FAP-CD3之牛痘病毒(FAP-CD3-VV)或共表現FAP-CD3及PD1-Ig之VV(PD1-Ig-FAP-CD3-VV)感染U87-GFP細胞。有關構築，參見實例1。在2.5% FBS培養基中用VV以MOI 1感染腫瘤細胞兩小時，隨後在完全培養基中培養。在37℃下將未刺激之人類PBMC(有關方案，參見實例3)培育兩小時以移除黏附細胞，且將非黏附PBMC以1:1或5:1之效應子:標靶(E:T)比率添加至U87-GFP細胞培養物中。如實例2中所描述，利用FACS針對CD3-APC及GFP分選細胞，並藉由GFP評估U87-GFP細胞活力。 預期當與陰性對照細胞(空病毒)相比較時，在兩種E:T比率下表現FAP-CD3之存活U87-GFP癌細胞之數量減少。預期在共表現PD1-Ig及FAP-CD3(PD1-Ig-FAP-CD3-VV)之U87-GFP細胞中GFP信號進一步減弱。此表明，共表現PD1-Ig及FAP-CD3可以增強在T細胞存在下FAP-CD3介導之U87腫瘤細胞溶解，且預期該作用在更高E:T比率下進一步加強。使用螢光顯微鏡檢查亦觀測到在T細胞存在下PD1-Ig誘導之腫瘤溶解作用增強及GFP信號減弱。預期相較於感染FAP-CD3-VV或空病毒之細胞，感染PD1-Ig-FAP-CD3-VV之U87-GFP細胞具有最弱的GFP信號。 為了進一步說明共表現PD1-Ig及FAP-CD3可以增強在T細胞存在下FAP-CD3介導之腫瘤細胞殺滅作用，使用FACS作圖，利用細胞凋亡標記物磷脂結合蛋白-V及碘化丙錠(PI)分析感染VV之U87細胞。簡言之，收集與PBMC在5:1之E:T下共培養的感染VV之U87細胞並將其與重組磷脂結合蛋白-V一起培育。在即將用流式細胞測量術分析之前，將PI添加至所有樣品中。分析細胞中之磷脂結合蛋白-V結合以及PI吸收，並指出呈磷脂結合蛋白-V陽性、PI陽性或雙重陽性之細胞的百分比。 預期感染對照病毒之U87細胞具有最低百分含量的PI/磷脂結合蛋白-V雙重陽性細胞，而感染FAP-CD3-VV之細胞具有中等含量的雙重陽性細胞。預期共表現PD1-Ig及FAP-CD3(PD1-Ig-FAP-CD3-VV)將磷脂結合蛋白-V/PI雙重陽性腫瘤細胞之百分比增大至較高水準，指示腫瘤溶解增加。該等資料可以指示，在U87腫瘤細胞中共表現PD1-Ig及FAP-CD3可以加強在T細胞存在下FAP-CD3介導之腫瘤溶解作用。 實例10：PD1-Ig及FAP-CD3共表現不影響T細胞表型 為表徵共表現之PD1-Ig及FAP-CD3(PD1-Ig-FAP-CD3-VV)對T細胞之影響，將未刺激之人類PBMC(1×106 個/孔)單獨培養或與感染VV之U87細胞(0.4×106 個/孔)一起在24孔盤中培育兩天。接著收集細胞並用針對細胞表面標記物CD3及CD69(CD69係T細胞活化標記物)或CD45RA及CCR7(用於區分CD4+與CD8+ T細胞之標記物)之螢光素結合抗體染色，隨後進行FACS分析，如實例2中所描述。使用針對CD45RA及CCR7之抗體作為陰性對照，因為PD-1主要在CD4/CD8陰性T細胞上表現。將PBMC單獨培養或與感染空病毒、編碼FAP-CD3之VV(FAP-CD3-VV)或共表現FAP-CD3及PD1-Ig之VV(PD1-Ig-FAP-CD3-VV)之U87細胞共培養。 當將PBMC與表現FAP-CD3之U87細胞共培養時，預期CD3+/CD69+細胞顯著增加。預期在與共表現FAP-CD3及PD1-Ig兩者(PD1-Ig-FAP-CD3-VV)之U87細胞共培養的PBMC中雙重陽性細胞之百分含量與僅表現FAP-CD3時的情形類似。因此，預期FACS偵測CD69(一種淋巴活化抗原)顯示在與FAP-CD3表現U87細胞一起培育之PBMC中T細胞活化之明顯增加；且在共表現PD1-Ig及FAP-CD3時，此數量並未顯著增加，表明PD1-Ig可能不影響T細胞表型。 實例11：PD1-Ig加強T細胞產生之細胞介素之FAP-CD3依賴性增加 為了研究共表現PD1-Ig及FAP-CD3是否增加T細胞產生之細胞介素，將PBMC與感染PD1-Ig-FAP-CD3-VV、空病毒或FAP-CD3-VV之U87細胞，或用抗PD-1或抗PD-L1抗體處理之U87細胞共培養。用VV以MOI 1感染U87細胞，如上文所描述添加PBMC，並在病毒感染後24小時至48小時，收集細胞培養物並使用ELISA分析促炎性細胞介素IFNγ、TNFα及IL-2之存在。當相較於與感染對照病毒之癌細胞一起培養的T細胞時，預期與感染FAP-CD3-VV之U87細胞一起培養的T細胞產生較高含量的IFNγ、TNFα及IL-2。預期當將T細胞與共表現FAP3-CD3及PD1-Ig(PD1-Ig-FAP-CD3-VV)之U87細胞一起培養時，IFNγ、TNFα及IL-2之釋放量進一步增大。將在FAP-CD3及PD1-Ig存在下IFNγ、TNFα及IL-2之釋放量與在FAP-CD3及抗PD-1抗體(目錄號10377-mhT28-200，Sino Biological)或FAP-CD3及抗PD-L1抗體(Clone 29E.2A3, BXCell)存在下之釋放量相比較。該等結果可以指示，使用PD1-Ig-FAP-CD3-VV，共表現PD1-Ig及FAP-CD3可以加強FAP-CD3誘導T細胞釋放細胞介素之能力。 實例12：PD1-Ig-FAP-CD3-VV在活體內抑制U87癌症腫瘤生長 在植入U87人類癌細胞之小鼠異種移植模型中評價PD1-Ig-FAP-CD3-VV之活體內功效。為了建立U87癌症之小鼠異種移植模型，將4×106 個U87人類癌細胞皮下接種至NSG小鼠之右側腹中。隨後在第8天，將PBS、1×108 pfu之對照VV、PD1-Ig-VV、FAP-CD3-VV或PD1-Ig-FAP-CD3-VV注射至右側腹腫瘤中，並在第11天靜脈內植入2×107 個未活化之人類PBMC細胞。僅接受PBS/PBMC之小鼠用作對照。在第21天之腫瘤體積分析中，預期僅接受PBS/PBMC之小鼠產生體積最高之腫瘤。預期接受對照VV之小鼠在PBMC存在下腫瘤體積中等地減小。預期注射PD1-Ig-VV或FAP-CD3-VV之小鼠在PBMC存在下在21天顯示甚至更小的腫瘤體積，而預期注射PD1-Ig-FAP-CD3-VV之小鼠可能由於腫瘤基質破壞而具有最小腫瘤體積。該等結果可以表明，共表現PD1-Ig及FAP-CD3可以在活體內抑制U87腫瘤生長，該抑制作用可能甚至優於單獨PD1-Ig-VV或FAP-CD3-VV。 序列表 SEQ ID NO: 1 (1H7e3 HVR-H1胺基酸序列)SEQ ID NO: 2 (1H7e3 HVR-H2胺基酸序列)SEQ ID NO: 3 (1H7e3 HVR-H3胺基酸序列)SEQ ID NO: 4 (1H7e3 HVR-L1胺基酸序列)SEQ ID NO: 5 (1H7e3 HVR-L2胺基酸序列)SEQ ID NO: 6 (1H7e3 HVR-L3胺基酸序列)SEQ ID NO: 7 (4F11C3 HVR-H1胺基酸序列)SEQ ID NO: 8 (4F11C3 HVR-H2胺基酸序列)SEQ ID NO: 9 (4F11C3 HVR-H3胺基酸序列)SEQ ID NO: 10 (4F11C3 HVR-:L1胺基酸序列)SEQ ID NO: 11 (4F11C3 HVR-L2胺基酸序列)SEQ ID NO: 12 (4F11C3 HVR-L3胺基酸序列)SEQ ID NO: 13 (1H7e3 VH胺基酸序列：HVR加下劃線)SEQ ID NO: 14 (1H7e3 VL胺基酸序列：HVR加下劃線)SEQ ID NO: 15 (4F11C3 VH胺基酸序列：HVR加下劃線)SEQ ID NO: 16 (4F11C3 VL胺基酸序列：HVR加下劃線)SEQ ID NO: 17 (編碼抗PD-1 1H7e3 VH之核酸：HVR序列加下劃線)SEQ ID NO: 18 (編碼抗PD-1 1H7e3 VL之核酸：HVR序列加下劃線)SEQ ID NO: 19 (編碼抗PD-1 4F11C3 VH之核酸：HVR序列加下劃線)SEQ ID NO: 20 (編碼抗PD-1 4F11C3 VL之核酸：HVR序列加下劃線)SEQ ID NO: 21 (1H7e3、4F11C3 VH信號肽之胺基酸序列)SEQ ID NO: 22 (編碼1H7e3、4F11C3 VH信號肽之核酸)SEQ ID NO: 23 (1H7e3, 4F11C3 VL信號肽之胺基酸序列)SEQ ID NO: 24 (編碼1H7e3、4F11C3 VL信號肽之核酸)SEQ ID NO: 25 (PD-1細胞外結構域胺基酸序列)SEQ ID NO: 26 (PD-1細胞外結構域核酸序列)SEQ ID NO: 27 (TMIGD2細胞外結構域胺基酸序列)SEQ ID NO: 28 (TMIGD2細胞外結構域-Fc融合蛋白之信號肽胺基酸序列)SEQ ID NO: 29 (SIRPα細胞外結構域胺基酸序列)SEQ ID NO: 30 (CXCL12 N末端序列)SEQ ID NO: 31 (SIRPα細胞外結構域與CXCL12之間之連接子(IgG1鉸鏈)之胺基酸序列)SEQ ID NO: 32 (SIRPα-CXCL12-Fc融合蛋白之信號肽胺基酸序列)SEQ ID NO: 33 (VH-VL連接子胺基酸序列)SEQ ID NO: 34 (VH-VL連接子胺基酸序列)SEQ ID NO: 35 (scFv-scFv連接子胺基酸序列)SEQ ID NO: 36 (FAP HVR-H1胺基酸序列)SEQ ID NO: 37 (FAP HVR-H2胺基酸序列)SEQ ID NO: 38 (FAP HVR-H3胺基酸序列)SEQ ID NO: 39 (FAP HVR-L1胺基酸序列)SEQ ID NO: 40 (FAP HVR-L2胺基酸序列)SEQ ID NO: 41 (FAP HVR-L3胺基酸序列)SEQ ID NO: 42 (FAP VH胺基酸序列：HVR序列加下劃線)SEQ ID NO: 43 (FAP VL胺基酸序列：HVR序列加下劃線)SEQ ID NO: 44 (FAP scFv胺基酸序列：HVR序列加下劃線，連接子序列為粗體)SEQ ID NO: 45 (全長FAP-人類CD3 TE胺基酸序列：HVR序列加下劃線，VH-VL連接子序列為粗體，scFv-scFv連接子序列為粗體且為斜體) Related Application This application requires priority of US Provisional Patent Application No. 62 / 385,930 and US Provisional Patent Application No. 62 / 385,933 filed on September 9, 2016, the contents of which are incorporated by reference in their entirety in. Sequence Listing Submitted in ASCII Text File The following content submitted in ASCII text file is incorporated into this article by full text citation: Computer-readable form (CRF) sequence table (file name: 768312000241SEQLIST.txt, record date: 2017 9 5th, size: 28 KB). The present invention provides an oncolytic vaccinia virus that encodes an immune checkpoint regulator (such as an immune checkpoint inhibitor) under the control of a late promoter, and encodes an immune checkpoint regulator (such as an immune checkpoint inhibitor) and bispecific junction Molecular (hereinafter also referred to as "bispecific molecule", "zygote" or "zygote") oncolytic viruses (such as oncolytic VV) as a new strategy to 1) promote T cell activation at the tumor site, 2) Effectively dissolve tumor cells infected with oncolytic virus or not infected with oncolytic virus (bystander killing), and / or 3) Minimize systemic adverse effects to achieve higher antitumor activity, especially against solid tumors. The present invention is based in part on the discovery that oncolytic viruses (such as oncolytic VV), immune checkpoint regulators and / or bispecific molecules that manifest at the tumor site can provide synergistic effects. In addition, the use of late promoters that are activated only after the virus infects tumor cells (such as the late vaccinia virus promoter F17R) to drive the expression of immune checkpoint regulators and / or bispecific molecules can avoid systemic toxicity and allow restricted delivery at the tumor site . Therefore, one aspect of the present invention provides an oncolytic vaccinia virus comprising a nucleic acid encoding an immune checkpoint regulator, wherein the nucleic acid is operably linked to a late promoter (such as F17R). Another aspect of the present invention provides an oncolytic virus (such as oncolytic VV), which includes a first nucleic acid encoding an immune checkpoint regulator (such as an immune checkpoint inhibitor), and a second nucleic acid encoding a bispecific molecule The bispecific molecule includes a first antigen binding domain that specifically recognizes a tumor antigen and a second antigen binding domain that specifically recognizes a cell surface molecule on an effector cell. Also provided are compositions (such as pharmaceutical compositions), and methods of using such oncolytic viruses (such as oncolytic VV) to treat cancer (such as solid cancer). The present invention also provides novel anti-PD-1 antibodies and methods of use thereof. I. Definitions Unless clearly indicated to the contrary, the practice of the present invention will use conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the skill of this technology, many of which It is described below for illustrative purposes. These techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, NY (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, Wiley & Sons, 1995; Sambrook and Russell , Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volumes I and II (edited by D. Glover); Oligonucleotide Synthesis (Edited by N. Gait, 1984); Nucleic Acid Hybridization (Edited by B. Hames and S. Higgins, 1985); Transcription and Translation (Edited by B. Hames and S. Higgins, 1984); Animal Cell Culture (Edited by R. Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other similar references. As used herein, "treatment (treating)" is a method for obtaining beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include (but are not limited to) one or more of the following: alleviate one or more symptoms caused by the disease, reduce the degree of the disease, stabilize the disease (eg, prevent or delay the deterioration of the disease) ), Preventing or delaying the spread of disease (eg, metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progress of the disease, improving the state of the disease, relieving the disease (partial or overall remission), reducing one of the treatments needed Or doses of many other drugs, delay disease progression, increase quality of life and / or prolong survival. "Treatment" also covers the reduction of cancer pathological results. The method of the present invention encompasses any one or more of these treatments. The term "prevent" and similar words, such as "prevented / preventing", indicate a method for preventing, inhibiting or reducing the likelihood of a disease or condition, such as cancer recurrence. It also refers to delaying the recurrence of a disease or condition, or delaying the recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it recurs. As used herein, "delaying" cancer development means delaying, hindering, slowing, retarding, stabilizing, and / or delaying the development of the disease. This delay may have different lengths of time depending on the disease being treated and / or the individual's medical history. The method of "delaying" cancer development is a method of reducing the probability of disease development within a given time frame and / or reducing the degree of disease within a given time range when compared to not using the method. These comparisons are usually based on clinical studies conducted using a statistically significant number of individuals. Cancer development can be detected using standard methods, including (but not limited to) computerized tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation test, arteriography, or biopsy. Development can also refer to cancer progression that may initially be undetectable and includes occurrence, recurrence, and attack. The term "effective amount" as used herein refers to an amount of an agent or combination of agents sufficient to treat a specified condition, condition or disease, such as ameliorating, alleviating, alleviating and / or delaying one or more symptoms thereof. For cancer, an effective amount includes an amount sufficient to shrink the tumor and / or reduce the tumor growth rate (thereby inhibiting tumor growth) or prevent or delay the proliferation of other unwanted cells. In some embodiments, the effective amount is an amount sufficient to delay development. In some embodiments, the effective amount is an amount sufficient to prevent or delay relapse. The effective amount can be administered in one or more doses. The effective amount of the drug or composition can: (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, block, slow down and preferably stop cancer cell infiltration in surrounding organs to a certain extent ; (Iv) inhibit (ie, slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) To some extent alleviate one or more cancer-related symptoms. As used herein, "individual" or "subject" refers to mammals, including (but not limited to) humans, bovines, horses, felines, canines, rodents, or primates. In some embodiments, each system is human. The terms "bispecific T cell zygote" or "BiTE" are used interchangeably herein to refer to antibodies or fragments thereof that have multiple epitope specificities and one specificity is against T cell surface molecules. The term "multispecific" used in conjunction with antibodies refers to having multiple epitope specificities (that is, two, three or more different epitopes that can specifically bind to a biomolecule or may be specific Antibodies that bind sexually to epitopes on two, three or more different biomolecules). The term "bispecific" used in conjunction with antibodies refers to antibodies that can specifically bind to two different epitopes on one biomolecule, or can specifically bind to epitopes on two different biomolecules. Unless otherwise indicated, the order in which the listed bispecific antibodies bind antigen is any order. That is, for example, the terms “anti-CD3 / EpCAM”, “anti-EpCAM / CD3”, “EpCAM × CD3”, “CD3 × EpCAM”, “CD3-EpCAM” and “EpCAM-CD3” are used interchangeably to refer to Bispecific antibody that specifically binds to both CD3 and EpCAM. As used herein, the term "immunological checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, or interferes with one or more inhibitory immune checkpoint molecules that can inhibit T cell activation and function. As used herein, the term "activator of stimulatory immune checkpoint molecules" refers to molecules that stimulate, activate, or increase the intensity of an immune response mediated by stimulatory immune checkpoint molecules. "Isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that usually contains a nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid molecule to which it is attached. The term includes vectors that exhibit self-replicating nucleic acid structures as well as vectors incorporated into the genome of the host cell into which they are introduced. Certain vectors can direct the expression of nucleic acids to which they are operably linked. Such carriers are referred to herein as "expression carriers". As used herein, the term "transfection" or "transformation" or "transduction" refers to a method of transferring or introducing exogenous nucleic acid into a host cell. "Transfected" or "transformed" or "transduced" cells are cells that have been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. The terms "host cell", "host cell strain" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from them, regardless of the number of subcultures. The nucleic acid content of the progeny may not be exactly the same as the parent cell, but may contain mutations. This article includes mutant progeny screened or selected for the same function or biological activity against the initial transformed cells. "Adjuvant setting" refers to a clinical situation in which an individual has a history of cancer and generally (but not necessarily) responds to therapy, including (but not limited to) surgery (eg, surgical resection), radiation therapy, and chemotherapy . However, due to a history of cancer, these individuals are considered to be at risk of developing the disease. The treatment or dosing in "adjuvant therapy" refers to the subsequent treatment mode. The degree of risk (for example, when an individual in adjuvant therapy is considered "high-risk" or "low-risk") depends on several factors, most often on the extent of the disease at first treatment. "Neoadjuvant therapy" refers to a clinical situation in which this method is performed before initial therapy / deterministic therapy. It should be understood that the embodiments of the present invention described herein include "consisting of embodiments" and / or "essentially consisting of embodiments". Reference herein to "about" includes (and describes) changes to the value or parameter itself. For example, a reference to "about X" includes a description about "X". As used herein, reference to a value or parameter that is "not" generally means and describes "except for a value or parameter." For example, the method is not used to treat type X cancer means that the method is used to treat types of cancer other than type X. The term "about X-Y" as used herein has the same meaning as "about X to about Y". Unless the context clearly indicates otherwise, the singular forms "a (an) (a / an)" and "the (the)" include plural (species) indicators as used in this document and the scope of the attached patent applications. II. Oncolytic viruses expressing immune checkpoint regulatorsOncolytic vaccinia virus expressing immune checkpoint regulator under late promoter The present invention provides an oncolytic vaccinia virus (VV) comprising a nucleic acid encoding an immune checkpoint regulator, wherein the nucleic acid is operably linked to a late promoter. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, the late promoter line is F17R. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Ankara Vaccinia Virus (MVA), Lister, King, IHD, Evans, USSR and Western Reserve (WR). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of the thymidine kinase (TK) gene and the vaccinia virus growth factor (VGF) gene. In some embodiments, the immune checkpoint regulator is an activator of an stimulatory immune checkpoint molecule (such as an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, immune checkpoint regulators are immune checkpoint inhibitors (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA -4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitors). In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is bound to an immune checkpoint molecule, such as a ligand of PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the oncolytic VV further comprises a second nucleic acid encoding a cytokine (such as GM-CSF). Late oncolytic vaccinia virus promoters include (but are not limited to) F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, the late promoter line is F17R. Therefore, in some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an immune checkpoint regulator, wherein the nucleic acid is operably linked to the F17R late promoter. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the immune checkpoint regulator is an activator of an stimulatory immune checkpoint molecule (such as an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, immune checkpoint regulators are immune checkpoint inhibitors (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA -4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitors). In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule, such as an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is bound to an immune checkpoint molecule, such as a ligand of PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). The immune checkpoint modulator may be an activator of stimulatory immune checkpoint molecules, including (but not limited to) activators of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS. Therefore, in some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an activator of a stimulatory immune checkpoint molecule (such as an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS), Wherein the nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an activator of an stimulatory immune checkpoint molecule (such as an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS), wherein the The nucleic acid line is operably linked to the F17R late promoter. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is a ligand that binds to the immune checkpoint molecule. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). The immune checkpoint regulator may be an immune checkpoint inhibitor, including (but not limited to) PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA -4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitors. Therefore, in some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an immune checkpoint inhibitor, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an immune checkpoint inhibitor, wherein the nucleic acid is operably linked to the F17R late promoter. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the immune checkpoint inhibitors are PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA , B7-H4, CD160, 2B4 or CD73 inhibitors. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is bound to a ligand of an immune checkpoint molecule, such as PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an antibody that specifically recognizes an inhibitory immune checkpoint molecule, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an antibody that specifically recognizes an inhibitory immune checkpoint molecule, wherein the nucleic acid is operably linked to the F17R late promoter. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the inhibitory immune checkpoint molecular lines PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA , B7-H4, CD160, 2B4 or CD73. In some embodiments, the inhibitory immune checkpoint molecular line is PD-1. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, there is provided an oncolytic vaccinia virus comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to the F17R late promoter. In some embodiments, the inhibitor of PD-1 is an antibody that specifically recognizes PD-1. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is Antibodies that specifically recognize PD-1. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, there is provided an oncolytic vaccinia virus comprising a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to an F17R late promoter, wherein the inhibitor of PD-1 is specific An antibody that recognizes PD-1. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is An antibody that specifically recognizes PD-1, the antibody includes: a heavy chain variable region (VH), which includes (1) HVR-H1 containing an amino acid sequence SEQ ID NO: 1; (2) an amino acid sequence HVR-H2 of SEQ ID NO: 2; and (3) HVR-H3 of amino acid sequence SEQ ID NO: 3; and / or light chain variable region (VL), including (1) amino acid HVR-L1 of sequence SEQ ID NO: 4; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 5; and (3) HVR-L3 containing amino acid sequence SEQ ID NO: 6. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late F17R promoter, wherein the inhibitor of PD-1 specifically recognizes PD -1 antibody, the antibody comprises: a heavy chain variable region (VH), which includes (1) HVR-H1 containing amino acid sequence SEQ ID NO: 1; (2) amino acid sequence SEQ ID NO: HVR-H2 of 2; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 3; and / or light chain variable region (VL), including (1) amino acid sequence SEQ ID NO : HVR-L1 of 4; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 5; and (3) HVR-L3 containing amino acid sequence SEQ ID NO: 6 In some embodiments, the anti-PD-1 antibody includes a heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 13, and / or a light chain variable containing the amino acid sequence SEQ ID NO: 14 District (VL). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is An antibody that specifically recognizes PD-1, the antibody includes a heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 13, and / or a light chain variable containing the amino acid sequence SEQ ID NO: 14 District (VL). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to an F17R late promoter, wherein the inhibitor of PD-1 is specifically recognized An antibody to PD-1, which includes a heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 13 and / or a light chain variable region (VL) containing the amino acid sequence SEQ ID NO: 14 ). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is specific An antibody that sexually recognizes PD-1, the antibody comprises: a heavy chain variable region (VH), which includes (1) HVR-H1 containing an amino acid sequence SEQ ID NO: 7; (2) an amino acid sequence SEQ ID NO: HVR-H2 of 8; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 9; and / or light chain variable region (VL), including (1) amino acid containing sequence HVR-L1 of SEQ ID NO: 10; (2) HVR-L2 containing the amino acid sequence SEQ ID NO: 11; and (3) HVR-L3 containing the amino acid sequence SEQ ID NO: 12. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late F17R promoter, wherein the inhibitor of PD-1 specifically recognizes PD -1 antibody, the antibody comprises: a heavy chain variable region (VH), which includes (1) HVR-H1 containing amino acid sequence SEQ ID NO: 7; (2) amino acid sequence SEQ ID NO: HVR-H2 of 8; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 9; and / or light chain variable region (VL), including (1) amino acid sequence SEQ ID NO : HVR-L1 of 10; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 11; and (3) HVR-L3 containing amino acid sequence SEQ ID NO: 12. In some embodiments, the anti-PD-1 antibody includes a heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 15 and / or a light chain variable containing the amino acid sequence SEQ ID NO: 16 District (VL). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to a late promoter (such as F17R), wherein the inhibitor of PD-1 is An antibody that specifically recognizes PD-1, the antibody includes a heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 15, and / or a light chain variable containing the amino acid sequence SEQ ID NO: 16 District (VL). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding an inhibitor of PD-1, wherein the nucleic acid is operably linked to an F17R late promoter, wherein the inhibitor of PD-1 is specifically recognized An antibody to PD-1, which includes a heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 15 and / or a light chain variable region (VL) containing the amino acid sequence SEQ ID NO: 16 ). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a ligand that binds to PD-L1 and / or PD-L2, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a ligand that binds to PD-L1 and / or PD-L2, wherein the nucleic acid is operably linked to the F17R late promoter. In some embodiments, the ligand binding to PD-L1 and / or PD-L2 comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to late initiation (Such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of a PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to late F17R Promoter. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to late initiation (Such as F17R), and wherein the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of a PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to late F17R Promoter, and wherein the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a ligand that binds to HHLA2, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a ligand that binds to HHLA2, wherein the nucleic acid is operably linked to the F17R late promoter. In some embodiments, the ligand that binds to HHLA2 comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter Such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to the F17R late promoter . In some embodiments, the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to a late promoter ( Such as F17R), and wherein the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to the F17R late promoter , And wherein the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a ligand that binds to CD47 and CXCR4, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a ligand that binds to CD47 and CXCR4, wherein the nucleic acid is operably linked to the F17R late promoter. In some embodiments, the ligand binding to CD47 and CXCR4 comprises SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the SIRPα extracellular domain comprises the amino acid sequence SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker, such as an IgG1 hinge, or a linker that includes the amino acid sequence SEQ ID NO: 31. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to the late Promoter (such as F17R). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided comprising a nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to F17R Late promoter. In some embodiments, the SIRPα extracellular domain comprises the amino acid sequence SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker, such as an IgG1 hinge, or a linker that includes the amino acid sequence SEQ ID NO: 31. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, an oncolytic VV is provided that includes a nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to the late stage A promoter (such as F17R), and wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO: 29 and the CXCL12 fragment contains the amino acid sequence SEQ ID NO: 30. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, an oncolytic VV is provided comprising a nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc), wherein the nucleic acid is operably linked to F17R Late promoter, and wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO: 29 and the CXCL12 fragment contains the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker, such as an IgG1 hinge, or a linker that includes the amino acid sequence SEQ ID NO: 31. In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV comprises a double deletion of TK and VGF genes. In some embodiments, the oncolytic VV further comprises a nucleic acid encoding a cytokine (such as GM-CSF). Oncolytic virus expressing immune checkpoint regulator and bispecific junction molecule The present invention also provides an oncolytic virus (such as oncolytic VV), which comprises a first nucleic acid encoding an immune checkpoint regulator and a bispecific molecule Second nucleic acid, the bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a cell surface molecule (such as T) that specifically recognizes the effector cell The second antigen binding domain of CD3 on lymphocytes (such as scFv). In some embodiments, the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Senegal Valley virus (SVV), adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2 ), Myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, measles virus, influenza virus, Sindby virus and New City Epidemic virus (NDV). In some embodiments, the oncolytic virus is oncolytic VV. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Ankara Vaccinia Virus (MVA), Lister, King, IHD, Evans, USSR and Western Reserve (WR). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic virus comprises a double deletion of the TK gene and the VGF gene. In some embodiments, the immune checkpoint regulator is an activator of an stimulatory immune checkpoint molecule (such as an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, the immune checkpoint regulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint regulator sub-line PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA , B7-H4, CD160, 2B4 or CD73 inhibitors. In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is a ligand that binds to the immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is a ligand of PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and phosphatidylinositol-3 (GPC3). In some embodiments, the tumor antigen EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell line is selected from the group consisting of T lymphocytes, B lymphocytes, natural killer (NK) cells, dendritic cells (DC), macrophages, monocytes, and neutrophils Ball and NKT cells. In some embodiments, the effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D. In some embodiments, the cell surface molecule line is CD3. In some embodiments, the first and / or second antigen binding domains are single chain variable fragments (scFv). In some embodiments, the first and second antigen binding domains are connected by a linker. In some embodiments, the first antigen binding domain is N-terminal to the second antigen binding domain. In some embodiments, the first antigen binding domain is at the C-terminus of the second antigen binding domain. In some embodiments, the first nucleic acid encoding the immune checkpoint regulator is operably linked to the late promoter. In some embodiments, the second nucleic acid encoding the bispecific molecule is operably linked to the late promoter. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, This late promoter line is F17R. In some embodiments, The oncolytic virus further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).  The oncolytic viruses described herein that express immune checkpoint regulators and bispecific junction molecules can: 1) Promote the activation of T cells at the tumor site; 2) Effectively dissolve tumor cells infected or not infected with oncolytic virus (bystander killing); 3) By selectively delivering and maintaining immune checkpoint regulators within the tumor, To minimize the side effects of systemic autoimmunity / spontaneous inflammation caused by non-tumor restricted blocking of immune checkpoint molecules 4) Minimize systemic adverse events by selectively delivering and maintaining bispecific junction molecules within the tumor; And / or 5) enhance tumor lysis activity mediated by bispecific junction molecules in the presence of T cells.  In some embodiments, Provide an oncolytic VV, It contains a first nucleic acid encoding an immune checkpoint regulator and a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, The oncolytic VV strain is WR strain. In some embodiments, Oncolytic VV contains the double deletion of TK and VGF genes. In some embodiments, Immune checkpoint regulators are immune checkpoint inhibitors. In some embodiments, The immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, The immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, The immune checkpoint regulator contains a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The oncolytic VV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF).  Immune checkpoint regulators can be activators of stimulatory immune checkpoint molecules, Including (but not limited to) CD27, CD28, CD40, CD122, CD137, OX40, Activator of GITR or ICOS.  therefore, In some embodiments, Provide an OV, It contains a molecular activator that encodes a stimulating immune checkpoint molecule (such as CD27, CD28, CD40, CD122, CD137, OX40, GITR or ICOS activator) the first nucleic acid and the second nucleic acid encoding the bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The immune checkpoint regulator is an antibody that specifically recognizes a stimulating immune checkpoint molecule. In some embodiments, The immune checkpoint regulator is a ligand that binds to the stimulatory immune checkpoint molecule. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  The immune checkpoint regulator can be an immune checkpoint inhibitor, Including (but not limited to) PD-1, PD-L1 PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor.  therefore, In some embodiments, Provide an oncolytic virus, It contains coded immune checkpoint inhibitors (such as PD-1, PD-L1 PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) the first nucleic acid, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint inhibitors are PD-1 inhibitors. In some embodiments, Immune checkpoint inhibitors are anti-PD-1 antibodies. In some embodiments, Immune checkpoint inhibitors comprise a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint inhibitor comprises a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Immune checkpoint inhibitors include SIRPα extracellular domains and fusions of CXCL12 fragments with immunoglobulin Fc fragments (such as IgG4 Fc). In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, PD-1 inhibitors are anti-PD-1 antibodies. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus,  It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first antigen-binding domain (first antigen-binding domain such as scFv) and second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) ), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen-binding domain (such as scFv) and the second antigen-binding domain that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO: 10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a ligand bound to PD-L1 and / or PD-L2, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, The ligand bound to PD-L1 and / or PD-L2 comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a ligand bound to HHLA2, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, The ligand that binds to HHLA2 contains a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a ligand that binds to CD47 and CXCR4, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, The ligand binding to CD47 and CXCR4 includes SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, The SIRPα extracellular domain contains the amino acid sequence SEQ ID NO: 29. In some embodiments, The CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, The CXCL12 fragment is through a linker, Such as IgG1 hinge, Or contains the amino acid sequence SEQ ID NO:  The linker 31 is connected to the SIRPα extracellular domain. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3. In some embodiments, Effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, The cell surface molecule is CD3. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  The tumor antigen may be a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, TAA or TSA is expressed on cells of solid tumors. Tumor antigens include (but are not limited to) EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2 and phosphoglypican-3. In some embodiments, The tumor antigen line EpCAM. In some embodiments, Tumor antigen line FAP. In some embodiments, The tumor antigen is EGFR. In some embodiments, Tumor antigen line GPC3.  Effector cells include (but are not limited to) T lymphocytes, B lymphocytes, Natural killer (NK) cells, Dendritic cells (DC), Macrophages, Monocytes, Neutrophil, NKT cells or similar cells. In some embodiments, Effector cell line T lymphocytes. In some embodiments, Effector cell line cytotoxic T lymphocytes.  Cell surface molecules on effector cells include (but are not limited to) CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, NKG2D or similar. In some embodiments, The cell surface molecule is CD3.  therefore, In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an immune checkpoint regulator and a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint regulators are immune checkpoint inhibitors. In some embodiments, The immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, The immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, The immune checkpoint regulator contains a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding the immune checkpoint regulator, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint regulators are immune checkpoint inhibitors. In some embodiments, The immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, The immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, The immune checkpoint regulator contains a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding the immune checkpoint regulator, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint regulators are immune checkpoint inhibitors. In some embodiments, The immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, The immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, The immune checkpoint regulator contains a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding the immune checkpoint regulator, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint regulators are immune checkpoint inhibitors. In some embodiments, The immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, The immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, The immune checkpoint regulator contains a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding the immune checkpoint regulator, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen-binding domain (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint regulators are immune checkpoint inhibitors. In some embodiments, The immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, The immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, The immune checkpoint regulator contains a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains coded immune checkpoint inhibitors (such as PD-1, PD-L1 PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) the first nucleic acid, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen-binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen-binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint inhibitors are antibodies that specifically recognize inhibitory immune checkpoint molecules. In some embodiments, Immune checkpoint inhibitors are PD-1 inhibitors. In some embodiments, Immune checkpoint inhibitors are anti-PD-1 antibodies. In some embodiments, Immune checkpoint inhibitors bind to inhibitory immune checkpoint molecules (such as PD-L1 / PD-L2, HHLA-2, CD47 or CXCR4) ligand. In some embodiments, Immune checkpoint inhibitors comprise a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint inhibitor comprises a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Immune checkpoint inhibitors include SIRPα extracellular domains and fusions of CXCL12 fragments with immunoglobulin Fc fragments (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains coded immune checkpoint inhibitors (such as PD-1, PD-L1 PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) the first nucleic acid, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint inhibitors are antibodies that specifically recognize inhibitory immune checkpoint molecules. In some embodiments, Immune checkpoint inhibitors are PD-1 inhibitors. In some embodiments, Immune checkpoint inhibitors are anti-PD-1 antibodies. In some embodiments, Immune checkpoint inhibitors bind to inhibitory immune checkpoint molecules (such as PD-L1 / PD-L2, HHLA-2, CD47 or CXCR4) ligand. In some embodiments, Immune checkpoint inhibitors comprise a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint inhibitor comprises a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Immune checkpoint inhibitors include SIRPα extracellular domains and fusions of CXCL12 fragments with immunoglobulin Fc fragments (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains coded immune checkpoint inhibitors (such as PD-1, PD-L1 PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) the first nucleic acid, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint inhibitors are antibodies that specifically recognize inhibitory immune checkpoint molecules. In some embodiments, Immune checkpoint inhibitors are PD-1 inhibitors. In some embodiments, Immune checkpoint inhibitors are anti-PD-1 antibodies. In some embodiments, Immune checkpoint inhibitors bind to inhibitory immune checkpoint molecules (such as PD-L1 / PD-L2, HHLA-2, CD47 or CXCR4) ligand. In some embodiments, Immune checkpoint inhibitors comprise a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint inhibitor comprises a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Immune checkpoint inhibitors include SIRPα extracellular domains and fusions of CXCL12 fragments with immunoglobulin Fc fragments (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains coded immune checkpoint inhibitors (such as PD-1, PD-L1 PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) the first nucleic acid, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint inhibitors are antibodies that specifically recognize inhibitory immune checkpoint molecules. In some embodiments, Immune checkpoint inhibitors are PD-1 inhibitors. In some embodiments, Immune checkpoint inhibitors are anti-PD-1 antibodies. In some embodiments, Immune checkpoint inhibitors bind to inhibitory immune checkpoint molecules (such as PD-L1 / PD-L2, HHLA-2, CD47 or CXCR4) ligand. In some embodiments, Immune checkpoint inhibitors comprise a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint inhibitor comprises a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Immune checkpoint inhibitors include SIRPα extracellular domains and fusions of CXCL12 fragments with immunoglobulin Fc fragments (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains coded immune checkpoint inhibitors (such as PD-1, PD-L1 PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) the first nucleic acid, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen-binding domain (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint inhibitors are antibodies that specifically recognize inhibitory immune checkpoint molecules. In some embodiments, Immune checkpoint inhibitors are PD-1 inhibitors. In some embodiments, Immune checkpoint inhibitors are anti-PD-1 antibodies. In some embodiments, Immune checkpoint inhibitors bind to inhibitory immune checkpoint molecules (such as PD-L1 / PD-L2, HHLA-2, CD47 or CXCR4) ligand. In some embodiments, Immune checkpoint inhibitors comprise a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint inhibitor comprises a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, Immune checkpoint inhibitors include SIRPα extracellular domains and fusions of CXCL12 fragments with immunoglobulin Fc fragments (such as IgG4 Fc). In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, PD-1 inhibitors are anti-PD-1 antibodies. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, PD-1 inhibitors are anti-PD-1 antibodies. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, PD-1 inhibitors are anti-PD-1 antibodies. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3, And a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, PD-1 inhibitors are anti-PD-1 antibodies. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen-binding domain (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, PD-1 inhibitors are anti-PD-1 antibodies. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  The light chain variable region of 16. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  The light chain variable region of 16. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It contains (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen-binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen-binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv). In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen-binding domain (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen-binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen-binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen-binding domain (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, TMIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen-binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen-binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen-binding domain (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, The SIRPα extracellular domain contains the amino acid sequence SEQ ID NO: 29. In some embodiments, The CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, The CXCL12 fragment is through a linker, Such as IgG1 hinge, Or contains the amino acid sequence SEQ ID NO:  The linker 31 is connected to the SIRPα extracellular domain. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EpCAM and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes EGFR and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes cell surface molecules on effector cells. scFv), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes GPC3 and a second antigen binding domain (such as CD3 on T lymphocytes) that specifically recognizes a cell surface molecule on effector cells. scFv), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first antigen binding domain (such as scFv) and the second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, The CXCL12 fragment is through a linker, Such as IgG1 hinge, Or contains the amino acid sequence SEQ ID NO:  The linker 31 is connected to the SIRPα extracellular domain. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  The bispecific molecules described herein can have any form. In some embodiments, The first antigen binding domain is scFv. In some embodiments, The second antigen binding domain is scFv. In some embodiments, Both the first and second antigen binding domains are scFv. In some embodiments, The first and second antigen binding domains are connected by a linker. In some embodiments, The first antigen binding domain is at the N-terminus of the second antigen binding domain. In some embodiments, The first antigen-binding domain is at the C-terminus of the second antigen-binding domain.  therefore, In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding the immune checkpoint regulator, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding the immune checkpoint regulator, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The first scFv is at the N-terminus of the second scFv. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, Immune checkpoint regulators are immune checkpoint inhibitors. In some embodiments, The immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, The immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, The immune checkpoint regulator contains a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The immune checkpoint regulator contains SIRPα extracellular domain and a fusion of CXCL12 fragment and immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, The first scFv and the second scFv are connected by a linker. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv, And a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, Provide an oncolytic virus, It contains the first nucleic acid encoding PD-1 inhibitor, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv, And a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The first scFv is at the N-terminus of the second scFv. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, PD-1 inhibitors are anti-PD-1 antibodies. In some embodiments, The first scFv and the second scFv are connected by a linker. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  1 of HVR-H1; (2) amino acid sequence SEQ ID NO:  2 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  3 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  4 of HVR-L1; (2) amino acid sequence SEQ ID NO:  5 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  6 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  13 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  14 Light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes: Heavy chain variable region (VH), It includes (1) amino acid sequence SEQ ID NO:  7 of HVR-H1; (2) amino acid sequence SEQ ID NO:  8 of HVR-H2; And (3) amino acid sequence SEQ ID NO:  9 of HVR-H3; And / or light chain variable region (VL), It includes (1) amino acid sequence SEQ ID NO:  10 of HVR-L1; (2) amino acid sequence SEQ ID NO:  11 of HVR-L2; And (3) amino acid sequence SEQ ID NO:  12 of HVR-L3. In some embodiments, Anti-PD-1 antibodies include amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding an antibody that specifically recognizes PD-1, And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes CD3 on T lymphocytes, The anti-PD-1 antibody includes amino acid sequence SEQ ID NO:  15 heavy chain variable region (VH), And / or amino acid sequence SEQ ID NO:  16 light chain variable region (VL). In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), The first scFv is at the N-terminus of the second scFv. In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes CD3 on T lymphocytes. In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes CD3 on T lymphocytes, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes CD3 on T lymphocytes. In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes CD3 on T lymphocytes, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes CD3 on T lymphocytes. In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes CD3 on T lymphocytes, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes CD3 on T lymphocytes. In some embodiments, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes CD3 on T lymphocytes, The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes CD3 on T lymphocytes. In some embodiments, TMIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes CD3 on T lymphocytes, Wherein the TGIGD2 extracellular domain contains the amino acid sequence SEQ ID NO: 27. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) first scFv and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes cell surface molecules on effector cells, such as CD3 on T lymphocytes. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes CD3 on T lymphocytes. In some embodiments, The SIRPα extracellular domain contains the amino acid sequence SEQ ID NO: 29. In some embodiments, The CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, The CXCL12 fragment is through a linker, Such as IgG1 hinge, Or contains the amino acid sequence SEQ ID NO:  The linker 31 is connected to the SIRPα extracellular domain. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EpCAM and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes FAP and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes EGFR and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule includes a first scFv that specifically recognizes GPC3 and a second scFv that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, Provide an oncolytic virus, It contains a first nucleic acid encoding a fusion of SIRPα extracellular domain and a CXCL12 fragment with an immunoglobulin Fc fragment (such as IgG4 Fc), And a second nucleic acid encoding a bispecific molecule, The bispecific molecule contains specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) the first scFv and the second scFv that specifically recognizes CD3 on T lymphocytes, Wherein the SIRPα extracellular domain contains the amino acid sequence SEQ ID NO:  29, And the CXCL12 fragment contains the amino acid sequence SEQ ID NO:  30. In some embodiments, The CXCL12 fragment is through a linker, Such as IgG1 hinge, Or contains the amino acid sequence SEQ ID NO:  The linker 31 is connected to the SIRPα extracellular domain. In some embodiments, This OV is an oncolytic VV. In some embodiments, This OV line is WR strain VV. In some embodiments, The OV contains double deletions of the TK and VGF genes. In some embodiments, The first scFv is at the N-terminus of the second scFv. In some embodiments, The first and / or second nucleic acid line is operably linked to a late promoter (such as F17R). In some embodiments, The OV further contains a third nucleic acid encoding a cytokine (such as GM-CSF).  The encoding bispecific molecules described herein, The nucleic acid of the immune checkpoint regulator and / or interleukin may be operably linked to the promoter. In some embodiments, Encoding bispecific molecules, At least two of the immune checkpoint regulator and the nucleic acid of the interleukin are operably linked to the same promoter. In some embodiments, Encoding bispecific molecules, The nucleic acids of the immune checkpoint regulator and interleukin are all operably linked to the same promoter. In some embodiments, Encoding bispecific molecules, The nucleic acids of the immune checkpoint regulator and interleukin are all operably linked to different promoters. In some embodiments, This promoter is the late promoter. In some embodiments, This promoter is a vaccinia virus promoter. In some embodiments, This promoter is the late VV promoter. In some embodiments, This promoter is F17R.  Oncolytic viruses Oncolytic viruses can selectively replicate in dividing cells (such as cancer cells), At the same time, non-dividing cells (such as normal cells) are not damaged. When the infected dividing cells are destroyed by lysis, It releases new infectious virus particles to infect surrounding dividing cells. Cancer cells are ideal hosts for many viruses, Because they have inactivated antiviral interferon pathways or have mutant tumor suppressor genes, Allow virus replication to proceed unimpeded (Chernajovsky et al.,  2006,  British Med.  J.  332: 170-2). Exemplary oncolytic viruses include, but are not limited to, vaccinia virus (VV), Senegal Valley virus (SVV), adenovirus (AdV), herpes simplex virus (HSV, such as HSV1 and HSV2), reovirus, mucus Oncovirus (MYXV), poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, measles virus, influenza virus, Sindby virus, Newcastle disease virus (NDV) Or similar viruses (see, for example, Kirn et al., Nat.  Med.  7: 781 (2001); Coffey et al., Science 282: 1332 (1998); Lorence et al., Cancer Res.  54: 6017 (1994); and Peng et al., Blood 98: 2002 (2001)). In some embodiments, the oncolytic virus described herein is oncolytic vaccinia virus (VV). Oncolytic viruses can use DNA or RNA as their genetic material. Oncolytic DNA viruses can have capsid symmetry, that is, icosahedral or complex. The icosahedral oncolytic DNA virus may be a naked virus or contain an envelope. Oncolytic DNA viruses include adenoviridae (eg adenovirus, genome size 36-38 kb), herpesvirus (eg HSV1, genome size 120-200 kb) and poxviridae (eg vaccinia virus and myxoma virus, The genome size is 130-280 kb). Oncolytic RNA viruses include RNA viruses with icosahedral or spiral capsid symmetry. The icosahedral oncolytic virus is a naked virus that does not contain an envelope and includes the reoviridae family (eg, reovirus, having a genome of 22-27 kb) and the small RNA virus family (eg, poliovirus, The genome size is 7. 2-8. 4 kb). Spiral oncolytic RNA viruses have an envelope and include Rhabdoviridae (eg VSV, genome size 13-16 kb) and Paramyxoviridae (eg MV and NDV, genome size 16-20 kb). In some embodiments, the oncolytic virus is vaccinia virus (VV). In some embodiments, the VV may be Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Ankara Vaccinia Virus (MVA), Lister, King, IHD, Evans, USSR, or Western Reserve (WR) strains. In some embodiments, the VV strain is a WR strain. In some embodiments, the oncolytic viruses of the invention (such as oncolytic VV) are modified by changing one or more viral genes. Such modifications preferably result in the synthesis of a defective protein that cannot ensure the activity (or lack of synthesis) of the protein produced by the unmodified gene under normal conditions. Modification encompasses the deletion, mutation and / or substitution of one or more nucleotides (adjacent or non-adjacent) within the viral gene or its regulatory elements. Modifications can be carried out using conventional recombination techniques in various ways known to those skilled in the art. Exemplary modifications are disclosed in the literature and alter DNA metabolism, host virulence, and IFN pathway (see, for example, Guse et al., 2011, Expert Opinion Biol.  Ther.  11 (5): 595-608) and the modification of viral genes involved in similar characteristics are particularly preferred. In some embodiments, an oncolytic virus (such as oncolytic VV) contains an inactive mutation of the thymidine kinase (TK) gene, thereby producing a negative TK phenotype. In some embodiments, the TK gene of VV is deleted. TK enzymes are involved in the synthesis of deoxyribonucleotides. TK is required for virus replication in normal cells, because these cells generally have low concentrations of nucleotides, but they are not necessary in dividing cells containing high nucleotide concentrations. Therefore, TK deletion clearly limits viral replication in dormant cells, so that effective viral replication occurs only in actively dividing cells (eg, cancer cells). Alternatively or in combination, other strategies can be implemented to further increase the tumor specificity of the virus. Representative examples of suitable modifications include disruption of the gene encoding VGF in the viral genome. VGF (representing VV growth factor) is a secreted protein that appears early after cell infection and its function appears to be important for the spread of the virus in normal cells. VGF-deficient vaccinia virus replication is greatly reduced in dormant (non-cancer) cells. In some embodiments, the VV of the present invention does not express functional acne growth factor (VGF). In some embodiments, the oncolytic virus of the present invention is a vaccinia virus lacking both TK and VGF activities. It has been shown that the effects of TK and VGF deletion are synergistic. Therefore, in some embodiments, an oncolytic vaccinia virus is provided, which comprises an immune checkpoint regulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc Fusion protein, or nucleic acid of SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein, wherein the nucleic acid is operably linked to a late promoter (such as F17R). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic VV includes a double deletion of the TK gene and the VGF gene. In some embodiments, an oncolytic virus (such as an oncolytic VV) is provided, which comprises an immune checkpoint regulator (such as an anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular structure Domain-Fc fusion protein, or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein) nucleic acids, further comprising nucleic acids encoding bispecific molecules, which contain specific recognition tumor antigens (such as EpCAM, FAP, EGFR or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, the oncolytic virus is oncolytic VV. In some embodiments, the oncolytic virus is a strain of WR. In some embodiments, the oncolytic virus comprises a double deletion of the TK gene and the VGF gene. It is envisaged that the present invention also relates to an oncolytic virus vector comprising the nucleic acid molecule described in the present invention. As used herein, the term "viral vector" is used according to its accepted meaning in the art. The term refers to a nucleic acid vector construct that includes at least one element of viral origin and can be packaged in viral vector particles. Viral vector particles can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells in vitro or in vivo. Oncolytic virus vectors include, but are not limited to, vaccinia virus (VV) vectors, Senegal Valley virus (SVV) vectors, adenovirus (AdV) vectors, herpes simplex virus vectors (such as HSV1 vectors), reovirus vectors, Myxoma virus (MYXV) vector, poliovirus vector, vesicular stomatitis virus (VSV) vector, measles virus (MV) vector, lentivirus vector, retrovirus vector, measles virus vector, influenza virus vector, Xin Derby virus vector and Newcastle disease virus (NDV) vector. In some embodiments, the oncolytic virus vector is an oncolytic VV vector. Therefore, the present invention relates to an oncolytic VV vector comprising an immune checkpoint regulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, Or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), wherein the nucleic acid is operably linked to a late promoter (such as F17R). An oncolytic virus vector (such as an oncolytic VV vector) is also provided, which contains an immune checkpoint regulator (such as an anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc Fusion protein, or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein) nucleic acid, further comprising a nucleic acid encoding a bispecific molecule that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3 ) Of a first antigen-binding domain (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). The present application also provides an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein). Such immune checkpoint regulators can be incorporated into oncolytic viruses, such as oncolytic VV, or can be provided in an isolated form. Immune checkpoint regulator Immune checkpoints are molecules in the immune system that increase (stimulating molecules) or decrease signals (inhibitory molecules). Immune checkpoint proteins regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses. Stimulus checkpoint molecules include, but are not limited to, CD27, CD40, OX40, GITR, and CD137 that belong to the tumor necrosis factor (TNF) receptor superfamily, and CD28 and ICOS that belong to the B7-CD28 superfamily. Inhibitory checkpoint molecules include (but are not limited to) planned death protein 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cells Immunoglobulin domain and mucin domain 3 (TIM-3, HAVCR2), T cell activated V domain Ig inhibitor (VISTA, B7-H5), B7-H3, B7-H4 (VTCN1), HHLA2 ( B7-H7), B and T lymphocyte attenuation factor (BTLA), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), adenosine A2A receptor (A2AR ), Ig and ITIM domain proteins T cell immune receptor (TIGIT), 2B4 (CD244) and its ligands. Various checkpoint proteins have been extensively studied, such as CTLA-4 and its ligands CD80 (B7-1) and CD86, and PD-1 and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC , CD273) (see, for example, Pardoll, Nature Reviews Cancer 12: 252-264 (2012)). The immune checkpoint regulator may be an immune checkpoint inhibitor (inhibitor of inhibitory immune checkpoint molecules) or an activator of stimulatory immune checkpoint molecules. Immune checkpoint inhibitors (inhibitors of inhibitory immune checkpoint molecules) have received much attention in the present invention, such as PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7-DC , CD273), LAG-3, TIM-3 (HAVCR2), BTLA, CTLA-4, TIGIT, VISTA (B7-H5), B7-H4 (VTCN1), CD160 (BY55), HHLA2 (B7-H7), CXCR4 , 2B4 (CD244), CD73, B7-1 (CD80), B7-H3 (CD276), KIR or IDO inhibitor. In some embodiments, immune checkpoint regulators are activators of stimulatory immune checkpoint molecules. In some embodiments, the activator of the stimulatory immune checkpoint molecule is a natural or engineered ligand of the stimulatory immune checkpoint molecule, including, for example, OX40 ligand (eg OX40L), CD28 ligand (eg CD80) , CD86), ICOS ligands (e.g. B7RP1), 4-1BB ligands (e.g. 4-1BBL, Ultra 4-1BBL), CD27 ligands (e.g. CD70), CD40 ligands (e.g. CD40L) and TCR Ligand (eg MHC class I or II molecules, IMCgp100). In some embodiments, the activator of the stimulatory immune checkpoint molecule is a secreted protein. In some embodiments, activators of stimulatory immune checkpoint molecules are antibodies (such as agonistic antibodies), such as anti-CD28, anti-OX40, anti-ICOS, anti-GITR, anti-4-1BB, anti-CD27, anti-CD40, anti- CD3 and anti-HVEM. In some embodiments, the immune checkpoint regulator is an immune checkpoint inhibitor. In some embodiments, immune checkpoint inhibitors target T cells. In some embodiments, immune checkpoint inhibitors target tumor cells. For example, in some cases, tumor cells can cut off activated T cells when they are connected to specific T cell receptors. However, immune checkpoint inhibitors can prevent tumor cells from connecting to T cells, thereby keeping T cells activated (see, for example, Howard West, JAMA Oncol.  1 (1): 115 (2015)). In some embodiments, immune checkpoint inhibitors are inhibitory immune checkpoint molecules that are natural or engineered ligands, including ligands such as CTLA-4 (eg B7. 1. B7. 2), TIM-3 ligands (such as Galectin-9), A2A receptor ligands (such as adenosine, Regadenoson), LAG-3 ligands (such as MHC class I) Or MHC class II molecules), BTLA ligands (e.g. HVEM, B7-H4), KIR ligands (e.g. class I MHC or class II MHC molecules), PD-1 ligands (e.g. PD-L1, PD- L2), IDO ligand (e.g. NKTR-218, Indoximod, NLG919), HHLA2 ligand (e.g. TMIGD2), CXCR4 ligand (e.g. CXCL12) and CD47 ligand (e.g. SIRP- α receptor). In some embodiments, the immune checkpoint inhibitor is secreted. In some embodiments, immune checkpoint inhibitors are antibodies (such as antagonistic antibodies) that target inhibitory immune checkpoint proteins, including (but not limited to) anti-CTLA-4, anti-TIM-3, anti-LAG-3, Anti-KIR, anti-PD-1, anti-PD-L1, anti-CD73, anti-B7-H3, anti-CD47, anti-BTLA, anti-VISTA, anti-A2AR, anti-B7-1, anti-B7-H4, anti-CD52, anti-IL-10 , Anti-IL-35 and anti-TGF-β. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an inhibitory checkpoint molecule selected from the group consisting of PD-1, PD-L1, LAG-3, TIM-3, HHLA2, CD47, CXCR4, CD160, CD73, BLTA, B7-H4, TIGIT and VISTA. In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes PD-1. In some embodiments, the immune checkpoint regulator is a ligand that binds to the immune checkpoint molecule. In some embodiments, the immune checkpoint modulator inhibitor is a ligand that binds to PD-L1 and / or PD-L2. In some embodiments, the immune checkpoint regulator is a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the immune checkpoint regulator is bound to the ligand of HHLA2. In some embodiments, the immune checkpoint regulator is a fusion of the extracellular domain of TMIGD2 and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator is bound to at least two different inhibitory immune checkpoint molecule ligands (eg, dual specific ligands), such as ligands that bind to both CD47 and CXCR4. In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). PD-1 PD-1 is part of a family of B7 / CD28 costimulatory molecules that regulate T cell activation and tolerance, and therefore antagonist anti-PD-1 antibodies can be used to overcome tolerance. PD-1 has been identified as a receptor for B7-4. B7-4 can inhibit the activation of immune cells when it binds to inhibitory receptors on immune cells. The junction of PD-1 / PD-L1 pathway leads to the inhibition of T cell effector function, cytokine secretion and proliferation. (Turnis et al., Onco Immunology 1 (7): 1172-1174, 2012). Higher PD-1 content is related to depleted or long-term stimulated T cells. In addition, increased PD-1 expression is associated with a shorter survival time for cancer patients. Agents used to down-regulate PD-1, B7-4, and the interaction between B7-4 and PD-1 inhibitory signals in immune cells can enhance the immune response. PD-L1 / PD-L2 PD-L1 (planned cell death ligand 1) is also called differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 is used as a ligand for PD-1 and plays a major role in suppressing the immune system during specific events such as pregnancy, tissue allographs, autoimmune diseases and other disease states such as hepatitis and cancer. The formation of the PD-1 receptor / PD-L1 ligand complex will transmit inhibitory signals, thereby reducing the proliferation of CD8 + T cells at the lymph nodes. PD-L2 (Planned Cell Death 1 Ligand 2) is also known as B7-DC. PD-L2 is used as a ligand for PD-1. In some cases, PD-L2 and its inhibitors can be used as substitutes for PD-L1 and its inhibitors, respectively. TMIGD2 HHLA2 is widely used in many human cancers of breast cancer, lung cancer, thyroid cancer, melanoma, pancreatic cancer, ovarian cancer, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer, esophageal cancer and hematological malignancies leukemia and lymphoma which performed. The HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and is an attractive target for human cancer therapy. TMIGD2 has been identified as a receptor for HHLA2. Blocking HHLA2 / TMIGD2 can be an effective strategy for cancer immunotherapy. CD47 CD47 is an anti-phagocytic ligand used by tumor cells, which weakens the antibody effect function by transmitting inhibitory signals through its receptor signaling regulatory protein α (SIRPα). Interfering with the CD47-SIRPα interaction can enhance the anti-tumor immune response. CXCR4 The chemokine CXCL12 and its receptor CXCR4 are widely expressed in human cancers including ovarian cancer. In human cancers, they are related to disease progression at the level of tumor cell proliferation, invasion and angiogenesis. CXCL12 produced by tumor tissue and surrounding stroma stimulates VEGF-mediated angiogenesis and recruitment of endothelial progenitor cells from bone marrow. In addition, it has been shown that CXCL12 recruits inhibitory CD11b + Gr1 + bone marrow cells and pDC at the tumor site, and induces the localization of T regulatory cells (Treg) within the tumor, thereby preventing the immune mechanism that destroys the tumor. Therefore, regulation of the CXCL12 / CXCR4 axis can affect many aspects of tumor pathogenesis, including abnormal immune regulation. Several CXCR4 antagonists have demonstrated antitumor efficacy in preclinical models and have been evaluated in early clinical trials. In some embodiments, oncolytic viruses of the invention (such as oncolytic VV) may comprise nucleic acids encoding any antibodies or antigen-binding fragments of the immune checkpoint molecules described herein. For example, the oncolytic viruses of the present invention (such as oncolytic VV) may comprise nucleic acids encoding scFv forms of the above-mentioned anti-PD-1 antibodies. In some embodiments, an oncolytic virus of the invention (such as oncolytic VV) may comprise a nucleic acid encoding a fusion protein comprising any antibody fragment described herein or any other functional variant or derivative of a full-length antibody. For example, the oncolytic virus of the present invention (such as oncolytic VV) may comprise a nucleic acid encoding a fusion of the antigen-binding domain of the above-mentioned anti-PD-1 antibody and an IgG4 fragment. The immune checkpoint regulators covered herein are proteins or peptides. In some embodiments, the immune checkpoint regulator comprises a single polypeptide chain. In some embodiments, the immune checkpoint regulator contains more than one (such as any of 2, 3, 4, or more) polypeptide chains. The polypeptide chain of the immune checkpoint regulator may be of any length, such as at least about any one of amino acid lengths of 10, 20, 50, 100, 200, 300, 500, or more. In the case of multi-chain immune checkpoint regulators, the nucleic acid sequences encoding the polypeptide chains can be operably linked to the same promoter or different promoters. In some embodiments, immune checkpoint regulators secrete proteins. In some embodiments, immune checkpoints modulate sub-line antibodies. Natural antibodies, such as immunoglobulin molecules that monoclonally react with specific antigens. In some embodiments, the anti-system agonist antibody. In some embodiments, the anti-systemic antagonist antibody. In some embodiments, anti-systemic monoclonal antibodies. In some embodiments, the anti-system full-length antibody. In some embodiments, the anti-system is selected from the group consisting of antigen-binding fragments consisting of: VH , VL , VNAR , VH H, Fab, Fab ', F (ab')2 , Fv, mini antibody, scFv, sc (Fv)2 , Three-chain antibody, four-chain antibody, scFv-scFv (such as BiTE® ), Miniantibodies, scFv-Fc, trifunctional antibodies and other antigen-binding sequences of full-length antibodies or their engineered combinations. In some embodiments, the anti-systemic human antibody, humanized antibody, or chimeric antibody. In some embodiments, the anti-systemic monovalent antibody. In some embodiments, anti-systemic multivalent antibodies, such as bivalent antibodies or tetravalent antibodies. In some embodiments, the anti-system bispecific antibody. In some embodiments, the anti-systemic multispecific antibody. In some embodiments, the anti-systemic single domain antibody (sdAb). In some embodiments, the anti-system contains only heavy chain antibodies, such as camel antibodies or derivatives thereof. In some embodiments, the anti-systemic single chain antibody. In some embodiments, the anti-system scFv. In some embodiments, the anti-system comprises antibody fragments (such as Fc-containing fusion proteins, such as PD-1 extracellular domain-Fc fusion protein) or fusion proteins of any other functional variants or derivatives of full-length antibodies. In some embodiments, immune checkpoint regulators include antibodies to heavy and light chains. In some embodiments, the heavy chain comprises VH Domain. In some embodiments, the heavy chain further comprises one or more constant domains, such as CH 1. CH 2. CH 3 or any combination thereof. In some embodiments, the light chain comprises VL Domain. In some embodiments, the light chain further comprises a constant domain, such as CL . In some embodiments, the heavy and light chains are connected to each other via a plurality of disulfide bonds. In some embodiments, the antibody comprises Fc, such as Fc fragments of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody does not comprise Fc fragments. In some embodiments, the immune checkpoint regulator is the Fab. In some embodiments, the immune checkpoint regulator is a full-length anti-PD-1 antibody. Oncolytic viruses (such as oncolytic VV) can express any number (such as any of 1, 2, 3, 4, 5, 6, or more) of immune checkpoint regulators. In some embodiments, the oncolytic virus comprises a nucleic acid encoding a single immune checkpoint regulator. In some embodiments, the oncolytic virus comprises one or more nucleic acids encoding at least two immune checkpoint regulators. In some embodiments, nucleic acids encoding at least two immune checkpoint regulators are operably linked to the same promoter. In some embodiments, nucleic acids encoding at least two immune checkpoint regulators are operably linked to different promoters. In some embodiments, the nucleic acids encoding the immune checkpoint modulators and bispecific molecules of the invention are operably linked to the same promoter. In some embodiments, nucleic acids encoding the immune checkpoint regulators and bispecific molecules of the invention are operably linked to different promoters. The heavy chain polypeptides and light chain polypeptides of multi-chain immunomodulatory antibodies are co-presented by a single nucleic acid or by two nucleic acids in the oncolytic viruses of the present invention (such as oncolytic VV). In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed in equal molar ratios. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed in approximately any ratio of 10: 1, 8: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 3 : 2, 4: 3, 5: 4, 1: 1, 4: 5, 3: 4, 2: 3, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 8 Or 1:10. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed in any ratio of about 1:10 to about 1: 5, about 1: 5 to about 1: 3, about 1: 4 to about 1: 2 , About 1: 2 to about 1: 1, about 1: 1 to about 2: 1, about 2: 1 to about 4: 1, about 3: 1 to about 5: 1, about 5: 1 to about 10: 1 , About 1: 2 to about 2: 1, about 1: 3 to about 3: 1, about 1: 5 to about 5: 1 or about 1:10 to about 10: 1. The optimal performance ratio between the heavy chain polypeptide and the light chain polypeptide can help the antibody folding and assembly process. See, for example, Schlatter S et al., Biotechnol Prog. 21 (1): 122-33 (2005). Various performance ratios between heavy chain and light chain polypeptides in multi-chain immunomodulatory antibodies can be achieved by manipulating the number of copies of heterologous nucleic acids and / or heavy chain and light chain nucleic acids, and / or inducing sequences and / or linkage The strength of the promoter to the nucleic acid encoding the heavy chain and the light chain is achieved. In some embodiments, the nucleic acid encoding the heavy chain and the nucleic acid encoding the light chain are operably linked to the same promoter. In some embodiments, the nucleic acid encoding the heavy chain and the nucleic acid encoding the light chain are operably linked to different promoters. In some embodiments, the promoter of the nucleic acid encoding the heavy chain and the promoter of the nucleic acid encoding the light chain can be induced simultaneously. In some embodiments, the promoter of the nucleic acid encoding the heavy chain and the promoter of the nucleic acid encoding the light chain can be induced in sequence. In some embodiments, the promoter of the nucleic acid encoding the heavy chain is induced before the promoter of the nucleic acid encoding the light chain is induced. In some embodiments, the promoter of the nucleic acid encoding the heavy chain is induced after the promoter of the nucleic acid encoding the light chain is induced. In some embodiments, the promoter of the nucleic acid encoding the heavy chain and the promoter of the nucleic acid encoding the light chain have approximately any of the following intensity ratios: 10: 1, 8: 1, 6: 1, 5: 1, 4: 1 , 3: 1, 2: 1, 3: 2, 4: 3, 5: 4, 1: 1, 4: 5, 3: 4, 2: 3, 1: 2, 1: 3, 1: 4, 1 : 5, 1: 6, 1: 8 or 1:10. In some embodiments, the promoter of the nucleic acid encoding the heavy chain and the promoter of the nucleic acid encoding the light chain have any of the following intensity ratios: about 1:10 to about 1: 5, about 1: 5 to about 1: 3, About 1: 4 to about 1: 2, about 1: 2 to about 1: 1, about 1: 1 to about 2: 1, about 2: 1 to about 4: 1, about 3: 1 to about 5: 1 About 5: 1 to about 10: 1, about 1: 2 to about 2: 1, about 1: 3 to about 3: 1, about 1: 5 to about 5: 1 or about 1:10 to about 10: 1. In some embodiments, the immune checkpoint regulator is a fusion protein comprising an antibody fragment or any other functional variant or derivative of a full-length antibody. In some embodiments, the immune checkpoint regulator is an Fc-containing fusion protein. In some embodiments, the immune checkpoint regulator comprises a fusion of an antigen binding domain of an antibody described herein (such as a fragment containing CDRs) and an Fc fragment. For example, the oncolytic virus of the present invention may comprise a nucleic acid encoding a fusion of an antigen-binding domain of an anti-PD-1 antibody and an IgG4 fragment. In some embodiments, the immune checkpoint regulator is a fusion of the extracellular domain (such as PD-1 extracellular domain) of the ligand of the inhibitory immune checkpoint molecule described herein and the Fc fragment. In some embodiments, the Fc fragment may be an Fc fragment of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-Fc fusion protein. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, the PD-1 extracellular domain is encoded by the nucleic acid sequence comprising SEQ ID NO: 26. In some embodiments, the immune checkpoint regulator sub-line TGIGD2 extracellular domain-Fc fusion protein. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, the TGIGD2 extracellular domain-Fc fusion protein further includes a signal peptide containing the amino acid sequence SEQ ID NO: 28. In some embodiments, the immune checkpoint regulator specifically binds two different immune checkpoint molecules, such as CD47 and CXCR4. In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the SIRPα extracellular domain comprises the amino acid sequence SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is connected to the SIRPα extracellular domain by a linker, such as an IgG1 hinge. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker that includes the amino acid sequence SEQ ID NO: 31. In some embodiments, the SIRPα-CXCL12-Fc fusion protein further comprises a signal peptide comprising the amino acid sequence SEQ ID NO: 32. In some embodiments, the SIRPα-CXCL12-Fc fusion protein has the structure of (N ′ to C ′) signal peptide-SIRPα extracellular domain-linker-CXCL12-Fc fragment. Cytokines As used herein, the term "cytokine / cytokines" refers to a general class of biomolecules that affect cells of the immune system. This definition is intended to include, but is not limited to, such biomolecules that act locally or can circulate in the blood, and when used in the present invention are used to regulate or modulate an individual's immune response to cancer. Exemplary cytokines for practicing the invention include, but are not limited to, interferons (such as IFN-α, IFN-β, IFN-γ), all interleukins (eg, IL-1 to IL-29, especially IL-1, IL-2, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, IL-23, IL-24 and IL-27), tumor necrosis factor (e.g. TNF-α and TNF-β), erythropoietin (EPO), MIP3a, ICAM, macrophage community stimulating factor (M-CSF), granulocyte community stimulating factor (G-CSF) and granulocyte-macrophage community Stimulating factor (GM-CSF). GM-CSF is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells, and fibroblasts that acts as an interleukin. GM-CSF induces the activation, proliferation and differentiation of various immunocompetent cell populations, thereby promoting the production of humoral and cell-mediated immunity (Warren and Weiner, 2000). In some embodiments, the cytokinin is GM-CSF. In some embodiments, an oncolytic vaccinia virus is provided, which comprises an immune checkpoint regulator (such as an anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein Or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), wherein the first nucleic acid encoding the immune checkpoint regulator is operably linked to a late promoter (such as F17R), further comprising an encoding cell The second nucleic acid of the element (such as GM-CSF). In some embodiments, nucleic acids encoding immune checkpoint regulators and cytokines are operably linked to the same late promoter (such as F17R). In some embodiments, nucleic acids encoding immune checkpoint regulators and interleukins are operably linked to different promoters. In some embodiments, the immune checkpoint regulator is an immune checkpoint inhibitor (such as an antibody that specifically recognizes an immune checkpoint molecule). In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is bound to a ligand of an immune checkpoint molecule, such as PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-Fc fusion protein. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the SIRPα extracellular domain comprises the amino acid sequence SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker, such as an IgG1 hinge, or a linker that includes the amino acid sequence SEQ ID NO: 31. In some embodiments, an oncolytic vaccinia virus is provided, which comprises a first nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof, wherein the first nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof may be It is operatively linked to a late promoter (such as F17R) and further contains a second nucleic acid encoding GM-CSF. In some embodiments, an oncolytic vaccinia virus is provided, which comprises a first nucleic acid encoding a PD-1 extracellular domain-Fc fusion protein, wherein the first nucleic acid encoding a PD-1 extracellular domain-Fc fusion protein It is operably linked to a late promoter (such as F17R) and further contains a second nucleic acid encoding GM-CSF. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, an oncolytic vaccinia virus is provided, which comprises a first nucleic acid encoding a TGIGD2 extracellular domain-Fc fusion protein, wherein the first nucleic acid encoding a TGIGD2 extracellular domain-Fc fusion protein is operable Linked to a late promoter (such as F17R), it further contains a second nucleic acid encoding GM-CSF. In some embodiments, the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, an oncolytic vaccinia virus is provided, which comprises a first nucleic acid encoding an extracellular domain of SIRPα and a CXCL12 fragment-Fc fusion protein, wherein A nucleic acid is operably linked to a late promoter (such as F17R), and further contains a second nucleic acid encoding GM-CSF. In some embodiments, the SIRPα extracellular domain comprises the amino acid sequence SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker, such as an IgG1 hinge, or a linker that includes the amino acid sequence SEQ ID NO: 31. In some embodiments, an oncolytic virus (such as an oncolytic VV) is provided, which comprises an immune checkpoint regulator (such as an anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular structure Domain-Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), and a second nucleic acid encoding a bispecific molecule, which contains a specific recognition tumor antigen (such as EpCAM, FAP, EGFR or GPC3) the first antigen-binding domain (such as scFv) and the second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), the The oncolytic virus further contains a third heterologous nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, at least two of the nucleic acids encoding immune checkpoint regulators, bispecific junction molecules and interleukins are operably linked to the same promoter. In some embodiments, nucleic acids encoding immune checkpoint regulators, bispecific junction molecules and interleukins are all operably linked to the same promoter. In some embodiments, nucleic acids encoding immune checkpoint regulators, bispecific junction molecules and interleukins are all operably linked to different promoters. In some embodiments, the immune checkpoint regulator is an immune checkpoint inhibitor (such as an antibody that specifically recognizes an immune checkpoint molecule). In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is bound to a ligand of an immune checkpoint molecule, such as PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-Fc fusion protein. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, the Fc fragment is IgG4 Fc. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the SIRPα extracellular domain comprises the amino acid sequence SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker, such as an IgG1 hinge, or a linker that includes the amino acid sequence SEQ ID NO: 31. In some embodiments, an oncolytic virus (such as oncolytic VV) is provided, which includes a first nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof, and a second nucleic acid encoding a bispecific molecule, the bispecific Sex molecules include the first antigen-binding domain (such as scFv) that specifically recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and the cell surface molecule (such as CD3 on T lymphocytes) that specifically recognizes effector cells. With a second antigen binding domain (such as scFv), the oncolytic virus further contains a third nucleic acid encoding GM-CSF. In some embodiments, an oncolytic virus (such as oncolytic VV) is provided, which comprises a first nucleic acid encoding a PD-1 extracellular domain-Fc fusion protein, and a second nucleic acid encoding a bispecific molecule, the double Specific molecules include a first antigen binding domain (such as scFv) that specifically recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and a cell surface molecule that specifically recognizes effector cells (such as CD3 on T lymphocytes) The second antigen binding domain (such as scFv), the oncolytic virus further comprises a third nucleic acid encoding GM-CSF. In some embodiments, the PD-1 extracellular domain comprises the amino acid sequence SEQ ID NO: 25. In some embodiments, an oncolytic virus (such as oncolytic VV) is provided, which comprises a first nucleic acid encoding a TGIGD2 extracellular domain-Fc fusion protein, and a second nucleic acid encoding a bispecific molecule, the bispecific The molecule contains the first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and the first that specifically recognizes a cell surface molecule on an effector cell (such as CD3 on T lymphocytes) Two antigen binding domains (such as scFv), the oncolytic virus further comprises a third nucleic acid encoding GM-CSF. In some embodiments, the TGIGD2 extracellular domain comprises the amino acid sequence SEQ ID NO: 27. In some embodiments, an oncolytic virus (such as an oncolytic VV) is provided, which includes a first nucleic acid encoding a SIRPα extracellular domain and a CXCL12 fragment-Fc fusion protein, and a second nucleic acid encoding a bispecific molecule, the Bispecific molecules include a first antigen binding domain (such as scFv) that specifically recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and a cell surface molecule (such as CD3 on T lymphocytes) that specifically recognizes effector cells ) Of the second antigen binding domain (such as scFv), the oncolytic virus further comprises a third nucleic acid encoding GM-CSF. In some embodiments, the SIRPα extracellular domain comprises the amino acid sequence SEQ ID NO: 29. In some embodiments, the CXCL12 fragment comprises the amino acid sequence SEQ ID NO: 30. In some embodiments, the CXCL12 fragment is linked to the SIRPα extracellular domain by a linker, such as an IgG1 hinge, or a linker that includes the amino acid sequence SEQ ID NO: 31. Regulatory sequences It is obvious to those skilled in the art that regulatory sequences can be added to the OV nucleic acid molecules included in the present invention. Such regulatory sequences (control elements) are known to the skilled person and may include promoters, for transcription (e.g. polyadenylation transcription termination sequences), mRNA transport (e.g. nuclear localization signal sequences), processing (e.g. splicing signals) , Stability (e.g. introns and non-coding 5 'and 3' sequences), appropriate initiation, regulation and / or translation (e.g. initiator Met, triple leader sequence, IRES ribosome binding site, signal peptide, etc.) / Or termination of additional elements, and insertion sites for introducing the insertion sequence into the viral vector. In some embodiments, the regulatory sequences are promoters, transcription enhancers, and / or sequences that allow appropriate expression of the bispecific molecules, immune checkpoint regulators, and cytokines of the invention. The term "regulatory sequence" refers to the DNA sequence required to affect the performance of the linked coding sequence. The nature of such control sequences varies depending on the host organism. In prokaryotes, control sequences generally include promoters, ribosome binding sites, and terminators. In eukaryotes, control sequences generally include promoters, terminators and in some cases enhancers, transactivator proteins or transcription factors. The term "control sequence" is intended to include, at a minimum, all components that need to be present and may also include additional advantageous components. The term "operably linked" refers to the concatenation of the described components in a relationship that allows them to function in their intended manner. The control sequences "operably linked" to the coding sequence are linked in such a way that the coding sequence behaves under conditions compatible with the control sequences. In the case where the control sequence is a promoter, it is obvious to those skilled in the art that a double-stranded nucleic acid is preferably used. As used herein, "promoter", promoter region or promoter element or regulatory region or regulatory element refers to a DNA or RNA segment that controls the transcription of DNA or RNA operatively linked thereto. The promoter region includes specific sequences involved in RNA polymerase recognition, binding, and transcription initiation. In addition, promoters include sequences that regulate RNA polymerase recognition, binding, and transcription initiation activity (ie, binding to one or more transcription factors). Such sequences may be cis-acting or may react to trans-acting factors. Depending on the nature of regulation, the promoter can be constitutive or regulatory. Regulatory promoters can be inducible or environmentally reactive (eg, responsive to cues such as pH, anaerobic conditions, osmotic agents, temperature, light, or cell density). Many such promoter sequences are known in the art. See, for example, U.S. Patent Nos. 4,980,285, 5,631,150, 5,707,928, 5,759,828, 5,888,783, 5,919,670, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press ( 1989). It is envisaged that the present invention also relates to an oncolytic virus vector (such as an oncolytic VV vector), which comprises the nucleic acid molecule described in the present invention. As used herein, the term "viral vector" is used according to its accepted meaning in the art. The term refers to a nucleic acid vector construct that includes at least one element of viral origin and can be packaged in viral vector particles. Viral vector particles can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells in vitro or in vivo. In an embodiment, the oncolytic virus vector is a VV vector. VV may be Wyeth or Western Reserve (WR) strain. VV may have a deletion in its genome or a mutation in one or more genes. The thymidine kinase gene of vaccinia virus may have been deleted. Vaccinia virus may have mutations in the gene encoding the vaccinia virus growth factor. In some embodiments, the oncolytic virus vector is a lentiviral vector. Lentiviral vectors are commercially available, including, for example, from Clontech (Mountain View, Calif.) Or GeneCopoeia (Rockville, Md.). "Expression vector" is a construct that can be used to transform a selected host and allow the coding sequence to be expressed in the selected host. The expression vector may be, for example, a selection vector, a binary vector or an integration vector. Expression includes the transcription of nucleic acid molecules, preferably transcribed into translatable mRNA. The regulatory elements that ensure performance in eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, it usually contains a promoter that ensures the start of transcription and, optionally, a polyadenylation signal that ensures the termination and stabilization of the transcription of the transcript. Examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast, or the CMV, SV40, RSV promoter (Rous sarcoma virus), CMV enhancement in mammalian and other animal cells Son, SV40 enhancer or hemoglobin intron. In addition to the elements responsible for the initiation of transcription, such regulatory elements may also include transcription termination signals downstream of the polynucleotide, such as the SV40-polyadenylation site or the tk-polyadenylation site. In addition, depending on the expression system used, a leader sequence capable of directing the polypeptide to the cell compartment or secreting it into the culture medium can be added to the coding sequence of the nucleic acid sequence and is well known in the art. The leader sequence, and preferably the leader sequence or part of it that can guide the secretion of the translated protein, are assembled in the periplasmic space or extracellular medium together with the translation, start and stop sequences at the appropriate stage. Optionally, the heterologous nucleic acid sequence may encode a fusion protein including an N-terminal marker peptide that confers desired characteristics, such as stabilization of the expressed recombinant product or simplified purification. Suitable expression vectors are known in the art, such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pEF-DHFR and pEF-ADA (Raum Et al., Cancer Immunol Immunother (2001) 50 (3), 141-150) or pSPORT1 (GIBCO BRL). Those skilled in the art should understand that the choice of regulatory sequences may depend on factors such as the nucleic acid molecule itself, the virus into which it is inserted, the host cell or subject, and the amount of performance required. The promoter is of particular importance. In the context of the present invention, it can constitutively guide the expression of nucleic acid molecules in many types of host cells or certain host cells (e.g. tumor-specific regulatory sequences) or in response to specific events or exogenous factors (e.g. temperature, nutritional additives , Hormones, etc.) or according to the stage of the viral cycle (such as late or early). In order to optimize virus production and avoid the potential toxicity of the expressed polypeptides, promoters that are repressed in response to specific events or exogenous factors during this production step can also be used. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells, but control sequences of prokaryotic hosts can also be used. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence, and the polypeptide of the present invention can be collected and purified as needed afterwards. The promoter may be natural or heterologous (relative to the oncolytic virus described herein). Any suitable promoter can be used, including synthetic and naturally occurring promoters and modified promoters. As used herein, a native promoter is a viral endogenous promoter and its nucleotide sequence and its position in the viral genome are unmodified compared to a wild-type viral line. The synthetic promoter is a heterologous promoter, and its nucleotide sequence is not found in nature. The synthetic promoter may be a nucleic acid molecule having a synthetic sequence or a sequence derived from a natural promoter or a portion thereof. The synthetic promoter may also be a hybrid promoter composed of different elements derived from different natural promoters. See, for example, US9005602 for exemplary vaccinia virus synthetic promoters. Viral promoters can include, but are not limited to, VV promoter, pox virus promoter, adenovirus late promoter, vaccinia ATI promoter, or T7 promoter. The promoter can be a vaccinia virus promoter, a synthetic promoter, a promoter that directs transcription during at least early infection, a promoter that directs transcription during at least mid-infection, a promoter that directs transcription during early / late infection, or at least during infection The promoter that directs transcription during the late stages. Promoters can be roughly classified as constitutive promoters or regulatory promoters, such as inducible promoters. Promoters suitable for constitutive expression in mammalian cells include, but are not limited to, cytomegalovirus (CMV) immediate early promoter (US 5,168,062), RSV promoter, adenovirus major late promoter, phosphoglycerate kinase ( PGK) promoter (Adra et al., 1987, Gene 60: 65-74), the thymidine kinase (TK) promoter of herpes simplex virus (HSV) -1, and the T7 polymerase promoter (WO98 / 10088). The vaccinia virus promoter is particularly suitable for expression in oncolytic poxviruses. Representative examples include (not limited to) acne 7.5K, H5R, 11K7.5 (Erbs et al., 2008, Cancer Gene Ther. 15 (1): 18-28), TK, p28, pll, pB2R, pA35R, and K1L activation And synthetic promoters, such as Chakrabarti et al. (1997, Biotechniques 23: 1094-7; Hammond et al., 1997, J. Virol Methods 66: 135-8; and Kumar and Boyle, 1990, Virology 179: 151-8 ) And the early / late chimeric promoters. Promoters suitable for oncolytic measles virus include (but are not limited to) any promoter that directs the expression of the measles transcription unit (Brandler and Tangy, 2008, CIMID 31: 271). Inducible promoters belong to the category of regulatory promoters. An inducible promoter can be induced under one or more conditions, such as physical conditions, the microenvironment of the host cell or the physiological state of the host cell, an inducing agent (ie, inducing agent), or a combination thereof. Promoters suitable for expression can be tested in vitro (eg, in a cell line suitable for culture) or in vivo (eg, in a suitable animal model or in a subject). When the encoded immune checkpoint regulator includes antibodies and especially mAb, examples of promoters suitable for expressing the heavier components of the immune checkpoint regulator include CMV, SV and vaccinia virus pH5R, F17R and pllK7.5 promoters; Examples of promoters suitable for expressing the lighter components of the immune checkpoint regulator include PGK, β-actin, and vaccinia virus p7.5K, F17R, and pA35R promoters. The promoter can be replaced with a stronger or weaker promoter, where the replacement causes changes in virus attenuation. As used herein, replacing a promoter with a stronger promoter refers to removing the promoter from the genome and replacing it with a promoter that increases the level of transcription initiation relative to the promoter being replaced. Generally, the ability of a stronger promoter to bind to the polymerase complex is improved relative to the promoter that is replaced. Therefore, an open reading frame operably linked to a stronger promoter has a higher level of gene expression. Similarly, replacing a promoter with a weaker promoter refers to removing the promoter from the genome and replacing it with a promoter that reduces the level of transcription initiation relative to the promoter being replaced. In general, the ability of weaker promoters to bind to the polymerase complex is reduced relative to the promoter that is replaced. Therefore, an open reading frame operably linked to a weaker promoter has a lower gene expression level. Viruses can exhibit differences in characteristics due to the use of stronger and weaker promoters, such as attenuation. For example, in vaccinia virus, the synthetic early / late promoter and late promoter are relatively strong promoters, while the acne synthetic early promoter, P7.5k early / late promoter, P7.5k early promoter and The late P28 promoter is a relatively weak promoter (see, for example, Chakrabarti et al. (1997)BioTechniques 23 (6) 1094-1097). In some embodiments, the promoter is a vaccinia virus promoter. Exemplary vaccinia virus promoters used in the present invention may include (but not limited to) P7.5k , P11k , PSE , PSEL , PSL , H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L , P4b or K1 promoter. In some embodiments, the promoter is a vaccinia virus natural promoter. As used herein, a native promoter is a viral endogenous promoter and its nucleotide sequence and its position in the viral genome are unmodified compared to a wild-type viral line. In some embodiments, the promoter is a vaccinia virus synthetic promoter (see, eg, US9005602). The synthetic promoter is a heterologous promoter, and its nucleotide sequence is not found in nature. The synthetic promoter may be a nucleic acid molecule having a synthetic sequence or a sequence derived from a natural promoter or a portion thereof. The synthetic promoter may also be a hybrid promoter composed of different elements derived from different natural promoters. Exemplary early, middle, and late promoters of acne include, for example, acne P7 . 5k Early / late promoter, acne PEL Early / late promoter, acne P13 Early promoter, acne P11k Late promoters and acne promoters listed elsewhere herein. Exemplary synthetic promoters include, for example, PSE Synthesis of early promoter, PSEL Synthesis of early / late promoter, PSL Late synthetic promoters, acne synthetic promoters listed elsewhere in this article (Patel et al.,Proc. Natl. Acad. Sci. USA 85: 9431-9435 (1988); Davison and Moss,J Mol Biol 210: 749-769 (1989); Davison et al.,Nucleic Acids Res. 18: 4285-4286 (1990); Chakrabarti et al.,BioTechniques 23: 1094-1097 (1997)). Different gene products can be expressed in the same virus or a combination of two different viruses using different promoters. In some embodiments, the promoter directs transcription during at least the late stage of infection (such as the F17R promoter). In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, a promoter (such as the F17R promoter) that directs transcription during at least the late stages of infection is used. The late vaccinia virus promoter F17R is activated only after VV infects tumor cells, so the use of the F17R promoter will further enhance the tumor-selective performance of the VV transgenic gene. The late manifestations of the immune checkpoint modulators, bispecific junction molecules and / or cytokines (such as GM-CSF) of the present invention will also allow sufficient virus replication before T cell activation and T cell mediated tumor lysis. Therefore, in some embodiments of the present invention, an oncolytic vaccinia virus is provided, which comprises an immune checkpoint regulator (such as an anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain -Nucleic acid of Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment (Fc fusion protein), wherein the nucleic acid encoding the immune checkpoint regulator is operably linked to the late promoter. In some embodiments, the late promoter line is F17R. In some embodiments, the oncolytic vaccinia virus that exhibits immune checkpoint regulators further comprises a second nucleic acid encoding an interleukin. In some embodiments, the nucleic acid encoding the interleukin can also be operably linked to the promoter. In some embodiments, the nucleic acid encoding the interleukin is linked to the late promoter. In some embodiments, the nucleic acid encoding the interleukin is linked to F17R. In some embodiments, the cytokinin is GM-CSF. In some embodiments, an oncolytic virus (such as an oncolytic VV) is provided, which comprises an immune checkpoint regulator (such as an anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular structure Domain-Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), and a second nucleic acid encoding a bispecific molecule, which contains a specific recognition tumor antigen (such as EpCAM, FAP, EGFR or GPC3) the first antigen-binding domain (such as scFv) and the second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes), wherein The nucleic acid encoding the immune checkpoint regulator and / or the nucleic acid encoding the bispecific molecule are operably linked to the promoter. In some embodiments, the promoter is an oncolytic vaccinia virus promoter. In some embodiments, the promoter is a late promoter. In some embodiments, the promoter is the F17R promoter. In some embodiments, the nucleic acid encoding the immune checkpoint regulator and the nucleic acid encoding the bispecific molecule are operably linked to a promoter. In some embodiments, the nucleic acid encoding the immune checkpoint regulator and the nucleic acid encoding the bispecific molecule are operably linked to two identical promoters. In some embodiments, the nucleic acid encoding the immune checkpoint regulator and the nucleic acid encoding the bispecific molecule are operably linked to different promoters. In some embodiments, nucleic acids encoding immune checkpoint regulators and nucleic acids encoding bispecific molecules are directed for transcription during the same or similar stages of viral infection. In some embodiments, nucleic acids encoding immune checkpoint regulators and nucleic acids encoding bispecific molecules are directed for transcription during different stages of viral infection. In some embodiments, the nucleic acid encoding the immune checkpoint regulator and the nucleic acid encoding the bispecific molecule are operably linked to promoters of the same or similar strength. In some embodiments, nucleic acids encoding immune checkpoint regulators and nucleic acids encoding bispecific molecules are operably linked to promoters of different strengths. In some embodiments, the nucleic acid encoding the bispecific molecule is operably linked to a promoter that is stronger than the promoter operably linked to the nucleic acid encoding the immune checkpoint regulator. In some embodiments, the nucleic acid encoding the bispecific molecule is operably linked to a promoter that is weaker than the promoter operably linked to the nucleic acid encoding the immune checkpoint regulator. Additional regulatory elements may include transcription and translation enhancers. Advantageously, the oncolytic virus vectors of the present invention described above contain selectable and / or scoreable markers. The selectable marker genes that can be used to select transformed cells are well known to those skilled in the art and include an antimetabolite resistance as a basis for selection, for example: dhfr, which confer resistance to methotrexate (Reiss, Plant Physiol. (Life-Sci. Adv.) 13 (1994), 143-149); npt, which confers aminoglycoside neomycin (neomycin), kanamycin (kanamycin) and Paromycin resistance (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confer resistance to hygromycin (Marsh, Gene 32 (1984) , 481-485). Another selectable gene has been described, namely trpB, which enables cells to use indole instead of tryptophan; hisD, which enables cells to use histamine instead of histidine (Hartman, Proc. Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase, which enables cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase), which confer inhibitors to ornithine decarboxylase 2- (difluoromethyl) -DL-ornithine DFMO resistance (McConlogue, 1987, In: Current Communications in Molecular Biology, edited by Cold Spring Harbor Laboratory) or deaminase from Aspergillus terreus Resistance to Blasicidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338). Useful scoreable markers are also known to those skilled in the art and are commercially available. Advantageously, the marker encodes luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47), YFP or β-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901-3907). Scorable markers are particularly suitable for simple and rapid screening of cells, tissues and organisms containing the vector. Where appropriate, it may be advantageous to include additional regulatory elements to facilitate insertion of at least one gene (ie, bispecific junction molecule, immune checkpoint regulator and / or cytokinin) into the viral genome of the oncolytic virus of the invention Performance, transportation and biological activity. For example, a signal peptide (or leader sequence) may be included to promote secretion of infected cells. The signal peptide is usually inserted into the N-terminus of the protein immediately after the Met promoter. The choice of signal peptides is wide and available to those skilled in the art. It is also conceivable to add a transmembrane domain to facilitate anchoring of the encoded protein in a suitable membrane (eg, plasma membrane) of infected cells. The transmembrane domain is usually inserted at the C-terminus of the protein, just before or immediately adjacent to the stop codon. Numerous transmembrane domains can be used in this technology (see for example WO99 / 03885). In some embodiments, the immune checkpoint regulator described herein further comprises a signal peptide fused at the N-terminus of the immune checkpoint regulator. In some embodiments, the immune checkpoint regulator comprises a signal peptide fused at the N-terminus of the VH domain, such as SEQ ID NO: 21. In some embodiments, the immune checkpoint regulator comprises a signal peptide fused at the N-terminus of the VL domain, such as SEQ ID NO: 23. In some embodiments, the signal peptide may be encoded by the nucleic acid SEQ ID NO: 22 or SEQ ID NO: 24. In some embodiments, the signal peptide comprises the amino acid sequence SEQ ID NO: 28 or SEQ ID NO: 32. As an additional example, peptide tags (usually short peptide sequences that can be recognized by available antisera or compounds) can also be added for subsequent expression, transportation, or purification of the encoded gene product. In the context of the present invention, a variety of tag peptides can be used, including (but not limited to) PK tags, FLAG octapeptides, MYC tags, HIS tags (usually segments of 4 to 10 histidine residues) and e tags US 6,686,152). When several tags are used, the tag peptides can be independently placed at the N-terminus of the protein, or alternatively at the C-terminus, or alternatively at any one of these positions. Tag peptides can be detected by immunodetection analysis using anti-tag antibodies. As another example, glycosylation can be altered to increase the biological activity (eg, increase) of the encoded gene product. Such modifications can be achieved, for example, by mutating one or more residues within the glycosylation site. Changing the glycosylation pattern can increase the ADCC capacity of the antibody and / or its affinity for its target. The described nucleic acid molecule or vector introduced into the host may be integrated into the host genome or it may remain extrachromosomal. The host can be any eukaryotic cell or prokaryotic cell. In particular, it is envisaged that the host may be a mammalian cell. Host cells include, but are not limited to, CV-1, BS-C-1, HuTK-143B, BHK-21, CEF, CHO cells, COS cells, myeloma cell lines such as SP2 / 0 or NS / 0 cells. Bispecific junction molecules In some embodiments, the present invention expresses immune checkpoint regulators (such as anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, Or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein) oncolytic viruses (such as oncolytic VV) further comprise a second nucleic acid encoding a bispecific molecule containing specific recognition of a tumor antigen (such as EpCAM , FAP, EGFR, or GPC3) the first antigen-binding domain (such as scFv) and the second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). The present invention discloses an oncolytic virus (such as oncolytic VV), which contains an immune checkpoint regulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion Protein, or the first nucleic acid of the extracellular domain of SIRPα and the CXCL12 fragment-Fc fusion protein), further comprising a second nucleic acid encoding a conjugation molecule, the conjugation molecule comprising specific recognition of one or more tumor antigens (such as EpCAM, FAP, EGFR Or GPC3) a first antigen binding domain (such as scFv) and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, the junction molecule is a bispecific molecule. In some embodiments, the cell surface molecule is a T lymphocyte surface marker (such as CD3), and thus the zygote is a T cell zygote (TE). In some embodiments, the present invention discloses an oncolytic virus (such as oncolytic VV) that expresses immune checkpoint regulators (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular Domain-Fc fusion protein, or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), further express bispecific T cell adaptor (BiTE). Once the second antigen binding domain of the junction molecule binds to the effector cell, the second antigen binding domain can activate the effector cell. In some embodiments, when the second antigen binding domain of the junction molecule binds to the cell surface molecule on the immune cell, and the first antigen binding domain binds to the tumor cell antigen, the immune cell kills the tumor cell. Tumor antigens In some embodiments, the antigen specifically recognized by the first antigen binding domain of the conjugation molecule is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In one embodiment, TAA or TSA lines are expressed on cancer cells. In some embodiments, TAA or TSA lines are expressed on blood cancer cells. In some embodiments, TAA or TSA is expressed on cells of solid tumors. As non-limiting examples, certain forms of solid tumor cancer include glioblastoma, non-small cell lung cancer, lung cancer other than small cell lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, liver cancer, colorectal cancer , Gastric cancer, spleen cancer, skin cancer (such as melanoma), brain cancer other than glioblastoma, kidney cancer, thyroid cancer, head and neck cancer, bladder cancer, esophageal cancer, or similar cancers. In some embodiments, TAA or TSA is one or more of the following, for example, the scFv on the adaptor is specific to one or more of the following: EphA2, HER2, GD2, GPC3, 5T4, 8H9, αv beta6 Integrin, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, κ light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD138, CD171, CEA, CSPG4 , EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, fetal AchR, folate receptor a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2 , KDR, λ (Lambda), Lewis-Y, MCSP, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1 TEM8, VEGFR2, carcinoembryonic antigen, HMW-MAA, VEGF receptor, and other exemplary antigens are antigens present in the extracellular matrix of tumors, such as carcinoembryonic variants of fibronectin, tendon protein, or tumor necrosis zone. In some embodiments, the antigen specifically recognized by the first antigen binding domain of the junction molecule is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and GPC3. In some embodiments, the tumor antigens are EpCAM, FAP, EGFR or GPC3. In some embodiments, the antigen specifically recognized by the first antigen binding domain of the junction molecule is an epithelial cell adhesion molecule (EpCAM, CD326), also known as 17-1A, ESA, AUA1, EGP40, etc., which consists of 314 Amino acids constitute a 40 kDa transmembrane glycoprotein. EpCAM is specifically expressed in various types of epithelial cells and most types of human malignant diseases. For example, EpCAM is abundantly expressed in colon cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, breast cancer, and ovarian cancer. In some embodiments, the antigen specifically recognized by the first antigen binding domain of the junction molecule is fibroblast activation protein (FAP). Fibroblast cells secrete connective tissue cells rich in extracellular matrix of collagen and other macromolecules. Fibroblasts in the tumor stroma (ie, tumor-bearing tissue) synthesize FAP, a type II transmembrane protein that acts as a serine protease. FAP is selectively overexpressed in more than 90% of stromal fibroblasts associated with colon cancer, breast cancer, and lung cancer. FAP is also expressed in some tumor cells, such as human malignant glioma cell line U87 and murine Lewis lung cancer cell line LL2 (Kraman et al.,Science 330: 827-830 (2010)). It has been reported that excessive performance of FAP makes it possible to promote tumor growth and increase metastasis, while treatment with anti-FAP antibodies inhibits tumor growth. In some embodiments, the antigen specifically recognized by the first antigen binding domain of the junction molecule is epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB family consisting of closely related receptors including EGFR (ErbB-1), Her2 / neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). The activation of EGFR causes the activation of receptor tyrosine kinase and a series of downstream signaling events that mediate cell proliferation, activity, adhesion, invasion and resistance to chemotherapy and inhibition of apoptosis, etc. The process is crucial for the continuous proliferation and survival of cancer cells. The performance of EGFR is related to the poor prognosis of various tumor types including stomach, colon, bladder, breast, prostate, endometrium, kidney and brain (such as glioma). EphA2 is called EPH receptor A2 (ephrin type-A receptor 2; EPHA2; ARCC2; CTPA; CTPP1; or ECK), which is the protein of the protein tyrosine kinase family in the human body. The protein encoded by the EPHA2 gene in the Lin receptor subfamily. The receptors in this subfamily generally include a single kinase domain and an extracellular region containing a Cys-rich domain and 2 fibronectin type III repeats; embodiments of the antibodies of the invention can target these domains Any of them. Aprelin receptors are divided into two groups due to the similarity of the sequences of their respective extracellular domains and their affinity for binding to Aprilin-A ligands and Aprilin-B ligands, and EphA2 encodes binding The protein of pregaline-A ligand. An exemplary human EphA2 nucleic acid sequence is in GenBank® accession number NM_004431, and an exemplary human EphA2 polypeptide sequence is in GenBank® accession number NP_004422, both of which are incorporated herein in their entirety. HER2 is called human epidermal growth factor receptor 2 (Neu, ErbB-2, CD340, or pi 85), which is a protein encoded by the ERBB2 gene in the epidermal growth factor receptor (EFR / ErbB) family in humans. HER2 contains an extracellular ligand binding domain, a transmembrane domain, and an intracellular domain that can interact with various signaling molecules. GD2 is a disialic ganglioside expressed on neuroectodermal-derived tumors, including human neuroblastoma and melanoma, and in normal tissues, mainly in human cerebellum and peripheral nerves is highly restricted . GD2 exists and is concentrated on the cell surface, and the two hydrocarbon chains of the ceramide portion are embedded in the plasma membrane and the oligosaccharide is located on the outer surface of the cell, where it exists extracellular molecules or recognition points adjacent to the cell surface. In some embodiments, the antigen specifically recognized by the first antigen-binding domain of the conjugation molecule is phosphoglypican-3 (GPC3). Glypican-3 (GPC3) is a carcinoembryonic antigen that reappears at a higher frequency in neoplastic hepatocytes. The GPC3 gene encodes a 70 kDa precursor core protein that can be cleaved by furin to produce a 40 kDa amine (N) terminal protein and a 30 kDa membrane-bound carboxyl (C) terminal protein. The C-terminus is connected to the cell membrane by a glycosylated phospholipid inositol (GPI) anchor. Vascular endothelial growth factor receptor 2 (VEGFR2, KDR3) is a VEGF receptor, which is one of the most effective specific positive regulators of angiogenesis. VEGFR2 is abundantly expressed in tumor-associated endothelial cells and promotes tumor growth, invasion and metastasis (Dias et al.,J Clin Invest. 106 (4): 511-521, 2000; Santos et al.,Blood 103 (10): 3883-3889, 2004; St. Croix et al.,Science 289: 1197-1202, 2000). In addition, VEGFR2 is also expressed on the surface of several tumor cells, including: B-cell lymphoma and leukemia, multiple myeloma, urothelial bladder cancer, breast cancer and lung cancer (El-Obeid et al.,Leuk Res. 28 (2): 133-137, 2004; Kumar et al.,Leukemia 17 (10): 2025-2031, 2003; Gakiopoulou-Givalou et al.,Histopathology 43 (3): 272-279, 2003; Kranz et al.,Int J Cancer 84 (3): 293-298, 1999; Decaussin et al.,J Pathol. 188 (4): 369-377, 1999). Compared with normal vascular endothelial cells, the relatively high expression level on tumor cells indicates that VEGFR2 is a suitable target for tumor therapy. Therefore, in some embodiments, the bispecific molecule comprises a first antigen-binding domain that specifically recognizes EpCAM (such as scFv) and a cell surface molecule that specifically recognizes effector cells (such as CD3 on T lymphocytes). Two antigen binding domains (such as scFv). In some embodiments, the bispecific molecule comprises a first antigen binding domain that specifically recognizes FAP (such as scFv) and a second antigen that specifically recognizes a cell surface molecule on effector cells (such as CD3 on T lymphocytes) Binding domain (such as scFv). In some embodiments, the bispecific molecule comprises a first antigen binding domain that specifically recognizes EGFR (such as scFv) and a second antigen that specifically recognizes a cell surface molecule on effector cells (such as CD3 on T lymphocytes) Binding domain (such as scFv). In some embodiments, the bispecific molecule comprises a first antigen binding domain that specifically recognizes GPC3 (such as scFv) and a second antigen that specifically recognizes a cell surface molecule on effector cells (such as CD3 on T lymphocytes) Binding domain (such as scFv). Exemplary effector cells on effector cell surface molecules include (but are not limited to) T lymphocytes, B lymphocytes, natural killer (NK) cells, dendritic cells (DC), macrophages, monocytes, neutrophils, NKT cells or similar cells. In some embodiments, the effector cell line is T lymphocytes. In some embodiments, the effector cell line is cytotoxic T lymphocytes. In some embodiments, the effector cell line is allogeneic. In some embodiments, the effector cell line is autologous. The cell surface molecules on the effector cells of the present invention are molecules found on the outer cell wall or plasma membrane of a specific cell type or a limited number of cell types. Examples of cell surface molecules include, but are not limited to, membrane proteins, such as receptors, transporters, ion channels, proton pumps, and G protein-coupled receptors; extracellular matrix molecules, such as adhesion molecules (eg, integrins, cadherins) , Selectin or NCAMS); see, eg, US Patent No. 7,556,928, which is incorporated herein by reference in its entirety. Cell surface molecules on effector cells include (but are not limited to) CD3, CD4, CD5, CD8, CD16, CD27, CD28, CD40, CD64, CD89, CD134, CD137, CD278, NKp46, NKp30, NKG2D and unchanged TCR. The cell surface molecular binding domain of the junction molecule can activate immune cells. Skilled artisans recognize that immune cells have different cell surface molecules. For example, CD3 is a cell surface molecule on T cells, while CD16, NKG2D or NKp30 is a cell surface molecule on NK cells, and CD3 or an unchanged TCR is a cell surface molecule on NKT cells. Therefore, the junction molecule of activated T cells may have a cell surface molecular binding domain different from that of activated NK cells. In some embodiments, such as some embodiments of immune cell line T cells, the activation molecule is one or more of the following: CD3, such as CD3γ, CD3δ, or CD3ε; or CD27, CD28, CD40, CD134, CD137, and CD278. In some other embodiments, for example in some embodiments of the immune cell line NK cell, the cell surface molecule line is CD16, NKG2D or NKp30, or in some embodiments of the immune cell line NKT cell, the cell surface molecule line is CD3 or not Change TCR. CD3 contains three different polypeptide chains (ε, δ, and γ chains), which are antigens expressed by T cells. The three CD3 polypeptide chains associate with the T cell receptor (TCR) and the ζ chain to form a TCR complex. The TCR complex has the function of activating the signaling cascade in T cells. Currently, many therapeutic strategies target TCR signal transduction to treat diseases using anti-human CD3 monoclonal antibodies. The CD3 specific antibody OKT3 is the first monoclonal antibody approved for human therapeutic use and is clinically used as an immunomodulator for the treatment of allograft rejection. In some embodiments, the second antigen binding domain specifically binds to CD3 on T lymphocytes. In some embodiments, the VH and VL regions of the human CD3 specific domain are derived from CD3 specific antibodies, such as X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5 , Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, WT31, F101.01 or Blinatumomab (Blincytomab)® , Namely CD19-CD3 bispecific antibody). Such CD3-specific antibodies are well known in the art and are described in particular in Tunnacliffe et al., Int Immunol. 1 (5): 546-50 (1989). In some embodiments, the VH and VL regions are derived from antibodies / antibody derivatives and analogs that can specifically recognize human CD3-ε chains or human CD3-ζ chains. In some embodiments, the VH and VL regions of the human CD3 specific domain are derived from brinomab (Blincyto®, ie CD19-CD3 bispecific antibody). In some embodiments, the second antigen binding domain specifically binds to an epitope within the human CD3-ε chain or human CD3-ζ chain. The skilled artisan should recognize that the TCR complex is an octamer complex with variable TCR α and β chains and three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD3ζ / ζ or ζ / η. Although in some cases, the conjugation molecules described herein target CD3ε with one scFv, the invention also covers targeting a specific scFv with other CD3 molecules, especially CD3ζ, or TCR α and β chains. In some embodiments, targeting molecules that are not part of the TCR complex (eg, CD27, CD28, CD40, CD134, CD137, and CD278) are covered in the present invention. Therefore, in some embodiments, the bispecific molecule contains a first antigen-binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and specifically recognizes CD3 on T lymphocytes A second antigen binding domain (such as scFv). In some embodiments, the bispecific molecule comprises a first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen that specifically recognizes CD16 on NK cells Binding domain (such as scFv). In some embodiments, the bispecific molecule comprises a first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen that specifically recognizes NKG2D on NK cells Binding domain (such as scFv). In some embodiments, the bispecific molecule comprises a first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a second antigen that specifically recognizes NKp30 on NK cells Binding domain (such as scFv). Conjugation Molecule Form The conjugation molecule described herein can be in any form known in the art. Such conjugation molecules generally include tumor antigen binding domains and effector cell surface molecular binding domains. The antigen binding domain of the junction molecule can be designed to bind to one or more tumor antigens present on the target cell, and the effector cell surface molecule binding domain of the junction molecule can be designed to bind to effector cells, such as T lymphocytes There are one or more cell surface molecules. Once the effector cell surface molecular binding domain of the binding molecule binds the effector cell, the effector cell surface molecular binding domain can activate the effector cell. In some embodiments, when the effector cell surface molecule binding domain of the adaptor binds to the cell surface molecule on the effector cell, and the tumor antigen binding domain binds to the tumor antigen on the tumor cell, the effector cell kills the tumor cell. In some embodiments, the conjugation molecule is composed of two parts (for example, including tumor antigen binding domain and effector cell surface molecular binding domain conjugated by a linker as appropriate), or may be composed of three or more parts Constituted (for example, including one or more tumor antigen binding domains and / or one or more effector cell surface molecule binding domains, or other domains, including one or more costimulatory domains and / or one or more Dimerization, tripolymerization or multipolymerization domains). In some embodiments, the adaptor is bispecific (eg, includes a tumor antigen binding domain and an effector cell surface molecule binding domain that are optionally joined by a linker, where the tumor antigen is different from the cell surface molecule). The conjugating molecule can also be multispecific (for example, including one tumor antigen binding domain and two effector cell surface molecule binding domains that are conjugated by a linker, where the tumor antigen is different from the two cell surface molecules ). Conjugation molecules can be in any form known in the art (see, for example, Weidle et al., Cancer Genomics Proteomics, 10 (1): 1-18, 2013; Geering and Fussenegger, Trends Biotechnol., 33 (2): 65- 79, 2015; Stamova et al., Antibodies, 1 (2): 172-198, 2012). The adaptor may be in the form of an "IgG-derived molecule" containing an Fc region. For example, the conjugation molecule may be in the following forms (but not limited to): shared LC (light chain), DAF (dual-acting Fab, which contains an evolved Fv with dual specificity), interchangeable Mab, IgG-dsscFv2 (disulfide Biostable scFv2), DVD (dual variable domain), IgG-dsFv (disulfide stabilized Fv), processed IgG-dsFv, IgG-scFab (single-chain Fab), scFab-dsscFv or Fv2-Fc. The Knobs-into-holes technique can be used for heterodimerization of different H chains in, for example, shared LC, interchangeable Mab, IgG-dsF, IgG-scFab, or Fv2-Fc. Adapters can also be in the form of "low Fc bispecific" forms, which usually include individual scFvs containing Fabs with different specificities fused together via linkers. For example, the zygote can be (but not limited to) the following forms: Fab-scFv2, Fab-scFv, scFv-scFv scFv (such as BiTE® ), Bifunctional antibody, scBsDb (single chain bispecific bifunctional antibody), DART (dual affinity retargeting molecule), TandAb (tetravalent tandem antibody), scBsTaFv (single chain bispecific tandem variable domain) DNL-F (ab)3 (Docking locked trivalent Fab), scFv-HSA-scFv (scFv-human serum albumin-scFv) or bssdAb (bispecific single domain antibody). The bivalent or trivalent Fab-Fv or Fab-Fv2 format is produced by fusing VH-CH1 and / or L chain with scFv. scFv-scFv molecules (such as BiTE® ) Is generated by fusing scFvs with different specificities. The linker peptide length can be adjusted so that VH and VL are paired correctly, such as in bifunctional antibodies, DART, and TandAb. These molecules can be further stabilized by interchain disulfide bonds (for example in DART, or between VH and VL of an antibody containing scFv). Conjugation molecules can also be in the form of antibody mimics, which include engineered small proteins (Geering and ussenegger, Trends Biotechnol., 33 (2): 65 -79, 2015). These molecules are derived from existing human scaffold proteins and contain a single polypeptide. Exemplary adaptors in the form of antibody mimics can be designed ankyrin repeat proteins (DARPin; containing 3-5 fully synthetic ankyrin repeat sequences flanked by N-terminal and C-terminal cap domains), Avid polymers (avimers; high-affinity proteins that contain multiple A domains, each of which has a lower affinity for the target) or anti-carrying proteins (lipoprotein-based scaffolds with four accessible loops, each The sequence can be randomized). The conjugation molecule employed in accordance with the present invention may be a chemically modified derivative of any of the foregoing conjugate forms, or it may comprise ligands, peptides or combinations thereof. The conjugating molecules used in accordance with the present invention can be further modified using known techniques known in the art, for example, by using amino acid deletions, insertions, substitutions, additions, and / or recombinations, alone or in combination, and / or Any other modifications known in the art (eg post-translational modifications and chemical modifications such as glycosylation and phosphorylation). Chemical / biochemical or molecular biology methods related to such modifications are known in the art and are described in particular in laboratory manuals (see Sambrook et al; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Section 2nd edition 1989 and 3rd edition 2001; Gerhardt et al; Methods for General and Molecular Bacteriology; ASM Press, 1994; Lefkovits; Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press, 1997; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual; Cold Spring Harbor Laboratory Press, 2002). In some embodiments, the bispecific ligation molecule of the invention is a bispecific single chain Fv (scFv). scFv generally contains VH and VL domains connected by a linker peptide. The secreted zygote is composed of two scFvs connected by a signal peptide (allowing secretion) from the cell and then connected by a linker peptide (Lx, Ly, Lz). The length and sequence of the linker may be sufficient to ensure that the first and second domains can maintain their different binding specificities independently of each other. Bispecific single chain molecules are known in the art and described in the following documents: WO 99/54440; Mack, J. Immunol. (1997), 158, 3965-3970; Mack, PNAS, (1995), 92 , 7021-7025; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197; Loffler, Blood, (2000), 95, 6, 2098-2103; and Bruhl, J. Immunol., (2001) , 166, 2420-2426. In some embodiments, an exemplary molecular form of the invention provides an oncolytic virus (such as an oncolytic VV) that includes a nucleic acid encoding a polypeptide containing a signal peptide followed by two scFvs, wherein the first scFv specifically recognizes a tumor antigen (Such as EpCAM, FAP, EGFR or GPC3), and the second scFv specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). scFv each contains a VH And a VL Area. The bispecific scFv can be a tandem bi-scFv or a bifunctional antibody. Bispecific scFv can be arranged in different forms: VH α-Lx-VL α-Ly-VH β-Lz-VL β, VL α-Lx-VH α-Ly-VH β-Lz-VL β, VL α-Lx-VH α-Ly-VL β-Lz-VH β, VH α-Lx-VL α-Ly-VL β-Lz-VH β, VH α-Lx-VL β-Ly-VH β-Lz-VL α, VL α-Lx-VL β-Ly-VH β-Lz-VH α, VH α-Lx-VH β-Ly-VL β-Lz-VL α, VL α-Lx-VH β-Ly-VL β-Lz-VH α, VH β-Lx-VL α-Ly-VH α-Lz-VL β, VL β-Lx-VL α-Ly-VH α-Lz-VH β, VH β-Lx-VH α-Ly-VL α-Lz-VL β, VL β-Lx-VH α-Ly-VL α-Lz-VH β. Therefore, the bispecific scFv with the above possible arrangement is a specific embodiment of the bispecific single-linked molecule. The linkers Lx, Ly and Lz may be the same or different. The linker can be a peptide linker of any length. In some embodiments, the peptide linker between the VH and VL of the antigen binding domain (such as scFv) is 1 amino acid to 20 amino acids long, 2 amino acids to 19 amino acids long , 3 amino acids to 18 amino acids long, 4 amino acids to 17 amino acids long, 5 amino acids to 17 amino acids long, 6 amino acids to 17 amino acids Acid length, 7 amino acids to 18 amino acids long, 8 amino acids to 17 amino acids long, 9 amino acids to 17 amino acids long, 10 amino acids to 17 Amino acids long, 11 amino acids to 16 amino acids long, 12 amino acids to 17 amino acids long, 13 amino acids to 16 amino acids long, 14 amino acids to 16 amino acids long or 14 amino acids to 15 amino acids long. In some embodiments, the peptide linker between VH and VL of an antigen binding domain (such as scFv) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19 or 20 of any amino acid length. In some embodiments, the peptide linker between the VH and VL of the antigen binding domain (such as scFv) is 14 or 15 amino acids long. In some embodiments, the peptide linkers between the first and second antigen binding domains (such as scFv) are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, Any one of 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, the peptide linker between the first and second antigen binding domains (such as scFv) is 5 amino acids long. The basic technical feature of such a peptide linker is that the peptide linker does not contain any polymerization activity. Features that include peptide linkers that do not have a secondary structure promoting effect are known in the art and described in, for example, Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (1992) 29, 21-30) and Raag and Whitlow (FASEB (1995) 9 (1), 73-80). In the case of "peptide linker", the preferred amino acid is Gly. In addition, peptide linkers that do not promote any secondary structure are preferred. The connection between the domains can be provided by genetic engineering, for example. Methods for preparing fused and operably linked bispecific single-stranded constructs and expressing them in mammalian cells or bacteria are well known in the art (eg WO 99/54440; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). The peptide linker may be a protease, especially a stable linker that is not cleavable by matrix metalloproteinase (MMP). The linker can also be a flexible linker. Exemplary flexible linkers include glycine polymer (G)n , Glycine-serine polymer (including for example (GS)n , (GSGGS)n And (GGGS)n , Where n is an integer of at least one), glycine-alanine polymer, alanine-serine polymer and other flexible linkers known in the art. Glycine and glycine-serine polymer systems are relatively unstructured and can therefore act as neutral tethers between components. Glycine is even more likely to enter the φ-ψ gap than alanine, and it is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those of ordinary skill should recognize that the design of bispecific antibody molecules can include a linker with full or partial flexibility, whereby the linker can include a flexible linker portion and cause a structure with lower flexibility One or more parts, thereby providing the desired bispecific antibody molecular structure. In some embodiments, the VH and VL domains of the first antigen binding domain (such as scFv) are folded by a length sufficient for each domain to allow binding to tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) Connectors are connected together. With regard to this embodiment, such linkers may comprise, for example, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 33), or GGGGSGGGGSGGSA (SEQ ID NO: 34). In some embodiments, the VH and VL domains of the second antigen binding domain (such as scFv) are of sufficient length to allow each domain to allow binding to cell surface molecules (such as T lymphocytes) on effector cells The connectors are folded together in the way of CD3). With regard to this embodiment, such linkers may comprise, for example, the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 33). In some embodiments, the first and second antigen binding domains (such as scFv) are cells that are long enough to allow each domain to bind to tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and effector cells The surface molecules (such as CD3 on T lymphocytes) are linked together by folding the linker in two ways. With regard to this embodiment, such linkers may comprise, for example, the amino acid sequence GGGGS (SEQ ID NO: 35). In some embodiments, the junction molecule additionally comprises one or more other domains, such as cytokines, costimulatory domains, domains of negative regulatory molecules that inhibit T cell activation, or one or more of their combinations. In some embodiments, the cytokines are IL-15, IL-2, and / or IL-7. In some embodiments, the costimulatory domain is CD27, CD80, CD83, CD86, CD134, or CD137. In some embodiments, the domain of the negative regulatory molecule that inhibits T cell activation is PD-1, PD-L1, CTLA4, or B7-H4. Thus, in some embodiments, the conjugation molecules described herein include a first antigen binding domain that recognizes EpCAM (such as scFv) and a second antigen binding domain that specifically recognizes CD3 on T lymphocytes (such as scFv). In some embodiments, the first antigen binding domain is scFv. In some embodiments, the second antigen binding domain is scFv. In some embodiments, both the first and second antigen binding domains are scFv. In some embodiments, the first and second antigen binding domains are connected by a linker. In some embodiments, the first antigen binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen binding domain is at the C-terminus of the bispecific molecule. In some embodiments, the junction molecule comprises a first antigen binding domain (such as scFv) that recognizes FAP and a second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, the first antigen binding domain is scFv. In some embodiments, the second antigen binding domain is scFv. In some embodiments, both the first and second antigen binding domains are scFv. In some embodiments, the first and second antigen binding domains are connected by a linker. In some embodiments, the first antigen binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen binding domain is at the C-terminus of the bispecific molecule. In some embodiments, the junction molecule comprises a first antigen binding domain that recognizes EGFR (such as scFv) and a second antigen binding domain that specifically recognizes CD3 on T lymphocytes (such as scFv). In some embodiments, the first antigen binding domain is scFv. In some embodiments, the second antigen binding domain is scFv. In some embodiments, both the first and second antigen binding domains are scFv. In some embodiments, the first and second antigen binding domains are connected by a linker. In some embodiments, the first antigen binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen binding domain is at the C-terminus of the bispecific molecule. In some embodiments, the junction molecule comprises a first antigen binding domain (such as scFv) that recognizes GPC3 and a second antigen binding domain (such as scFv) that specifically recognizes CD3 on T lymphocytes. In some embodiments, the first antigen binding domain is scFv. In some embodiments, the second antigen binding domain is scFv. In some embodiments, both the first and second antigen binding domains are scFv. In some embodiments, the first and second antigen binding domains are connected by a linker. In some embodiments, the first antigen binding domain is at the N-terminus of the bispecific molecule. In some embodiments, the first antigen binding domain is at the C-terminus of the bispecific molecule. III. Methods of treating cancer Pharmaceutical compositions The present application further provides pharmaceutical compositions comprising an effective amount of any of the oncolytic vaccinia viruses described herein and optionally pharmaceutically acceptable carriers, the oncolytic Vaccinia virus contains an immune checkpoint regulator (such as anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or SIRPα extracellular domain and CXCL12 fragment-Fc Fusion protein), wherein the nucleic acid encoding an immune checkpoint regulator is operably linked to a late promoter (such as F17R). The present application also provides a pharmaceutical composition comprising an effective amount of any of oncolytic viruses (such as oncolytic VV) and optionally pharmaceutically acceptable carriers, the oncolytic virus comprising an immune checkpoint regulator The first nucleic acid (such as an anti-PD-1 antibody, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), and A second nucleic acid encoding the bispecific molecule described herein. In some embodiments, the present application also provides a pharmaceutical composition comprising: a first OV comprising a first nucleic acid encoding an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein) (E.g. oncolytic VV); and a second OV (e.g. oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific molecule comprising The first antigen binding domain (such as scFv) that specifically recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and the second antigen that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Binding domains (such as scFv); and optionally pharmaceutically acceptable carriers. In some embodiments, a pharmaceutical composition is provided that includes: a first OV (eg, oncolytic) comprising a first nucleic acid encoding an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein) VV); and a second OV (eg, oncolytic VV) containing a second nucleic acid encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF); and optionally medically available Accepted carrier. In some embodiments, a pharmaceutical composition is provided that includes: a first OV (eg, oncolytic) comprising a first nucleic acid encoding an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein) VV); a second OV (eg, oncolytic VV) containing a second nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific molecule contains a specific recognition tumor antigen (Such as EpCAM, FAP, EGFR or GPC3) the first antigen-binding domain (such as scFv) and the second antigen-binding domain (such as CD3 on T lymphocytes) that specifically recognizes the cell surface molecule on the effector cell (such as scFv) scFv); and a third OV (eg, oncolytic VV) containing a third nucleic acid encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF); and optionally medically available Accepted carrier. In some embodiments, the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Senegal Valley virus (SVV), adenovirus, herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2 ), Myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, measles virus, influenza virus, Sindby virus and New City Epidemic virus (NDV). In some embodiments, the OV is an oncolytic VV. In some embodiments, the oncolytic VV is selected from the group consisting of Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, Modified Ankara Vaccinia Virus (MVA), Lister, King, IHD, Evans, USSR and Western Reserve (WR). In some embodiments, the oncolytic VV strain is a WR strain. In some embodiments, the oncolytic virus comprises a double deletion of the TK gene and the VGF gene. In some embodiments, the immune checkpoint regulator is an activator of an stimulatory immune checkpoint molecule (such as an activator of CD27, CD28, CD40, CD122, CD137, OX40, GITR, or ICOS). In some embodiments, the immune checkpoint regulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint regulator sub-line PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA , B7-H4, CD160, 2B4 or CD73 inhibitors. In some embodiments, the immune checkpoint regulator is an inhibitor of PD-1. In some embodiments, the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is a ligand that binds to the immune checkpoint molecule. In some embodiments, the immune checkpoint regulator is a ligand of PD-L1 / PD-L2, HHLA-2, CD47, or CXCR4. In some embodiments, the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and phosphatidylinositol-3 (GPC3). In some embodiments, the tumor antigen is EpCAM. In some embodiments, the tumor antigen is FAP. In some embodiments, the tumor antigen is EGFR. In some embodiments, the tumor antigen is GPC3. In some embodiments, the effector cell line is selected from the group consisting of T lymphocytes, B lymphocytes, natural killer (NK) cells, dendritic cells (DC), macrophages, monocytes, and neutrophils Ball and NKT cells. In some embodiments, the effector cell line T lymphocytes (such as cytotoxic T lymphocytes). In some embodiments, the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D. In some embodiments, the cell surface molecule line is CD3. In some embodiments, the first and / or second antigen binding domains are single chain variable fragments (scFv). In some embodiments, the first and second antigen binding domains are connected by a linker. In some embodiments, the first antigen binding domain is N-terminal to the second antigen binding domain. In some embodiments, the first antigen binding domain is at the C-terminus of the second antigen binding domain. In some embodiments, the first nucleic acid encoding the immune checkpoint regulator is operably linked to the late promoter. In some embodiments, the second nucleic acid encoding the bispecific molecule is operably linked to the late promoter. In some embodiments, the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters, and PSL The late promoter is synthesized. In some embodiments, the late promoter line is F17R. As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is not toxic to cells or mammals exposed to it at the dose and concentration used. Generally physiologically acceptable carriers are aqueous pH buffer solutions. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition containing such a carrier can be formulated by well-known conventional methods. The solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, physiological saline (such as sodium chloride), Ringer's solution, glucose, trehalose or sucrose solution, Hank's solution, and other physiologically balanced aqueous salts Solution (see, for example, the latest version of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins). The pharmaceutical composition of the present invention can be administered locally (such as in a tumor) or systemically. Administration is generally parenteral, such as intravenous administration; DNA can also be administered directly to the target site, such as by gene gun delivery to internal or external target sites, or via catheters to sites in the arteries . In some embodiments, the pharmaceutical composition is administered subcutaneously. In some embodiments, the pharmaceutical composition is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous vehicles include water, alcohol / aqueous solutions, emulsions or suspensions, including physiological saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oil. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as Ringer's dextrose-based electrolyte supplements), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention may contain a protein carrier, preferably a protein carrier of human origin, such as, for example, serum albumin or immunoglobulin. Various virus formulations in frozen, liquid or lyophilized forms (for example, WO98 / 02522, WO01 / 66137, WO03 / 053463, WO2007 / 056847, WO2008 / 114021, etc.) can be used in this technology. A solid (eg dry powder or lyophilized) composition can be obtained by methods involving vacuum drying and freeze drying (see for example WO2014 / 053571). It is envisaged that in addition to immune checkpoint regulators (and / or bispecific junction molecules, cytokines) or their encoding nucleic acid molecules or vectors (as described in the present invention), depending on the intended use of the pharmaceutical composition, The pharmaceutical composition of the present invention may also contain other bioactive agents. In some embodiments, the pharmaceutical compositions described herein are suitably buffered for human use. Suitable buffers include, but are not limited to, phosphate buffers (eg PBS), bicarbonate buffers and / or Tris buffers capable of maintaining physiological or slightly alkaline pH (eg about pH 7 to about pH 9). In some embodiments, the pharmaceutical composition can also be made isotonic with blood by adding a suitable tonicity modifier, such as glycerin. In some embodiments, the pharmaceutical composition is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in a container as a whole. In some embodiments, the pharmaceutical composition is cryopreserved. Therapeutic use of oncolytic viruses One aspect of this application relates to a method of treating cancer, which comprises administering an effective amount of a pharmaceutical composition to an individual, the pharmaceutical composition comprising an oncolytic vaccinia virus and optionally pharmaceutically acceptable Carrier, the oncolytic vaccinia virus contains coding immune checkpoint regulators (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA- 4. TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) nucleic acid, wherein the nucleic acid encoding an immune checkpoint regulator is operably linked to a late promoter (such as F17R). In some embodiments, patients with cancer in this system are either susceptible or suspected of having cancer. The invention includes nucleic acid sequences encoding immune checkpoint regulators, and vectors encoding immune checkpoint regulators (such as oncolytic virus vectors) covered by and / or produced by the methods covered herein. Another aspect of the present application relates to a method of treating cancer, which comprises administering an effective amount of a pharmaceutical composition to an individual, the pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) and optionally pharmaceutically acceptable Carrier, the oncolytic virus contains coding immune checkpoint regulators (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4 , TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73 inhibitor) and the second nucleic acid encoding a bispecific molecule, which contains a specific recognition tumor antigen (such as EpCAM, FAP , EGFR or GPC3) the first antigen-binding domain (such as scFv) and the second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, patients with cancer in this system are either susceptible or suspected of having cancer. The invention includes nucleic acid sequences encoding immune checkpoint regulators, and vectors encoding immune checkpoint regulators (such as oncolytic virus vectors) covered by and / or produced by the methods covered herein. Part of the invention covers viruses, protein constructs (such as bispecific molecules or immune checkpoint regulators), nucleic acid molecules and / or vectors (such as oncolytic virus vectors), which can be administered alone or with any of the other therapies Combined and administered with a pharmaceutically acceptable carrier or excipient in at least some aspects. In some embodiments, prior to administration of the viral or protein construct, it can be combined with suitable pharmaceutical carriers and excipients well known in the art. The composition prepared according to the present invention can be used to treat or delay the onset or worsening of cancer. In some embodiments, a method of treating cancer in an individual (such as a human) is provided, which comprises administering to the individual an effective amount of a pharmaceutical composition and optionally a pharmaceutically acceptable carrier, the pharmaceutical composition Immune checkpoint regulators (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160 , 2B4 or CD73 inhibitors) oncolytic vaccinia virus, wherein the nucleic acid encoding an immune checkpoint regulator is operably linked to a late promoter (such as F17R). In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting cancer cell proliferation; (3) inducing tumors Immune response; (4) reducing tumor size; (5) reducing one or more symptoms of individuals with cancer; (6) inhibiting tumor metastasis; (7) prolonging survival period; (8) prolonging cancer development time; (9) Prevent, inhibit or reduce the possibility of cancer recurrence; (10) induce redistribution of surrounding T cells; and (11) reduce the incidence or burden of preexisting tumor metastases (such as lymph node metastasis). In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher The tumor cell mortality rate of any of the percentages. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 95% or higher percentage of bystander tumor cells (tumor cells not infected with oncolytic VV) mortality. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce tumor size by at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%). In some embodiments, a method of inhibiting tumor metastasis mediated by a pharmaceutical composition described herein can inhibit at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80% , Any of 90% or 100%). In some embodiments, the method of prolonging the survival period of an individual (such as a human) mediated by the pharmaceutical composition described herein may extend the survival period of the individual by at least 1, 2, 3, 4, 5, 6, 7, Any of 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging cancer progression time mediated by the pharmaceutical composition described herein can prolong cancer progression time by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Either 11 or 12 weeks. In some embodiments, a method of treating cancer in an individual (such as a human) is provided, which comprises administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as an oncolytic VV) and optionally medicine Academically acceptable carrier, the oncolytic virus contains coded immune checkpoint regulators (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA , CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) and the second nucleic acid encoding a bispecific molecule, the bispecific molecule contains a specific recognition tumor antigen ( First antigen-binding domain (such as scFv) such as EpCAM, FAP, EGFR or GPC3 and second antigen-binding domain (such as scFv) that specifically recognize cell surface molecules on effector cells (such as CD3 on T lymphocytes) ). In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting cancer cell proliferation; (3) inducing peripheral T Redistribution of cells; (4) induce immune response in tumor; (5) reduce tumor size; (6) reduce one or more symptoms of individuals with cancer; (7) inhibit tumor metastasis; (8) prolong survival Period; (9) prolong the time of cancer progression; (10) prevent, inhibit or reduce the possibility of cancer recurrence; (11) induce stromal destruction or kill tumor stromal cells in tumors; (12) promote oncolytic viruses Spread through the tumor; (13) promote T cell infiltration in the tumor; and (14) reduce the incidence or burden of preexisting tumor metastases (such as lymph node metastases). In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher The tumor cell mortality rate of any of the percentages. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 95% or higher percentage of bystander tumor cells (tumor cells not infected with oncolytic VV) mortality. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce tumor size by at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%). In some embodiments, a method of inhibiting tumor metastasis mediated by a pharmaceutical composition described herein can inhibit at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80% , Any of 90% or 100%). In some embodiments, the method of prolonging the survival period of an individual (such as a human) mediated by the pharmaceutical composition described herein may extend the survival period of the individual by at least 1, 2, 3, 4, 5, 6, 7, Any of 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging cancer development time mediated by the pharmaceutical composition described herein can prolong cancer development time by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Either 11 or 12 weeks. In some embodiments, there is also provided a method of treating cancer in an individual (such as a human), which comprises administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising: comprising an immune checkpoint regulator (such as described herein) Any of the described immune checkpoint regulators, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, fusion of PD-1 extracellular domain with IgG4 Fc, fusion of TGIGD2 extracellular domain with IgG4 Fc Substance, or the first OV of the first nucleic acid of the SIRPα extracellular domain and the fusion of the CXCL12 fragment and IgG4 Fc) (eg, oncolytic VV); and contains a coding bispecific molecule (such as the bispecific molecule described herein) The second OV of the second nucleic acid of any one of them) (eg oncolytic VV), the bispecific molecule contains a first antigen binding domain (such as EpCAM, FAP, EGFR or GPC3) that specifically recognizes a tumor antigen (such as scFv) and a second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes); and optionally pharmaceutically acceptable carriers. In some embodiments, there is also provided a method of treating cancer in an individual (such as a human), which comprises administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising: comprising an immune checkpoint regulator (such as described herein) Any of the described immune checkpoint regulators, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, fusion of PD-1 extracellular domain with IgG4 Fc, fusion of TGIGD2 extracellular domain with IgG4 Fc Substance, or the first OV of the first nucleic acid of the SIRPα extracellular domain and the fusion of the CXCL12 fragment and IgG4 Fc) (eg, oncolytic VV); and contains a coding cytokine (such as the cytokines described herein) Any one, such as the second OV of the second nucleic acid of GM-CSF); and optionally a pharmaceutically acceptable carrier. In some embodiments, there is also provided a method of treating cancer in an individual (such as a human), which comprises administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising: comprising an immune checkpoint regulator (such as described herein) Any of the described immune checkpoint regulators, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, fusion of PD-1 extracellular domain with IgG4 Fc, fusion of TGIGD2 extracellular domain with IgG4 Fc Substance, or the first OV of the first nucleic acid of the extracellular domain of SIRPα and the fusion of the CXCL12 fragment and IgG4 Fc) (eg, oncolytic VV); contains a coding bispecific molecule (such as the bispecific molecule described herein) The second OV of any second nucleic acid (eg oncolytic VV), the bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) ) And a second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes); and contains encoding cytokines (such as those in the cytokines described herein) Either , Such as a third OV GM-CSF) of the third nucleic acid (e.g. VV of oncolytic); and optionally a pharmaceutically acceptable carrier. In some embodiments, there is also provided a method of treating cancer in an individual, such as a human, comprising administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a first OV (eg, oncolytic VV) and optionally a pharmaceutically acceptable carrier, the first OV contains an encoded immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as immune checkpoint inhibitors, such as anti- PD-1 antibody, fusion of PD-1 extracellular domain and IgG4 Fc, fusion of TGIGD2 extracellular domain and IgG4 Fc, or fusion of SIRPα extracellular domain and CXCL12 fragment and IgG4 Fc) Nucleic acid; and an effective amount of a second pharmaceutical composition comprising a second OV (eg oncolytic VV) and optionally a pharmaceutically acceptable second carrier, the second OV comprising a coded bispecific Second nucleic acid of a sexual molecule (such as any of the bispecific molecules described herein), the bispecific molecule comprising a first antigen binding domain that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) (Such as scFv) and specificity Cell surface molecules (such as CD3 on the T-lymphocytes) on effector cells of other antigen-binding domain of a second (such as a scFv). In some embodiments, a method of treating cancer in an individual (such as a human) is provided, which comprises administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a first OV (eg, oncolytic VV ) And optionally a pharmaceutically acceptable first carrier, the first OV contains an encoded immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, for example, immune checkpoint inhibitors, for example Anti-PD-1 antibody, fusion of PD-1 extracellular domain and IgG4 Fc, fusion of TGIGD2 extracellular domain and IgG4 Fc, or fusion of SIRPα extracellular domain and CXCL12 fragment and IgG4 Fc) A nucleic acid; and an effective amount of a second pharmaceutical composition comprising a second OV (eg oncolytic VV) and optionally a pharmaceutically acceptable second carrier, the second OV comprising coding cells The second nucleic acid of an interleukin (such as any of the cytokines described herein, such as GM-CSF). In some embodiments, a method of treating cancer in an individual (such as a human) is provided, which comprises administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a first OV (eg, oncolytic VV ) And optionally a pharmaceutically acceptable first carrier, the first OV contains an encoded immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, for example, immune checkpoint inhibitors, for example Anti-PD-1 antibody, fusion of PD-1 extracellular domain and IgG4 Fc, fusion of TGIGD2 extracellular domain and IgG4 Fc, or fusion of SIRPα extracellular domain and CXCL12 fragment and IgG4 Fc) A nucleic acid; an effective amount of a second pharmaceutical composition comprising a second OV (eg oncolytic VV) and optionally a pharmaceutically acceptable carrier, the second OV comprising a bispecific molecule A second nucleic acid (such as any of the bispecific molecules described herein) that contains a first antigen binding domain (such as EpCAM, FAP, EGFR, or GPC3) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) scFv) and specific recognition A second antigen binding domain (such as scFv) of a cell surface molecule on a cell (such as CD3 on T lymphocytes); and an effective amount of a third pharmaceutical composition comprising a third OV (for example Oncolytic VV) and optionally a pharmaceutically acceptable third carrier, the third OV contains a third nucleic acid encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF) . In some embodiments, the method of treating cancer has one or more of the following biological activities: (1) killing cancer cells (including bystander killing); (2) inhibiting cancer cell proliferation; (3) inducing peripheral T Redistribution of cells; (4) induce immune response in tumor; (5) reduce tumor size; (6) reduce one or more symptoms of individuals with cancer; (7) inhibit tumor metastasis; (8) prolong survival Period; (9) prolong the time of cancer progression; (10) prevent, inhibit the recurrence of cancer or reduce the possibility of cancer recurrence; (11) induce stromal destruction in tumors or kill tumor stromal cells (for example when expressing FAP-CD3 T Cell zygote); (12) promote the spread of oncolytic viruses through the tumor (e.g. when expressing FAP-CD3 T cell zygote); (13) promote T cell infiltration in the tumor (e.g. when expressing FAP-CD3 T cell conjugate (Time); and (14) reduce the incidence or burden of preexisting tumor metastases (such as lymph node metastases). In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher The tumor cell mortality rate of any of the percentages. In some embodiments, the method of killing cancer cells mediated by the pharmaceutical composition described herein can achieve at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%, 95% or higher percentage of bystander tumor cells (tumor cells not infected with OV) mortality. In some embodiments, the method of reducing tumor size mediated by the pharmaceutical composition described herein can reduce tumor size by at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80%, 90% or 100%). In some embodiments, a method of inhibiting tumor metastasis mediated by a pharmaceutical composition described herein can inhibit at least about 10% (including, for example, at least about 20%, 30%, 40%, 60%, 70%, 80% , Any of 90% or 100%). In some embodiments, the method of prolonging the survival period of an individual (such as a human) mediated by the pharmaceutical composition described herein may extend the survival period of the individual by at least 1, 2, 3, 4, 5, 6, 7, Any of 8, 9, 10, 11, 12, 18, or 24 months. In some embodiments, the method of prolonging cancer progression time mediated by the pharmaceutical composition described herein can prolong cancer progression time by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, Either 11 or 12 weeks. The efficacy of an oncolytic vaccinia virus encoding an immune checkpoint regulator, and the efficacy of an oncolytic virus (such as an oncolytic VV) expressing the immune checkpoint inhibitor and bispecific conjugate molecule described herein may be due to the spread of the tumor matrix as a virus And the physical barrier of T cells is restricted. Preclinical studies have shown that co-targeting tumor cells and tumor stroma significantly enhances the anti-tumor activity of immunotherapy. In some embodiments, an oncolytic virus of the invention (such as oncolytic VV) comprises a first nucleic acid encoding an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein), and encoding a bispecific The second nucleic acid of a sex molecule. The bispecific molecule contains a first antigen binding domain that specifically recognizes the tumor antigen FAP (eg, scFv) and a cell surface molecule that specifically recognizes on effector cells (eg, CD3 on T lymphocytes). The second antigen binding domain (eg scFv). In some embodiments, the oncolytic virus that co-expresses the immune checkpoint regulator and the bispecific molecule is oncolytic VV. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-Fc fusion protein. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). Therefore, in some embodiments, a method of inducing stromal destruction or killing tumor stromal cells in a tumor of an individual (such as a human) is provided, which comprises administering to the individual an effective amount of an oncolytic virus (such as oncolytic VV) And optionally a pharmaceutical composition of a pharmaceutically acceptable carrier, the oncolytic virus contains coding immune checkpoint regulators (such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3 , TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) first nucleic acid, and a second nucleic acid encoding a bispecific molecule, the bispecific The molecule contains a first antigen binding domain (such as scFv) that specifically recognizes tumor antigen FAP and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) . In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, there is provided a method of inducing stromal destruction or killing tumor stromal cells in a tumor of an individual, such as a human, comprising administering to the individual: a first pharmaceutical composition, the first pharmaceutical composition comprising a code Immune checkpoint regulators (such as any of the immune checkpoint regulators described herein, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular Domain-Fc fusion protein, or the first OV of SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), and optionally a pharmaceutically acceptable first carrier; and a second pharmaceutical composition, the second The pharmaceutical composition comprises an OV encoding a bispecific molecule (such as any of the bispecific molecules described herein), and optionally a pharmaceutically acceptable second carrier, the bispecific molecule comprises specific recognition The first antigen-binding domain of FAP (such as scFv) and the second antigen-binding domain (such as scFv) that specifically recognize cell surface molecules on effector cells (such as CD3 on T lymphocytes) A pharmaceutical composition comprising a third OV encoding a cytokine (such as any of the cytokines described herein), and optionally a pharmaceutically acceptable third carrier). In some embodiments, a method of inducing stromal destruction or killing tumor stromal cells in a tumor of an individual (such as a human) is provided, which comprises administering a pharmaceutical composition to the individual, the pharmaceutical composition comprising: encoding immune checkpoint modulation (Such as any of the immune checkpoint regulators described herein, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc The first OV of the fusion protein, or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein; and the second OV encoding a bispecific molecule (such as any of the bispecific molecules described herein) Specific molecules include a first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) ; And / or a third OV encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF); and optionally a pharmaceutically acceptable carrier. In some embodiments, the oncolytic virus is vaccinia virus. In some embodiments, the method further induces bystander matrix destruction or kills bystander tumor stromal cells (cells not infected with the oncolytic virus described herein) in the presence of T cells. In some embodiments, the method further comprises administering an effective amount of another pharmaceutical composition to the individual, the pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) and optionally a pharmaceutically acceptable carrier, the Oncolytic viruses contain a nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein) that contains a tumor antigen that specifically recognizes not FAP (eg EpCAM, EGFR, or GPC3) The first antigen-binding domain (eg scFv) and the second antigen-binding domain (eg scFv) that specifically recognize cell surface molecules on effector cells (eg CD3 on T lymphocytes). In some embodiments, the oncolytic virus in the other pharmaceutical composition is vaccinia virus. In some embodiments, the oncolytic virus with FAP-T cell zygote (FAP-TEA-OV) enhances non-FAP-TEA-OV, such as EpCAM-TEA-OV (also referred to as EpCAM-CD3-OV hereinafter) , EGFR-TEA-OV (hereinafter also referred to as EGFR-CD3-OV) or GPC3-TEA-OV (hereinafter also referred to as GPC3-CD3-OV) antitumor activity. In some embodiments, immune checkpoint regulators expressed by oncolytic viruses that co-express FAP-TE (hereinafter also referred to as FAP-CD3) further enhance the anti-tumor effects of FAP-TE and / or non-FAP-TE. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-IgG4 Fc. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, there is also provided a method for promoting the spread of oncolytic viruses through a tumor in an individual (such as a human), which comprises administering to the individual an effective amount of an oncolytic virus (such as an oncolytic VV) and optionally medicine A pharmaceutical composition of an acceptable carrier, the oncolytic virus contains an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as PD-1, PD-L1, PD-L2 , CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73 inhibitor), and encode bispecific The second nucleic acid of the molecule, the bispecific molecule contains a first antigen binding domain that specifically recognizes the tumor antigen FAP (such as scFv) and a cell surface molecule that specifically recognizes effector cells (such as CD3 on T lymphocytes) A second antigen binding domain (such as scFv). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, a method of promoting the spread of an oncolytic virus through a tumor in an individual (such as a human) is provided, which comprises administering to the individual: a first pharmaceutical composition comprising an encoded immune checkpoint modulation (Such as any of the immune checkpoint regulators described herein, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc Fusion protein, or the first OV of SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), and optionally a pharmaceutically acceptable first carrier; and a second pharmaceutical composition, the second pharmaceutical composition comprising OV encoding a bispecific molecule (such as any of the bispecific molecules described herein), and optionally a pharmaceutically acceptable second carrier, the bispecific molecule includes a first specific recognition FAP Antigen binding domains (such as scFv) and second antigen binding domains (such as scFv) that specifically recognize cell surface molecules on effector cells (such as CD3 on T lymphocytes) (and optionally third pharmaceutical compositions, which Contains a third OV encoding a cytokine (such as any of the cytokines described herein), and optionally a pharmaceutically acceptable third carrier). In some embodiments, a method of promoting the spread of an oncolytic virus through a tumor in an individual (such as a human) is provided, which comprises administering to the individual a pharmaceutical composition, the pharmaceutical composition comprising: encoding an immune checkpoint regulator (such as herein) Any of the described immune checkpoint regulators, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or SIRPα extracellular domain and the first OV of the CXCL12 fragment-Fc fusion protein; and the second OV encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific molecule comprising A first antigen binding domain (such as scFv) that specifically recognizes FAP and a second antigen binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes); and / or A third OV encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF); and optionally a pharmaceutically acceptable carrier. In some embodiments, the oncolytic virus is vaccinia virus. In some embodiments, the method further comprises administering an effective amount of another pharmaceutical composition to the individual, the pharmaceutical composition comprising an oncolytic virus and optionally a pharmaceutically acceptable carrier, the oncolytic virus comprising A nucleic acid of a specific molecule (such as any of the bispecific molecules described herein) that includes a first antigen binding structure that specifically recognizes a tumor antigen that is not FAP (eg, EpCAM, EGFR, or GPC3) Domain (eg, scFv) and a second antigen-binding domain (eg, scFv) that specifically recognizes cell surface molecules on effector cells (eg, CD3 on T lymphocytes). In some embodiments, the oncolytic virus in the other pharmaceutical composition is vaccinia virus. In some embodiments, OV with FAP-T cell zygote (FAP-TEA-OV) enhances non-FAP-TEA-OV, such as EpCAM-TEA-OV, EGFR-TEA-OV, or GPC3-TEA-OV diffusion. In some embodiments, immune checkpoint regulators expressed by oncolytic viruses that co-express FAP-TE further enhance the effects of FAP-TE and / or non-FAP-TE. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-IgG4 Fc. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, a method of increasing T cell tumor infiltration in an individual (such as a human) is provided, which comprises administering to the individual an effective amount of an oncolytic virus (such as oncolytic VV) and optionally pharmaceutically acceptable The pharmaceutical composition of the carrier, the oncolytic virus contains an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4, or CD73 inhibitor) and the first nucleic acid encoding a bispecific molecule Two nucleic acids, the bispecific molecule contains a first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and a cell surface molecule (such as T lymphocytes) that specifically recognizes effector cells The second antigen binding domain of CD3 on the cell (such as scFv). In some embodiments, the OV further comprises a third nucleic acid encoding a cytokine (such as GM-CSF). In some embodiments, a method of increasing T cell tumor infiltration in an individual (such as a human) is provided, which comprises administering to the individual: a first pharmaceutical composition comprising an immune checkpoint regulator ( Such as any of the immune checkpoint regulators described herein, for example, immune checkpoint inhibitors, such as anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein , Or the first OV of SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein), and optionally a pharmaceutically acceptable first carrier; and a second pharmaceutical composition, the second pharmaceutical composition OV of a specific molecule (such as any of the bispecific molecules described herein), and optionally a pharmaceutically acceptable second carrier, the bispecific molecule contains a specific recognition tumor antigen (such as EpCAM, FAP, EGFR or GPC3) the first antigen-binding domain (such as scFv) and the second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) (and As The third condition pharmaceutical composition comprising a cytokine encoding (described in this document of any cytokine, such as in the one kind) of the OV third, and optionally a third pharmaceutically acceptable carrier). In some embodiments, a method of increasing T cell tumor infiltration in an individual (such as a human) is provided, which comprises administering to the individual a pharmaceutical composition comprising: encoding an immune checkpoint regulator (such as described herein Any of the immune checkpoint regulators, such as immune checkpoint inhibitors, such as anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or SIRPα cells The first OV of the outer domain and the CXCL12 fragment-Fc fusion protein; and the second OV encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific molecule contains specificity Identify the first antigen-binding domain (such as scFv) of a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) and the second antigen-binding structure that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Domain (such as scFv); and / or a third OV encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF); and optionally a pharmaceutically acceptable carrier. In some embodiments, the oncolytic virus is vaccinia virus. In some embodiments, the method induces a T cell immune response in bystander tumor cells (cells not infected with the oncolytic virus described herein) in the presence of T cells. In some embodiments, the method further comprises administering an effective amount of another pharmaceutical composition to the individual, the pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) and optionally a pharmaceutically acceptable carrier, the Oncolytic viruses include nucleic acids encoding bispecific molecules (such as any of the bispecific molecules described herein) that include a first antigen-binding domain (eg, scFv) that specifically recognizes tumor antigen FAP And a second antigen binding domain (eg, scFv) that specifically recognizes cell surface molecules on effector cells (eg, CD3 on T lymphocytes). In some embodiments, the oncolytic virus in the other pharmaceutical composition is vaccinia virus. In some embodiments, the FAP-TEA-VV of the other pharmaceutical composition (hereinafter also referred to as FAP-CD3-VV) promotes tumor cells infected with the oncolytic virus described herein and / or is not infected with the described herein Onlookers of oncolytic viruses infiltrate T cells in tumor cells. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-IgG4 Fc. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). The methods described herein are suitable for treating a variety of cancers, including both solid and liquid cancers. These methods are applicable to all stages of cancer, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer or remission cancer. The method described herein can be used as a first therapy, a second therapy, a third therapy, or using this technology in adjuvant therapy or neoadjuvant therapy (ie, the method can be performed before initial therapy / deterministic therapy) Combination therapy of other types of cancer therapy known such as chemotherapy, surgery, hormone therapy, radiation, gene therapy, immunotherapy (such as T cell therapy), bone marrow transplantation, stem cell transplantation, targeted therapy, Ultra-low temperature therapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation or similar therapy. In some embodiments, the method is used to treat previously treated individuals. In some embodiments, the cancer is difficult to treat with previous therapies. In some embodiments, the method is used to treat individuals who have not previously been treated. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cutaneous adenocarcinoma, AIDS-related cancer (eg, AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (eg, cerebellar and cerebral astrocytic cells) Tumor), basal cell carcinoma, cholangiocarcinoma (e.g. extrahepatic cholangiocarcinoma), bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g. glioma, brain stem glioma, cerebellum or brain Astrocytoma (eg hair cell astrocytoma, diffuse astrocytoma, pleomorphic (malignant) astrocytoma), malignant glioma, ependymoma, oligodendroglioma, meninges Tumors, craniopharyngiomas, hemangioblastomas, neuroblastomas, primitive neuroectodermal tumors, visual pathways and hypothalamic gliomas, and glioblastomas), breast cancer, bronchial adenoma / class Carcinoma, carcinoid (e.g. gastrointestinal carcinoid), cancer of unknown primary site, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g. uterine cancer), Ependymoma, esophagus , Ewing's family of tumor, eye cancer (such as intraocular melanoma and retinoblastoma), gallbladder cancer, gastric / stomach cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (GIST) , Germ cell tumors (such as extracranial, extragonadal, ovarian tumors), trophoblastic tumors during pregnancy, head and neck cancer, hepatocellular (liver) cancers (such as hepatocellular carcinoma and hepatocellular tumors), hypopharyngeal carcinoma, islet cell carcinoma ( Endocrine pancreatic cancer), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cancer, oral cancer, liver cancer, lung cancer (such as small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), lymphangioma (E.g. lymphoma), neuroblastoma, melanoma, mesothelioma, metastatic squamous cell neck cancer, oral cancer, multiple endocrine tumor syndrome, myelodysplastic syndrome, myelodysplasia / myeloproliferative diseases , Nasal cavity and sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer (such as ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant tumor), pancreatic cancer, parathyroid cancer, penile cancer, peritoneum Cancer, pharynx , Pheochromocytoma, pineal blastoma and suprasuperior primitive neuroectodermal tumor, pituitary tumor, pleural lung blastoma, lymphoma, primary central nervous system lymphoma (small glioma), rectum Cancer, kidney cancer, renal pelvis and ureteral cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary adenocarcinoma, skin cancer (e.g. non-melanoma (e.g. squamous cell carcinoma), melanoma and Merkel cell carcinoma) ), Small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and lymphoproliferative hyperplasia after transplantation Sexual disorders (PTLD), abnormal vascular proliferation associated with macular spot disease, edema (such as edema associated with brain tumors), and Meigs' syndrome. The pharmaceutical compositions described herein can be used for all stages and types of cancer, including minimal residual disease, early solid tumors, late solid tumors, and / or metastatic solid tumors. In some embodiments, the method is suitable for treating cancers with abnormal PD-1 or PD-L1 / PD-L2 expressive activity and / or signaling, including (as a non-limiting example) blood cancers and / or solid tumors. Some cancers in which the antibodies of the present invention can be used to inhibit growth include cancers that generally respond to immunotherapy. Non-limiting examples of cancers suitable for treatment include melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer And lung cancer (eg, non-small cell lung cancer). In addition, the present invention includes refractory or recurrent malignant diseases that can inhibit growth using the antibodies of the present invention. Examples of other cancers that can be treated with the antibodies of the present invention include bone cancer; pancreatic cancer; skin cancer; head and neck cancer; malignant melanoma of the skin or eye; uterine cancer; ovarian cancer; rectal cancer; anal cancer; stomach cancer; Cancer; Uterine cancer; Tubal cancer; Endometrial cancer; Cervical cancer; Vaginal cancer; Vulvar cancer; Hodgkin's disease; Non-Hodgkin's lymphoma; Esophageal cancer; Small bowel cancer; Endocrine system cancer; Thyroid cancer; Parathyroid cancer; Adrenal cancer; Soft tissue sarcoma; Urethral cancer; Penile cancer; Chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytes Leukemia; solid tumors in children; lymphocytic lymphoma; bladder cancer; renal or urinary tract cancer; renal pelvis cancer; central nervous system (CNS) neoplasms; primary CNS lymphoma; tumor angiogenesis; spinal axis tumors; brain Stem glioma; pituitary adenoma; Kaposi's sarcoma; epidermoid carcinoma; squamous cell carcinoma; T-cell lymphoma; environmentally-induced Cancer, including cancer induced by asbestos; and a combination of these cancers. The present invention can also be used to treat metastatic cancer, especially metastatic cancer that exhibits PD-L1 (Iwai et al. (2005)Int. Immunol. 17: 133-144). Therefore, in some embodiments, an immunotherapy-responsive solid tumor (such as a carcinoma or adenocarcinoma, such as an abnormal PD-1 or PD-L1 / PD-L2 manifestation, activity, and / or signaling is provided for the treatment of an individual Cancer), which comprises administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus and optionally a pharmaceutically acceptable carrier, the oncolytic vaccinia virus comprising an immune checkpoint regulator (such as PD -1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) Nucleic acid, wherein the nucleic acid encoding an immune checkpoint regulator is operably linked to a late promoter (such as F17R). In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-Fc fusion protein. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; (3) inducing immune response in tumors; (4) reducing tumors Size; (5) reduce one or more symptoms of individuals with cancer; (6) inhibit tumor metastasis; (7) reduce the incidence or burden of preexisting tumor metastasis (such as lymph node metastasis); (8) prolong survival ; And / or (9) extend the time for cancer to progress. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer, colon cancer, or liver cancer. In some embodiments, an immunotherapy responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancer with abnormal PD-1 or PD-L1 / PD-L2 performance, activity, and / or signaling) is provided to treat an individual Method, which comprises administering to a subject an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) and optionally a pharmaceutically acceptable carrier, the oncolytic virus comprising: encoding an immune checkpoint regulator (Such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 Inhibitor) a first nucleic acid; and a second nucleic acid encoding a bispecific molecule comprising a first antigen binding domain (such as scFv) that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) ) And a second antigen-binding domain (such as scFv) that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes). In some embodiments, an immunotherapy responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancer with abnormal PD-1 or PD-L1 / PD-L2 performance, activity, and / or signaling) is provided to treat an individual The method comprises administering an effective amount of a pharmaceutical composition to an individual, the pharmaceutical composition comprising: comprising an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as PD-1, (PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) the first A first OV of nucleic acid (eg oncolytic VV); and a second OV (eg oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein), the Bispecific molecules include a first antigen binding domain (such as scFv) that specifically recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and a cell surface molecule (such as CD3 on T lymphocytes) that specifically recognizes effector cells ) Of the second antigen-binding domain (such as scFv); and optionally medically Accepted carrier. In some embodiments, the pharmaceutical composition further comprises a third OV encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF). In some embodiments, an immunotherapy responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancer with abnormal PD-1 or PD-L1 / PD-L2 performance, activity, and / or signaling) is provided to treat an individual The method comprising administering to an individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising an immunological checkpoint regulator encoding (such as any of the immune checkpoint regulators described herein, for example Inhibitors of PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 ) Of the first nucleic acid of the first nucleic acid (for example, oncolytic VV), and optionally a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition The second OV (eg, oncolytic VV) of the second nucleic acid of a specific molecule (such as any of the bispecific molecules described herein), and a pharmaceutically acceptable second carrier as appropriate, the bispecific The sex molecule contains a first antigen junction that specifically recognizes a tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) Domains (such as scFv) and cell surface molecules (such as CD3 on the T-lymphocytes) on effector cells specifically recognizing the antigen binding domain of a second (such as a scFv). In some embodiments, the method further comprises administering to the individual an effective amount of a third pharmaceutical composition, the third pharmaceutical composition comprising an encoded cytokine (such as any of the cytokines described herein, such as GM -The third OV of CSF), and a pharmaceutically acceptable third carrier as appropriate. In some embodiments, the immune checkpoint regulator is an anti-PD-1 antibody. In some embodiments, the immune checkpoint regulator is the PD-1 extracellular domain-Fc fusion protein. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; (3) inducing immune response in tumors; (4) reducing tumors Size; (5) reduce one or more symptoms of individuals with cancer; (6) inhibit tumor metastasis; (7) reduce the incidence or burden of preexisting tumor metastasis (such as lymph node metastasis); (8) prolong survival ; (9) prolong the time for cancer progression; (10) increase T cell tumor infiltration; and / or (11) enhance any of the mentioned effects. For example, the co-expression of PD-1 extracellular domain-Fc fusion protein can enhance the killing effect of tumor cells activated by T cells in the presence of co-expressed bispecific junction molecules, and increase the production of T cells. Or eliminate or reduce the tumor escape effect caused by inhibitory immune checkpoint molecules expressed by tumor cells. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer, colon cancer, or liver cancer. In some embodiments, the method is suitable for treating cancers with abnormal HHLA2 and / or TMIGD2 performance, activity, and / or signaling, including (but not limited to) breast cancer, lung cancer, thyroid cancer, melanoma, pancreatic cancer, Ovarian cancer, liver cancer, bladder cancer, colon cancer, prostate cancer, kidney cancer, esophageal cancer, and hematological malignancies leukemia and lymphoma. Therefore, in some embodiments, a method of treating an immunotherapy-responsive solid tumor of an individual (such as carcinoma or adenocarcinoma, such as cancer with abnormal HHLA2 and / or TMIGD2 performance, activity, and / or signaling) is provided, which Comprising administering to the individual an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus and optionally a pharmaceutically acceptable carrier, the oncolytic vaccinia virus comprising an immune checkpoint regulator (such as the immune checkpoint described herein) Any of the regulators, such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) nucleic acid, wherein the nucleic acid encoding an immune checkpoint regulator is operably linked to a late promoter (such as F17R). In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; (3) inducing immune response in tumors; (4) reducing tumors Size; (5) reduce one or more symptoms of individuals with cancer; (6) inhibit tumor metastasis; (7) reduce the incidence or burden of preexisting tumor metastasis (such as lymph node metastasis); (8) prolong survival ; And / or (9) extend the time for cancer to progress. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer, colon cancer, or liver cancer. In some embodiments, a method of treating an individual's immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancer with abnormal HHLA2 and / or TMIGD2 performance, activity, and / or signaling) is provided, which includes The individual administers an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as an oncolytic VV) and optionally a pharmaceutically acceptable carrier, the oncolytic virus comprising: encoding an immune checkpoint regulator (such as described herein Any of the immune checkpoint regulators, such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7 -A first nucleic acid of an inhibitor of H4, CD160, 2B4 or CD73); and a second nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific molecule comprising specific The first antigen binding domain (such as scFv) that sexually recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and the second antigen binding that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Domain (such as scFv). In some embodiments, a method of treating an individual's immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancer with abnormal HHLA2 and / or TMIGD2 performance, activity, and / or signaling) is provided, which includes An individual administers an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising: comprising an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as PD-1, PD-L1, PD- Inhibitor of L1, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73) (Eg, oncolytic VV); and a second OV (eg, oncolytic VV) comprising a second nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific molecule comprising specific The first antigen binding domain (such as scFv) that sexually recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and the second antigen binding that specifically recognizes cell surface molecules on effector cells (such as CD3 on T lymphocytes) Domains (such as scFv); and optionally medically Accepted carrier. In some embodiments, the pharmaceutical composition further comprises a third OV encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF). In some embodiments, a method of treating an individual's immunotherapy-responsive solid tumor (such as carcinoma or adenocarcinoma, such as cancer with abnormal HHLA2 and / or TMIGD2 performance, activity, and / or signaling) is provided, which includes Individual administration: an effective amount of a first pharmaceutical composition, which includes an encoded immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as PD-1, PD- Inhibitors of L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73) A first OV (for example, oncolytic VV), and optionally a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition comprising an encoding bispecific molecule (such as herein) The second OV of the second nucleic acid of any of the bispecific molecules described) (eg oncolytic VV), and optionally a pharmaceutically acceptable second carrier, the bispecific molecule contains specific recognition Tumor antigen (such as EpCAM, FAP, EGFR, or GPC3) first antigen junction Domains (such as scFv) and cell surface molecules (such as CD3 on the T-lymphocytes) on effector cells specifically recognizing the antigen binding domain of a second (such as a scFv). In some embodiments, the method further comprises administering to the individual an effective amount of a third pharmaceutical composition, the third pharmaceutical composition comprising an encoded cytokine (such as any of the cytokines described herein, such as GM -The third OV of CSF), and a pharmaceutically acceptable third carrier as appropriate. In some embodiments, the immune checkpoint regulator comprises a fusion of the TGIGD2 extracellular domain and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; (3) inducing immune response in tumors; (4) reducing tumor size; (5) Reduce one or more symptoms of individuals with cancer; (6) Inhibit tumor metastasis; (7) Reduce the incidence or burden of preexisting tumor metastasis (such as lymph node metastasis); (8) Prolong survival period; 9) Prolong the time for cancer progression; (10) increase T cell tumor infiltration; and / or (11) enhance any of the mentioned effects. For example, the co-expression of the TGIGD2 extracellular domain-Fc fusion protein can enhance the effect of activated T cells in killing tumor cells in the presence of co-expressed bispecific junction molecules, and increase the production of T cells by cytokines, or Eliminate or reduce tumor evasion caused by inhibitory immune checkpoint molecules expressed by tumor cells. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer, colon cancer, or liver cancer. In some embodiments, the method is suitable for treating cancers that include abnormal CD47 / SIRPα performance, activity, and / or signaling, and / or abnormal CXCL12 / CXCR4 performance, activity, and / or signaling, including (but not limited to) ovaries Cancer, melanoma, prostate cancer, ovarian cancer, multiple myeloma, breast cancer, lung cancer, liver cancer. Therefore, in some embodiments, an immunotherapy responsive solid tumor (such as a carcinoma or adenocarcinoma, such as a cancer with abnormal CD47 / SIRPα and / or CXCL12 / CXCR4 performance, activity, and / or signaling) is provided to treat an individual Method, which comprises administering to a subject an effective amount of a pharmaceutical composition comprising an oncolytic vaccinia virus and optionally a pharmaceutically acceptable carrier, the oncolytic vaccinia virus comprising an immune checkpoint regulator (such as described herein) Any of the immune checkpoint regulators, such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor) nucleic acid, wherein the nucleic acid encoding an immune checkpoint regulator is operably linked to a late promoter (such as F17R). In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; (3) inducing immune response in tumors; (4) reducing tumors Size; (5) reduce one or more symptoms of individuals with cancer; (6) inhibit tumor metastasis; (7) reduce the incidence or burden of preexisting tumor metastasis (such as lymph node metastasis); (8) prolong survival ; And / or (9) extend the time for cancer to progress. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer or liver cancer. In some embodiments, a method of treating an immunotherapy-responsive solid tumor of an individual (such as carcinoma or adenocarcinoma, such as cancer with abnormal CD47 / SIRPα and / or CXCL12 / CXCR4 performance, activity, and / or signaling) is provided , Which comprises administering an effective amount of a pharmaceutical composition comprising an oncolytic virus (such as oncolytic VV) and optionally pharmaceutically acceptable carriers to the individual, the oncolytic virus comprising: an immune checkpoint regulator (such as Any of the immune checkpoint regulators described herein, such as PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT , VISTA, B7-H4, CD160, 2B4 or CD73 inhibitor); and a second nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific Sex molecules include the first antigen-binding domain (such as scFv) that specifically recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and the cell surface molecule (such as CD3 on T lymphocytes) that specifically recognizes effector cells. A second antigen binding domain (such as scFv). In some embodiments, a method of treating an immunotherapy-responsive solid tumor of an individual (such as carcinoma or adenocarcinoma, such as cancer with abnormal CD47 / SIRPα and / or CXCL12 / CXCR4 performance, activity, and / or signaling) is provided , Which comprises administering an effective amount of a pharmaceutical composition to an individual, the pharmaceutical composition comprising: comprising an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as PD-1, PD- Inhibitors of L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73) A first OV (eg oncolytic VV); and a second OV (eg oncolytic VV) containing a second nucleic acid encoding a bispecific molecule (such as any of the bispecific molecules described herein), the bispecific Sex molecules include the first antigen-binding domain (such as scFv) that specifically recognizes tumor antigens (such as EpCAM, FAP, EGFR, or GPC3) and the cell surface molecule (such as CD3 on T lymphocytes) that specifically recognizes effector cells. Second antigen binding domain (such as scFv); and as appropriate A pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a third OV encoding a cytokine (such as any of the cytokines described herein, such as GM-CSF). In some embodiments, a method of treating an immunotherapy-responsive solid tumor of an individual (such as carcinoma or adenocarcinoma, such as cancer with abnormal CD47 / SIRPα and / or CXCL12 / CXCR4 performance, activity, and / or signaling) is provided , Which comprises administering to an individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising an immune checkpoint regulator (such as any of the immune checkpoint regulators described herein, such as PD- 1.Inhibitors of PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73) The first OV of the first nucleic acid (e.g. oncolytic VV), and optionally a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition including The second OV (eg oncolytic VV) of the second nucleic acid of the molecule (such as any of the bispecific molecules described herein), and optionally a pharmaceutically acceptable second carrier, the bispecific molecule Contains the specific recognition of tumor antigens (such as EpCAM, FAP, EGFR or GPC3) Antigen-binding domain (such as a scFv), and a cell surface molecule on the effector cells that specifically recognize an antigen of a second (such as a-CD3 on the T lymphocytes) binding domain (such as a scFv). In some embodiments, the method further comprises administering to the individual an effective amount of a third pharmaceutical composition, the third pharmaceutical composition comprising an encoded cytokine (such as any of the cytokines described herein, such as GM -CSF) and optionally a pharmaceutically acceptable third carrier. In some embodiments, the immune checkpoint regulator comprises a SIRPα extracellular domain and a fusion of a CXCL12 fragment and an immunoglobulin Fc fragment (such as IgG4 Fc). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; (3) inducing immune response in tumors; (4) reducing tumors Size; (5) reduce one or more symptoms of individuals with cancer; (6) inhibit tumor metastasis; (7) reduce the incidence or burden of preexisting tumor metastasis (such as lymph node metastasis); (8) prolong survival ; (9) prolong the time for cancer progression; (10) increase T cell tumor infiltration; and / or (11) enhance any of the mentioned effects. For example, the co-expression of SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein can enhance the killing effect of tumor cells that activate T cells in the presence of co-expressed bispecific conjugate molecules, and increase the production of T cells. , Or eliminate or reduce the tumor evasion caused by inhibitory immune checkpoint molecules expressed by tumor cells. In some embodiments, the immunotherapy-responsive solid tumor is breast cancer or liver cancer. In some embodiments, the cancer line is, for example, EpCAM positive, FAP positive, EGFR positive, or GPC3 positive. In some embodiments, the cancer is positive for any of the tumor-associated antigens or tumor-specific antigens listed herein, for example, displayed on its cell surface. In some embodiments, the methods described herein are suitable for treating cancers that overexpress EpCAM on the surface of cancer cells, such as EpCAM-positive solid cancers. For example, EpCAM expressed by cancer cells in an individual (such as a human) may be at least about any of normal cells that exceeds 2, 5, 10, 20, 50, 100, 200, 500, 1000 times, or higher multiples . EpCAM-positive solid cancer can be carcinoma or adenocarcinoma. EpCAM positive solid cancers include (but are not limited to) small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer (such as melanoma), kidney cancer, bladder cancer, thyroid cancer, prostate Cancer, ovarian cancer, breast cancer, cholangiocarcinoma and head and neck cancer. In some embodiments, the methods described herein are suitable for treating cancers that overexpress FAP on tumor cells or tumor stromal fibroblasts, such as FAP positive solid cancers. For example, tumor stromal fibroblasts in individuals (such as humans) exhibit a FAP that is at least about 2, 5, 10, 20, 50, 100, 200, 500, 1000 times, or higher than normal fibroblasts. Any of them. FAP-positive solid cancer can be carcinoma or adenocarcinoma. In some embodiments, the FAP positive solid cancer is selected from the group consisting of colorectal cancer, breast cancer, brain cancer, lung cancer, and skin cancer (such as melanoma). In some embodiments, the methods described herein are suitable for treating cancers that overexpress EGFR on the surface of cancer cells, such as EGFR positive solid cancers. For example, cancer cells in individuals (such as humans) exhibit EGFR that is at least about any of normal cells that exceeds 2, 5, 10, 20, 50, 100, 200, 500, 1000 times, or higher. The EGFR positive solid cancer may be carcinoma or adenocarcinoma. EGFR-mediated cancers include (but are not limited to) glioblastoma, head and neck cancer, pancreatic cancer, lung cancer, nervous system cancer, gastrointestinal cancer, prostate cancer, ovarian cancer, breast cancer, kidney cancer, retinal cancer, skin cancer, Liver cancer, urogenital cancer and bladder cancer. In some embodiments, the methods described herein are suitable for treating cancers that overexpress GPC3 on the surface of cancer cells, such as GPC3-positive solid cancers. For example, GPC3 expressed by cancer cells in an individual (such as a human) may be at least about any of normal cells that exceeds 2, 5, 10, 20, 50, 100, 200, 500, 1000 times, or higher multiples . The GPC3-positive solid cancer may be, for example, squamous cell carcinoma or hepatocellular carcinoma (HCC). In some embodiments, the methods described herein are suitable for treating colorectal cancer, such as adenocarcinoma, gastrointestinal carcinoid, gastrointestinal stromal tumor, leiomyosarcoma, melanoma, or squamous cell carcinoma. In some embodiments, the methods described herein are suitable for the treatment of liver cancer, such as hepatocellular carcinoma, fibrous lamellar hepatocellular carcinoma, or mixed hepatocellular cholangiocarcinoma. In some embodiments, the methods described herein are suitable for the treatment of breast cancer, such as early breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, remission breast cancer, breast cancer in adjuvant therapy or Breast cancer in neoadjuvant therapy. In some embodiments, the breast cancer is fibroadenoma or intraductal papilloma. In some embodiments, the breast cancer line is HER2 positive or HER2 negative. In some specific examples, the breast cancer is triple negative breast cancer. The effective amount of the virus (such as VV) used in the pharmaceutical composition described herein will depend on a variety of circumstances, such as the purpose of introduction, the specific type and stage of the cancer being treated, the regimen used (e.g. the number of doses and Approach), the stability of the virus, the activity of the encoded bispecific molecules and immune checkpoint regulators, and similar situations. The dosage regimen of the pharmaceutical composition described herein will be determined by the attending physician and clinical factors. As is well-known in medical technology, for any patient, the dose depends on many factors, including the patient's physique, body surface area, age, specific compound to be administered, gender, time and route of administration, general health status, and simultaneous administration Of other drugs. In some embodiments, the effective amount of the pharmaceutical composition described herein is lower than the amount that induces toxicological effects (ie, effects that exceed clinically acceptable toxicity levels) or when the pharmaceutical composition is administered to an individual The amount of potential side effects can be controlled or tolerated. In some embodiments, an effective amount of an oncolytic virus (such as an oncolytic VV) in the pharmaceutical composition described herein is about 105 Up to about 1013 pfu, including, for example, any of the following: about 105 Up to about 1012 pfu, about 106 Up to about 1013 pfu, about 106 Up to about 1012 pfu, about 107 Up to about 1013 pfu, about 107 Up to about 1012 pfu, about 107 Up to about 1011 pfu, about 107 Up to about 1010 pfu, about 107 Up to about 109 pfu, about 108 Up to about 1013 pfu, about 108 Up to about 1012 pfu, about 108 Up to about 1011 pfu, about 108 Up to about 1010 pfu, about 108 Up to about 109 pfu, about 109 Up to about 1013 pfu, about 109 Up to about 1012 pfu, about 109 Up to about 1011 pfu, about 109 Up to about 1010 pfu, about 1010 Up to about 1013 pfu, about 1011 Up to about 1013 pfu, or about 1012 Up to about 1013 pfu. In some embodiments, the effective amount of OV (such as oncolytic VV) in the pharmaceutical composition described herein is about 1013 pfu, about 1012 pfu, about 1011 pfu, about 1010 pfu, about 109 pfu, about 108 pfu, about 107 pfu, about 106 pfu or about 105 pfu. In some embodiments, the effective amount of OV (such as oncolytic VV) in the pharmaceutical composition described herein is about 105 Up to about 1013 pfu. In some embodiments, the effective amount of OV (such as oncolytic VV) in the pharmaceutical composition described herein is about 107 Up to about 109 pfu. In some embodiments, the effective amount of OV (such as oncolytic VV) in the pharmaceutical composition described herein is about 109 pfu. In some embodiments, the pharmaceutical composition is administered at one time (eg, bolus injection). In some embodiments, the pharmaceutical composition is administered multiple times (such as any of 2, 3, 4, 5, 6, or more times). If the drug is administered multiple times, it can be performed by the same or different routes and can be performed at the same site or at an alternative site. The pharmaceutical composition can be twice a week, 3 times a week, 4 times a week, 5 times a week, once a day, once a day uninterrupted, once a week, once a week uninterrupted, once every 2 weeks, every 3 Once a week, once a month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, every Once every 9 months, once every 10 months, once every 11 months or once a year. The time interval between administrations can be about 24 hours to 48 hours, 2 days to 3 days, 3 days to 5 days, 5 days to 1 week, 1 week to 2 weeks, 2 weeks to 1 month, 1 month to 2 Any one of month, 2 months to 3 months, 3 months to 6 months or 6 months to one year. The time interval can also be irregular (e.g. following tumor progression). In some embodiments, there is no break in the time course of administration. In some embodiments, the pharmaceutical composition comprising the oncolytic virus described herein may be in the range of about 105 pfu to about 1013 pfu range (such as about 107 pfu to about 109 pfu, or about 109 pfu) is administered once or several times (eg 2, 3, 4, 5, 6, 7, or 8 times, etc.). The time interval between each administration can vary from about 1 day to about 8 weeks, from about 2 days to about 6 weeks, from about 3 days to about 4 weeks, from about 1 week to about 3 weeks, or every two weeks. In some embodiments, a pharmaceutical composition containing an oncolytic virus described herein (such as an oncolytic VV) is administered intravenously or intratumorally at a time interval of about 1 or 2 weeks at about 105 pfu to about 1013 pfu range (such as about 107 pfu to about 109 pfu, or about 109 pfu) 2 to 5 times (for example 3 times). Those skilled in medicine can easily determine the optimal dose and treatment plan for a specific patient by monitoring the patient's disease symptoms and adjusting the treatment accordingly. The pharmaceutical compositions described herein may be suitable for multiple modes of administration, including, for example, systemic or local administration. In some embodiments, the pharmaceutical composition is administered parenterally, transdermally (in the dermis), intraductally, intraarterially (in the artery), intramuscularly (in the muscle), intrathecally, or intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously (under the skin). In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition described herein is administered to the individual via infusion or injection. In some embodiments, the pharmaceutical composition is injected directly into the tumor site (intratumor, ie directly into or near the tumor). Pharmaceutical compositions comprising oncolytic virus vectors encoding bispecific molecules described herein and pharmaceutically acceptable carriers are also encompassed by the present invention. Oncolytic virus vectors can be delivered directly to the target site, for example, by gene gun delivery to an internal or external target site or via a catheter to a site in the artery. Administration can use conventional syringes and needles (eg Quadrafuse injection needles) or any compound or device available in the art that can facilitate or improve the delivery of the active agent in the subject. In some embodiments, a systemic mammal to be treated. Examples of mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cattle, and similar mammals. In some embodiments, each system is human. IV. Antibodies Various forms of this application use antibodies. The present application provides novel antibodies, antibody fragments or antigen-binding fragments thereof. These antibodies can be used independently, or can be incorporated into any of the OV (such as oncolytic VV) or oncolytic virus vectors (such as oncolytic VV vector) described herein. The term "antibody" is used in the broadest sense and covers various antibody structures, including (but not limited to) monoclonal antibodies, multiple antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, as long as they display the desired antigen Just combine the activity. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. The IgM antibody is composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contains 10 antigen binding sites, while the IgA antibody contains 2 to 5 basic 4 chain units, which can be polymerized to Combine with J chain to form multivalent clusters. In the case of IgG, the 4-chain unit is generally about 150,000 Daltons. Each L chain is connected to the H chain via a covalent disulfide bond, and depending on the H chain isotype, the two H chains are connected to each other via one or more disulfide bonds. The H chain and the L chain each also have regularly spaced in-chain disulfide bridges. Each H chain has a variable domain (VH ), For each of the α and γ chains there are then three constant domains (CH ) And four C for the μ and ε isotypesH Domain. Each L chain has a variable domain (VL ), Followed by a constant domain at the other end. VL With VH Aligned, and CL With heavy chain (CH 1) The first constant domain is aligned. Xianxin specific amino acid residues form an interface between the light chain and heavy chain variable domains. VH And VL Pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see for example,Basic and Clinical Immunology , 8th edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, Conn, 1994, page 71 and chapter 6. The L chain from any vertebrate species can be assigned to one of two distinct types called κ and λ based on the amino acid sequence of its constant domain. Depending on the constant domain of the heavy chain of the immunoglobulin (CH ) Of the amino acid sequence, immunoglobulin can be assigned to different classes or isotypes. There are 5 classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, which have heavy chains designated α, δ, ε, γ, and μ, respectively. Based on CH Relatively small differences in sequence and function, the γ and α categories are further divided into subclasses, for example, humans exhibit the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2. A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or prepared using any of the human antibody preparation techniques disclosed herein. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues. Human antibodies can be manufactured using various techniques known in the art, including phage display libraries. Hoogenboom and Winter,J. Mol. Biol., 227: 381 (1991); Marks et al.,J. Mol. Biol., 222: 581 (1991). In addition, methods that can be used to prepare human monoclonal antibodies are described in Cole et al.,Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, page 77 (1985); Boerner et al.,J. Immunol., 147 (1): 86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering antigens to transgenic animals that have been modified to produce such antibodies in response to antigen challenge but have endogenous loci disabled, such as immunized xenomice (about XENOMOUSE ™ technology (See, for example, US Patent Nos. 6,075,181 and 6,150,584). For human antibodies produced by human B cell fusion tumor technology, see also Li et al.,Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as from a host cell such as NS0 or CHO cells or from the transfer of human immunoglobulin genes An antibody isolated from a genetic animal (eg, a mouse), or an antibody expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in rearranged form. The recombinant human antibody according to the present invention has undergone somatic hypermutation in vivo. Therefore, the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the human reproductive VH and VL sequences, but may not be sequences that naturally exist in the human antibody reproductive lineage in vivo. "Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, the humanized anti-body system is a human immunoglobulin (recipient antibody) in which residues from the recipient's HVR (defined below) are derived from those with the desired specificity, affinity, and / or ability Non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate HVR residue replacement. In some cases, human immunoglobulin framework ("FR") residues are replaced with corresponding non-human residues. In addition, the humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications can be made to further optimize antibody performance, such as binding affinity. Generally speaking, a humanized antibody will comprise at least one and generally two variable domains of substantially all of which all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin sequences, and all or substantially All the above FR regions are FR regions of human immunoglobulin sequences, but these FR regions may include one or more individual FR residue substitutions that improve antibody performance (such as binding affinity, isomerization, immunogenicity, etc.). The number of such amino acid substitutions in FR is usually not more than 6 in the H chain and not more than 3 in the L chain. The humanized antibody will also optionally contain an immunoglobulin constant region (Fc), usually at least a part of the human immunoglobulin constant region. For more details, see for example, Jones et al.,Nature 321: 522-525 (1986); Riechmann et al.,Nature 332: 323-329 (1988); and Presta,Curr. Op. Struct. Biol. 2: 593-596 (1992). See also, for example, Vaswani and Hamilton,Ann. Allergy, Asthma &Immunol. 1: 105-115 (1998); Harris,Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5: 428-433 (1994); and US Patent Nos. 6,982,321 and 7,087,409. The term "chimeric" anti-system refers to antibodies that are usually prepared using recombinant DNA technology, where part of the heavy chain and / or light chain is derived from a specific source or species, and the rest of the heavy chain and / or light chain is derived from different Source or species. Chimeric antibodies can include murine variable regions and human constant regions. A "chimeric antibody" may also be an antibody whose constant region has been modified or altered from the original antibody to produce the characteristics according to the invention, especially C1q binding and / or Fc receptor (FcR) binding. Such chimeric antibodies are also called "class-switching antibodies." The chimeric anti-system comprises the expression products of immunoglobulin genes of DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques well known in the art. See, for example, Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Patent Nos. 5,202,238 and 5,204,244. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies that make up the population, except for possible naturally occurring mutations and / or post-translational modifications that may exist in small amounts (For example, isomerization, amidation) The rest are the same. Monoclonal antibodies are highly specific and target a single antigenic site. In contrast to multiple antibody preparations that usually include different antibodies against different determinants (antigenic determinants), each monoclonal antibody system is directed against a single determinant on the antigen. In addition to specificity, monoclonal antibodies are also advantageous in that they are synthesized by fusion tumor cultures and are not contaminated with other immunoglobulins. The modifier "single plant" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and should not be understood as requiring the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including, for example, fusion tumor methods (such as Kohler and Milstein.,Nature, 256: 495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995); Harlow et al.,Antibodies: A Laboratory Manual , (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al.,Monoclonal Antibodies and T -Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, eg, US Patent No. 4,816,567), phage display technology (see, eg, Clackson et al.,Nature, 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Sidhu et al.,J. Mol. Biol. 338 (2): 299-310 (2004); Lee et al.,J. Mol. Biol. 340 (5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284 (1-2): 119-132 (2004), and techniques for producing human or human-like antibodies in animals with part or all of human immunoglobulin loci or genes encoding human immunoglobulin sequences (see For example, WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al.,Nature 362: 255-258 (1993); Bruggemann et al.,Year in Immunol. 7:33 (1993); US Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al.,Bio / Technology 10: 779-783 (1992); Lonberg et al.,Nature 368: 856-859 (1994); Morrison,Nature 368: 812-813 (1994); Fishwild et al.,Nature Biotechnol. 14: 845-851 (1996); Neuberger,Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13: 65-93 (1995). The terms "full-length antibody", "intact antibody" or "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, as opposed to an antibody fragment. Specifically, full-length 4-chain antibodies include those having heavy and light chains, including the Fc region. The constant domain may be a natural sequence constant domain (for example, a human natural sequence constant domain) or an amino acid sequence variant thereof. In some cases, intact antibodies may have one or more effector functions. "Antibody fragment" includes a part of an intact antibody, preferably the antigen binding region and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')2 And Fv fragments; bifunctional antibodies; linear antibodies (see US Patent No. 5,641,870, Example 2; Zapata et al.,Protein Eng. 8 (10): 1057-1062 [1995]); single chain antibody molecules and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments; and residual "Fc" fragments, the name reflects the ability to crystallize easily. The Fab fragment consists of the complete L chain and the variable region domain of the H chain (VH ) And the first constant domain of a heavy chain (CH 1) Composition. Each Fab fragment is monovalent in terms of antigen binding, that is, it has a single antigen binding site. Pepsin treatment of antibodies produces a single larger F (ab ')2 A fragment, which roughly corresponds to two Fab fragments with different antigen binding activities connected by disulfide bonds and still capable of cross-linking antigens. The difference between Fab 'fragment and Fab fragment is that Fab' fragment is in CH 1 The carboxy terminus of the domain has several additional residues, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the name of the Fab 'carrying free thiol groups for the cysteine residues of the constant domain. F (ab ')2 Antibody fragments were originally produced as a pair of Fab 'fragments with hinged cysteines in between. Other chemical couplings of antibody fragments are also known. The term "constant domain" refers to a portion of an immunoglobulin molecule that has an amino acid sequence that is conserved other than an immunoglobulin that contains an antigen binding site, ie, a variable domain. The constant domain contains the heavy chain CH 1. CH 2 and CH 3 domain (collectively referred to as CH) and the CHL (or CL) domain of the light chain. The "light chain" of an antibody (immunoglobulin) from any mammalian species can be designated as one of two distinct types called κ and λ based on the amino acid sequence of its constant domain. The term "bifunctional antibody" refers to antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable structure linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL) Domain (VH). By using a linker that is too short to match between the two domains on the same chain, these domains are forced to pair with the complementary domains of the other chain, and two antigen binding sites are created. Bifunctional antibodies can be bivalent or bispecific. Bifunctional antibodies are more fully described in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Trifunctional antibodies and tetrafunctional antibodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003). The "Fc" fragment contains the carboxy-terminal portions of two H chains held together by disulfide bonds. The effector function of an antibody is determined by the sequence in the Fc region, which is also recognized by the Fc receptor (FcR) found on certain types of cells. "Fv" is the smallest antibody fragment that contains complete antigen recognition and antigen binding sites. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain that are tightly and non-covalently associated. The folding of these two domains results in six hypervariable loops (3 loops each from the H chain and the L chain) that provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even if a single variable domain (or only half of the Fv containing three HVRs specific for an antigen) can recognize and bind the antigen, its affinity is lower than that of the complete binding site. "Single-chain Fv" is also abbreviated as "sFv" or "scFv", which includes V linked to a single polypeptide chainH And VL Antibody fragments of antibody domains. In some embodiments, the scFv polypeptide is at VH With VL There are further polypeptide linkers between the domains, which enable the scFv to form the structure required for antigen binding. scFv is known in the art, see for example Pluckthun,The Pharmacology of Monoclonal Antibodies , Volume 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pages 269-315 (1994). The "variable region" or "variable domain" of an antibody refers to the amine terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains can be called "VH "And" VL ". These domains are generally the most variable part of antibodies (relative to other antibodies of the same kind) and contain antigen binding sites. The term "variable" refers to the fact that the sequences of certain segments of the variable domain vary widely among antibodies. The V domain mediates antigen binding and determines the specificity of a specific antibody for its specific antigen. However, the variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVR) in the light chain variable domain and the heavy chain variable domain. The more conserved part of the variable domain is called the framework region (FR). The variable domains of the natural heavy chain and the light chain each include four FR regions, which are connected by three HVRs, generally in a β-sheet configuration, and these HVRs form a loop connecting the β-sheet structure And in some cases form part of the β-sheet structure. The HVRs in each chain are held together by the FR region and are in close proximity to HVRs from other chains, thereby promoting the formation of antibody antigen binding sites (see Kabat et al.,Sequences of Immunological Interest , Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in the binding of antibody to antigen, but exhibits various effector functions, such as the participation of antibody in antibody-dependent cytotoxicity. As used herein, the term "hypervariable region", "HVR", or "HV" refers to the region of the variable domain of an antibody that has a highly variable sequence and / or forms a structured loop. Generally speaking, a 4-chain antibody contains six HVRs; three in VH (H1, H2, H3), and three in VL (L1, L2, L3). Among the natural 4-chain antibodies, H3 and L3 display most of the diversity of these six HVRs, and Xianxin especially H3 plays a unique role in conferring fine specificity on antibodies. See for example, Xu et al.,Immunity 13: 37-45 (2000); Johnson and Wu,Methods in Molecular Biology 248: 1-25 (Ed. Lo, Human Press, Totowa, N.J., 2003). In fact, naturally-occurring camel antibodies composed only of heavy chains are also functional and stable in the absence of light chains. See for example, Hamers-Casterman et al.,Nature 363: 446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3: 733-736 (1996). Various HVR descriptions have been used and are covered herein. The Kabat `` complementarity determining region '' (or `` CDR '') is based on sequence variability and is the most commonly used (Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia mentioned the position of the structural ring (Chothia and Lesk,J. Mol. Biol. 196: 901-917 (1987)). AbM HVR represents a compromise between Kabat HVR and Chothia structural loop, and is used by Oxford Molecular's AbM antibody simulation software. "Contact" HVR is based on the analysis of available complex crystal structures. The residues from each of these HVRs are described in Table 1 below. Table 1. HVR description. HVR can include the following "Extended HVR": VL Of 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3), and VH Among them, 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3). Variable domain residues are numbered according to each of these definitions in Kabat et al. (Same as above). The expression "numbering of variable domain residues as in Kabat" or "numbering of amino acid positions as in Kabat" and variations thereof refer to the variable structure of the heavy chain used for antibody compilation in Kabat et al. (Same as above) Numbering system for the domain or light chain variable domain. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or HVR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (based on residue 52a of Kabat) and a residue inserted after residue 82 of heavy chain FR (eg according to Kabat Residues 82a, 82b, 82c, etc.). For a given antibody, the Kabat numbering of residues can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbering sequence. "Framework" or "FR" residues are variable domain residues other than HVR residues as defined herein. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, HVR and FR sequences generally appear in VH (or VL) in the following order: FR1-H1 (L1) -FR2-H2 (L2) -FR3-H3 (L3) -FR4. Unless otherwise indicated herein, the numbering of residues in the immunoglobulin heavy chain is the numbering of the EU index as in Kabat et al. (Same as above). "EU index in Kabat" refers to the residue number of human IgG1 EU antibody. As used herein, the terms "binding", "specific binding to", "specific recognition" or "specificity" refer to determining the target in the presence of a group of heterogeneous molecules (including biomolecules) Measurable and reproducible interactions of the presence of the target, such as the binding between the target and antibody. For example, the affinity, affinity, ease, and / or duration of binding to, or specifically recognizing, or specifically binding to a target (which may be an epitope) of the anti-body binding to the target is stronger than the binding Antibodies to other targets. In some embodiments, as measured by, for example, radioimmunoassay (RIA), the degree of binding of the antibody to the unrelated target is about 10% lower than the binding of the antibody to the target. In some embodiments, the antibody specifically binds to an epitope on the protein, which epitope is conserved among proteins from different species. In another embodiment, specific binding may include exclusive binding, but it is not required. As used herein, "percent amino acid sequence identity (%)" and "homology" with respect to peptide, polypeptide, or antibody sequences are defined as after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage , And does not consider any conservative substitution as part of the sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific peptide or polypeptide sequence. Alignment for the purpose of determining the percent amino acid sequence identity can be achieved by various means within the skill of the technology, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN ™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences. In some embodiments, an antibody, antibody fragment or antigen-binding domain that specifically binds to PD-1 (hereinafter also referred to as "anti-PD-1 antibody", "anti-PD-1 antibody fragment", "PD -1 binding fragment "or" PD-1 binding domain "). The two exemplary anti-PD-1 antibodies or antigen-binding fragments thereof are 1H7e3 and 4F11C3. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as a full-length antibody or scFv) includes a heavy chain variable region (VH) containing one, two, or three HVRs from SEQ ID NO: 13, And / or containing the light chain variable region (VL) from one, two or three HVRs of SEQ ID NO: 14. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as a full-length antibody or scFv) includes a heavy chain variable region (VH) containing three HVRs from SEQ ID NO: 13, and / or contains The light chain variable region (VL) of the three HVRs of SEQ ID NO: 14. In some embodiments, the anti-PD-1 antibody includes an antigen binding domain (such as scFv), which comprises: a heavy chain variable region (VH), the heavy chain variable region comprising an amino acid sequence SEQ ID NO: 1 HVR-H1; HVR-H2 containing amino acid sequence SEQ ID NO: 2; and HVR-H3 containing amino acid sequence SEQ ID NO: 3; and / or light chain variable region (VL), the light The chain variable region includes HVR-L1 containing the amino acid sequence SEQ ID NO: 4; HVR-L2 containing the amino acid sequence SEQ ID NO: 5; and HVR-L3 containing the amino acid sequence SEQ ID NO: 6 . In some embodiments, the anti-PD-1 antibody comprises an antigen-binding domain (such as scFv), which comprises: comprising at least about 85%, at least about 86%, at least about 87% of the amino acid sequence SEQ ID NO: 13; At least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, A heavy chain variable region (VH) of an amino acid sequence that is at least about 98% or at least about 99% identical; and / or contains at least about 85%, at least about 86%, at least About 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least The light chain variable region (VL) of about 97%, at least about 98%, or at least about 99% identical amino acid sequences. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as scFv) includes the heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 13, and / or the amino acid sequence SEQ ID NO: 14 is the light chain variable region (VL). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as a full-length antibody or scFv) includes a heavy chain variable region (VH) containing one, two, or three HVRs from SEQ ID NO: 15, And / or containing the light chain variable region (VL) from one, two or three HVRs of SEQ ID NO: 16. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as a full-length antibody or scFv) includes a heavy chain variable region (VH) containing three HVRs from SEQ ID NO: 15, and / or contains The light chain variable region (VL) of the three HVRs of SEQ ID NO: 16. In some embodiments, the anti-PD-1 antibody includes an antigen binding domain (such as scFv), which comprises: a heavy chain variable region (VH), the heavy chain variable region comprising an amino acid sequence SEQ ID NO: 7 HVR-H1; HVR-H2 containing amino acid sequence SEQ ID NO: 8; and HVR-H3 containing amino acid sequence SEQ ID NO: 9; and / or light chain variable region (VL), the light The chain variable region includes HVR-L1 containing the amino acid sequence SEQ ID NO: 10; HVR-L2 containing the amino acid sequence SEQ ID NO: 11; and HVR-L3 containing the amino acid sequence SEQ ID NO: 12 . In some embodiments, the anti-PD-1 antibody comprises an antigen-binding domain (such as scFv), which includes: comprising at least about 85%, at least about 86%, at least about 87% of the amino acid sequence SEQ ID NO: 15; At least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, A heavy chain variable region (VH) of an amino acid sequence that is at least about 98% or at least about 99% identical; and / or includes at least about 85%, at least about 86%, at least About 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least The light chain variable region (VL) of about 97%, at least about 98%, or at least about 99% identical amino acid sequences. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof (such as scFv) includes the heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 15, and / or the amino acid sequence SEQ ID NO: 16 is the light chain variable region (VL). In some embodiments, the anti-PD-1 anti-system full-length antibody. In some embodiments, the full-length anti-PD-1 antibody comprises Fc sequences from immunoglobulins, such as IgA, IgD, IgE, IgG, and IgM. In some embodiments, the full-length anti-PD-1 antibody comprises the Fc sequence of any IgG, such as any of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the full-length anti-PD-1 antibody comprises the Fc sequence of human immunoglobulin. In some embodiments, the full-length anti-PD-1 antibody comprises an Fc sequence that has been altered or otherwise altered to have an enhanced antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) effector function. Also provided is an isolated antibody or antigen-binding fragment thereof that competes with any of the anti-PD-1 antibodies described herein for binding to PD-1. In some embodiments, an isolated antibody or antigen-binding fragment thereof that binds to the same epitope as any of the anti-PD-1 antibodies described herein is provided. Methods for screening antibodies with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immune-mediated techniques known in the art. Those skilled in the art should recognize that, without undue experimentation, it is possible to determine whether a monoclonal antibody prevents the monoclonal antibody of the invention (for example, having a variable heavy chain containing the amino acid sequence SEQ ID NO: 13 and / or Anti-PD-1 antibody containing the variable light chain of the amino acid sequence SEQ ID NO: 14, or the variable heavy chain having the amino acid sequence SEQ ID NO: 15 and / or the amino acid sequence SEQ ID NO : Anti-PD-1 antibody with variable light chain of 16) bound to PD-1 to determine whether the monoclonal antibody has the same specificity as the antibody of the present invention. If the test monoclonal antibody competes with the monoclonal antibody of the present invention as shown by the reduced binding of the monoclonal antibody of the present invention, the two monoclonal antibodies bind to the same epitope or closely related epitopes. An alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody of the present invention is to pre-incubate the monoclonal antibody of the present invention with a soluble PD-1 protein (a protein that is usually reactive with the monoclonal antibody), And then add a test monoclonal antibody to determine whether the test monoclonal antibody's ability to bind PD-1 is inhibited. If the test monoclonal antibody is inhibited, it is highly likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. In some embodiments, the anti-PD-1 anti-system monoclonal antibody, such as a monovalent antibody. In some embodiments, the anti-PD-1 anti-system full-length antibody. In some embodiments, the anti-PD-1 antigen binding fragment is Fab, Fab ', F (ab')2 , Single chain Fv (scFv), Fv fragment, bifunctional antibody or linear antibody form. In some embodiments, the anti-PD-1 anti-system binds to PD-1 and also binds to one or more other targets and optionally multispecific antibodies that inhibit its function. A multispecific antibody system is a single, preferably human or humanized antibody that has binding specificity for two or more different antigens (eg, a bispecific antibody that has binding specificity for at least two antigens). In some embodiments, the anti-PD-1 anti-system bispecific molecule, wherein the bispecific molecule further comprises a molecule that specifically recognizes another inhibitory immune checkpoint described herein (eg, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73) second antigen-binding fragment. In some embodiments, the anti-PD-1 anti-system bispecific molecule, wherein the bispecific molecule further comprises specific recognition and activation of the stimulatory immune checkpoint molecules described herein (eg, OX40L, CD80, CD86, B7RP1 4-1BBL, Ultra 4-1BBL, CD70, CD40L, or Class I or II MHC molecules, IMCgp100) second antigen-binding fragment. In some embodiments, multispecific anti-PD-1 molecular systems such as bifunctional antibodies (Db), single chain bifunctional antibodies (scDb), tandem scDb (Tandab), linear dimerized scDb (LD-scDb), circular dimers Poly-scDb (CD-scDb), bi-bifunctional antibody, tandem scFv, tandem two scFv (e.g. bispecific T cell adaptor), tandem tri scFv, trifunctional antibody, bispecific Fab2 , Two minibodies, four-functional antibodies, scFv-Fc-scFv fusions, dual affinity retargeting (DART) antibodies, dual variable domain (DVD) antibodies, IgG-scFab, scFab-ds-scFv, Fv2-Fc , IgG-scFv fusion, docking locking (DNL) antibody, well-knob-into-hole (KiH) antibody (bispecific IgG prepared by KiH technology), DuoBody (bispecific prepared by Duobody technology) IgG), heteromultimeric antibodies or heteroconjugate antibodies. In some embodiments, the multispecific anti-PD-1 molecule is a tandem scFv (eg, a tandem two scFv, such as a bispecific T cell zygote). Further provided are fusion proteins, conjugates, or isolated cells containing any of the above-mentioned anti-PD-1 antibodies or antigen-binding fragments thereof. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof is bound to a therapeutic agent (eg, cytotoxic agent, radioisotope, and chemotherapeutic agent) or detects PD in a patient sample or in vivo by imaging -1 label (such as radioisotopes, fluorescent dyes and enzymes). In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof binds to the toxin. The anti-PD-1 antibodies or antigen-binding fragments described herein can be used in a variety of therapeutic and diagnostic methods. Further provided is a method of treating cancer in an individual, which comprises administering to the individual an effective amount of the above-mentioned anti-PD-1 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof. For example, anti-PD-1 antibodies (or antigen-binding fragments thereof) can be used alone or in combination with other agents to treat diseases characterized by abnormal PD-L1 or PD-1 manifestations, or cancers that respond to immunotherapy , Including (but not limited to) melanoma (eg, metastatic malignant melanoma), kidney cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer, liver cancer, and lung cancer ( (For example, non-small cell lung cancer). The anti-PD-1 antibody or antigen-binding fragment of the present invention can also be used to treat metastatic cancer, especially metastatic cancer that exhibits PD-L1 (Iwai et al. (2005)Int. Immunol. 17: 133-144). The antibodies provided herein can also be used to detect PD-1 protein in patients or patient samples. Further provide an isolated nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof, an OV (such as an oncolytic VV) containing the nucleic acid encoding the anti-PD-1 antibody or antigen-binding fragment thereof, expressing the anti-PD-1 antibody or An isolated cell of an antigen-binding fragment thereof, a pharmaceutical composition containing any of the anti-PD-1 antibody or antigen-binding fragment thereof, an OV encoding it (such as oncolytic VV), a host cell expressing it, use this Method for treating cancer of an individual with a pharmaceutical-like composition. In some embodiments, the pharmaceutical composition is administered intravenously to the individual to be treated. In some embodiments, the pharmaceutical composition is administered intratumorally to the individual to be treated. In some embodiments, the system to be treated is human. Monoclonal antibodies The screening of monoclonal antibodies of the invention can also be performed, for example, by measuring PD-1 mediated signaling and determining whether the test monoclonal antibodies can regulate, block, inhibit, reduce, antagonize, neutralize or Other methods interfere with PD-1 mediated signal transduction. Such analysis methods may include competitive binding analysis methods. In addition, these analytical methods can measure biological readout. Monoclonal antibodies directed against PD-1 or against derivatives, fragments, analogs, homologs or orthologs thereof can be made using various procedures known in the art. See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference. The complete sequence of the light chain and heavy chain (including HVR) of the fully human anti-system is derived from the antibody molecule of human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Human monoclonal antibody systems are prepared, for example, using the procedures described in the examples provided below. Human monoclonal antibodies can also be produced by using three-source fusion tumor technology; human B-cell fusion tumor technology (see Kozbor et al., 1983 Immunol Today 4: 72); and EBV fusion tumor technology to produce human monoclonal antibodies (see Cole et al. People, 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pages 77-96). Human monoclonal antibodies can be utilized and can be used by using human fusion tumors (see Cote et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by using Epstein Barr Virus (Epstein Barr Virus) in vitro ) Transformed human B cells (see Cole et al., 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pages 77-96). Antibody systems are purified by well-known techniques, such as affinity chromatography using protein A or protein G, thereby mainly providing IgG dissociated fractions of immune serum. Subsequently or alternatively, the specific antigen or epitope that is the target of the immunoglobulin search can be fixed on the column to purify the immunospecific antibody by immunoaffinity chromatography. The purification of immunoglobulins is discussed in, for example, D. Wilkinson (The Scientist, The Scientist, Inc., Philadelphia PA, Volume 14, Issue 8 (April 17, 2000), pages 25-28). The PD-1 anti-system monoclonal antibody of the present invention. Monoclonal antibody systems that modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with PD-1 mediated cell signaling, for example, by using membrane-bound and / or soluble PD-1, such as human PD- 1 or its immunogenic fragments, derivatives or variants are produced by immunizing animals. Alternatively, animals are immunized with cells transfected with a vector containing a nucleic acid molecule encoding PD-1, whereby PD-1 is expressed and associated with the surface of the transfected cells. Alternatively, antibodies can be obtained by screening a library containing antibody- or antigen-binding domain sequences bound to PD-1. This library is prepared, for example, in the form of a fusion of a protein or peptide and a phage coat protein in a phage, which is expressed on the surface of the assembled phage particles and the coding DNA sequence is contained within the phage particles (ie, "phage display") library"). Next, fusion tumors produced by myeloma / B cell fusion were screened for reactivity with PD-1. Monoclonal antibody systems are prepared, for example, using fusion tumor methods, such as those described by Kohler and Milstein, Nature, 256: 495 (1975). In the fusion tumor method, mice, hamsters, or other suitable host animals are usually immunized with an immunizing agent to enable lymphocytes to produce or be able to produce antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. Immunological agents will generally include protein antigens, fragments thereof or fusion proteins thereof. In general, if cells of human origin are required, peripheral blood lymphocytes ("PBL") are used; or if cells of non-human mammal origin are required, spleen cells or lymph node cells are used. The lymphocytes are then fused with the immortalized cell line using suitable fusion agents (such as polyethylene glycol) to form fusion tumor cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pages 59-103). Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. Fused tumor cells can be cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of immortalized cells that are not fused. For example, if the parent cell lacks the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the medium used for fusion tumors will usually include hypoxanthine, aminopterin, and thymidine (" HAT medium ”), these substances prevent the growth of cells lacking HGPRT. Preferably, the immortalized cell lines are efficiently fused to support the selected antibody-producing cells to stably express the antibodies at a high level and are sensitive to media such as HAT medium. A better immortalized cell line is the murine myeloma cell line, which can be obtained from, for example, Salk Institute Cell Distribution Center (San Diego, California) and American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human hybrid myeloma cell lines for the production of human monoclonal antibodies have been described (see Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pages 51-63)). The media for culturing the fusion tumor cells can then be analyzed for the presence of monoclonal antibodies directed against the antigen. The binding specificity of the monoclonal antibodies produced by the fusion tumor cells is preferably determined by immunoprecipitation or by an in vitro binding analysis method such as radioimmunoassay (RIA) or enzyme-linked immunosorbent analysis (ELISA). These techniques and analytical methods are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980). In addition, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies that are highly specific for the target antigen. After identifying the desired fusion tumor cells, the pure lines can be sub-cloned by restrictive dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) page 59 -103 pages). Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, fusion tumor cells can grow in mammals in the form of ascites in vivo. Monoclonal antibodies secreted by sub-pure lines can be separated from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography Or purification. Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). After isolation, the DNA can be put into expression vectors, and then these expression vectors are transfected into host cells that do not additionally produce immunoglobulin proteins, such as Chinese hamster ovary (COS) cells, human embryonic kidney (HEK) 293 cells , Monkey COS cells, PER.C6®, NS0 cells, SP2 / 0, YB2 / 0 or myeloma cells to synthesize monoclonal antibodies in recombinant host cells. It is also possible, for example, by replacing homologous murine sequences with coding sequences of human heavy and light chain constant domains (see US Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by immunizing All or part of the globulin polypeptide coding sequence is covalently joined to the immunoglobulin coding sequence to modify the DNA. Such non-immunoglobulin polypeptides can replace the constant domain of the antibody, or can replace the variable domain of an antigen combination site of the antibody to produce a chimeric bivalent antibody. Human antibodies and humanization of antibodies The monoclonal antibodies of the present invention include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without causing human immune responses to the administered immunoglobulins. Anti-PD-1 antibodies can be produced using any procedure known in the art. For example, a modified multi-site repeated immunization (Repetitive Immunization Multiple Sites, RIMMS) immunization strategy can be used in mice and subsequent generation of fusion tumors to identify anti-PD-1 antibodies. In other alternative methods, such as using phage display methods, antibodies containing only human sequences are used to generate anti-PD-1 antibodies. Such methods are well known in the art, for example, see WO92 / 01047 and US Patent No. 6,521,404, which are incorporated herein by reference. In this method, PD-1 or fragments thereof of natural or recombinant origin are used to screen a combinatorial library of phage carrying any pair of light and heavy chains. In another method, an anti-PD-1 antibody can be produced by the following method, wherein at least one step of the procedure includes immunizing the transgenic non-human animal with human PD-1 protein. In this method, part of the endogenous heavy chain and / or kappa light chain locus of this heterologous non-human animal has been disabled and is unable to perform the rearrangements required to produce genes encoding immunoglobulins in response to antigens. In addition, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into animals. Therefore, in response to the administered antigen, the human loci are rearranged to provide genes encoding human variable regions that are immunospecific for the antigen. Therefore, after immunization, xenomouse produces B cells that secrete fully human immunoglobulins. Various techniques for producing heterogeneous non-human animals are well known in this technology. For example, see US Patent Nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety. This general strategy was confirmed by the creation of the first XenoMouse ™ strain released in 1994. See Green et al., Nature Genetics 7: 13-21 (1994), which is incorporated herein by reference in its entirety. See also US Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598; and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2; and Europe Patent No. EP 0 463 151 B1, and International Patent Application Nos. WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and related family members. In an alternative method, a "minilocus" method is used in which an exogenous Ig locus is simulated by including fragments (individual genes) from the Ig locus. Therefore, one or more VH genes, one or more DH Gene, one or more JH The gene, the μ constant region and the second constant region (preferably the γ constant region) form a construct for insertion into the animal. See, for example, U.S. Patent Nos. 5,545,806, 5,545,807, 5,591,669, 5,612,205, 5,625,825, 5,625,126, 5,633,425, 5,643,763, 5,661,016, 5,721,367, 5,770,429, No. 5,789,215, No. 5,789,650, No. 5,814,318, No. 5,877,397, No. 5,874,299, No. 6,023,010 and No. 6,255,458; and European Patent No. EP 0 546 073 B1; and International Patent Application No. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, article WO 97/13852 and WO 98/24884 and related family members. It has also been shown to produce human antibodies from mice in which larger chromosomal fragments or whole chromosomes are introduced via microcell fusion. See European Patent Application Nos. 773 288 and 843 961. The human anti-mouse antibody (HAMA) response has led the industry to prepare chimeric or other humanized antibodies. Although the chimeric antibody has a human constant region and an immune variable region, it is expected that certain human anti-chimeric antibody (HACA) reactions will be observed, especially when the antibody is used for long-term or multiple doses. Therefore, in order to reduce or otherwise alleviate the problems and / or effects of the HAMA or HACA response, it is necessary to provide fully human antibodies against PD-1. An appropriate library is also used to produce antibodies with reduced immunogenicity through humanization, chimerism, and display techniques. It should be understood that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See, for example, Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al., Crit, Reviews in Immunol. 12125-168 (1992). The antibody of interest can be engineered by recombinant DNA technology to replace CH1, CH2, CH3, hinge domains and / or framework domains with corresponding human sequences (see WO 92102190 and US Patent Nos. 5,530,101, 5,585,089, 5,693,761 No. 5,693,792, 5,714,350 and 5,777,085). In addition, Ig cDNA is used to construct chimeric immunoglobulin gene lines known in the art (Liu et al., P.N.A.S. 84: 3439 (1987) and J. Immunol. 139: 3521 (1987)). MRNA is isolated from fusion tumors or other cells producing the antibody and used to make cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (US Patent Nos. 4,683,195 and 4,683,202). Alternatively, a library is prepared and screened to isolate the sequence of interest. Next, the DNA sequence encoding the variable region of the antibody is fused to the human constant region sequence. The sequence of human constant region genes can be found in Kabat et al. (1991) Sequences of Proteins of immunological Interest, N.I.H. publication number 91-3242. Human C region genes are easily obtained from known pure lines. The choice of isotype will be guided by the desired effector function, such as complement binding or activity in antibody-dependent cytotoxicity. The preferred isotypes are IgG1, IgG2, IgG3 and IgG4. Either the human light chain constant region κ or λ can be used. Next, the chimeric humanized antibody is expressed using conventional methods. Antibody fragments, such as Fv, F (ab ')2 And Fab can be prepared by cleaving intact proteins, for example by protease or chemical cleavage. Or, design truncated genes. For example, encode a part of F (ab ')2 The chimeric gene of the fragment will include the DNA sequence encoding the H chain CH1 domain and the hinge region, followed by the translation stop codon, thereby obtaining a truncated molecule. The common sequence of the H region, the L region, and the J region can be used to design an oligonucleotide used as a primer to introduce useful restriction sites into the J region, and then connect the V region segment to the human C region segment. C-region cDNA can be modified by inducing site-directed mutations by placing restriction sites at similar positions in the human sequence. Expression vectors include plastids, retroviruses, YAC, EBV-derived episomes and the like. Suitable vectors are those that encode fully functional human CH or CL immunoglobulin sequences and have been engineered to allow for easy insertion and expression of appropriate restriction sites for any VH or VL sequences. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site before the human C region, and at the splice region that occurs within the exon of human CH. Polyadenylation and transcription termination occur at natural chromosomal sites downstream of the coding region. The resulting chimeric antibody can be conjugated to any strong promoter, including retroviral LTR, such as the SV-40 early promoter (Okayama et al., Mol. Cell. Bio. 3: 280 (1983)), Rous sarcoma virus LTR ( Gorman et al., PNAS 79: 6777 (1982)) and Moloney murine leukemia (moloney murine leukemia) virus LTR (Grosschedl et al., Cell 41: 885 (1985)). In addition, it should be understood that native Ig promoters and the like can be used. In addition, human antibodies or antibodies from other species can be produced using display technologies, including but not limited to phage display, retrovirus display, ribosome display, and other technologies, using techniques well known in the art, and the resulting molecules Additional maturity can be experienced, such as affinity maturation, and such techniques are also well known in the art. Wright et al, Crit, Reviews in Immunol. 12125-168 (1992); Hanes and Plückthun PNAS USA 94: 4937-4942 (1997) (ribosome display); Parmley and Smith Gene 73: 305-318 (1988) (phage display); Scott , TIBS, Vol. 17: 241-245 (1992); Cwirla et al., PNAS USA 87: 6378-6382 (1990); Russel et al., Nucl. Acids Research 21: 1081-1085 (1993); Hoganboom et al., Immunol. Reviews 130: 43-68 (1992); Chiswell and McCafferty TIBTECH; 10: 80-8A (1992); and US Patent No. 5,733,743. If display technology is used to generate non-human antibodies, such antibodies can be humanized as described above. Using these techniques, antibodies can be generated against PD-1 expressing cells, soluble forms of PD-1, their epitopes or peptides, and their expression libraries (see, eg, US Patent No. 5,703,057), which can then be described as above , Screening these performance libraries for the activity described herein. The anti-PD-1 antibody of the present invention can be expressed by a vector containing a DNA segment encoding the above single-chain antibody. Such carriers may include carriers, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, and the like. Vectors include chemical conjugates with targeting moieties (such as ligands for cell surface receptors) and nucleic acid binding moieties (such as polyamic acid), such as described in WO 93/64701; viral vectors (such as DNA or RNA viruses) Carrier); fusion proteins, such as those described in PCT / US95 / 02140 (WO 95/22618), which contain target portions (such as antibodies specific for target cells) and nucleic acid binding portions (such as fish) Protamine) fusion protein; plastid; phage and so on. Such vectors can be chromosomal, non-chromosomal, or synthetic. Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA virus vectors are preferred. Such vectors include pox vectors such as orthopox or fowlpox vectors; herpes virus vectors such as herpes simplex virus I (HSV) vectors (see Geller, AI et al., J. Neurochem, 64: 487 (1995); Lim, F . Et al., DNA Cloning: Mammalian Systems, edited by D. Glover (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci .: USA 90: 7603 (1993); Geller , AI et al., Proc Natl. Acad. Sci USA 87: 1149 (1990)), adenovirus vectors (see LeGal LaSalle et al., Science, 259: 988 (1993); Davidson et al., Nat. Genet 3: 219 ( 1993); Yang et al., J. Virol. 69: 2004 (1995)) and adeno-associated viral vectors (see Kaplitt, MG et al., Nat. Genet. 8: 148 (1994)). Poxvirus vectors introduce genes into the cytoplasm of cells. Fowlpox virus vectors only cause short-term performance of nucleic acids. For introducing nucleic acids into nerve cells, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred. The performance time caused by adenovirus vector (about 2 months) is shorter than that of adeno-associated virus (about 4 months), and adeno-associated virus is shorter than HSV vector. Vaccinia virus vectors can multiply in many different types of cells. The specific vector selected will depend on the target cell and the condition being treated. Introduction can be by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer models include, for example, naked DNA, CaPO4 Precipitation, DEAE dextran, electroporation, protoplast fusion, liposome transfection, cell microinjection and viral vectors. Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to guide the vector (eg adenovirus, HSV) to the desired location. In addition, particles can be delivered by intraventricular (icv) infusion using a small pump infusion system, such as the SynchroMed infusion system. A method based on total flow (referred to as convection) has also proven effective in delivering macromolecules to extended areas of the brain and can be used to deliver vectors to target cells (see Bobo et al., Proc. Natl. Acad. Sci. USA 91: 2076-2080 (1994); Morrison et al., Am. J. Physiol. 266: 292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal, and subcutaneous injections, as well as oral or other known routes of administration. Such vectors can be used to express large amounts of antibodies, which can be used in a variety of ways. For example, to detect the presence of PD-1 in a sample. Anti-PD-1 antibodies can also be used to try to bind to PD-1 and disrupt PD-1 mediated signaling. The technology may be suitable for the production of single-chain antibodies specific for the antigen protein of the present invention (see, for example, US Patent No. 4,946,778). In addition, the method may be suitable for constructing Fab expression libraries (see, for example, Huse et al., 1989 Science 246: 1275-1281) in order to quickly and efficiently identify a protein or its derivatives, fragments, analogs, or homologues that have the desired Specific single plant Fab fragment. Antibody fragments containing individual genotypes against protein antigens can be manufactured by techniques known in the art, including (but not limited to): (i) F (ab ') produced by pepsin digestion of antibody molecules2 Fragment; (ii) by reducing F( ab ') 2 Fab fragments generated by disulfide bridges of the fragments; (iii) Fab fragments produced by treating antibody molecules with papain and reducing agents; and (iv) Fv Fragment. The invention also includes Fv , Fab, Fab 'and F (ab')2 Anti-PD-1 fragments, single-chain anti-PD-1 antibodies, single-domain antibodies (eg, nanobody antibodies or VHH), multispecific (such as bispecific) anti-PD-1 antibodies, and hetero-binding anti-PD-1 antibodies. Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant manufacturing of bispecific antibodies is based on the presentation of two immunoglobulin heavy chain / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305: 537-539 (1983 )). Due to the random classification of immunoglobulin heavy and light chains, these fusion tumors (four-source fusion tumors) produce a potential mixture of ten different antibody molecules, only one of which has an appropriate bispecific structure. Purification of appropriate molecules is usually achieved by affinity chromatography steps. Similar procedures were disclosed in WO 93/08829 and Traunecker et al., EMBO J., 10: 3655-3659 (1991) published on May 13, 1993. An antibody variable domain (antibody-antigen combination site) with the desired binding specificity can be fused to an immunoglobulin constant domain sequence. Preferably, it is fused with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one fusion. The DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. For additional details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986). According to another method described in WO 96/27011, the interface between pairs of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface includes at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg, tyrosine or tryptophan). At the interface of the second antibody molecule, by replacing the larger amino acid side chain with a smaller amino acid side chain (such as alanine or threonine), a compensatory size equal to or similar to the larger side chain is generated Cavity ". This provides a mechanism to increase the yield of heterodimers over other undesired end products, such as homodimers. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to produce F (ab ')2 Fragment. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The Fab 'fragments generated are then converted to thionitrobenzoate (TNB) derivatives. Next, one of the Fab'-TNB derivatives is converted back to Fab'-thiol by mercaptoethylamine reduction and mixed with an equal molar amount of other Fab'-TNB derivatives to form a bispecific antibody. The bispecific antibodies produced can be used as reagents for the selective immobilization of enzymes. In addition, Fab 'fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describe a fully humanized bispecific antibody F (ab ')2 Molecular manufacturing. Each Fab 'fragment is independently secreted from E. coli and undergoes directed chemical coupling in vitro to form a bispecific antibody. Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zippers have been used to generate bispecific antibodies. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). The leucine zipper peptides from Fos and Jun proteins are linked to the Fab 'part of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. The "bifunctional antibody" technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. These fragments comprise a light chain variable domain (VL ) Of the heavy chain variable domain (VH ), The linker is too short to allow pairing between the two domains on the same chain. Therefore, to force a fragment of VH And VL The complement of the domain and another fragment VL And VH The domains are paired, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single chain Fv (scFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994). Covers antibodies with more than two valences. For example, trispecific antibodies can be prepared. See, for example, Tutt et al., J. Immunol. 147: 60 (1991). Exemplary bispecific antibodies can bind to two different epitopes, at least one of which is derived from the protein antigen of the present invention. Alternatively, the anti-antigen arm of an immunoglobulin molecule can be combined with a trigger molecule that binds to white blood cells, such as a T cell receptor molecule (eg, CD2, CD3, CD28, or B7), or an Fc receptor of IgG (FcγR), such as FcγRI (CD64), Fc [gamma] RII (CD32) and Fc [gamma] RIII (CD16) arms are combined to concentrate the cell defense mechanism on cells expressing specific antigens. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing specific antigens. These antibodies have an antigen-binding arm and an arm that binds a cytotoxic agent or a radionuclear chelating agent (such as EOTUBE, DPTA, DOTA, or TETA). Heteroconjugate antibodies are also within the scope of the present invention. The heteroconjugate anti-system consists of two covalently joined antibodies. For example, such antibodies have been proposed to target immune system cells to unwanted cells (see US Patent No. 4,676,980) and are used to treat HIV infection (see WO 91/00360, O 92/200373, EP 03089). It is expected that these antibodies can be prepared in vitro using known methods in synthetic protein chemistry (including methods involving cross-linking agents). For example, an immunotoxin can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of reagents suitable for this purpose include iminothiol esters and methyl 4-mercaptobutylimide and reagents disclosed in, for example, US Patent No. 4,676,980. It may be necessary to modify the antibodies of the invention for effector functions in order to enhance the effectiveness of the antibodies in treating diseases and disorders related to abnormal PD-1 signaling, for example. For example, cysteine residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibody can have improved internalization capabilities and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC) (see Caron et al., J. Exp Med., 176 : 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, antibodies can be engineered to have dual Fc regions and therefore can have enhanced complement solubilization and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)). The present invention also relates to immunoconjugates, which include antibodies and cytotoxic agents such as toxins (eg, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), radioactive isotopes (ie, radioactive conjugates), or by Imaging detects combinations of labels (eg, radioisotopes, fluorescent dyes, and enzymes) of target antigens (such as PD-1) in patient samples or in vivo. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), gabatoxin A chain, Acacia toxin A chain, Mo Modeccin A chain, alpha-sarcin, Aleurites fordii protein, carnation protein, Phytolaca americana protein (PAPI, PAPII and PAP-S) , Momordica charantia inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictedocin ), Phenolmycin, enomycin (tricomecene) and mycotoxin (tricothecene). A variety of radioactive nuclear species can be used to produce radioactively bound antibodies. Examples include212 Bi,131 I,131 In,90 Y and186 Re. The combination of antibody and cytotoxic agent is prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyl dithiol) propionate (SPDP) , Bifunctional derivatives of iminothiolane (IT), imidate (such as dimethyl diimino adipate hydrochloride), active esters (such as disuccinic acid subsuccinate Amine esters), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzyl) hexamethylenediamine), double azinium derivatives (such as bis- (p-diazonium benzene (Methanyl) -ethylenediamine), diisocyanate (such as 2,6-diisocyanate tolyl), and dual-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin toxin immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for binding radionucleotide to antibodies. (See WO94 / 11026). Those of ordinary skill in the art should recognize that many possible parts can be coupled to the resulting antibodies of the invention (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference). Coupling can be achieved by any chemical reaction that binds two molecules, as long as the antibody and the other part retain their corresponding activities. This linkage may include many chemical mechanisms, such as covalent binding, affinity binding, insertion, coordination binding, and mismatch. However, the preferred binding system is covalent binding. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporating external bridging molecules. Many bivalent or multivalent linking agents can be used to couple protein molecules, such as antibodies of the invention, to other molecules. For example, representative coupling agents may include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylene diamine. It is expected that this list is not an exhaustive list of the various types of coupling agents known in the art, but is actually an example of more common coupling agents (see Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al. People, Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987)). Preferred linkers are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44: 201-208 (1984) describes the use of m-cis-butadienyliminobenzyl-N-hydroxysuccinyl Urethane (MBS)). See also U.S. Patent No. 5,030,719 describing the use of halogenated acetyl hydrazide derivatives to couple to antibodies by means of oligopeptide linkers. Particularly preferred linkers include: (i) EDC (1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidooxy) Carbonyl-α-methyl-α- (2-pyridyl-dithio) -toluene (Pierce Chem. Co., catalog number (21558G); (iii) SPDP (succinimide-6 [3- (2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., catalog number 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimide amide 6 [3- (2-pyridyldithio) -propionamide] hexanoate (Pierce Chem. Co., catalog number 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-) bonded to EDC Succinimide: Pierce Chem. Co., catalog number 24510). The above linker contains components with different properties, resulting in conjugates with different physical and chemical properties. For example, the sulfo group of alkyl carboxylic acid -NHS ester is more stable than sulfo-NHS ester of aromatic carboxylic acid. The linker containing NHS-ester has lower solubility than sulfo-NHS ester. In addition, the linker SMPT contains sterically hindered disulfide bond and can form Conjugates with increased stability. The disulfide linkage is generally less stable than other linkages because the disulfide linkage is active External cleavage makes it difficult to obtain conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide coupling. When carbodiimide coupling (such as EDC) is used in combination with sulfo-NHS, ester pairs are formed The resistance to hydrolysis is greater than the carbodiimide coupling reaction alone. The antibodies disclosed herein can also be formulated in the form of immunoliposomes. Lipid systems containing antibodies are prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and US Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in US Patent No. 5,013,556. Particularly useful liposomes can be phospholipid ethanolamine (PEG-PE) derived from phospholipid choline, cholesterol and PEG by reverse phase evaporation. The liposome composition is produced. The liposomes are extruded through a filter with a defined pore size to produce liposomes with the desired diameter. The use of antibodies against PD-1 should be understood that the administration of therapeutic entities according to the present invention will Suitable for Carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and the like are administered together. A variety of suitable formulations can be found where all medicinal chemists know: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, PA (1975)), in particular Chapter 87 of Blaug, Seymour. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, vesicle-containing lipids (cationic or anionic) (such as Lipofectin ™), DNA conjugates, anhydrous absorption pastes, Oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycol with various molecular weights), semi-solid gels and semi-solid mixtures containing carbs wax. Any of the aforementioned mixtures can be used in the treatment and therapy according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and can tolerate administration way. For additional information on formulations, excipients, and carriers well known to pharmaceutical chemists, see also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32 (2): 210-8 ( 2000); Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203 (1-2): 1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts. "J Pharm Sci. 89 (8): 967-78 (2000); Powell et al.," Compendium of excipients for parenteral formulations "PDA J Pharm Sci Technol. 52: 238-311 (1998) and the quotes in it . In some embodiments, the anti-PD-1 antibodies of the invention can be used as therapeutic agents. Such agents will generally be used for diagnosis, prognosis, monitoring, treatment, remission, and / or prevention of a subject's disease or pathology related to abnormal PD-1 or PD-L1 / PD-L2 performance, activity, and / or signaling . Treatment options are by using standard methods to identify a disease or condition (or risk of developing the disease or condition) that is associated with abnormal PD-1 or PD-L1 / PD-L2 performance, activity, and / or signaling, such as Subjects with cancer or other neoplastic disorders, such as human patients. The antibody preparation, preferably an antibody preparation having high specificity and high affinity for its target antigen, is administered to the subject, and generally will have an effect due to its binding to the target. The administration of the antibody can abolish or inhibit or interfere with the performance, activity and / or signaling function of the target (eg PD-1). The administration of the antibody can abolish or inhibit or interfere with the binding of the target (eg PD-1) to its naturally bound endogenous ligand. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, or otherwise interferes with PD-1 performance, activity, and / or signaling. Diseases or disorders associated with abnormal PD-1 or PD-L1 / PD-L2 performance, activity, and / or signaling include (as non-limiting examples) blood cancers and / or solid tumors. Cancers that can use the antibodies of the present invention to inhibit growth include cancers that generally respond to immunotherapy. Examples of cancers for treatment include, but are not limited to, melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), breast cancer, colon cancer , Liver cancer and lung cancer (such as non-small cell lung cancer). The present invention can also be used to treat metastatic cancer, especially metastatic cancer that exhibits PD-L1 (Iwai et al. (2005) Int. Immunol. 17: 133-144). As appropriate, antibodies against PD-1 can be combined with the following immunogenic reagents: such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells and encoded immunostimulatory cytokines Gene-transfected cells (He et al. (2004) J. Immunol. 173: 4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigen, Trp-2, MART1 and / or tyrosinase, or transfected to express cytokine GM- CSF tumor cells. The anti-PD-1 antibody of the present invention can also be combined with the bispecific binding molecules described herein, such as a first antigen-binding domain (such as scFv) containing a specific recognition tumor antigen (such as EpCAM, FAP, EGFR or GPC3) and A bispecific molecule combination that specifically recognizes a second antigen binding domain (such as scFv) of a cell surface molecule on effector cells (such as CD3 on T lymphocytes). Symptoms associated with cancer and other neoplastic conditions include, for example, inflammation, fever, general malaise, fever, pain (often limited to inflamed areas), loss of appetite, weight loss, edema, headache, fatigue, rash, anemia, muscle weakness, muscle Fatigue, and abdominal symptoms such as abdominal pain, diarrhea, or constipation. The therapeutically effective amount of the antibody of the present invention generally refers to the amount required to achieve therapeutic purposes. As indicated above, this may be a binding interaction between the antibody and its target antigen, which in some cases interferes with the function of the target. The amount required for administration will additionally depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which the administered antibody depletes from its free volume and other subjects. A common range of therapeutically effective doses of antibodies or antibody fragments of the invention may be (as a non-limiting example) about 0.1 mg / kg body weight to about 100 mg / kg body weight. The usual administration frequency may be, for example, in the range of twice a day to once a week or once every two weeks. The effectiveness of treatment is determined in conjunction with any known method for diagnosing or treating specific inflammatory-related conditions. Remission of one or more symptoms of the inflammatory-related disorder indicates that the antibody provides clinical benefit. In another embodiment, antibodies directed against PD-1 can be used in methods known in the art to be related to the localization and / or quantification of PD-1 (eg, to measure the content of PD-1 in appropriate physiological samples , Used in diagnostic methods, for imaging proteins and similar methods). In a given embodiment, an antibody or derivative, fragment, analog or homolog thereof specific for PD-1 containing an antibody-derived antigen-binding domain is used as a pharmacologically active compound (hereinafter referred to as " Therapeutic agent "). In another embodiment, antibodies specific for PD-1 can be used to isolate the PD-1 polypeptide by standard techniques such as immunoaffinity chromatography or immunoprecipitation. Antibodies against PD-1 protein (or fragments thereof) can be used diagnostically to monitor protein content in tissues as part of clinical testing procedures, for example to determine the efficacy of a given treatment regimen. The detection can be aided by coupling the antibody to a detectable substance (ie, physically connected). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin Protein / biotin; examples of suitable fluorescent substances include umbelliferone, luciferin, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin Examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, luciferin, and luminescent proteins; and examples of suitable radioactive substances include125 I,131 I,35 S or3 H. In some embodiments, the anti-PD-1 antibody according to the present invention can be used as a reagent to detect the presence of PD-1 (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. Multiple antibodies against the system, or more preferably, single antibodies. Use whole antibodies or fragments thereof (eg scFv). The term "labeling" with respect to probes or antibodies is intended to cover the direct labeling of probes or antibodies by coupling (ie, physically connecting) detectable substances to the probes or antibodies, as well as The probe or antibody of another reagent reaction is indirectly labeled. Examples of indirect labeling include the detection of primary antibodies using fluorescently labeled secondary antibodies, and the labeling of the ends of DNA probes with biotin so that they can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from the subject, as well as tissues, cells and fluids present in the subject. Therefore, the use of the term "biological sample" includes blood and blood components or components, including serum, plasma, or lymph. That is, the detection method of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques used to detect analyte proteins include enzyme-linked immunosorbent analysis (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described in, for example, "ELISA: Theory and Practice: Methods in Molecular Biology", Volume 42, JR Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis And T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In addition, in vivo techniques for detecting analyte proteins include introducing labeled anti-analyte protein antibodies into the subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques. In some embodiments, a system mammal. Examples of mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cattle, and similar mammals. In some embodiments, each system is human. Also provided is a pharmaceutical composition comprising an effective amount of an anti-PD-1 antibody described herein, a host cell expressing the anti-PD-1 antibody described herein, or an oncolytic virus encoding the anti-PD-1 antibody described herein (Such as oncolytic VV), and pharmaceutically acceptable carriers as appropriate. Also provided is a method of treating cancer in an individual, comprising administering to the individual an effective amount of a pharmaceutical composition comprising the anti-PD-1 antibody described herein, a host expressing the anti-PD-1 antibody described herein Cells or oncolytic viruses encoding anti-PD-1 antibodies described herein (such as oncolytic VV), and optionally pharmaceutically acceptable carriers. Therapeutic effects can include, but are not limited to, killing cancer cells, inhibiting cancer cell proliferation, inducing redistribution of surrounding T cells, inducing immune responses in tumors, reducing tumor size, inhibiting tumor metastasis, and reducing preexisting tumor metastasis ( Such as the incidence or burden of lymph node metastasis), prolonging the survival time of individuals, prolonging the time of cancer progression, etc. In some embodiments, the pharmaceutical composition is administered intravenously or intratumorally to the individual. In some embodiments, each system is human. V. Preparation method The anti-PD-1 antibody (antigen-binding fragment thereof) described herein, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment -The Fc fusion protein can be prepared by any of the protein expression and purification methods known in the art. In some embodiments, the present application provides isolated nucleic acids encoding one or more of the following polypeptide chains: anti-PD-1 antibodies (or antigen-binding fragments thereof), PD-1 extracellular structures described herein Domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein. In some embodiments, the isolated nucleic acid comprises a first nucleic acid sequence SEQ ID NO: 17 and a second nucleic acid sequence SEQ ID NO: 18. The isolated nucleic acids can be DNA or RNA. In some embodiments, the isolated nucleic acid described herein is operably linked to a promoter. In some embodiments, the promoter is a late promoter. In some embodiments, the promoter is a VV promoter. In some embodiments, the promoter is a VV late promoter. In some embodiments, the promoter is F17R. In some embodiments, the isolated nucleic acid comprises a first nucleic acid sequence SEQ ID NO: 19 and a second nucleic acid sequence SEQ ID NO: 20. The isolated nucleic acids can be DNA or RNA. In some embodiments, the isolated nucleic acid described herein is operably linked to a promoter. In some embodiments, the promoter is a late promoter. In some embodiments, the promoter is a VV promoter. In some embodiments, the promoter is a VV late promoter. In some embodiments, the promoter is F17R. In some embodiments, the isolated nucleic acid is inserted into a vector, such as an expression vector, a viral vector (such as an oncolytic VV vector), or a selection vector. To express the nucleic acids, a vector can be introduced into the host cell to allow the nucleic acids to be expressed in the host cell. Such expression vectors may contain various elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. Where appropriate, these components can be selected by those skilled in the art. For example, the promoter sequence can be selected to facilitate transcription of the polynucleotide in the vector. Suitable promoter sequences include (but are not limited to) T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF1a promoter, CMV promoter, SV40 promoter and vaccinia virus promoter (such as F17R) . The enhancer sequence can be selected to enhance the transcription of the nucleic acid. The selectable marker can be selected to allow selection of the inserted host cell containing the vector from host cells that do not contain the vector. For example, the selectable marker can be a gene that confers antibiotic resistance. The signal sequence may be selected to allow the expressed polypeptide to be transported out of the host cell. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding a signal peptide. In some embodiments, the oncolytic virus line of vaccinia virus that encodes an immune checkpoint regulator (and / or bispecific junction molecule, cytokines, such as those described herein). Vaccinia virus has attracted attention in cancer gene therapy due to several characteristics. It has a natural tendency to cancer cells and can significantly enhance selectivity by deleting part of viral genes. In some embodiments, oncolytic viruses (such as oncolytic VV) encoding immune checkpoint regulators (and / or bispecific ligation molecules, cytokines, such as those described herein) contain a thymidine kinase (TK) gene And double deletion of vaccinia virus growth factor (VGF) gene (vvDD virus strain). The TK and VGF genes are required for virus replication in normal cells, but they are not required for replication in cancer cells. It can be engineered to be deficient in TK or VGF in the TK or VGF regions that confer activity, respectively. For example, this can be achieved by recombining pSEM-1 shuttle plastids containing immune checkpoint regulators (and / or bispecific junction molecules, cytokines, such as those described herein) into Western Reserve Vaccinia Virus (WR VV) was generated from the TK gene of the VSC20 virus strain (VGF-deficient virus strain). The VSC20 virus strain can be constructed by inserting the lacZ gene under the control of the p11 promoter into two copies of the viral VGF gene, thereby inactivating VGF. The constructed shuttle vector pSEM-1 can allow full viral replication before T cells are activated, such as the F17R late promoter under the transcriptional control of immune checkpoint regulators (and / or bispecific junction molecules, cytokines, (Such as those described herein). In some embodiments, the VV may further express markers, such as DsRed2, YFP, GFP, or YFP-GFP to allow virus selection. In some embodiments, the infectivity monitoring marker can be expressed under the transcriptional control of the same promoter that drives the immune checkpoint regulator (and / or the bispecific junction molecule described herein). In some embodiments, viral selection markers can be expressed under the transcriptional control of different promoters, such as the Pse / I promoter or the P7.5 promoter. In some embodiments, viral selection markers can be flanked by loxP sites in the same orientation. In order to construct recombinant viruses encoding immune checkpoint regulators (and / or bispecific junction molecules, cytokines, such as those described herein), an immune checkpoint regulator (and / or bispecific junction molecules, Cytokines, such as the shuttle vector pSEM-1 described herein, are transfected into human 143 TK cells. Next, the cells can be infected with the virus VSC20 with an infection rate (MOI) of 0.1. After 3 to 5 (e.g. five) rounds of plaque selection and amplification and confirmation of the performance of immune checkpoint regulators (and / or bispecific junction molecules, cytokines, such as those described herein), a pure line can be selected Perform amplification and purification. In some embodiments, the selection marker can be removed from the recombinant virus, such as the YFP-GFP cassette. For example, in some embodiments, the virus can be passaged on a U2OS cell line (U2OS-Cre) expressing the cytoplasmic form of Cre recombinase. After 3 to 5 (e.g. five) rounds of plaque selection and amplification to confirm the performance of immune checkpoint regulators (and / or bispecific ligation molecules, interleukins, such as those described herein), one can choose Select pure lines with negative markers (eg, YFP-GFP negative) for amplification and purification. Those skilled in the art should realize that any suitable method can be used to generate inactive mutations in the gene of interest, including mutation induction, polymerase chain reaction, homologous recombination, or any other genetic engineering known to those skilled in the art technology. Mutations can involve modification of nucleotide sequences, single genes, or arrays of genes. Mutation may involve a single nucleotide (such as point mutation, which involves the removal, addition, or substitution of a single nucleotide base within the DNA sequence) or it may involve the insertion or deletion of a large number of nucleotides. Mutations can occur spontaneously as a result of events such as errors in the fidelity of DNA replication, or can be induced after exposure to chemical or physical mutagens. Mutations can also be made site-specific using specific targeting methods well known to those skilled in the art. The obtained virus of the present invention can be replicated by a conventional method for virus replication, for example, by infecting a host cell (such as 293 cell) with the virus. It is envisaged that for other purposes, oncolytic virus nucleic acid molecules may contain, for example, thioester bonds and / or nucleotide analogs. Modifications can be used to stabilize nucleic acid molecules against endonucleases and / or exonucleases in cells. The nucleic acid molecule can be transcribed by an appropriate oncolytic vector containing a chimeric gene that allows the nucleic acid molecule to be transcribed in the cell. In this regard, it should also be understood that such polynucleotides can be used in "gene targeting" or "gene therapy" methods. Nucleic acid molecules can also be labeled. Methods for detecting nucleic acids are well known in the art, such as Southern and Northern blotting, PCR, or primer extension. This embodiment can be used in screening methods to verify the successful introduction of the nucleic acid molecules described above, for example during gene therapy methods. In some embodiments, there is provided an isolated host cell comprising an isolated nucleic acid encoding the above-mentioned anti-PD-1 antibody (or antigen-binding fragment thereof), encoding the above-mentioned PD-1 extracellular domain-Fc fusion protein, TMIGD2 cell Isolated nucleic acids of the outer domain-Fc fusion protein or SIRPα extracellular domain and the CXCL12 fragment-Fc fusion protein. Host cells containing the isolated nucleic acids described herein can be used to express or colonize the anti-PD-1 antibodies (or antigen-binding fragments thereof) described herein, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular structure Domain-Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. This technique fully determines the performance of antibodies and antigen-binding fragments in prokaryotic cells such as E. coli. For reviews, see, for example, Pluckthun, A. BioTechnology 9: 545-551 (1991). Those skilled in the art can also use it to be expressed in eukaryotic cells in culture as an option for the production of antibodies or antigen-binding fragments, see recent reviews such as Ref, ME (1993) Curr. Opinion Biotech. 4: 573-576; JJ et al. (1995) Curr. Opinion Biotech 6: 553-560. Advanced eukaryotic cells, especially those derived from multicellular organisms, can be used to express glycosylated polypeptides. Suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells, as well as vertebrate cells. The vector can be introduced into the host cell using any suitable method known in the art, including (but not limited to) DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated Transfection, electroporation, microprojectile bombardment, receptor-mediated gene delivery, delivery mediated by polyamic acid, histone, chitosan and peptide. Standard methods for transfecting and transforming cells to express the vector of interest are well known in the art. In some embodiments, the host cell includes a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cell includes a single vector that includes isolated nucleic acids encoding the first polypeptide and the second polypeptide. In some embodiments, the present application provides expression of the anti-PD-1 antibody (or antigen-binding fragment thereof) described herein, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein or The method of any one of SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein and / or bispecific junction molecule, which comprises culturing isolated host cells containing a vector and recovering the anti-PD-1 antibody from the cell culture ( Or its antigen-binding fragments), PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein, and / or bispecific junction molecules . These isolated host cells are cultured under conditions that allow expression of the isolated nucleic acid inserted into the vector. Suitable conditions for expressing polynucleotides may include, but are not limited to, suitable medium, suitable host cell density in the medium, presence of required nutrients, presence of supplementary factors, suitable temperature and humidity, and absence of microbial contaminants . Generally, those skilled in the art can choose suitable conditions suitable for the performance purpose. In some embodiments, the polypeptides expressed in the host cell can form dimers and thus produce anti-PD-1 antibodies (or antigen-binding fragments thereof) and / or bispecific ligation molecules described herein. In some embodiments, the polypeptide expressed in the host cell can form a polypeptide complex, which is a homodimer. In some embodiments where the host cell expresses the first polynucleotide and the second polynucleotide, the first polynucleotide and the second polynucleotide may form a polypeptide complex, the complex is heterodimeric Aggregate. In some embodiments, a polypeptide complex (such as a bispecific junction molecule or anti-PD-1 antibody or antigen-binding fragment thereof) can be formed inside the host cell. For example, dimers can be formed inside the host cell by means of related enzymes and / or cofactors. In some embodiments, the polypeptide complex can be secreted from the cell. In some embodiments, the first polypeptide and the second polypeptide can be secreted from the host cell and form a dimer outside the host cell. In some embodiments, the first polypeptide and the second polypeptide can be expressed separately and allowed to dimerize under suitable conditions to form a bispecific junction molecule or anti-PD-1 antibody (or antigen-binding fragment thereof). For example, the first polypeptide and the second polypeptide can be combined in a suitable buffer and dimerize the first protein monomer and the second protein monomer via appropriate interactions, such as hydrophobic interactions. In some embodiments, the first polypeptide and the second polypeptide may be combined in a suitable buffer containing enzymes and / or cofactors that promote dimerization of the first polypeptide and the second polypeptide. In some embodiments, the first polypeptide and the second polypeptide can be combined in a suitable vehicle and allowed to react with each other in the presence of a suitable reagent and / or catalyst. The expressed polypeptides and / or polypeptide complexes can be collected using any suitable method. Polypeptides and / or polypeptide complexes can be expressed intracellularly, in the periplasmic space, or secreted extracellularly into the culture medium. If the polypeptide and / or polypeptide complex is expressed intracellularly, the host cell containing the polypeptide and / or polypeptide complex can be lysed and unwanted debris can be removed by centrifugation or ultrafiltration to isolate the polypeptide from the lysate And / or peptide complexes. If the polypeptide and / or polypeptide complex is secreted into the periplasmic space of E. coli, the cell paste can be thawed in the presence of reagents such as sodium acetate (pH 3.5), EDTA, and benzylsulfonyl fluoride (PMSF) For about 30 minutes, and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10: 163-167 (1992)). If the polypeptide and / or polypeptide complex is secreted into the culture medium, a commercially available protein concentration filter, such as an Amincon or Millipore Pellicon ultrafiltration device can be used to collect and concentrate the cell culture supernatant. Protease inhibitors and / or antibiotics can be included in the collection and concentration steps to inhibit protein degradation and / or the growth of contaminated microorganisms. The expressed polypeptides and / or polypeptide complexes can be further purified by suitable methods such as (but not limited to) affinity chromatography, hydroxyapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, Separation of ion exchange fractions on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica chromatography, chromatography on heparin agarose, on anion or cation exchange resins (such as polyaspartic acid) Chromatography, chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation on an acid column : principles, high resolution methods and applications, published by Wiley-VCH, 1998). In some embodiments, the polypeptide and / or polypeptide dimer complex can be purified by affinity chromatography. In some embodiments, protein A chromatography or protein A / G (fusion protein of protein A and protein G) chromatography can be used to purify polypeptides comprising components derived from the CH2 domain and / or CH3 domain of the antibody And / or polypeptide complexes (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)); Zettlit, KA, Antibody Engineering, Part V, 531-535, 2010). In some embodiments, protein G chromatography can be used to purify polypeptides and / or polypeptide complexes containing IgG γ3 heavy chains (Guss et al., EMBO J. 5: 1567 1575 (1986)). In some embodiments, protein L chromatography can be used to purify polypeptides and / or polypeptide complexes containing kappa light chains (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, BHK Et al., J. Biol. Chem., 267, 2234-2239 (1992)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Compared to what can be achieved with agarose, mechanically stable matrices such as controlled microporous glass or poly (styrenedivinyl) benzene allow faster flow rates and shorter processing times. In the case where the antibody contains a CH3 domain, Bakerbond ABX resin (J.T. Baker, Phillipsburg, N.J.) can be used for purification. VI. Articles and kits Any of the compositions described herein can be included in kits (eg, express immune checkpoint regulators (eg, anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein or SIRPα extracellular domain and CXCL12 fragment-Fc fusion protein) oncolytic vaccinia virus, oncolytic virus expressing immune checkpoint regulator and bispecific junction molecule, anti-PD-1 Antibody composition, host cells expressing anti-PD-1 antibody or oncolytic virus encoding anti-PD-1 antibody). In a non-limiting example, one or more viruses and / or reagents used to generate or manipulate viruses may be included in the kit. The components of the kit are provided in suitable container components. Some of the components of these kits can be packaged in aqueous media or in lyophilized form. The container components of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container component in which components can be placed and preferably aliquoted appropriately. In the case where there is more than one component in the set, the set will generally also contain a second, third or other additional container into which additional components can be placed separately. Various component combinations can also be included in vials. These kits usually also include components for containing closed restricted components for commercial sale. Such containers may include injection molded or blow molded plastic containers that hold the desired vials. When the components of the kit are provided in the form of one or more liquid solutions, the liquid solution is an aqueous solution, and a sterile aqueous solution is particularly useful. In some cases, the container member itself may be a syringe, dropper, and / or other such devices, from which the formulation can be applied to an infected area of the body, injected into an animal, and / or even applied to other groups of kits And / or mixed with other components of the kit. The components of the kit are also available in dry powder form. When the reagents and / or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is envisioned that the solvent can also be provided in another container member. The kits may also contain a second container component for containing sterile, pharmaceutically acceptable buffers and / or other diluents. In some embodiments, the virus used in therapy is provided in the kit, and in some cases the virus is essentially the only component of the kit. The kit can contain reagents and materials to improve the required virus. In specific embodiments, the reagents and materials include expression constructs, primers for amplifying the desired sequence, restriction enzymes, one or more DNAs for inclusion in the virus, nucleotides, suitable buffers or buffers Reagents, salts, and the like, and in some cases, such reagents include vectors and / or DNA that encode the junction molecules and / or their corresponding regulatory elements as described herein. In some embodiments, there are one or more devices suitable for extracting one or more samples from the individual in the kit. Such devices may be syringes, scrapers, and the like. In some embodiments, in addition to viral implementation, the kit also includes a second cancer therapy, such as chemotherapy, hormone therapy, and / or immunotherapy. The kit can be customized for an individual's specific cancer and contain a corresponding second cancer therapy for that individual. The kit described herein may further include other materials needed from a business and user point of view, including other buffers, diluents, filters, needles, syringes, and instructions with instructions for performing any of the methods described herein Drug Instructions. The present application further provides articles comprising the compositions described herein, such as pharmaceutical compositions, in a suitable packaging form. For use in the compositions described herein (such as expressing immune checkpoint regulators (e.g., anti-PD-1 antibodies, PD-1 extracellular domain-Fc fusion protein, TMIGD2 extracellular domain-Fc fusion protein, or SIRPα extracellular structure Domain and CXCL12 fragment-Fc fusion protein) oncolytic vaccinia virus, oncolytic viruses expressing immune checkpoint regulators and bispecific junction molecules, anti-PD-1 antibody compositions, host cells expressing anti-PD-1 antibodies or Suitable packaging for oncolytic viruses encoding anti-PD-1 antibodies is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, cans, flexible packaging (such as sealed polyester Film (Mylar) or plastic bags) and the like. These products can be further sterilized and / or sealed. Illustrative Examples Example 1. An oncolytic vaccinia virus containing a nucleic acid encoding an immune checkpoint regulator, wherein the nucleic acid is operably linked to a late promoter. Example 2. Oncolytic vaccinia virus as in Example 1, wherein the late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters and PSL The late promoter is synthesized. Example 3. Oncolytic vaccinia virus as in Example 2, wherein the late promoter is F17R. Embodiment 4. The oncolytic vaccinia virus according to any one of embodiments 1 to 3, wherein the oncolytic vaccinia virus is selected from the group consisting of: Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, modified Ankara Vaccinia virus (MVA), Lister, King, IHD, Evans, USSR and Western Reserve (WR). Example 5. Oncolytic vaccinia virus as in Example 4, wherein the oncolytic vaccinia virus is WR strain. Embodiment 6. The oncolytic vaccinia virus as in embodiment 5, wherein the oncolytic vaccinia virus contains a double deletion of thymidine kinase (TK) gene and acne growth factor (VGF) gene. Embodiment 7. The oncolytic vaccinia virus according to any one of embodiments 1 to 6, wherein the immune checkpoint regulator is an activator of stimulatory immune checkpoint molecules. Embodiment 8. The oncolytic vaccinia virus according to any one of embodiments 1 to 6, wherein the immune checkpoint regulator is an immune checkpoint inhibitor. Example 9. Oncolytic vaccinia virus as in Example 8, wherein the immune checkpoint inhibitors are PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, Inhibitors of BTLA, CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73. Example 10. Oncolytic vaccinia virus as in Example 9, wherein the immune checkpoint inhibitor is an inhibitor of PD-1. Embodiment 11. The oncolytic vaccinia virus according to any one of embodiments 1 to 10, wherein the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. Example 12. Oncolytic vaccinia virus as in Example 11, wherein the immune checkpoint regulator is an anti-PD-1 antibody, which comprises: a heavy chain variable region (VH), the heavy chain variable region including (1) HVR-H1 containing amino acid sequence SEQ ID NO: 1; (2) HVR-H2 containing amino acid sequence SEQ ID NO: 2; and (3) HVR- containing amino acid sequence SEQ ID NO: 3 H3; and the light chain variable region (VL), which includes (1) HVR-L1 containing the amino acid sequence SEQ ID NO: 4; (2) amino acid sequence SEQ ID NO: 5 HVR-L2; and (3) HVR-L3 containing the amino acid sequence SEQ ID NO: 6. Embodiment 13. The oncolytic vaccinia virus as in Embodiment 11, wherein the immune checkpoint regulator is an anti-PD-1 antibody, which comprises: a heavy chain variable region (VH), the heavy chain variable region including (1) HVR-H1 containing amino acid sequence SEQ ID NO: 7; (2) HVR-H2 containing amino acid sequence SEQ ID NO: 8; and (3) HVR- containing amino acid sequence SEQ ID NO: 9 H3; and the light chain variable region (VL), which includes (1) HVR-L1 containing the amino acid sequence SEQ ID NO: 10; (2) amino acid sequence SEQ ID NO: 11 HVR-L2; and (3) HVR-L3 containing the amino acid sequence SEQ ID NO: 12. Embodiment 14. Oncolytic vaccinia virus according to any one of embodiments 1 to 9, wherein the immune checkpoint regulator is bound to the ligand of the immune checkpoint molecule. Example 15. Oncolytic vaccinia virus as in Example 14, wherein the immune checkpoint molecule is PD-L1, PD-L2, HHLA-2, CD47 or CXCR4. Embodiment 16. Oncolytic vaccinia virus as in embodiment 15, wherein the immune checkpoint regulator comprises a fusion of PD-1 extracellular domain and immunoglobulin Fc fragment, TMIGD2 extracellular domain and immunoglobulin Fc fragment Fusion, or fusion of SIRPα extracellular domain and CXCL12 fragment with immunoglobulin Fc fragment. Example 17. Oncolytic vaccinia virus as in Example 16, wherein the Fc fragment is IgG4 Fc. Embodiment 18. The oncolytic vaccinia virus according to any one of embodiments 1 to 17, which further comprises a second nucleic acid encoding cytokine. Example 19. Oncolytic vaccinia virus as in Example 18, wherein the interleukin is GM-CSF. Embodiment 20. An oncolytic virus comprising a first nucleic acid encoding an immune checkpoint regulator and a second nucleic acid encoding a bispecific molecule, the bispecific molecule comprising a first antigen binding structure that specifically recognizes a tumor antigen Domain and a second antigen binding domain that specifically recognizes cell surface molecules on effector cells. Example 21. The oncolytic virus as in Example 20, wherein the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Senegal valley virus (SVV), adenovirus, herpes simplex virus 1 (HSV1 ), Herpes simplex virus 2 (HSV2), myxoma virus, reovirus, poliovirus, vesicular stomatitis virus (VSV), measles virus (MV), lentivirus, retrovirus, measles virus, Influenza virus, Sindby virus and Newcastle disease virus (NDV). Example 22. The oncolytic virus as in Example 21, wherein the oncolytic virus is an oncolytic vaccinia virus. Embodiment 23. The oncolytic virus of embodiment 22, wherein the oncolytic vaccinia virus is selected from the group consisting of: Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, modified Ankara vaccinia virus (MVA), Lister , King, IHD, Evans, USSR and Western Reserve (WR). Example 24. Oncolytic virus as in Example 23, wherein the oncolytic vaccinia virus is WR strain. Embodiment 25. The oncolytic virus of embodiment 24, wherein the oncolytic vaccinia virus contains a double deletion of the thymidine kinase (TK) gene and the acne growth factor (VGF) gene. Embodiment 26. The oncolytic virus according to any one of embodiments 20 to 25, wherein the immune checkpoint regulator is an activator of stimulatory immune checkpoint molecules. Embodiment 27. The oncolytic virus according to any one of embodiments 20 to 25, wherein the immune checkpoint regulator is an immune checkpoint inhibitor. Embodiment 28. Oncolytic virus as in embodiment 27, wherein the immune checkpoint inhibitors are PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA , CTLA-4, TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitors. Embodiment 29. The oncolytic virus of embodiment 28, wherein the immune checkpoint inhibitor is an inhibitor of PD-1. Embodiment 30. The oncolytic virus according to any one of embodiments 20 to 29, wherein the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. Embodiment 31. The oncolytic virus of embodiment 30, wherein the immune checkpoint regulator is an anti-PD-1 antibody, which comprises: a heavy chain variable region (VH), the heavy chain variable region including (1) containing HVR-H1 of amino acid sequence SEQ ID NO: 1; (2) HVR-H2 containing amino acid sequence SEQ ID NO: 2; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 3 ; And a light chain variable region (VL), which includes (1) HVR-L1 containing the amino acid sequence SEQ ID NO: 4; (2) the amino acid sequence SEQ ID NO: 5 HVR-L2; and (3) HVR-L3 containing the amino acid sequence SEQ ID NO: 6. Embodiment 32. The oncolytic virus of embodiment 30, wherein the immune checkpoint regulator is an anti-PD-1 antibody, which comprises: a heavy chain variable region (VH), the heavy chain variable region including (1) containing Amino acid sequence HVR-H1 of SEQ ID NO: 7; (2) HVR-H2 containing amino acid sequence SEQ ID NO: 8; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 9 ; And a light chain variable region (VL), which includes (1) HVR-L1 containing the amino acid sequence SEQ ID NO: 10; (2) amino acid sequence SEQ ID NO: 11 HVR-L2; and (3) HVR-L3 containing the amino acid sequence SEQ ID NO: 12. Embodiment 33. The oncolytic virus according to any one of embodiments 20 to 28, wherein the immune checkpoint regulator is bound to the ligand of the immune checkpoint molecule. Embodiment 34. The oncolytic virus of embodiment 33, wherein the immune checkpoint molecule is PD-L1, PD-L2, HHLA-2, CD47, or CXCR4. Embodiment 35. The oncolytic virus of embodiment 34, wherein the immune checkpoint regulator comprises a fusion of PD-1 extracellular domain and immunoglobulin Fc fragment, TGIGD2 extracellular domain and immunoglobulin Fc fragment Fusion, or fusion of SIRPα extracellular domain and CXCL12 fragment with immunoglobulin Fc fragment. Embodiment 36. Oncolytic virus as in embodiment 35, wherein the Fc fragment is IgG4 Fc. Embodiment 37. The oncolytic virus according to any one of embodiments 20 to 36, wherein the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and phospholipid inositol protein Glycan-3 (GPC3). Example 38. The oncolytic virus of Example 37, wherein the tumor antigen is EpCAM. Embodiment 39. The oncolytic virus as in Embodiment 37, wherein the tumor antigen is FAP. Embodiment 40. The oncolytic virus of embodiment 37, wherein the tumor antigen is EGFR. Example 41. Oncolytic virus as in Example 37, wherein the tumor antigen is GPC3. Embodiment 42. The oncolytic virus according to any one of embodiments 20 to 41, wherein the effector cell line is selected from the group consisting of T lymphocytes, B lymphocytes, natural killer (NK) cells, dendritic cells (DC), macrophages, monocytes, neutrophils and NKT cells. Example 43. Oncolytic virus as in Example 42, wherein the effector cell is T lymphocytes. Example 44. The oncolytic virus as in Example 43, wherein the T lymphocytes are cytotoxic T lymphocytes. Embodiment 45. The oncolytic virus according to any one of embodiments 20 to 44, wherein the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46 and NKG2D. Embodiment 46. The oncolytic virus of embodiment 45, wherein the cell surface molecule is CD3. Embodiment 47. The oncolytic virus according to any one of embodiments 20 to 46, wherein the first antigen binding domain is a single chain variable fragment (scFv). Embodiment 48. The oncolytic virus according to any one of embodiments 20 to 47, wherein the second antigen-binding domain is scFv. Embodiment 49. The oncolytic virus according to any one of embodiments 20 to 48, wherein the first antigen-binding domain and the second antigen-binding domain are connected by a linker. Embodiment 50. The oncolytic virus according to any one of embodiments 20 to 49, wherein the first antigen-binding domain is at the N-terminus of the second antigen-binding domain. Embodiment 51. The oncolytic virus according to any one of embodiments 20 to 49, wherein the first antigen-binding domain is at the C-terminus of the second antigen-binding domain. Embodiment 52. The oncolytic virus according to any one of embodiments 20 to 51, wherein the first nucleic acid encoding the immune checkpoint regulator is operably linked to the late promoter. Embodiment 53. The oncolytic virus according to any one of embodiments 20 to 52, wherein the second nucleic acid encoding the bispecific molecule is operably linked to the late promoter. Embodiment 54. The oncolytic virus of embodiment 52 or 53, wherein the late promoter that drives the expression of immune checkpoint regulators and / or bispecific molecules is selected from the group consisting of: F17R, I2L late promoter , L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters and PSL The late promoter is synthesized. Example 55. The oncolytic virus of Example 54 wherein the late promoter is F17R. Embodiment 56. The oncolytic virus according to any one of embodiments 20 to 55, wherein the oncolytic virus further comprises a third nucleic acid encoding cytokine. Embodiment 57. The oncolytic virus as in embodiment 56, wherein the interleukin is GM-CSF. Embodiment 58. A pharmaceutical composition comprising the oncolytic virus according to any one of embodiments 1 to 57, and a pharmaceutically acceptable carrier. Embodiment 59. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition as in embodiment 58. Embodiment 60. The method of embodiment 59, wherein the effective amount is about 105 Up to about 1013 pfu. Embodiment 61. The method of embodiment 60, wherein the effective amount is about 109 pfu. Embodiment 62. The method of any one of embodiments 59 to 61, wherein the pharmaceutical composition is administered systemically. Embodiment 63. The method of embodiment 62, wherein the pharmaceutical composition is administered intravenously. Embodiment 64. The method of any one of embodiments 59 to 61, wherein the pharmaceutical composition is administered locally. Embodiment 65. The method of embodiment 64, wherein the pharmaceutical composition is administered intratumorally. Embodiment 66. The method of any one of embodiments 59 to 65, wherein the cancer is a solid tumor. Embodiment 67. The method of embodiment 66, wherein the cancer is selected from the group consisting of colorectal cancer, liver cancer, and breast cancer. Embodiment 68. The method of any one of embodiments 59 to 67, further comprising administering additional cancer therapy to the individual. Embodiment 69. The method of embodiment 68, wherein the additional cancer therapy is surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or a combination thereof. Embodiment 70. The method of any one of embodiments 59 to 69, wherein the system is human. Example 71. An anti-PD-1 antibody comprising: a heavy chain variable region (VH), the heavy chain variable region comprising (1) HVR-H1 containing the amino acid sequence SEQ ID NO: 1; (2 ) HVR-H2 containing amino acid sequence SEQ ID NO: 2; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 3; and light chain variable region (VL), the light chain variable The region includes (1) HVR-L1 containing amino acid sequence SEQ ID NO: 4; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 5; and (3) amino acid sequence SEQ ID NO : 6 of HVR-L3. Embodiment 72. The anti-PD-1 antibody of Embodiment 71, wherein the antigen-binding domain of the construct includes the heavy chain variable region (VH) containing the amino acid sequence SEQ ID NO: 13, and / or contains an amine The light chain variable region (VL) of the acid sequence SEQ ID NO: 14. Example 73. An anti-PD-1 antibody comprising: a heavy chain variable region (VH), the heavy chain variable region comprising (1) HVR-H1 containing the amino acid sequence SEQ ID NO: 7; ) HVR-H2 containing amino acid sequence SEQ ID NO: 8; and (3) HVR-H3 containing amino acid sequence SEQ ID NO: 9; and light chain variable region (VL), the light chain variable The region includes (1) HVR-L1 containing amino acid sequence SEQ ID NO: 10; (2) HVR-L2 containing amino acid sequence SEQ ID NO: 11; and (3) amino acid sequence SEQ ID NO : 12 of HVR-L3. Embodiment 74. The anti-PD-1 antibody of Embodiment 73, wherein the antigen-binding domain of the construct includes the amino acid sequence-containing heavy chain variable region (VH) of SEQ ID NO: 15 and / or contains an amino group The light chain variable region (VL) of the acid sequence SEQ ID NO: 16. Embodiment 75. The construct of any one of embodiments 71 to 74, wherein the anti-PD-1 anti-system full-length antibody. Embodiment 76. The construct of any one of embodiments 71 to 74, wherein the anti-PD-1 anti-system scFv. Embodiment 77. An isolated nucleic acid encoding the anti-PD-1 antibody of any one of Embodiments 71 to 76. Embodiment 78. The isolated nucleic acid of Embodiment 77, which comprises a first nucleic acid sequence SEQ ID NO: 17 and a second nucleic acid sequence SEQ ID NO: 18. Embodiment 79. The isolated nucleic acid of embodiment 77, which comprises a first nucleic acid sequence SEQ ID NO: 19 and a second nucleic acid sequence SEQ ID NO: 20. Embodiment 80. The isolated nucleic acid of any one of embodiments 77 to 79, wherein the isolated nucleic acid is operably linked to a promoter. Embodiment 81. The isolated nucleic acid of Embodiment 80, wherein the promoter is a late promoter. Embodiment 82. The isolated nucleic acid of embodiment 81, wherein the promoter is the late vaccinia virus promoter. Embodiment 83. The isolated nucleic acid of embodiment 82, wherein the vaccinia virus late promoter is selected from the group consisting of: F17R, I2L late promoter, L4R late promoter, P7 . 5k Early / late promoter, PEL Early / late promoter, P11k Late promoter, PSEL Synthesis of early / late promoters and PSL The late promoter is synthesized. Embodiment 84. The isolated nucleic acid of Embodiment 83, wherein the vaccinia virus late promoter is F17R. Embodiment 85. An isolated host cell comprising the isolated nucleic acid of any one of embodiments 77 to 84. Embodiment 86. An oncolytic virus comprising the nucleic acid according to any one of embodiments 77 to 84. Embodiment 87. A pharmaceutical composition comprising the anti-PD-1 antibody according to any one of embodiments 71 to 76, the isolated host cell according to embodiment 85 or the oncolytic virus according to embodiment 86, and medicine Acceptable carrier. Embodiment 88. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition as in Embodiment 87. Embodiment 89. The method of embodiment 88, wherein the pharmaceutical composition is administered to the individual intravenously or intratumorally. Embodiment 90. The method of embodiment 88 or 89, wherein the system is human. Embodiment 91. A pharmaceutical composition comprising: a first OV comprising a first nucleic acid encoding an immune checkpoint regulator; a second OV comprising a second nucleic acid encoding a bispecific molecule, the bispecific molecule comprising specific The first antigen-binding domain that sexually recognizes tumor antigens and the second antigen-binding domain that specifically recognizes cell surface molecules on effector cells; and a pharmaceutically acceptable carrier. Embodiment 92. The pharmaceutical composition as in embodiment 91, comprising: a first OV comprising a first nucleic acid encoding the immune checkpoint regulator of any one of embodiments 20 to 36, 52, 54 and 55; comprising The second OV of the second nucleic acid encoding the bispecific molecule of any one of Examples 20, 37 to 51, and 53 to 55; and a pharmaceutically acceptable carrier. Embodiment 93. A pharmaceutical composition comprising: a first OV including a first nucleic acid encoding an immune checkpoint regulator, a second OV including a second nucleic acid encoding an interleukin, and a pharmaceutically acceptable carrier . Embodiment 94. The pharmaceutical composition as in embodiment 93, comprising: a first OV comprising a first nucleic acid encoding the immune checkpoint regulator of any one of embodiments 20 to 36, 52, 54 and 55; comprising The second OV encoding the second nucleic acid as in Example 56 or 57 interleukin; and a pharmaceutically acceptable carrier. Embodiment 95. A pharmaceutical composition comprising: a first OV comprising a first nucleic acid encoding an immune checkpoint regulator; a second OV comprising a second nucleic acid encoding a bispecific molecule, the bispecific molecule comprising specific The first antigen-binding domain that sexually recognizes tumor antigens and the second antigen-binding domain that specifically recognizes cell surface molecules on effector cells; a third OV that includes a third nucleic acid encoding an interleukin; and a pharmaceutically acceptable The carrier. Embodiment 96. The pharmaceutical composition as in embodiment 95, comprising: a first OV comprising a first nucleic acid encoding the immune checkpoint regulator of any one of embodiments 20 to 36, 52, 54 and 55; comprising The second OV encoding the second nucleic acid of the bispecific molecule according to any one of embodiments 20, 37 to 51 and 53 to 55; the third OV including the third nucleic acid encoding the interleukin according to embodiment 56 or 57 ; And pharmaceutically acceptable carriers. Embodiment 97. A method of treating cancer in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition according to any one of embodiments 91 to 96. Embodiment 98. A method of treating cancer in an individual, comprising administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a first nucleic acid comprising a first nucleic acid encoding an immune checkpoint regulator OV and a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition comprising a second OV containing a second nucleic acid encoding a bispecific molecule and a pharmaceutically acceptable For the second carrier, the bispecific molecule includes a first antigen binding domain that specifically recognizes a tumor antigen and a second antigen binding domain that specifically recognizes a cell surface molecule on an effector cell. Embodiment 99. The method as in embodiment 98, which comprises administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a code such as in embodiments 20 to 36, 52, 54 and 55 The first OV of the first nucleic acid of the immune checkpoint regulator of any one, and a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition including The second OV of the second nucleic acid of the bispecific molecule of any one of Examples 20, 37 to 51 and 53 to 55, and a pharmaceutically acceptable second carrier. Embodiment 100. A method of treating cancer in an individual, comprising administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a first nucleic acid comprising a first nucleic acid encoding an immune checkpoint regulator OV, and a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition including a second OV containing a second nucleic acid encoding an interleukin, and a pharmaceutically acceptable Accepted second carrier. Embodiment 101. The method as in embodiment 100, which comprises administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a code such as in embodiments 20 to 36, 52, 54 and 55 The first OV of the first nucleic acid of the immune checkpoint regulator of any one, and a pharmaceutically acceptable first carrier; and an effective amount of a second pharmaceutical composition, the second pharmaceutical composition including The second OV of the second nucleic acid of the interleukin of Example 56 or 57 and the second pharmaceutically acceptable carrier. Embodiment 102. A method of treating cancer in an individual, comprising administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition including a first OV including a first nucleic acid encoding an immune checkpoint regulator , And a pharmaceutically acceptable first carrier; an effective amount of a second pharmaceutical composition, the second pharmaceutical composition including a second OV containing a second nucleic acid encoding a bispecific molecule, and a pharmaceutically acceptable A second carrier, the bispecific molecule includes a first antigen binding domain that specifically recognizes a tumor antigen and a second antigen binding domain that specifically recognizes a cell surface molecule on an effector cell; and an effective amount of a third medicine The composition, the third pharmaceutical composition includes a third OV including a third nucleic acid encoding cytokine, and a pharmaceutically acceptable third carrier. Embodiment 103. The method as in embodiment 102, which comprises administering to the individual: an effective amount of a first pharmaceutical composition, the first pharmaceutical composition comprising a code such as in embodiments 20 to 36, 52, 54 and 55 The first OV of the first nucleic acid of any one of the immune checkpoint regulators, and a pharmaceutically acceptable first carrier; an effective amount of a second pharmaceutical composition, the second pharmaceutical composition including The second OV of the second nucleic acid of the bispecific molecule of any of Examples 20, 37 to 51 and 53 to 55, and a pharmaceutically acceptable second carrier; and an effective amount of the third pharmaceutical composition, The third pharmaceutical composition includes a third OV containing a third nucleic acid encoding the interleukin as in Example 56 or 57, and a pharmaceutically acceptable third carrier. Examples The following examples are only intended to be examples of the invention, and therefore should not be considered as limiting the invention in any way. The following examples and detailed description are provided by way of illustration and not by way of limitation. In the present invention, oncolytic vaccinia virus (hereinafter referred to as "PD1-Ig-VV") expressing recombinant PD1-IgG4-Fc fusion is used to block PD1 ligand on cancer cells and PD1 receptor on T cells The co-suppression interaction between them, thereby enhancing the anti-tumor immune response. Production of PD1-IgG4-Fc recombinant protein (hereinafter referred to as "PD1-Ig"), in which the extracellular domain of PD1 is fused to the constant (Fc) domain of immunoglobulin G4, effectively generating the relationship between PD1 and its ligand Interacting inhibitors. Example 1: Construction of PD1-Ig-VV, GPC3-CD3-VV, FAP-CD3-VV, and PD1-Ig-FAP-CD3-VV to produce oncolytic vaccinia virus (VV) construct PD1-Ig-VV to express Recombinant protein of fusion of PD-1 extracellular domain and constant (Fc) domain of immunoglobulin G4 (IgG4). The PD-1 extracellular domain contains the amino acid sequence SEQ ID NO: 25. GPC3-CD3 (hereinafter also referred to as GPC3-TE (GPC3 T cell zygote)) targets hepatocellular carcinoma (HCC) tumor antigen phosphoinosin-3 (GPC3) and CD3 on T cells Bispecific molecules. The oncolytic VV construct GPC3-CD3-VV (also referred to as GPC3-TEA-VV hereinafter) was generated to express secreted GPC3-scFv-human CD3-scFv (GPC3-CD3). FAP-CD3 (hereinafter also referred to as FAP-TE) is a bispecific molecule that targets fibroblast activation protein (FAP) antigen on cancer-associated fibroblasts and CD3 on T cells. Oncolytic VV construct FAP-CD3-VV (also referred to as FAP-TEA-VV hereinafter) was generated to express secreted FAP-scFv-human CD3-scFv (FAP-CD3 or FAP-TE). Produce oncolytic VV construct PD1-Ig-FAP-CD3-VV (hereinafter also referred to as PD1-Ig-FAP-TEA-VV (PD1-Ig-FAP-T cell zygote VV)) to co-express PD1-IgG4-Fc and secreted FAP-scFv-human CD3-scFv (FAP-CD3). The nucleic acid sequences encoding PD1-IgG4-Fc and FAP-CD3 were linked by a T2A self-cleavage sequence. The anti-FAP scFv comprises: a heavy chain variable region (VH) including (1) HVR-H1 containing the amino acid sequence SEQ ID NO: 36; (2) amino acid containing sequence SEQ ID NO : 37 of HVR-H2; and (3) amino acid sequence SEQ ID NO: 38 of HVR-H3; and light chain variable region (VL), the light chain variable region includes (1) amino acid HVR-L1 of sequence SEQ ID NO: 39; (2) HVR-L2 containing the amino acid sequence SEQ ID NO: 40; and (3) HVR-L3 containing the amino acid sequence SEQ ID NO: 41. The anti-FAP scFv includes VH containing the amino acid sequence SEQ ID NO: 42 and VL containing the amino acid sequence SEQ ID NO: 43. The anti-FAP scFv contains the amino acid sequence SEQ ID NO: 44. The FAP-CD3 bispecific molecule contains the amino acid sequence SEQ ID NO: 45. The co-presented PD1-IgG4-Fc includes the PD-1 extracellular domain containing the amino acid sequence SEQ ID NO: 25. The code was generated by recombining the pSEM-1 plastid form containing PD1-Ig, T cell zygote (TE) or PD1-Ig and FAP-CD3 into the TK gene of the VSC20 strain of WR Vaccinia virus (WR VV) Secreted PD1-IgG4-Fc (PD1-Ig-VV), GPC3-CD3 (GPC3-CD3-VV or GPC3-TEA-VV), FAP-CD3 (FAP-CD3-VV or FAP-TEA-VV) or a total of Vaccinia virus (Western Reserve strain) expressing secreted PD1-IgG4-Fc and FAP-CD3 (PD1-Ig-FAP-CD3-VV or PD1-Ig-FAP-TEA-VV). First, the shuttle vector pSEM-1 containing PD1-Ig, T cell zygote, or both PD1-Ig and FAP-CD3 was constructed (Figure 1). The inserted PD1-Ig, TE or both PD1-Ig and FAP-CD3 are expressed under the transcriptional control of the F17R late promoter to allow sufficient viral replication before T cell activation. These VVs also exhibit YFP-GFP markers to allow virus selection. YFP-GFP was expressed under the transcriptional control of the P7.5 promoter, and the loxP site was flanked by the selectable marker YFP-GFP in the same orientation (Figure 1). To construct recombinant viruses encoding PD1-Ig, TE or both PD1-Ig and FAP-CD3, the shuttle vector pSEM-1 was first transfected into human 143 TK cells. The cells were then infected with the virus VSC20 at an infection rate (MOI) of 0.1. After five rounds of plaque selection and amplification to confirm the performance of PD1-Ig, TE or both PD1-Ig and FAP-CD3, a pure line was selected for amplification and purification. In order to remove the YFP-GFP cassette from the recombinant virus, the virus was passaged on the U2OS cell line (U2OS-Cre) expressing the cytoplasmic form of Cre recombinase. After five rounds of plaque selection and amplification to confirm the performance of PD1-Ig, TE or both PD1-Ig and FAP-CD3, a YFP-GFP negative pure line was selected for amplification and purification. Example 2: Expression of vaccinia virus encoding PD1-Ig and testing its binding to PD-L1 on Huh7 cells To test PD1-Ig-VV performance of PD1-Ig, collect PD1-Ig-VV (as described in Example 1 ) Or VV-GFP control (Vaccinia virus encoding GFP) infected human osteosarcoma 143 TK cells and the supernatant was analyzed by Western blot method. The PD1-Ig was detected to be 85-90 KDa (Figure 2A). To test the binding of PD1-Ig expressed by VV to the PD-L1 ligand on Huh7 cells, Huh7 cells were incubated with supernatant from human 143 TK cells infected with VV-GFP or PD1-Ig-VV for two hours. FACS analysis of cells against APC-anti-Fc was performed to detect PD1-IgG4-Fc (for FACS protocol, see below). Huh7 cells incubated with PD1-Ig supernatant showed an increase in Fc positive cells when compared to cells incubated with GFP, indicating that PD1-Ig binds to PD-L1 on Huh7 cells (Figure 2B).FACS Perform FACS according to the general protocol. Briefly, cells were collected and washed once with PBS containing 1% FBS (Sigma, St. Louis, MO; FACS buffer), and then antibodies were added. The cells were then incubated in the dark for 30 minutes on ice, washed once, and incubated with secondary antibodies to luciferin (if applicable) in the dark for 30 minutes, and then washed once. The washed cells were then fixed in 0.5% paraformaldehyde / FACS buffer and then analyzed. For each sample, 10,000 cells were analyzed using the FACSCalibur instrument (BD, Becton Dickinson, Mountain View, CA), using Cell Quest software (Becton Dickinson) or using FCS Express software (De Novo Software, Los Angeles, CA). Example 3: PD1-Ig enhances GPC3-CD3-dependent Huh7-GFP cell killing mediated by activated T cells. To test the ability of PD1-Ig to enhance the anti-tumor effect of bispecific T cell zygote, PD1-Ig and GPC3 -CD3, which is a bispecific molecule targeting hepatic carcinoma (HCC) tumor antigen phospholipid inositol-3 (GPC3) and CD3 on T cells (GPC3-scFv-CD3-scFv; see examples for construction 1) Perform together. Infected with empty virus (-GPC3-CD3), vaccinia virus expressing GPC3-CD3 (+ GPC3-CD3) or infected with vaccinia virus encoding GPC3-CD3 and vaccinia virus encoding PD1-Ig (GPC3-CD3 + PD1-Ig) Huh7-GFP cells were co-infected (Figure 3A). Tumor cells were infected with MOI 1 with VV in 2.5% FBS medium for two hours, and then cultured in complete medium. Incubate unstimulated human PBMC at 37 ° C (see below for protocol) for two hours to remove adherent cells and the non-adherent PBMC with an effector: target (E: T) ratio of 1: 1 (Figure 3A, top panel) ) Or 5: 1 (Figure 3A lower panel) was added to Huh7-GFP cell culture. As described in Example 2, cells were sorted against CD3-APC and Huh7-GFP using FACS, and Huh7 cell viability was evaluated by GFP.Peripheral blood mononuclear cells ( PBMC ) To generate PBMC groups, blood samples were obtained from healthy donors according to a protocol approved by the Institutional Review Board of Baylor College Medicine. The peripheral blood was Ficoll gradient treated, and the resulting PBMC was supplemented with 10% heat-inactivated FCS and 2 mmol / L GLUTAMAX at the Roswell Park Memorial Institute (Roswell Park Memorial Institute) 1640 (Thermo Scientific HyClone, Waltham, MA; Lonza , Basel, Switzerland). As can be seen from FIG. 3A, when compared with the negative control cell (-GPC3-CD3), the number of surviving Huh7-GFP cancer cells exhibiting GPC3-CD3 was reduced at two E: T ratios. In Huh7 cells expressing both PD1-Ig and GPC3-CD3 (GPC3-CD3 + PD1-Ig), the GFP signal was further attenuated. This indicates that PD1-Ig enhances GPC3-CD3-mediated Huh7 tumor cell lysis in the presence of T cells, and the effect is further enhanced at a higher E: T ratio (compare FIG. 3A lower panel with upper panel). Examination using fluorescent microscopy also observed an increase in PD1-Ig-induced tumor lysis and a decrease in GFP signal in the presence of T cells (Figure 3B). To further illustrate that PD1-Ig can enhance GPC3-CD3-mediated tumor cell killing in the presence of T cells, FACS mapping was used to analyze apoptosis markers phospholipid binding protein-V and propidium iodide (PI) Huh7 cells infected with VV. Briefly, VV-infected Huh7 cells co-cultured with PBMC at 5: 1 E: T were collected and incubated with recombinant phospholipid binding protein-V. Immediately before analysis by flow cytometry, PI was added to all samples. Cells were analyzed for phospholipid-binding protein-V binding and PI uptake, and the percentage of cells that were phospholipid-binding protein-V positive, PI positive, or double positive was indicated (Figure 3C). Huh7 cells infected with the control virus (-GPC3-CD3) only had 8.94% PI / phospholipid binding protein-V double positive cells (Figure 3C left panel), while cells expressing GPC3-CD3 had 24.1% double positive cells (Figure 3C middle picture). Co-infection with VV encoding PD1-Ig and VV encoding GPC3-CD3 increased the percentage of phospholipid-binding protein-V / PI double positive Huh7 tumor cells to 44.2% (Figure 3C right panel), indicating increased tumor lysis. These data indicate that co-expression of PD1-Ig in Huh7 human liver cancer cells can enhance GPC3-CD3-mediated tumor lysis in the presence of T cells. Example 4: PD1-Ig does not affect T cell phenotype in response to GPC3-CD3 To characterize the effect of PD1-Ig on T cells, unstimulated human PBMC (1 × 106 Cells / well) cultured alone or with VV-infected Huh7 cells (0.4 × 106 Cells / well) were incubated together in a 24-well dish for two days. The cells were then collected and stained with luciferin-binding antibodies against cell surface markers CD3 and CD69 (CD69 T cell activation markers) (Figure 4A) or CD45RA and CCR7 (Figure 4B), followed by FACS analysis, as in Example 2 Described. The percentage of cell population is shown. Antibodies against CD45RA and CCR7 (markers used to distinguish between CD4 + and CD8 + T cells) were used as negative controls because PD-1 is mainly expressed on CD4- / CD8-negative T cells (Figure 4B). Incubate PBMC alone (Figure 4A and 4B, left panel) or use empty virus (second panel from left, top and bottom, -GPC3-CD3), and use VV encoding GPC3-CD3 (second panel from right) , GPC3-CD3 / medium), or co-infected Huh7 tumor cells co-infected with vaccinia virus encoding GPC3-CD3 and vaccinia virus encoding PD1-Ig (right, GPC3-CD3 / PD1-Ig). When PBMC were co-cultured with Huh7 cells expressing GPC3-CD3, CD3 + / CD69 + cells increased significantly to 57.9% (Figure 4A). The percentage of double positive cells in PBMC co-cultured with Huh7 cells expressing both GPC3-CD3 and PD1-Ig increased slightly to 59.7% (Figure 4A, right panel). Therefore, FACS detection of CD69 (a lymphatic activation antigen) showed a significant increase in T cell activation in PBMC incubated with GPC3-CD3 expressing Huh7 cells; however, after adding PD1-Ig, this number did not increase significantly, indicating that PD1-Ig does not affect the T cell phenotype. Example 5: PD1-Ig enhances the GPC3-CD3 dependent increase of T cell cytokine production. To investigate whether the presence of PD1-Ig increases T cell cytokine production, PBMC was expressed with GPC3-CD3 only or GPC3- CD3 and PD1-Ig Huh7 cells were co-cultured. For co-expression of GPC3-CD3 and PD1-Ig in Huh7 cells, Huh7 cells were co-infected with VV encoding GPC3-CD3 (GPC3-CD3-VV) and VV encoding PD1-Ig (PD1-Ig-VV). Infect Huh7 cells with MOI 1 with VV, add PBMC as described above, and collect the cell culture 24 hours to 48 hours after virus infection and analyze the pro-inflammatory cytokine IFNγ using enzyme-linked immunosorbent assay (ELISA) , The presence of TNFα and IL-2 (Figures 5A, 5B and 5C, respectively). When compared to T cells cultured with Huh7 infected with control virus, T cells cultured with GPC3-CD3 expressing Huh7 cells produced higher amounts of IFNγ, TNFα, and IL-2 (Figures 5A to 5C, respectively, " "Media" relative to "-GPC3-CD3"). When T cells were cultured with Huh7 cells expressing both GPC3-CD3 and PD1-Ig, the release of IFNγ, TNFα, and IL-2 was further increased (Figures 5A to 5C, "PD1-Ig", respectively). The release amount of IFNγ, TNFα and IL-2 in the presence of GPC3-CD3 and PD1-Ig is even greater than in the presence of GPC3-CD3 and anti-PD-1 antibody (Figures 5A to 5C, "αPD1", respectively; catalog number 10377- mhT28-200, Sino Biological) or in the presence of GPC3-CD3 and anti-PD-L1 antibodies (Figures 5A to 5C, "αPD-L1"; Clone29E.2A3, BXCell), respectively. This indicates that PD1-Ig can enhance the ability of GPC3-CD3 to induce T cells to release cytokines. Example 6: PD1-Ig-VV inhibits SK-Br-3 breast cancer tumor growth in vivo. The in vivo efficacy of PD1-Ig-VV was evaluated in a mouse xenograft model implanted with human tumor cells. In order to establish a mouse xenograft model of breast cancer, the 4 × 106 SK-Br-3 human breast cancer cells were inoculated subcutaneously into the right flank of NSG mice. Then on the 8th day, the PBS, 1 × 108 Pfu control VV or PD1-Ig-VV was injected into the right abdominal tumor and implanted 2 × 10 intravenously on the 11th day7 Unactivated human PBMC cells (Figure 6A). Mice receiving only PBS / PBMC served as controls. In tumor volume analysis, mice receiving PBS / PBMC produced approximately 3600 mm by day 213 The tumor volume (Figure 6B and Figure 6C left panel). Mice receiving control VV had a moderate decrease in tumor volume in the presence of PBMC (Figure 6B and 6C middle panel). PD1-Ig-VV-injected mice showed a significant reduction in tumor volume in the presence of PBMC on day 21 (Figure 6B and Figure 6C right panel), indicating that PD1-Ig-VV can inhibit SK-Br-3 breast cancer in vivo Tumor growth. Example 7: PD1-Ig-VV inhibits the growth of HT-29 colorectal adenocarcinoma tumors in vivo . In order to build this model, the 4 × 106 HT-29 colorectal adenocarcinoma cells were inoculated subcutaneously into the right abdomen of NSG mice, then on the 8th day, PBS, 1 × 108 Pfu control VV or PD1-Ig-VV was injected into the right abdominal tumor, followed by intravenous implantation of 2 × 10 on day 117 Unactivated human PBMC cells (Figure 7A). Examination of tumor volume over the course of 24 days showed that control mice injected with PBS only or PBS in the presence of PBMC produced an average of approximately 1000 mm on day 243 Tumor (Figure 7B). Tumor volume of mice injected with control virus GFP-VV and implanted with PBMC was slightly reduced (average about 350 mm3 ). However, mice injected with PD1-Ig-VV and implanted with PBMC showed significant inhibition of tumor growth, and the average tumor size was close to 0 mm3 . These results indicate that PD1-Ig-VV can strongly inhibit tumor growth in vivo in various mouse xenograft models. Example 8: Vaccinia virus co-expressing PD1-Ig and FAP-CD3 and its binding to PD-L1 and FAP on U87 cells To test PD1-Ig-FAP-CD3-VV co-expression PD1-Ig and FAP- CD3, collect human osteosarcoma 143 TK cells infected with PD1-Ig-FAP-CD3-VV (as described in Example 1) or VV-GFP control (VV encoding GFP) and analyze the supernatant by Western blot analysis liquid. To test the binding of PD1-Ig expressed by VV to PD-L1 ligand on U87 (human glioblastoma) cells, U87 cells were combined with human 143 from VV-GFP or PD1-Ig-FAP-CD3-VV The supernatants of TK cells were incubated together for two hours. FACS analysis of cells against APC-anti-Fc was performed to detect PD1-IgG4-Fc (see Example 2 for FACS protocol). When compared to cells incubated with GFP supernatant, U87 cells incubated with PD1-Ig supernatant are expected to show an increase in Fc-positive cells, indicating that PD1-Ig binds to PD-L1 on U87 cells. To test the binding of FAP CD3 expressed by VV to FAP antigen on FAP positive U87 cells, U87 cells were incubated with supernatant from human 143 TK cells infected with VV-GFP or PD1-Ig-FAP-CD3-VV for two hours . The cells were subjected to FACS analysis against anti-CD3 scFv antibodies to detect FAP-CD3 (FAP-TE) (see Example 2 for FACS protocol). When compared to cells incubated with GFP supernatant, U87 cells incubated with FAP-CD3 supernatant are expected to show an increase in FAP-CD3 positive cells, indicating that FAP-CD3 binds to FAP on U87 cells. Example 9: PD1-Ig enhances the activation of FAP-CD3-dependent U87 cell killing mediated by activated T cells. To test the ability of PD1-Ig to enhance the antitumor effect of bispecific T cell zygote, an empty virus, encoding only FAP- Vaccinia virus (FAP-CD3-VV) of CD3 or VV (PD1-Ig-FAP-CD3-VV) co-expressing FAP-CD3 and PD1-Ig infected U87-GFP cells. For construction, see Example 1. Tumor cells were infected with MOI 1 with VV in 2.5% FBS medium for two hours, and then cultured in complete medium. Incubate the unstimulated human PBMC (see Example 3 for the protocol for two hours) at 37 ° C to remove adherent cells, and the non-adherent PBMC with 1: 1 or 5: 1 effector: target (E: T ) Ratio is added to U87-GFP cell culture. As described in Example 2, cells were sorted against CD3-APC and GFP using FACS, and U87-GFP cell viability was evaluated by GFP. It is expected that the number of surviving U87-GFP cancer cells showing FAP-CD3 at two E: T ratios is reduced when compared to negative control cells (empty virus). It is expected that the GFP signal will be further attenuated in U87-GFP cells that jointly express PD1-Ig and FAP-CD3 (PD1-Ig-FAP-CD3-VV). This indicates that co-expression of PD1-Ig and FAP-CD3 can enhance FAP-CD3-mediated lysis of U87 tumor cells in the presence of T cells, and this effect is expected to be further enhanced at higher E: T ratios. Fluorescence microscopy also observed an increase in PD1-Ig-induced tumor lysis and a decrease in GFP signal in the presence of T cells. It is expected that U87-GFP cells infected with PD1-Ig-FAP-CD3-VV have the weakest GFP signal compared to cells infected with FAP-CD3-VV or empty virus. To further illustrate that co-expression of PD1-Ig and FAP-CD3 can enhance FAP-CD3 mediated tumor cell killing in the presence of T cells, FACS mapping was used, using apoptosis markers phospholipid binding protein-V and iodination Propidium (PI) analysis of U87 cells infected with VV. Briefly, VV-infected U87 cells co-cultured with PBMC at 5: 1 E: T were collected and incubated with recombinant phospholipid binding protein-V. Immediately before analysis by flow cytometry, PI was added to all samples. Analyze the phospholipid-binding protein-V binding and PI uptake in the cells, and indicate the percentage of cells that are phospholipid-binding protein-V positive, PI positive, or double positive. The U87 cells infected with the control virus are expected to have the lowest percentage of PI / phospholipid binding protein-V double positive cells, while the cells infected with FAP-CD3-VV have moderate double positive cells. It is expected that PD1-Ig and FAP-CD3 (PD1-Ig-FAP-CD3-VV) will increase the percentage of phospholipid-binding protein-V / PI dual positive tumor cells to a higher level, indicating increased tumor lysis. These data can indicate that co-expression of PD1-Ig and FAP-CD3 in U87 tumor cells can enhance FAP-CD3-mediated tumor lysis in the presence of T cells. Example 10: Co-expression of PD1-Ig and FAP-CD3 does not affect the T cell phenotype. To characterize the co-expression of PD1-Ig and FAP-CD3 (PD1-Ig-FAP-CD3-VV) on T cells, it will not be stimulated Human PBMC (1 × 106 Cells / well) cultured alone or with VV-infected U87 cells (0.4 × 106 Cells / well) were incubated together in a 24-well dish for two days. Cells were then collected and stained with luciferin-binding antibodies against cell surface markers CD3 and CD69 (CD69 T cell activation markers) or CD45RA and CCR7 (markers used to distinguish CD4 + and CD8 + T cells), followed by FACS analysis , As described in Example 2. Antibodies against CD45RA and CCR7 were used as negative controls because PD-1 is mainly expressed on CD4 / CD8 negative T cells. PBMC were cultured alone or with U87 cells infected with empty virus, VV encoding FAP-CD3 (FAP-CD3-VV), or VV expressing FAP-CD3 and PD1-Ig (PD1-Ig-FAP-CD3-VV) to cultivate. When PBMC were co-cultured with U87 cells expressing FAP-CD3, CD3 + / CD69 + cells were expected to increase significantly. It is expected that the percentage of double positive cells in PBMC co-cultured with U87 cells co-expressing both FAP-CD3 and PD1-Ig (PD1-Ig-FAP-CD3-VV) is similar to the situation when only expressing FAP-CD3 . Therefore, FACS detection of CD69 (a lymphatic activation antigen) is expected to show a significant increase in T cell activation in PBMCs incubated with FAP-CD3 expressing U87 cells; and when PD1-Ig and FAP-CD3 are co-expressed, this number is not No significant increase, indicating that PD1-Ig may not affect the T cell phenotype. Example 11: PD1-Ig enhances the FAP-CD3 dependent increase of cytokines produced by T cells U87 cells of FAP-CD3-VV, empty virus or FAP-CD3-VV, or U87 cells treated with anti-PD-1 or anti-PD-L1 antibodies were co-cultured. Infect U87 cells with VV at MOI 1, add PBMC as described above, and collect cell cultures 24 to 48 hours after virus infection and analyze the presence of proinflammatory interleukins IFNγ, TNFα, and IL-2 using ELISA . When compared to T cells cultured with cancer cells infected with control viruses, T cells cultured with U87 cells infected with FAP-CD3-VV are expected to produce higher levels of IFNγ, TNFα, and IL-2. It is expected that when T cells are cultured together with U87 cells co-expressing FAP3-CD3 and PD1-Ig (PD1-Ig-FAP-CD3-VV), the release amount of IFNγ, TNFα and IL-2 will further increase. The amount of IFNγ, TNFα and IL-2 released in the presence of FAP-CD3 and PD1-Ig and FAP-CD3 and anti-PD-1 antibody (Cat. No. 10377-mhT28-200, Sino Biological) or FAP-CD3 and anti- The release amount of PD-L1 antibody (Clone 29E.2A3, BXCell) was compared. These results can indicate that the use of PD1-Ig-FAP-CD3-VV, showing that PD1-Ig and FAP-CD3 can enhance the ability of FAP-CD3 to induce T cells to release interleukins. Example 12: PD1-Ig-FAP-CD3-VV inhibits U87 cancer tumor growth in vivo The in vivo efficacy of PD1-Ig-FAP-CD3-VV was evaluated in a mouse xenograft model implanted with U87 human cancer cells. In order to establish a mouse xenograft model of U87 cancer, 4 × 106 U87 human cancer cells were inoculated subcutaneously into the right flank of NSG mice. Then on the 8th day, the PBS, 1 × 108 pfu control VV, PD1-Ig-VV, FAP-CD3-VV or PD1-Ig-FAP-CD3-VV was injected into the right abdominal tumor and implanted 2 × 10 intravenously on the 11th day7 An unactivated human PBMC cell. Mice receiving only PBS / PBMC served as controls. In the analysis of tumor volume on day 21, the mice receiving only PBS / PBMC were expected to produce the tumor with the highest volume. Mice receiving control VV are expected to have a moderate decrease in tumor volume in the presence of PBMC. Mice injected with PD1-Ig-VV or FAP-CD3-VV are expected to show an even smaller tumor volume in the presence of PBMC at 21 days, while mice injected with PD1-Ig-FAP-CD3-VV may be due to tumor matrix The destruction has the smallest tumor volume. These results can indicate that co-expression of PD1-Ig and FAP-CD3 can inhibit U87 tumor growth in vivo, and this inhibitory effect may even be superior to PD1-Ig-VV or FAP-CD3-VV alone. Sequence Listing SEQ ID NO: 1 (1H7e3 HVR-H1 amino acid sequence)SEQ ID NO: 2 (1H7e3 HVR-H2 amino acid sequence)SEQ ID NO: 3 (1H7e3 HVR-H3 amino acid sequence)SEQ ID NO: 4 (1H7e3 HVR-L1 amino acid sequence)SEQ ID NO: 5 (1H7e3 HVR-L2 amino acid sequence)SEQ ID NO: 6 (1H7e3 HVR-L3 amino acid sequence)SEQ ID NO: 7 (4F11C3 HVR-H1 amino acid sequence)SEQ ID NO: 8 (4F11C3 HVR-H2 amino acid sequence)SEQ ID NO: 9 (4F11C3 HVR-H3 amino acid sequence)SEQ ID NO: 10 (4F11C3 HVR-: L1 amino acid sequence)SEQ ID NO: 11 (4F11C3 HVR-L2 amino acid sequence)SEQ ID NO: 12 (4F11C3 HVR-L3 amino acid sequence)SEQ ID NO: 13 (1H7e3 VH amino acid sequence: HVR underlined)SEQ ID NO: 14 (1H7e3 VL amino acid sequence: HVR underlined)SEQ ID NO: 15 (4F11C3 VH amino acid sequence: HVR underlined)SEQ ID NO: 16 (4F11C3 VL amino acid sequence: HVR underlined)SEQ ID NO: 17 (nucleic acid encoding anti-PD-1 1H7e3 VH: HVR sequence is underlined)SEQ ID NO: 18 (nucleic acid encoding anti-PD-1 1H7e3 VL: HVR sequence is underlined)SEQ ID NO: 19 (nucleic acid encoding anti-PD-1 4F11C3 VH: HVR sequence is underlined)SEQ ID NO: 20 (nucleic acid encoding anti-PD-1 4F11C3 VL: HVR sequence is underlined)SEQ ID NO: 21 (amino acid sequence of 1H7e3, 4F11C3 VH signal peptide)SEQ ID NO: 22 (nucleic acid encoding 1H7e3, 4F11C3 VH signal peptide)SEQ ID NO: 23 (amino acid sequence of 1H7e3, 4F11C3 VL signal peptide)SEQ ID NO: 24 (nucleic acid encoding 1H7e3, 4F11C3 VL signal peptide)SEQ ID NO: 25 (PD-1 extracellular domain amino acid sequence)SEQ ID NO: 26 (PD-1 extracellular domain nucleic acid sequence)SEQ ID NO: 27 (TMIGD2 extracellular domain amino acid sequence)SEQ ID NO: 28 (TMIGD2 extracellular domain-Fc fusion protein signal peptide amino acid sequence)SEQ ID NO: 29 (SIRPα extracellular domain amino acid sequence)SEQ ID NO: 30 (CXCL12 N-terminal sequence)SEQ ID NO: 31 (amino acid sequence of the linker (IgG1 hinge) between SIRPα extracellular domain and CXCL12)SEQ ID NO: 32 (signal peptide amino acid sequence of SIRPα-CXCL12-Fc fusion protein)SEQ ID NO: 33 (VH-VL linker amino acid sequence)SEQ ID NO: 34 (VH-VL linker amino acid sequence)SEQ ID NO: 35 (scavv-scFv linker amino acid sequence)SEQ ID NO: 36 (FAP HVR-H1 amino acid sequence)SEQ ID NO: 37 (FAP HVR-H2 amino acid sequence)SEQ ID NO: 38 (FAP HVR-H3 amino acid sequence)SEQ ID NO: 39 (FAP HVR-L1 amino acid sequence)SEQ ID NO: 40 (FAP HVR-L2 amino acid sequence)SEQ ID NO: 41 (FAP HVR-L3 amino acid sequence)SEQ ID NO: 42 (FAP VH amino acid sequence: HVR sequence is underlined)SEQ ID NO: 43 (FAP VL amino acid sequence: HVR sequence is underlined)SEQ ID NO: 44 (FAP scFv amino acid sequence: HVR sequence is underlined, linker sequence is bold)SEQ ID NO: 45 (full-length FAP-human CD3 TE amino acid sequence: HVR sequence is underlined, VH-VL linker sequence is bold, scFv-scFv linker sequence is bold and italic)

圖1描繪編碼PD1(細胞外結構域)-IgG4-Fc之表現卡匣PD1-Ig-VV、編碼雙特異性接合分子GPC3-CD3(GPC3-TE)之表現卡匣GPC3-TEA-VV、編碼雙特異性接合分子FAP-CD3(FAP-TE)之表現卡匣FAP-TEA-VV以及共編碼雙特異性接合分子FAP-CD3(FAP-TE)及PD1-IgG4-Fc之表現卡匣PD1-Ig-FAP-TEA-VV的例示性方案，所有該等表現卡匣均處於晚期啟動子F17R控制下。在確定PD1-Ig、GPC3-CD3、FAP-CD3或FAP-CD3及PD1-Ig之表現之後，移除YFP-GFP可選擇標記物。 圖2A描繪在感染VV-GFP或PD1-Ig-VV之人類143 TK細胞之上清液中的PD1-Ig之西方墨點(Western blot)。圖2B描繪PD1-Ig成功結合於Huh7腫瘤細胞上。將Huh7細胞與來自感染VV之143 TK細胞之上清液一起培育且藉由FACS及APC-抗Fc評估結合。使用同型對照作為陰性對照。 圖3A-3C描繪PD1-Ig能夠增進T細胞對GPC3-CD3依賴性Huh7-GFP腫瘤細胞之溶解。圖3A描繪存活Huh7-GFP細胞之FACS圖。指示在效應子:標靶比率為1:1(上圖)及5:1(下圖)下CD3+/GFP+雙重陽性細胞之百分含量。圖3B描繪存活Huh7-GFP細胞之免疫螢光分析。圖3C描繪如使用細胞凋亡標記物磷脂結合蛋白-V及碘化丙錠(PI)，藉由FACS分析所量測，經GPC3-CD3-VV及PD1-Ig-VV共感染之Huh7-GFP細胞中細胞溶解增加。 圖4A展示Huh7細胞中PD1-Ig之表現不影響共培養之PBMC細胞中之的T細胞表型。將PBMC單獨培養，或與經GPC3-CD3-VV感染或經GPC3-CD3-VV及PD1-Ig-VV共感染之Huh7細胞一起培養。使用CD3 T細胞標記物及CD69 T細胞活化標記物，藉由FACS分析T細胞。指出CD3+/CD69+雙重陽性細胞之百分含量。圖4B描繪使用CCR7及CD45RA標記物進行之T細胞之陰性對照FACS分析。 圖5A-5C描繪PD1-Ig能夠加強T細胞之細胞介素產生之GPC3-CD3依賴性增加。將PBMC與經GPC3-CD3-VV感染、經GPC3-CD3-VV及PD1-Ig-VV共感染，或經GPC3-CD3-VV感染組合針對PD-1或PD-L1之抗體的Huh7細胞共培養。用MOI為1之VV感染Huh7細胞，添加PBMC且在病毒感染後48小時，收集細胞培養物進行酶聯結免疫吸附劑分析法(ELISA)。ELISA結果顯示與PD1-Ig及GPC3-CD3表現性Huh7細胞共培養的T細胞產生之IFNγ(圖5A)、TNFα(圖5B)及IL-2(圖5C)細胞介素增加。 圖6A-6C描繪PD1-Ig能夠在活體內抑制SK-BR3腫瘤生長。圖6A描繪以下方案：在第0天，將4×106 個SK-BR3細胞植入NSG小鼠之右側腹中，隨後在第8天，腹膜內注射1×108 PFU之病毒或PBS對照且在第11天，靜脈內植入2×107 個PBMC。圖6B描繪在21天過程中小鼠體內的腫瘤體積(mm3 )。圖6C描繪在代表性小鼠中之腫瘤影像。 圖7A-7B描繪PD1-Ig能夠在活體內抑制HT-29腫瘤生長。圖7A描繪以下方案：在第0天，將4×106 個HT-29細胞植入NSG小鼠之右側腹中，隨後在第8天，腹膜內注射1×108 PFU之病毒或PBS對照且在第11天，靜脈內植入2×107 個PBMC。圖7B描繪在24天過程中小鼠體內的腫瘤體積(mm3 )。Figure 1 depicts the performance cassette encoding PD1 (extracellular domain) -IgG4-Fc PD1-Ig-VV, the performance cassette encoding the bispecific junction molecule GPC3-CD3 (GPC3-TE) GPC3-TEA-VV, encoding The performance cassette FAP-TEA-VV of the bispecific junction molecule FAP-CD3 (FAP-TE) and the performance cassette PD1-IgG4-Fc of the co-coding bispecific junction molecule FAP-CD3 (FAP-TE) and PD1-IgG4-Fc In the exemplary scheme of Ig-FAP-TEA-VV, all such performance cassettes are under the control of the late promoter F17R. After determining the performance of PD1-Ig, GPC3-CD3, FAP-CD3 or FAP-CD3 and PD1-Ig, the YFP-GFP selectable marker was removed. Figure 2A depicts Western blot of PD1-Ig in the supernatant of human 143 TK cells infected with VV-GFP or PD1-Ig-VV. Figure 2B depicts the successful binding of PD1-Ig to Huh7 tumor cells. Huh7 cells were incubated with the supernatant from 143 TK cells infected with VV and evaluated for binding by FACS and APC-anti-Fc. Use the isotype control as a negative control. Figures 3A-3C depict that PD1-Ig can enhance T cell lysis of GPC3-CD3-dependent Huh7-GFP tumor cells. Figure 3A depicts a FACS chart of surviving Huh7-GFP cells. Indicates the percentage of CD3 + / GFP + double positive cells at an effector: target ratio of 1: 1 (upper graph) and 5: 1 (lower graph). Figure 3B depicts immunofluorescence analysis of surviving Huh7-GFP cells. Fig. 3C depicts Huh7-GFP co-infected with GPC3-CD3-VV and PD1-Ig-VV as measured by FACS analysis using the apoptosis markers phospholipid binding protein-V and propidium iodide (PI) Cell lysis is increased in the cells. Figure 4A shows that the expression of PD1-Ig in Huh7 cells does not affect the T cell phenotype in co-cultured PBMC cells. PBMC were cultured alone or together with Huh7 cells infected with GPC3-CD3-VV or co-infected with GPC3-CD3-VV and PD1-Ig-VV. T cells were analyzed by FACS using CD3 T cell markers and CD69 T cell activation markers. Point out the percentage of CD3 + / CD69 + double positive cells. Figure 4B depicts a negative control FACS analysis of T cells using CCR7 and CD45RA markers. Figures 5A-5C depict that PD1-Ig can enhance the GPC3-CD3 dependent increase in T cell cytokine production. Co-culture of PBMC with Huh7 cells infected with GPC3-CD3-VV, co-infected with GPC3-CD3-VV and PD1-Ig-VV, or combined with GPC3-CD3-VV infection against antibodies against PD-1 or PD-L1 . Huh7 cells were infected with VV with MOI of 1, PBMC was added and 48 hours after virus infection, cell cultures were collected for enzyme-linked immunosorbent analysis (ELISA). ELISA results showed that T cells co-cultured with PD1-Ig and GPC3-CD3 expressing Huh7 cells produced increased interleukins of IFNγ (Figure 5A), TNFα (Figure 5B) and IL-2 (Figure 5C). 6A-6C depict that PD1-Ig can inhibit SK-BR3 tumor growth in vivo. FIG. 6A depicts the following scheme: on day 0, 4 × 10 6 SK-BR3 cells were implanted into the right abdomen of NSG mice, and then on day 8, 1 × 10 8 PFU virus or PBS control was injected intraperitoneally And on the 11th day, 2 × 10 7 PBMCs were implanted intravenously. Figure 6B depicts the tumor volume (mm 3 ) in mice during 21 days. Figure 6C depicts tumor images in representative mice. 7A-7B depict that PD1-Ig can inhibit HT-29 tumor growth in vivo. Fig. 7A depicts the following scheme: on day 0, 4 × 10 6 HT-29 cells were implanted into the right abdomen of NSG mice, and then on day 8, 1 × 10 8 PFU virus or PBS control was injected intraperitoneally And on the 11th day, 2 × 10 7 PBMCs were implanted intravenously. 7B depicts 24 days in vivo in mouse tumor volume (mm 3).

Claims (35)

一種包含編碼免疫檢查點調節子之核酸的溶瘤牛痘病毒，其中該核酸係可操作地連接至晚期啟動子。An oncolytic vaccinia virus containing a nucleic acid encoding an immune checkpoint regulator, wherein the nucleic acid is operably linked to a late promoter. 如請求項1之溶瘤牛痘病毒，其中該晚期啟動子係選自由以下組成之群：F17R、I2L晚期啟動子、L4R晚期啟動子、P7 . 5k 早期/晚期啟動子、PEL 早期/晚期啟動子、P11k 晚期啟動子、PSEL 合成早期/晚期啟動子，及PSL 合成晚期啟動子。Group as requested item oncolytic 1 of vaccinia virus, wherein the late promoter selected from the group consisting of:. F17R, I2L late promoter, L4R late promoter, P 7 5k early / late promoter, P EL early / late Promoter, P 11k late promoter, P SEL synthetic early / late promoter, and P SL synthetic late promoter. 如請求項1之溶瘤牛痘病毒，其中該溶瘤牛痘病毒係選自由以下組成之群：Elstree、Wyeth、Copenhagen、Tiantan、Tash Kent、Patwadangar、改良型安卡拉牛痘病毒(MVA)、Lister、King、IHD、Evans、USSR及Western Reserve(WR)。The oncolytic vaccinia virus according to claim 1, wherein the oncolytic vaccinia virus is selected from the group consisting of: Elstree, Wyeth, Copenhagen, Tiantan, Tash Kent, Patwadangar, modified Ankara vaccinia virus (MVA), Lister, King, IHD, Evans, USSR and Western Reserve (WR). 如請求項1之溶瘤牛痘病毒，其中該免疫檢查點調節子係刺激性免疫檢查點分子之活化劑。The oncolytic vaccinia virus according to claim 1, wherein the immune checkpoint regulator is an activator of a stimulatory immune checkpoint molecule. 如請求項1之溶瘤牛痘病毒，其中該免疫檢查點調節子係免疫檢查點抑制劑。The oncolytic vaccinia virus according to claim 1, wherein the immune checkpoint regulator is an immune checkpoint inhibitor. 如請求項5之溶瘤牛痘病毒，其中該免疫檢查點抑制劑係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。The oncolytic vaccinia virus according to claim 5, wherein the immune checkpoint inhibitor is PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA- 4. TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitors. 如請求項6之溶瘤牛痘病毒，其中該免疫檢查點抑制劑係PD-1之抑制劑。The oncolytic vaccinia virus according to claim 6, wherein the immune checkpoint inhibitor is an inhibitor of PD-1. 如請求項1之溶瘤牛痘病毒，其中該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。The oncolytic vaccinia virus of claim 1, wherein the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. 如請求項1之溶瘤牛痘病毒，其中該免疫檢查點調節子係結合至該免疫檢查點分子之配體。The oncolytic vaccinia virus of claim 1, wherein the immune checkpoint regulator is bound to the ligand of the immune checkpoint molecule. 如請求項9之溶瘤牛痘病毒，其中該免疫檢查點分子係PD-L1。The oncolytic vaccinia virus according to claim 9, wherein the immune checkpoint molecule is PD-L1. 如請求項10之溶瘤牛痘病毒，其中該免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段的融合物。The oncolytic vaccinia virus according to claim 10, wherein the immune checkpoint regulator comprises a fusion of PD-1 extracellular domain and immunoglobulin Fc fragment. 如請求項1之溶瘤牛痘病毒，其進一步包含編碼細胞介素之第二核酸。The oncolytic vaccinia virus according to claim 1, further comprising a second nucleic acid encoding cytokine. 一種溶瘤病毒，其包含編碼免疫檢查點調節子之第一核酸，及編碼雙特異性分子之第二核酸，該雙特異性分子包含特異性識別腫瘤抗原之第一抗原結合結構域及特異性識別效應細胞上之細胞表面分子的第二抗原結合結構域。An oncolytic virus comprising a first nucleic acid encoding an immune checkpoint regulator and a second nucleic acid encoding a bispecific molecule, the bispecific molecule comprising a first antigen binding domain that specifically recognizes a tumor antigen and specificity Identify the second antigen binding domain of the cell surface molecule on the effector cell. 如請求項13之溶瘤病毒，其中該溶瘤病毒係選自由以下組成之群：牛痘病毒(VV)、塞內加谷病毒(SVV)、腺病毒、單純疱疹病毒1(HSV1)、單純疱疹病毒2(HSV2)、黏液瘤病毒、呼腸孤病毒、脊髓灰白質炎病毒、水泡性口炎病毒(VSV)、麻疹病毒(MV)、慢病毒、反轉錄病毒、麻疹病毒(morbillivirus)、流感病毒、辛德比病毒(Sindbis virus)及新城疫病毒(NDV)。The oncolytic virus of claim 13, wherein the oncolytic virus is selected from the group consisting of vaccinia virus (VV), Senegal valley virus (SVV), adenovirus, herpes simplex virus 1 (HSV1), herpes simplex Virus 2 (HSV2), Myxoma virus, Reovirus, Poliovirus, Vesicular stomatitis virus (VSV), Measles virus (MV), Lentivirus, Retrovirus, Morbillivirus, Influenza Viruses, Sindbis virus and Newcastle disease virus (NDV). 如請求項14之溶瘤病毒，其中該溶瘤病毒係溶瘤牛痘病毒。The oncolytic virus according to claim 14, wherein the oncolytic virus is an oncolytic vaccinia virus. 如請求項13之溶瘤病毒，其中該免疫檢查點調節子係免疫檢查點抑制劑。The oncolytic virus according to claim 13, wherein the immune checkpoint regulator is an immune checkpoint inhibitor. 如請求項16之溶瘤病毒，其中該免疫檢查點抑制劑係PD-1、PD-L1、PD-L2、CD47、CXCR4、CSF1R、LAG-3、TIM-3、HHLA2、BTLA、CTLA-4、TIGIT、VISTA、B7-H4、CD160、2B4或CD73之抑制劑。The oncolytic virus of claim 16, wherein the immune checkpoint inhibitor is PD-1, PD-L1, PD-L2, CD47, CXCR4, CSF1R, LAG-3, TIM-3, HHLA2, BTLA, CTLA-4 , TIGIT, VISTA, B7-H4, CD160, 2B4 or CD73 inhibitors. 如請求項13之溶瘤病毒，其中該免疫檢查點調節子係特異性識別免疫檢查點分子之抗體。The oncolytic virus of claim 13, wherein the immune checkpoint regulator is an antibody that specifically recognizes an immune checkpoint molecule. 如請求項13之溶瘤病毒，其中該免疫檢查點調節子係結合至該免疫檢查點分子之配體。The oncolytic virus of claim 13, wherein the immune checkpoint regulator is bound to the ligand of the immune checkpoint molecule. L1。L1. 如請求項19之溶瘤病毒，其中該免疫檢查點調節子包含PD-1細胞外結構域與免疫球蛋白Fc片段的融合物。The oncolytic virus of claim 19, wherein the immune checkpoint regulator comprises a fusion of the PD-1 extracellular domain and an immunoglobulin Fc fragment. 如請求項13之溶瘤病毒，其中該腫瘤抗原係選自由以下組成之群：EpCAM、FAP、EphA2、HER2、GD2、EGFR、VEGFR2及磷脂醯肌醇蛋白聚糖-3(GPC3)。The oncolytic virus according to claim 13, wherein the tumor antigen is selected from the group consisting of EpCAM, FAP, EphA2, HER2, GD2, EGFR, VEGFR2, and phosphatidylinositol-3 (GPC3). 如請求項13之溶瘤病毒，其中該效應細胞係選自由以下組成之群：T淋巴細胞、B淋巴細胞、自然殺手(NK)細胞、樹突狀細胞(DC)、巨噬細胞、單核細胞、嗜中性球及NKT細胞。The oncolytic virus of claim 13, wherein the effector cell line is selected from the group consisting of T lymphocytes, B lymphocytes, natural killer (NK) cells, dendritic cells (DC), macrophages, and monocytes Cells, neutrophils and NKT cells. 如請求項23之溶瘤病毒，其中該效應細胞係T淋巴細胞。The oncolytic virus of claim 23, wherein the effector cell is a T lymphocyte. 如請求項13之溶瘤病毒，其中該細胞表面分子係選自由以下組成之群：CD3、CD4、CD5、CD8、CD16、CD28、CD40、CD64、CD89、CD134、CD137、NKp46及NKG2D。The oncolytic virus of claim 13, wherein the cell surface molecule is selected from the group consisting of CD3, CD4, CD5, CD8, CD16, CD28, CD40, CD64, CD89, CD134, CD137, NKp46, and NKG2D. 如請求項25之溶瘤病毒，其中該細胞表面分子係CD3。The oncolytic virus according to claim 25, wherein the cell surface molecule is CD3. 如請求項13之溶瘤病毒，其中該第一結合結構域及/或該第二抗原結合結構域係單鏈可變片段(scFv)。The oncolytic virus according to claim 13, wherein the first binding domain and / or the second antigen binding domain are single-chain variable fragments (scFv). 如請求項13之溶瘤病毒，其中該溶瘤病毒進一步包含編碼細胞介素之第三核酸。The oncolytic virus according to claim 13, wherein the oncolytic virus further comprises a third nucleic acid encoding cytokine. 如請求項28之溶瘤病毒，其中該細胞介素係GM-CSF。The oncolytic virus according to claim 28, wherein the interleukin is GM-CSF. 一種醫藥組合物，其包含如請求項1至29中任一項之溶瘤病毒，及醫藥學上可接受之載劑。A pharmaceutical composition comprising the oncolytic virus according to any one of claims 1 to 29, and a pharmaceutically acceptable carrier. 一種如請求項1至29中任一項之溶瘤病毒的用途，其係用於製造供治療個體之癌症用的藥劑。A use of the oncolytic virus according to any one of claims 1 to 29 for the manufacture of a medicament for the treatment of cancer in an individual. 如請求項31之用途，其中該醫藥組合物係全身性投與。The use according to claim 31, wherein the pharmaceutical composition is administered systemically. 如請求項31之用途，其中該醫藥組合物係局部投與。The use according to claim 31, wherein the pharmaceutical composition is administered locally. 如請求項31之用途，其中該癌症係實體腫瘤。The use according to claim 31, wherein the cancer is a solid tumor. 如請求項31之用途，其中該個體係人類。For the purpose of claim 31, the system is human.
TW106130884A 2016-09-09 2017-09-08 Oncolytic virus expressing immune checkpoint modulators TW201825511A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201662385930P 2016-09-09 2016-09-09
US201662385933P 2016-09-09 2016-09-09
US62/385,933 2016-09-09
US62/385,930 2016-09-09

Publications (1)

Publication Number Publication Date
TW201825511A true TW201825511A (en) 2018-07-16

Family

ID=59930791

Family Applications (2)

Application Number Title Priority Date Filing Date
TW106130884A TW201825511A (en) 2016-09-09 2017-09-08 Oncolytic virus expressing immune checkpoint modulators
TW106130883A TW201825674A (en) 2016-09-09 2017-09-08 Oncolytic virus expressing bispecific engager molecules

Family Applications After (1)

Application Number Title Priority Date Filing Date
TW106130883A TW201825674A (en) 2016-09-09 2017-09-08 Oncolytic virus expressing bispecific engager molecules

Country Status (2)

Country Link
TW (2) TW201825511A (en)
WO (2) WO2018049248A1 (en)

Families Citing this family (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2977660A1 (en) 2015-02-25 2016-09-15 Memorial Sloan-Kettering Cancer Center Use of inactivated nonreplicating modified vaccinia virus ankara (mva) as monoimmunotherapy or in combination with immune checkpoint blocking agents for solid tumors
KR20180006916A (en) 2015-04-17 2018-01-19 메모리얼 슬로안-케터링 캔서 센터 Use of MVA or MVA delta E3L as an immunotherapeutic agent for solid tumors
CN116904404A (en) 2016-01-27 2023-10-20 昂克诺斯公司 Oncolytic viral vectors and uses thereof
BR112018016949A2 (en) 2016-02-25 2019-01-08 Memorial Sloan Kettering Cancer Center replication-competent attenuated vaccinia virus as deletion of thymidine kinase with or without expression of human flt3l or gm-csf for cancer immunotherapy
CA3015818A1 (en) 2016-02-25 2017-08-31 Memorial Sloan Kettering Cancer Center Recombinant mva or mva.delta.e3l expressing human flt3l and use thereof as immuno-therapeutic agents against solid tumors
WO2018006005A1 (en) 2016-06-30 2018-01-04 Oncorus, Inc. Pseudotyped oncolytic viral delivery of therapeutic polypeptides
US11242509B2 (en) 2017-05-12 2022-02-08 Memorial Sloan Kettering Cancer Center Vaccinia virus mutants useful for cancer immunotherapy
JP7249323B2 (en) 2017-07-26 2023-03-30 オンコラス， インコーポレイテッド Oncolytic virus vector and use thereof
EP3731850A4 (en) * 2017-12-29 2021-12-01 Oncorus, Inc. Oncolytic viral delivery of therapeutic polypeptides
EP3796891A4 (en) * 2018-04-11 2022-04-20 Precision Molecular Inc. Therapeutic constructs for treating cancer
CN108796123B (en) * 2018-05-04 2022-09-23 中农威特生物科技股份有限公司 Kit for rapidly detecting Seneca virus and detection method thereof
TW202024326A (en) * 2018-07-13 2020-07-01 美國亞利桑那州立大學亞利桑那州評議委員會 Methods of treating cancer with tnf expressing myxoma virus
MX2021001883A (en) * 2018-08-16 2021-07-15 Musc Found For Res Dev Recombinant myxoma viruses and uses thereof.
CA3112788A1 (en) * 2018-09-05 2020-03-12 Arizona Board Of Regents On Behalf Of Arizona State University Oncolytic virus platform to treat hematological cancer
WO2020052551A1 (en) * 2018-09-10 2020-03-19 Genesail Biotech (Shanghai) Co. Ltd. A modified oncolytic virus, composition and use thereof
AU2019337603A1 (en) * 2018-09-13 2021-05-13 Nkarta, Inc. Natural killer cell compositions and immunotherapy methods for treating tumors
WO2020067085A1 (en) * 2018-09-26 2020-04-02 アステラス製薬株式会社 Cancer therapy where oncolytic vaccinia virus and immune checkpoint inhibitor are used in combination, and pharmaceutical composition and combination drug used therein
GB201816547D0 (en) * 2018-10-10 2018-11-28 Univ London Queen Mary Oncolytic virus for the treatment of cancer
CN109182278B (en) * 2018-10-12 2021-08-06 河南省动物疫病预防控制中心 Seikaga valley virus strain and application thereof
JP7487112B2 (en) 2019-03-05 2024-05-20 ンカルタ・インコーポレイテッド CD19-directed chimeric antigen receptors and their use in immunotherapy - Patents.com
CN114127107A (en) * 2019-04-01 2022-03-01 Immetas治疗股份有限公司 Bispecific binding molecules targeting tumor microenvironment and immune checkpoint proteins
CN112225780B (en) * 2019-06-24 2022-02-11 中国动物疫病预防控制中心(农业农村部屠宰技术中心) Polypeptide for detecting Seneca virus antibody and application thereof
US20220380457A1 (en) * 2019-08-23 2022-12-01 City Of Hope Igg antibody compositions and methods of making the same
WO2021071534A1 (en) * 2019-10-08 2021-04-15 Icell Kealex Therapeutics Mutant vaccinia viruses and use thereof
MX2022004323A (en) * 2019-10-10 2022-08-02 Univ Arizona State Oncolytic viruses that express multi-specific immune cell engagers.
US20220064672A1 (en) * 2020-08-27 2022-03-03 University Of Southern California Engineered oncolytic viruses expressing pd-l1 inhibitors and uses thereof
US20240025971A1 (en) * 2020-10-23 2024-01-25 Atreca, Inc. Antibodies to coronavirus sars-cov-2
WO2022147480A1 (en) 2020-12-30 2022-07-07 Ansun Biopharma, Inc. Oncolytic virus encoding sialidase and multispecific immune cell engager
WO2022151078A1 (en) * 2021-01-13 2022-07-21 嘉兴允英医学检验有限公司 Oncolytic virus and application thereof
CN115161293A (en) * 2021-04-01 2022-10-11 南京惟亚德生物医药有限公司 Oncolytic vaccinia virus for coding bispecific T cell adaptor and preparation method and application thereof
CN113912023B (en) * 2021-10-08 2023-05-23 常熟理工学院 Preparation method of negative electrode material of sodium ion battery
CN114934065A (en) * 2021-11-25 2022-08-23 浙江理工大学绍兴生物医药研究院有限公司 Oncolytic adenovirus construction method carrying immune checkpoint molecule TIM-3 antibody gene and application thereof
WO2023122882A1 (en) * 2021-12-27 2023-07-06 上海鑫湾生物科技有限公司 Bispecific t-cell engager, recombinant oncolytic virus thereof, and use thereof
CN114249836A (en) * 2021-12-27 2022-03-29 上海鑫湾生物科技有限公司 Bispecific T cell engagers, recombinant oncolytic viruses thereof and uses thereof
CN117004579A (en) * 2023-06-29 2023-11-07 苏州恩立维生物科技有限公司 Recombinant oncolytic vaccinia virus and uses thereof

Family Cites Families (101)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2413974A1 (en) 1978-01-06 1979-08-03 David Bernard DRYER FOR SCREEN-PRINTED SHEETS
US4485045A (en) 1981-07-06 1984-11-27 Research Corporation Synthetic phosphatidyl cholines useful in forming liposomes
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4544545A (en) 1983-06-20 1985-10-01 Trustees University Of Massachusetts Liposomes containing modified cholesterol for organ targeting
JPH0746994B2 (en) 1984-10-04 1995-05-24 味の素株式会社 Method for producing L-amino acid by fermentation method
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US6548640B1 (en) 1986-03-27 2003-04-15 Btg International Limited Altered antibodies
EP0279862B1 (en) 1986-08-28 1993-11-03 Teijin Limited Cytocidal antibody complex and process for its preparation
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5204244A (en) 1987-10-27 1993-04-20 Oncogen Production of chimeric antibodies by homologous recombination
US5202238A (en) 1987-10-27 1993-04-13 Oncogen Production of chimeric antibodies by homologous recombination
GB8823869D0 (en) 1988-10-12 1988-11-16 Medical Res Council Production of antibodies
US5175384A (en) 1988-12-05 1992-12-29 Genpharm International Transgenic mice depleted in mature t-cells and methods for making transgenic mice
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
DE3920358A1 (en) 1989-06-22 1991-01-17 Behringwerke Ag BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE
AU641673B2 (en) 1989-06-29 1993-09-30 Medarex, Inc. Bispecific reagents for aids therapy
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6673986B1 (en) 1990-01-12 2004-01-06 Abgenix, Inc. Generation of xenogeneic antibodies
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
EP0463151B1 (en) 1990-01-12 1996-06-12 Cell Genesys, Inc. Generation of xenogeneic antibodies
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
CA2090473A1 (en) 1990-08-29 1992-03-01 Robert M. Kay Homologous recombinatin in mammalian cells
US6255458B1 (en) 1990-08-29 2001-07-03 Genpharm International High affinity human antibodies and human antibodies against digoxin
US5789650A (en) 1990-08-29 1998-08-04 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5874299A (en) 1990-08-29 1999-02-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5877397A (en) 1990-08-29 1999-03-02 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
ES2108048T3 (en) 1990-08-29 1997-12-16 Genpharm Int PRODUCTION AND USE OF LOWER TRANSGENIC ANIMALS CAPABLE OF PRODUCING HETEROLOGICAL ANTIBODIES.
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US6300129B1 (en) 1990-08-29 2001-10-09 Genpharm International Transgenic non-human animals for producing heterologous antibodies
WO1992010210A1 (en) 1990-12-14 1992-06-25 E.I. Du Pont De Nemours And Company Inhibition of angiogenesis by il-1
WO1992020373A1 (en) 1991-05-14 1992-11-26 Repligen Corporation Heteroconjugate antibodies for treatment of hiv infection
WO1992022670A1 (en) 1991-06-12 1992-12-23 Genpharm International, Inc. Early detection of transgenic embryos
WO1992022645A1 (en) 1991-06-14 1992-12-23 Genpharm International, Inc. Transgenic immunodeficient non-human animals
GB9114948D0 (en) 1991-07-11 1991-08-28 Pfizer Ltd Process for preparing sertraline intermediates
AU2515992A (en) 1991-08-20 1993-03-16 Genpharm International, Inc. Gene targeting in animal cells using isogenic dna constructs
GB9304200D0 (en) 1993-03-02 1993-04-21 Sandoz Ltd Improvements in or relating to organic compounds
JPH07500820A (en) 1991-09-05 1995-01-26 ユニバーシティ オブ コネティカット Targeted delivery of polynucleotides or oligonucleotides to cells
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
JPH07501450A (en) 1991-11-29 1995-02-16 モンサント　カンパニー Cyclic diguanylate metabolizing enzyme
ES2341666T3 (en) 1991-12-02 2010-06-24 Medimmune Limited PRODUCTION OF AUTHORTIC BODIES OF REPERTORIES OF ANTIQUE RPOS SEGMENTS EXPRESSED ON THE FAGOS SURFACE.
EP0746609A4 (en) 1991-12-17 1997-12-17 Genpharm Int Transgenic non-human animals capable of producing heterologous antibodies
US5777085A (en) 1991-12-20 1998-07-07 Protein Design Labs, Inc. Humanized antibodies reactive with GPIIB/IIIA
ATE208374T1 (en) 1992-02-18 2001-11-15 Otsuka Kagaku Kk BETA-LACTAM AND CEPHAM COMPOUNDS AND THEIR PRODUCTION
US5714350A (en) 1992-03-09 1998-02-03 Protein Design Labs, Inc. Increasing antibody affinity by altering glycosylation in the immunoglobulin variable region
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
NZ253943A (en) 1992-06-18 1997-01-29 Genpharm Int Transfering polynucleotides into eukaryotic cells using co-lipofection complexes of a cationic lipid and the polynucleotide
AU675661B2 (en) 1992-07-24 1997-02-13 Abgenix, Inc. Generation of xenogeneic antibodies
EP0672161B1 (en) 1992-11-05 1999-09-22 Xyrofin Oy Recombinant method and host for manufacture of xylitol
ATE196606T1 (en) 1992-11-13 2000-10-15 Idec Pharma Corp THERAPEUTIC USE OF CHIMERIC AND LABELED ANTIBODIES DIRECTED AGAINST A DIFFERENTIATION ANTIGEN WHICH EXPRESSION IS RESTRICTED TO HUMAN B LYMPHOCYTES, FOR THE TREATMENT OF B-CELL LYMPHOMA
JPH08509612A (en) 1993-04-26 1996-10-15 ジェンファーム インターナショナル インコーポレイテッド Transgenic non-human animal capable of producing heterologous antibody
US5707928A (en) 1993-09-28 1998-01-13 American Cyanamid Company Emulsifiable suspension concentrate compositions of imidazolinyl benzoic acids, esters and salts thereof, and dinitroaniline herbicides
US5625825A (en) 1993-10-21 1997-04-29 Lsi Logic Corporation Random number generating apparatus for an interface unit of a carrier sense with multiple access and collision detect (CSMA/CD) ethernet data network
WO1995022618A1 (en) 1994-02-22 1995-08-24 Dana-Farber Cancer Institute Nucleic acid delivery system, method of synthesis and uses thereof
CN1124340C (en) 1994-08-30 2003-10-15 味之素株式会社 Process for producing L-valine and L-leucine
US5643763A (en) 1994-11-04 1997-07-01 Genpharm International, Inc. Method for making recombinant yeast artificial chromosomes by minimizing diploid doubling during mating
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5703057A (en) 1995-04-07 1997-12-30 Board Of Regents The University Of Texas System Expression library immunization
CA2219361C (en) 1995-04-27 2012-02-28 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
AU718138B2 (en) 1995-08-29 2000-04-06 Kyowa Hakko Kirin Co., Ltd. Chimeric animal and method for constructing the same
FR2751343B1 (en) 1996-07-16 1998-12-18 Transgene Sa PROCESS FOR THE PRESERVATION OF INFECTIOUS RECOMBINANT VIRUSES, AQUEOUS VIRAL SUSPENSION, AND USE AS A MEDICAMENT
IL128780A0 (en) 1996-09-06 2000-01-31 Univ Pennsylvania An inducible method for production of recombinant adeno-associated viruses utilizing T7 polymerase
EP1500329B1 (en) 1996-12-03 2012-03-21 Amgen Fremont Inc. Human antibodies that specifically bind human TNF alpha
JP3966583B2 (en) 1997-06-23 2007-08-29 協和醗酵工業株式会社 Method for producing L-amino acid by fermentation
FR2766091A1 (en) 1997-07-18 1999-01-22 Transgene Sa ANTITUMOR COMPOSITION BASED ON MODIFIED IMMUNOGENIC POLYPEPTIDE WITH CELL LOCATION
EP1034298B1 (en) 1997-12-05 2011-11-02 The Scripps Research Institute Humanization of murine antibody
PT1071752E (en) 1998-04-21 2003-11-28 Micromet Ag SPECIFIC POLYMPEPTIDES FOR CD19XCD3 AND ITS USES
US6410319B1 (en) 1998-10-20 2002-06-25 City Of Hope CD20-specific redirected T cells and their use in cellular immunotherapy of CD20+ malignancies
US6322980B1 (en) 1999-04-30 2001-11-27 Aclara Biosciences, Inc. Single nucleotide detection using degradation of a fluorescent sequence
US6833268B1 (en) 1999-06-10 2004-12-21 Abgenix, Inc. Transgenic animals for producing specific isotypes of human antibodies via non-cognate switch regions
CA2799545A1 (en) 2000-03-07 2001-09-13 Merck Sharp & Dohme Corp. Adenovirus formulations
EP1334188B1 (en) 2000-11-07 2006-08-30 City of Hope Cd19-specific redirected immune cells
US7070995B2 (en) 2001-04-11 2006-07-04 City Of Hope CE7-specific redirected immune cells
US7514537B2 (en) 2001-04-30 2009-04-07 City Of Hope Chimeric immunoreceptor useful in treating human gliomas
DE60229246D1 (en) 2001-05-26 2008-11-20 One Cell Systems Inc
IL161590A0 (en) 2001-12-10 2004-09-27 Bavarian Nordic As Poxvirus containing formulations and process for preparing stable poxvirus containing compositions
AU2004242847B2 (en) * 2003-05-31 2011-06-09 Amgen Research (Munich) Gmbh Pharmaceutical composition comprising a bispecific antibody for EpCAM
ATE289514T1 (en) 2003-11-03 2005-03-15 Peter-Hansen Volkmann VAGINAL CARE COMPOSITION
WO2006116181A2 (en) 2005-04-25 2006-11-02 Trustees Of Dartmouth College Regulatory t cell mediator proteins and uses thereof
US8231872B2 (en) 2005-04-25 2012-07-31 The Trustees Of Dartmouth College Regulatory T cell mediator proteins and uses thereof
EP1951865A4 (en) 2005-11-21 2010-06-23 Sanofi Pasteur Ltd Stabilizing formulations for recombinant viruses
EP1880729A1 (en) 2006-07-20 2008-01-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Use of soluble CD160 to suppress immunity
US20090098529A1 (en) 2006-10-16 2009-04-16 Nanhai Chen Methods for attenuating virus strains for diagnostic and therapeutic uses
GB0705245D0 (en) 2007-03-19 2007-04-25 Stabilitech Ltd Stable biological products
KR101319499B1 (en) 2008-02-22 2013-10-17 엘지디스플레이 주식회사 Method for forming layer and pattern of nanowire or carbon nanotube using chemical self assembly and fabricating method in liquid crystal display device thereby
JP2010031968A (en) 2008-07-29 2010-02-12 Aisin Seiki Co Ltd Fluid coupling device
WO2011120013A2 (en) 2010-03-26 2011-09-29 Trustees Of Dartmouth College Vista regulatory t cell mediator protein, vista binding agents and use thereof
TWI690322B (en) 2012-10-02 2020-04-11 法商傳斯堅公司 Virus-containing formulation and use thereof
JP2016512199A (en) * 2013-03-05 2016-04-25 ベイラー カレッジ オブ メディスンＢａｙｌｏｒ Ｃｏｌｌｅｇｅ Ｏｆ Ｍｅｄｉｃｉｎｅ Oncolytic virus
DK3169341T3 (en) * 2014-07-16 2019-08-05 Transgene Sa ONCOLYTIC VIRUS FOR EXPRESSION OF IMMUN CHECKPOINT MODULATORS
US11485791B2 (en) * 2015-03-17 2022-11-01 Tilt Biotherapeutics Oy Oncolytic adenoviruses coding for bi-specific antibodies

Also Published As

Publication number Publication date
TW201825674A (en) 2018-07-16
WO2018049248A1 (en) 2018-03-15
WO2018049261A1 (en) 2018-03-15

Similar Documents

Publication Publication Date Title
TW201825511A (en) Oncolytic virus expressing immune checkpoint modulators
JP6799101B2 (en) Agents for treating claudin-expressing cancer diseases
JP7034489B2 (en) Multispecific Fab fusion protein and its use
ES2966992T3 (en) Chimeric antigen receptors directed at BCMA and methods of their use
US20210130459A1 (en) Antibodies specific to human nectin4
JP2022116230A (en) Modified cells for immunotherapy
KR20210057705A (en) Various antigen binding domains, novel platforms and other enhancements for cell therapy
AU2018360800A1 (en) Chimeric antigen receptors specific for B-cell maturation antigen (BCMA)
JP2023010753A (en) Single domain antibodies to programmed cell death (pd-1)
KR20180116215A (en) Cytotoxicity-inducing therapeutic agent for treating cancer
SA518392058B1 (en) Chimeric Antigen Receptors Targeting Epidermal Growth Factor Receptor Variant III
US20210024630A1 (en) Guidance and navigation control proteins and method of making and using thereof
KR20180002653A (en) Antigen binding complexes having an agonistic activity activity and methods of use
JP6175590B1 (en) Cytotoxicity-inducing therapeutic agent for use in cancer treatment
TW201932490A (en) Single-domain antibodies and variants thereof against PD-1
TW202019968A (en) Ox40-binding polypeptides and uses thereof
US20230183342A1 (en) Antibodies to nkp46 and constructs thereof for treatment of cancers and infections
CN114040927A (en) Polypeptide binding to CD33 and application thereof
CN115812081A (en) anti-CTLA-4 antibodies and uses thereof
JP2023554422A (en) Multispecific antibodies for cancer treatment
TW202334233A (en) Gamma delta t-cell-binding polypeptides and uses thereof
TW202334193A (en) Gamma delta t-cell-targeted modified il-2 polypeptides and uses thereof
WO2019196117A1 (en) Anti-cd27 antibodies and use thereof
CA3223375A1 (en) Combination dosage regime of cd137 and pd-l1 binding agents
TW202328171A (en) Nkp46-targeted modified il-2 polypeptides and uses thereof