阅读说明：本技术 抗肿瘤治疗的方法 (Method of antitumor therapy ) 是由 塔马·拉赫米尔维茨·米内伊 伊扎克·门德尔 尼瓦·雅各布 埃亚勒·布赖特巴特 于 2020-04-13 设计创作，主要内容包括：本公开提供了治疗有需要的受试者的肿瘤的方法,包括向受试者施用有效剂量的载体和有效剂量的免疫检查点抑制剂的组合,所述载体包含与内皮细胞特异性启动子可操作地连接的Fas-嵌合体基因。在本公开的一些方面,免疫检查点抑制剂是PD-1拮抗剂或PD-L1拮抗剂。(The present disclosure provides a method of treating a tumor in a subject in need thereof comprising administering to the subject an effective dose of a combination of a vector comprising a Fas-chimera gene operably linked to an endothelial cell specific promoter and an effective dose of an immune checkpoint inhibitor. In some aspects of the disclosure, the immune checkpoint inhibitor is a PD-1 antagonist or a PD-L1 antagonist.)

1. A vector comprising a Fas-chimera gene operably linked to an endothelial cell specific promoter for reducing tumor size or inhibiting tumor growth or eliminating tumors in a subject in need thereof, characterized in that (a) an effective dose of the vector is to be administered to the subject, and (b) an effective dose of an immune checkpoint inhibitor is to be administered to the subject.

2. A vector comprising a Fas-chimera gene operably linked to an endothelial cell specific promoter for treating a tumor or metastasis thereof in a subject in need thereof, wherein (a) an effective dose of the vector is administered to the subject, and (b) an effective dose of an immune checkpoint inhibitor is administered to the subject.

3. The vector of claim 1 or 2, wherein the tumor is derived from or associated with: leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung cancer, non-small cell lung cancer, primary brain tumors (including glioblastoma multiforme), gastrointestinal cancer (GI) (including but not limited to esophageal, gallbladder, biliary tract, liver, pancreas, stomach, small intestine, large intestine, colon, rectum, and anus cancers), malignant pancreatic island tumors, malignant carcinoid cancers, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, papillary thyroid cancer, neuroblastoma, glioblastoma multiforme, neuroendocrine cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Muller's cancer, Michelle's cancer, cervical cancer, adrenal cortex cancer, prostate cancer, Michelle's cancer, pancreatic cancer, gastric carcinoma, pancreatic carcinoma, bladder carcinoma, and other, Ovarian cancer, peritoneal cancer, fallopian tube cancer, or papillary serous carcinoma of the uterus.

4. The vector of any one of claims 1 to 3, wherein the vector is present at about 1 x 10¹⁰To about 1X 10¹⁶About 1X 10¹¹To about 1X 10¹⁵About 1X 10¹¹To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁵About 1X 10¹²To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁴About 5X 10¹²To about 1X 10¹⁶About 5X 10¹²To about 1X 10¹⁵About 5X 10¹²To about 1X 10¹⁴About 1X 10¹²To about 1X 10¹³Or about 1X 10¹³To about 1X 10¹⁴An effective dose of each viral particle is administered with the vector.

5. The vector of any one of claims 1 to 4, wherein the vector is present at about 1 x 10¹⁶、1×10¹⁵、1×10¹⁴、5×10¹³、4×10¹³、3×10¹³、2×10¹³、1×10¹³、9×10¹²、8×10¹²、7×10¹²、6×10¹²、5×10¹²、4×10¹²、3×10¹²、2×10¹²、1×10¹²、9×10¹¹、8×10¹¹、7×10¹¹、6×10¹¹、5×10¹¹、4×10¹¹、3×10¹¹、2×10¹¹、1×10¹¹、9×10¹⁰、8×10¹⁰、7×10¹⁰、6×10¹⁰、5×10¹⁰、4×10¹⁰、3×10¹⁰、2×10¹⁰Or 1X 10¹⁰An effective dose of each viral particle is administered with the vector.

6. The vector of any one of claims 1 to 5, wherein the vector and the immune checkpoint inhibitor are administered sequentially.

7. The vector of any one of claims 1 to 6, wherein the vector is administered repeatedly.

8. The vector of claim 7, wherein the vector is repeatedly administered daily, once on about 2 days, once on about 3 days, once on about 4 days, once on about 5 days, once on about 6 days, once on about 7 days, once on about 2 weeks, once on about 3 weeks, once on about 4 weeks, once on about 5 weeks, once on about 6 weeks, once on about 7 weeks, once on about 2 months, or once on about 6 months.

9. The vector of any one of claims 1 to 8, wherein the administration of the immune checkpoint inhibitor is repeated.

10. The vector of claim 9, wherein the administration of the immune checkpoint inhibitor is repeated once about 7 days, once about 2 weeks, once about 3 weeks, once about 4 weeks, once about 2 months, once about 3 months, once about 4 months, once about 5 months, or once about 6 months.

11. The vector of any one of claims 1 to 11, wherein the immune checkpoint inhibitor is a PD-1 antagonist administered in an effective amount of less than about 15mg/kg, less than about 14mg/kg, less than about 13mg/kg, less than about 12mg/kg, less than about 11mg/kg, less than about 10mg/kg, less than about 9mg/kg, less than about 8mg/kg, less than about 7mg/kg, less than about 6mg/kg, less than about 5mg/kg, less than about 4mg/kg, less than about 3mg/kg, less than about 2mg/kg, or less than about 1 mg/kg.

12. The vector of claim 11, wherein the PD-1 antagonist is administered in an effective amount at a fixed dose of about 100mg to about 600mg, about 120mg to about 500mg, about 140mg to about 460mg, about 180mg to about 420mg, about 200mg to about 380mg, about 220mg to about 340mg, about 230mg to about 300mg, or about 230mg to about 260 mg.

13. The vector of claim 11 or 12, wherein the PD-1 antagonist is selected from the group consisting of antibodies to nivolumab, pembrolizumab, carprilizumab, cimiralizumab, sillizumab, and PDR 001.

14. The vector of any one of claims 1 to 13, wherein the vector is administered in combination with an effective dose of one or more chemotherapeutic agents.

15. The vector of claim 14, wherein the one or more chemotherapeutic agents are selected from the group consisting of: acivicin; aclarubicin; (ii) aristozole hydrochloride; (ii) abelmoscine; doxorubicin; (ii) Alexanox; aldesleukin; ainingda; altretamine; an apramycin; amenthraquinone acetate; aminoglutethimide; amsacrine; anastrozole; anthranilic acid; an asparaginase enzyme; a triptyline; azacitidine; azatepa; (ii) azomycin; batimastat; benzotepa; bicalutamide; bisantrene hydrochloride; bisnefaede dimesylate; bevacizumab; bizelesin; bleomycin sulfate; brequinar sodium; briprimine; busulfan; actinomycin; (ii) carroterone; a carbimide; a carbapenem; carboplatin; carmustine (BiCNU); a doxorubicin hydrochloride; folding to get new; cediogo, and cediogo; chlorambucil; a sirolimus; cisplatin; cladribine; cllinaltol mesylate; cyclophosphamide; cytarabine; dacarbazine; actinomycin D; daunorubicin hydrochloride; decitabine; (ii) dexomaplatin; 2, dizagutanin; 1, dizagutinine mesylate; diazaquinone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; drotandrosterone propionate; daptomycin; edatrexae; eflornithine hydrochloride; elsamitrucin; enloplatin; an enpu urethane; epinastine; eprosamine hydrochlorideComparing stars; (ii) ebuzole; isosbacin hydrochloride; estramustine; estramustine sodium phosphate; etanidazole; etoposide; etoposide phosphate; etophenine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; (iii) flucitabine; a phosphorus quinolone; fostrexasin sodium; gemcitabine; gemcitabine hydrochloride;slicing; a hydroxyurea; idarubicin hydrochloride; ifosfamide; ilofovir dipivoxil; interferon alpha-2 a; interferon alpha-2 b; interferon alpha-nl; interferon alpha-n 3; interferon beta-Ia; interferon gamma-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprorelin acetate; liazole hydrochloride; lometrexol sodium; lomustine (CCNU); losoxantrone hydrochloride; (ii) maxolone; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; (ii) a melanoril; mercaptopurine; methotrexate; methotrexate sodium; chlorpheniramine; meltupipide; mitodomide; mitokacin; mitorubin; schizophyllan; mitomacin; mitomycin; mitospirane culturing; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; a noramycin; ormaplatin; oshuzuren; pazopanib; paclitaxel; a pemetrexed; a pelithromycin; pentazocine; pellomycin sulfate; hyperphosphamide; pipobroman; piposulfan; piroxantrone hydrochloride; (ii) a plicamycin; pramipexole; porfimer sodium; a podomycin; deltemustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazole furan rhzomorph; (ii) lybodenosine; ludwimine; safrog; safrog hydrochloride; semustine; octreozine; sorafenib; sodium salt of sparadronate; a sparamycin; germanospiramine hydrochloride; spiromustine; spiroplatinum; a puromycin; a streptozocin; a sulfochlorophenylurea; sunitinib; talimox; taxol; sodium tegafur; tegafur; tiloxanthraquinone hydrochloride; temoporfin; temozolomide; (ii) teniposide; a tiroxiron; a testosterone ester; (ii) a thiopurine; thioguanine; thiotepa; thiazolfurin; tirapazamine; topotecan hydrochloride; toremifene citrate; triton acetate; quxi phosphoric acidStanding; trimetrexate; tritrexate glucuronate; triptorelin; tobramzole hydrochloride; uracil mustard; uretipi; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinblastine epoxy sulfate; vinorelbine tartrate; vinblastine sulfate; vinzolidine sulfate; (ii) vorozole; zeniplatin; netastatin and zorubicin hydrochloride.

16. The vector of any one of claims 1 to 15, wherein the vector comprises, consists of, or consists essentially of SEQ ID NO 19.

17. The vector of any one of claims 1 to 16, wherein the vector is an isolated virus having european animal cell collection (ECACC) accession number 1302201.

Background

Angiogenesis is a common major feature of several pathologies. Among these diseases are diseases in which angiogenesis may improve the disease condition (such as ischemic heart disease) and diseases in which excessive angiogenesis is part of the pathology and thus should be eliminated. These latter diseases include diabetes (diabetic retinopathy), cardiovascular disease (atherosclerosis), chronic inflammation (rheumatoid arthritis) and cancer. Angiogenesis occurs in tumors and allows their growth, invasion and metastasis. In 1971, Folkman proposed that tumor growth and metastasis were angiogenesis-dependent, and therefore inhibition of angiogenesis could be used as a strategy to prevent tumor growth.

There are several molecules, from transcription factors to growth factors, involved in angiogenesis. Hypoxia is an important environmental factor that leads to neovascularization, and it induces the release of several cytokines that are pro-angiogenic factors. These include Vascular Endothelial Growth Factor (VEGF) and its receptors, members of the angiopoietin family, basic fibroblast growth factor (bFGF), and endothelin-1 (ET-1). These factors are involved in the induction of angiogenesis through the activation, proliferation and migration of endothelial cells.

Recombinant forms of endogenous angiogenesis inhibitors have been tested for the treatment of cancer. The potential pharmacokinetic, biotechnological and economic drawbacks of long-term delivery of these recombinant inhibitors have prompted scientists to develop additional approaches.

The development of the anti-VEGF monoclonal antibody bevacizumab has validated the anti-angiogenic approach as a complementary treatment modality to chemotherapy. Several small molecule inhibitors, including second generation multi-targeted tyrosine kinase inhibitors, have also shown promise as anti-angiogenic agents for cancer.

Immune checkpoints also play a role in tumor growth and development. For example, tumor cells can evade the host immune system by naturally stimulating immune checkpoints through receptor/ligand interactions. Thus, detection of molecules that block immune checkpoints (e.g., immune checkpoint inhibitors) are useful in the treatment of cancer. However, these inhibitors only work in subjects with only a few types of tumors. In addition, the subject's response to immune checkpoint therapy is often accompanied by relapse and disease progression.

The potential pharmacokinetic and economic drawbacks of long-term delivery of recombinant inhibitors, antibodies and small molecules, as well as the limited activity exhibited when applied as monotherapy, have prompted scientists to evaluate gene therapy. However, there are a number of obstacles that limit successful gene therapy, including the duration of expression, induction of immune responses, vector cytotoxicity, and tissue specificity. Two general strategies for cancer gene therapy are proposed: tumor-targeted or systemic gene therapy. Targeting gene therapy products to cancer cells or their environment by systemic therapy has been unsuccessful, resulting in most therapies being administered to the tumor itself.

Disclosure of Invention

The present disclosure also provides a method of reducing or inhibiting tumor size or eliminating a tumor in a subject in need thereof, the method comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. The present disclosure also provides a method of treating a tumor or metastasis thereof in a subject in need thereof, the method comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. The present disclosure also provides a method of inducing or improving T cell activation in a subject having a tumor, the method comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. The present disclosure also provides a method of inducing or improving the efficacy of an immune checkpoint inhibitor in a subject having a tumor, the method comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. The present disclosure also provides a method of converting a cold tumor to a hot tumor in a subject in need thereof, the method comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor.

In some aspects, the tumor is derived from or associated with: leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung cancer, non-small cell lung cancer, primary brain tumors (including glioblastoma multiforme), Gastrointestinal (GI) cancer (including but not limited to cancers of the esophagus, gall bladder, biliary tract, liver, pancreas, stomach, small intestine, large intestine, colon, rectum, and anus), malignant pancreatic island tumors, malignant carcinoids, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Muller's cancer, ovarian cancer, peritoneal cancer, and combinations thereof, Fallopian tube carcinoma or uterine papillary serous carcinoma.

In some aspects, the Fas chimera gene encodes a polypeptide comprising the extracellular domain of a TNF receptor 1(TNFR1) polypeptide fused to the transmembrane and intracellular domains of a Fas polypeptide. In some aspects, the extracellular domain of TNFR1 comprises an amino acid sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 4, wherein the extracellular domain of TNFR1 is capable of binding TNF-a. In some aspects, the transmembrane domain and intracellular domain of the Fas polypeptide comprise an amino acid sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 8, wherein the transmembrane domain and intracellular domain of the Fas polypeptide are capable of inducing Fas-mediated apoptosis. In some aspects, the Fas-chimera gene comprises a first nucleotide sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID No. 3; and a second nucleotide sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO. 7.

In some aspects of the disclosure, the endothelial cell specific promoter comprises the PPE-1 promoter. In some aspects, the endothelial cell specific promoter further comprises a cis-acting regulatory element. In some aspects, the cis-acting regulatory element comprises a nucleotide sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 15 or SEQ ID NO 16. In some aspects, the cis-acting regulatory element comprises SEQ ID NO 11 or SEQ ID NO 12. In some aspects, the cis-acting regulatory element further comprises SEQ ID NO 13 or SEQ ID NO 14.

In some aspects of the disclosure, the endothelial cell specific promoter is the PPE-1-3X promoter. In some aspects, the PPE-1-3X promoter comprises a nucleotide sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 18, wherein the PPE-1-3X promoter is capable of directing Fas chimera gene expression in endothelial cells.

In some aspects of the present invention, the first and second electrodes are,at about 1X 10¹⁰To about 1X 10¹⁶About 1X 10¹¹To about 1X 10¹⁵About 1X 10¹¹To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁵About 1X 10¹²To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁴About 5X 10¹²To about 1X 10¹⁶About 5X 10¹²To about 1X 10¹⁵About 5X 10¹²To about 1X 10¹⁴About 1X 10¹²To about 1X 10¹³Or about 1X 10¹³To about 1X 10¹⁴The amount of each viral particle is administered in an effective dose of the vector. In some aspects, at about 1 × 10¹⁶、1×10¹⁵、1×10¹⁴、5×10¹³、4×10¹³、3×10¹³、2×10¹³、1×10¹³、9×10¹²、8×10¹²、7×10¹²、6×10¹²、5×10¹²、4×10¹²、3×10¹²、2×10¹²、1×10¹²、9×10¹¹、8×10¹¹、7×10¹¹、6×10¹¹、5×10¹¹、4×10¹¹、3×10¹¹、2×10¹¹、1×10¹¹、9×10¹⁰、8×10¹⁰、7×10¹⁰、6×10¹⁰、5×10¹⁰、4×10¹⁰、3×10¹⁰、2×10¹⁰Or 1X 10¹⁰The amount of each viral particle is administered in an effective dose of the vector.

In some aspects of the disclosure, the vector and immune checkpoint inhibitor are administered sequentially. In some aspects, the vector is administered prior to administration of the immune checkpoint inhibitor. In a particular aspect, the vector is administered prior to administration of the immune checkpoint inhibitor, and the immune checkpoint inhibitor is administered as the tumor progresses. In other aspects, the immune checkpoint inhibitor is administered prior to administration of the vector.

In some aspects, the vector is administered repeatedly. In some aspects, the vehicle is repeatedly administered daily, once per about 2 days, once per about 3 days, once per about 4 days, once per about 5 days, once per about 6 days, once per about 7 days, once per about 2 weeks, once per about 3 weeks, once per about 4 weeks, once per about 5 weeks, once per about 6 weeks, once per about 7 weeks, once per about 2 months, or once per about 6 months.

In some aspects of the disclosure, the administration of the immune checkpoint inhibitor is repeated. In some aspects, the administration of the immune checkpoint inhibitor is repeated once about 7 days, once about 2 weeks, once about 3 weeks, once about 4 weeks, once about 2 months, once about 3 months, once about 4 months, once about 5 months, or once about 6 months.

In some aspects of the disclosure, the immune checkpoint inhibitor is a PD-1 antagonist. In some aspects, the PD-1 antagonist is administered in an effective amount of less than about 15mg/kg, less than about 14mg/kg, less than about 13mg/kg, less than about 12mg/kg, less than about 11mg/kg, less than about 10mg/kg, less than about 9mg/kg, less than about 8mg/kg, less than about 7mg/kg, less than about 6mg/kg, less than about 5mg/kg, less than about 4mg/kg, less than about 3mg/kg, less than about 2mg/kg, or less than about 1 mg/kg. In other aspects, the PD-1 antagonist is administered in a fixed dose effective amount of about 100mg to about 600mg, about 120mg to about 500mg, about 140mg to about 460mg, about 180mg to about 420mg, about 200mg to about 380mg, about 220mg to about 340mg, about 230mg to about 300mg, or about 230mg to about 260 mg. In some aspects, the PD-1 antagonist is administered in an effective amount at a fixed dose of about 400mg to about 600mg, about 450mg to about 520mg, about 460mg to about 510mg, or about 470mg to about 500 mg. In some aspects, the PD-1 antagonist is administered in a fixed dose effective amount of about 60mg, about 80mg, about 100mg, about 120mg, about 140mg, about 160mg, about 180mg, about 200mg, about 220mg, about 240mg, about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg, about 500mg, about 520mg, about 540mg, about 560mg, about 580mg, or about 600 mg.

In a particular aspect, the PD-1 antagonist is an antibody that binds PD-1. In some aspects, the antibody is a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody, or a chimeric antibody. In a more specific aspect, the antibody is selected from the group consisting of nivolumab, pembrolizumab, camrelizumab (camrelizumab), cimiralizumab, sillimumab, and PDR 001. In a particular aspect, the PD-1 antagonist is nivolumab.

In some aspects of the disclosure, at 3 × 10¹²To 3X 10¹³Administering an effective amount of each viral particle with the vector, and nivolumab in an effective amount of 2mg/kg to 12 mg/kg. In other aspects at 3 × 10¹²To 3X 10¹³An effective amount of each viral particle is administered with the vector, and nivolumab is administered at a fixed dose of 460mg to 500 mg.

In some aspects, the vehicle is administered once every 2 months and nivolumab is administered once every 2 weeks. In other aspects, the vector is administered once every 2 months and nivolumab is administered once every 2 months. In some aspects, nivolumab is administered one month after each administration of the vehicle.

In some aspects of the disclosure, the PD-1 antagonist is an antibody that binds PD-L1. In some aspects, the antibody is a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody, or a chimeric antibody. In particular aspects, the antibody is selected from the group consisting of atilizumab, avizumab, devoluumab, and BMS-936559.

Some aspects of the disclosure include further administering to the subject an effective dose of one or more chemotherapeutic agents. In some aspects, the one or more chemotherapeutic agents are selected from: acivicin, aclarubicin, acarbozole, alchlorline, adriamycin, adolesin, aldesleukin, eindacine, altretamine, ambroxycin, acertan acetate, aminoglutethimide, amsacrine, anastrozole, amrubicin, asparaginase, clindamycin, azacitidine, azatepa, azomycin, batistat, benzotepa, bicalutamide, bisamide hydrochloride, bisnefad dimesylate, bevacizumab, bizelesin, bleomycin sulfate, brequinar sodium, bripirimid, busulfan, actinomycin, carroterone, carbitamine, carbimide, carboplatin, carmustine (BiCNU), carubin hydrochloride, carzelesin, sildenafil, gazelesin, cisplatin, rickettine mesylate, cyclophosphamide, arabinose, dacarbazine hydrochloride, amitocin, dacarbazine hydrochloride, dacarbazine, dactinomycin hydrochloride, dactinomycin, dacarbazine, sildenafine, dacarbazine, dactyline, dactinomycin, dactyline, and a, Daunorubicin hydrochloride, decitabine, dexomaplatin, dizagutanine mesylate, disazoquinone, docetaxel, doxorubicin hydrochloride, droloxifeneDroloxifene citrate, drostasterone propionate, daptomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, empretamine, epipipidine, epirubicin hydrochloride, ibutrole, esorubicin hydrochloride, estramustine sodium phosphate, etanidazole, etoposide phosphate, etoposide, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flucitabine, fequinone, fostricin sodium, gemcitabine hydrochloride, doxycycline hydrochloride, fludarabine hydrochloride, fludarcinonide hydrochloride, fludarcinolone hydrochloride, fludarcinoxathicin, gemcitabine, fludarabine hydrochloride, fludarcinolone hydrochloride, fludarabine hydrochloride, fludarcinoxathixathixathixathixathixathixathixathixathixatin, fludarone, fludaruss-D, fludaruss-N, fludaruss-D, fludarabine hydrochloride, fludaruss-D, fludarabine, fludaruss-D, fludarabine, fludaruss-D, and a, fludaruss-D, fludaruss-,Tablets, hydroxyurea, idarubicin hydrochloride, ifosfamide, imofovir, interferon alpha-2 a, interferon alpha-2 b, interferon alpha-nl, interferon alpha-n 3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprorelin acetate, liazole hydrochloride, lometrexol sodium, lomustine (CCNU), loxinoxantrone hydrochloride, maxol, maytansine, meclizine hydrochloride, megestrol acetate, melphalan, melnolium, mercaptopurine, methotrexate sodium, chlorphenidine, metoteracil, mitodomide, mitoxantrone hydrochloride, mycophenolate mofetil, norgestimatinib, mitomycin, mitoxantrone, mitomycin, mitotane hydrochloride, mitoxantrone, medroxy, norgestrel, norgestimate hydrochloride, ipratropium, Ormaplatin, oxsulam, pazopanib (pazotinib), paclitaxel, pemetrexed, pellitorin, pentazocine, pellomycin sulfate, perphosphamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, pluriment, porfimer sodium, podofycin, bronimostine, procarbazine hydrochloride, puromycin hydrochloride, pyrazolofuranin, lydenosine, roglucimine, safrog, saffipogo hydrochloride, semustine, octreozine, sorafenib, sodium spartamate, spidromycin hydrochloride, germanospiromustine hydrochloride, spiromustine, spiroplatin, puromycin, streptozocin, sulfochlorbenzuron, sunitinib, talirosin, taxol, tegorale sodium (tecolalan sodium), tegafur, tilloxanone hydrochloride, temozolone, temoporfin, temozolomide, temozotamide, tizotinib, paclitaxelNipagin, tirofilone, testolactone, thioprine, thioguanine, thiotepa, thiazolfurin (tiazofurin), tirapazamine, topotecan hydrochloride, toremifene citrate, tritulone acetate, triciribine phosphate, trimetrexate, tritrexate glucuronate, triptorelin, tobrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinpocetine sulfate, vincristine sulfate, vindesine sulfate, vinpocetine sulfate, vinglycinate, vinblastine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, Zeniplatin (zeiplatin), setastatin, and zorubicin hydrochloride.

In particular aspects of the disclosure, the vector comprises, consists of, or consists essentially of SEQ ID No. 19. In some aspects, the vector is an isolated virus having european animal cell collection (ECACC) accession number 13021201.

Drawings

FIG. 1 shows the study design of the combination therapy of anti-PD-L1 antibody and AD5-PPE-1-3X-Fas-c vector VB-111. Domestication for 3 days: mice were acclimated in their cages for 3 days prior to disease induction; induction of diseases: mice were footpad inoculated with D122 cells (metastatic lung tumor model) and tumor growth was monitored until tumors reached 7mm³(ii) a Day 0 of the dissection: when the tumor reached the target size, the tumor was excised by resection, beginning "day 0"; VB-111 I.V: 5 days after tumor resection, mice were treated with intravenous VB-111 vector; anti-PD-L1 antibody: for some groups of mice, anti-PD-L1 antibody was administered intraperitoneally on days 5, 8, and 11, and VB-111 was administered intravenously on day 5.

FIG. 2 shows VB-111 (1X 10) alone in normal saline (control)¹¹Or 1X 10⁹Individual viral particles), anti-PD-L1 antibody alone (200. mu.g), or VB-111 (1X 10)¹¹Individual viral particles) mouse lung weight (grams) after treatment in combination with anti-PD-L1 antibody (200 μ g);

FIG. 3 shows VB-111 (1X 10) alone in saline (control)¹¹Or 1X 10⁹Individual virus particles), anti-PD-L1 antibody alone (200. mu.g), or VB-111(1×10¹¹Individual viral particles) and anti-PD-L1 antibody (200 μ g) in combination.

FIG. 4 shows the results obtained with physiological saline (square) at 1X 10¹¹VB-111 alone (round), 200. mu.g of anti-PD-L1 antibody alone (triangle), or at 1X 10 for each viral particle¹¹Mouse melanoma tumor size (mm) after VB-111 used with individual viral particles in combination (star) with 200 μ g of anti-PD-L1 antibody³). Arrows indicate treatment days 9, 12 and 14. I.v. intravenously; i.p. intraperitoneally.

FIG. 5 shows the study design of the first part of a phase I/phase II clinical trial with VB-111 treatment in combination with an anti-PD-1 antibody (e.g., nivolumab). In this design, VB-111 (3X 10) will be administered to the subject¹²Individual virus particles or 1X 10¹³Individual viral particles) in combination with nivolumab (3 mg/kg). DLT: dose limiting toxicity.

Figure 6 shows the study design of the second part of a phase I/phase II clinical trial with VB-111 treatment in combination with an anti-PD-1 antibody (e.g., nivolumab). In the second part, the subject will be administered: VB-111 (1X 10)¹³Individual virus particles) were combined with nivolumab (3mg/kg) (arm 1); or nivolumab (3mg/kg) (arm 2). DLT: dose limiting toxicity.

Figure 7 shows the study design of an open-label, one-armed phase II study for the combination of VB-111 with anti-PD 1 antibody (nivolumab) in advanced, refractory metastatic colorectal cancer patients. Patients will have a pre-treatment biopsy on day 1 of cycle 2 or day 1 of cycle 4 and a post-treatment biopsy.

Detailed description of the preferred embodiments

1. A method of reducing tumor size or inhibiting tumor growth or eliminating a tumor in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter, and (b) administering to the subject an effective dose of an immune checkpoint inhibitor.

2. A method of treating a tumor or metastasis thereof in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter, and (b) administering to the subject an effective dose of an immune checkpoint inhibitor.

3. A method of inducing or improving T cell activation in a subject having a tumor, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter, and (b) administering to the subject an effective dose of an immune checkpoint inhibitor.

4. A method of inducing or enhancing the therapeutic effect of an immune checkpoint inhibitor in a subject having a tumor, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter, and (b) administering to the subject an effective dose of an immune checkpoint inhibitor.

5. A method of converting a cold tumor to a hot tumor in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter, and (b) administering to the subject an effective dose of an immune checkpoint inhibitor.

6. The method according to any one of embodiments 1 to 5, wherein the tumor is derived from or associated with: leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, small cell lung cancer, non-small cell lung cancer, primary brain tumors (including glioblastoma multiforme), Gastrointestinal (GI) cancers (including but not limited to cancers of the esophagus, gall bladder, biliary tract, liver, pancreas, stomach, small intestine, large intestine, colon, rectum, and anus), malignant pancreatic islet tumors, malignant carcinoids, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, papillary thyroid cancer, neuroblastoma, glioblastoma multiforme, neuroendocrine cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Muller's cancer, Ovarian cancer, peritoneal cancer, fallopian tube cancer, or papillary serous carcinoma of the uterus.

7. The method according to any one of embodiments 1 to 6, wherein the Fas chimera gene encodes a polypeptide comprising the extracellular domain of a TNF receptor 1(TNFR1) polypeptide fused to the transmembrane and intracellular domains of a Fas polypeptide.

8. The method according to embodiment 7, wherein the extracellular domain of TNFR1 comprises an amino acid sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 4, wherein the extracellular domain of TNFR1 is capable of binding TNF-a.

9. The method according to embodiment 8, wherein the transmembrane domain and intracellular domain of the Fas polypeptide comprise an amino acid sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 8, wherein the transmembrane domain and intracellular domain of the Fas polypeptide are capable of inducing Fas-mediated apoptosis.

10. The method according to any one of embodiments 1 to 9, wherein the Fas-chimera gene comprises a first nucleotide sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 3; and a second nucleotide sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO. 7.

11. The method according to any one of embodiments 1 to 10, wherein the endothelial cell specific promoter comprises the PPE-1 promoter.

12. The method according to any one of embodiments 1 to 11, wherein the endothelial cell specific promoter further comprises a cis-acting regulatory element.

13. The method of embodiment 12, wherein said cis-acting regulatory element comprises a nucleotide sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 15 or SEQ ID NO 16.

14. The method according to embodiment 13, wherein the cis-acting regulatory element comprises SEQ ID NO 11 or SEQ ID NO 12.

15. The method according to embodiment 14, wherein said cis-acting regulatory element further comprises SEQ ID NO 13 or SEQ ID NO 14.

16. The method according to any one of embodiments 1 to 15, wherein the endothelial cell specific promoter is the PPE-1-3X promoter.

17. The method according to embodiment 16, wherein the PPE-1-3X promoter comprises a nucleotide sequence having at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 18, wherein the PPE-1-3X promoter is capable of directing Fas chimera gene expression in endothelial cells.

18. The method according to any one of embodiments 1 to 17, wherein the concentration is about 1 x 10¹⁰To about 1X 10¹⁶About 1X 10¹¹To about 1X 10¹⁵About 1X 10¹¹To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁵About 1X 10¹²To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁴About 5X 10¹²To about 1X 10¹⁶About 5X 10¹²To about 1X 10¹⁵About 5X 10¹²To about 1X 10¹⁴About 1X 10¹²To about 1X 10¹³Or about 1X 10¹³To about 1X 10¹⁴The amount of each viral particle is administered in an effective dose of the vector.

19. The method of any one of embodiments 1 to 18, wherein the concentration is about 1 x 10¹⁶、1×10¹⁵、1×10¹⁴、5×10¹³、4×10¹³、3×10¹³、2×10¹³、1×10¹³、9×10¹²、8×10¹²、7×10¹²、6×10¹²、5×10¹²、4×10¹²、3×10¹²、2×10¹²、1×10¹²、9×10¹¹、8×10¹¹、7×10¹¹、6×10¹¹、5×10¹¹、4×10¹¹、3×10¹¹、2×10¹¹、1×10¹¹、9×10¹⁰、8×10¹⁰、7×10¹⁰、6×10¹⁰、5×10¹⁰、4×10¹⁰、3×10¹⁰、2×10¹⁰Or 1X 10¹⁰The amount of each viral particle is administered in an effective dose of the vector.

20. The method according to any one of embodiments 1 to 19, wherein the vector and immune checkpoint inhibitor are administered sequentially.

21. The method according to embodiment 20, wherein the vector is administered prior to administration of the immune checkpoint inhibitor.

22. The method according to embodiment 21, wherein the immune checkpoint inhibitor is administered at the time of tumor progression.

23. The method according to embodiment 20, wherein the immune checkpoint inhibitor is administered prior to administration of the vector.

24. The method according to any one of embodiments 1 to 23, wherein the administration of the carrier is repeated.

25. The method of embodiment 24, wherein the vector is administered repeatedly daily, once on about 2 days, once on about 3 days, once on about 4 days, once on about 5 days, once on about 6 days, once on about 7 days, once on about 2 weeks, once on about 3 weeks, once on about 4 weeks, once on about 5 weeks, once on about 6 weeks, once on about 7 weeks, once on about 2 months, or once on about 6 months.

26. The method according to any one of embodiments 1 to 25, wherein the administration of the immune checkpoint inhibitor is repeated.

27. The method according to embodiment 26, wherein the administration of the immune checkpoint inhibitor is repeated once about 7 days, once about 2 weeks, once about 3 weeks, once about 4 weeks, once about 2 months, once about 3 months, once about 4 months, once about 5 months, or once about 6 months.

28. The method according to any one of embodiments 1 to 27, wherein the immune checkpoint inhibitor is a PD-1 antagonist.

29. The method of embodiment 28, wherein the PD-1 antagonist is administered in an effective amount of less than about 15mg/kg, less than about 14mg/kg, less than about 13mg/kg, less than about 12mg/kg, less than about 11mg/kg, less than about 10mg/kg, less than about 9mg/kg, less than about 8mg/kg, less than about 7mg/kg, less than about 6mg/kg, less than about 5mg/kg, less than about 4mg/kg, less than about 3mg/kg, less than about 2mg/kg, or less than about 1 mg/kg.

30. The method according to embodiment 28, wherein the PD-1 antagonist is administered in an effective amount at a fixed dose of about 100mg to about 600mg, about 120mg to about 500mg, about 140mg to about 460mg, about 180mg to about 420mg, about 200mg to about 380mg, about 220mg to about 340mg, about 230mg to about 300mg, or about 230mg to about 260 mg.

31. The method according to embodiment 28, wherein the PD-1 antagonist is administered in an effective amount at a fixed dose of about 400mg to about 600mg, about 450mg to about 520mg, about 460mg to about 510mg, or about 470mg to about 500 mg.

32. The method according to embodiment 28, wherein the PD-1 antagonist is administered in an effective amount of about 60mg, about 80mg, about 100mg, about 120mg, about 140mg, about 160mg, about 180mg, about 200mg, about 220mg, about 240mg, about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg, about 500mg, about 520mg, about 540mg, about 560mg, about 580mg, or about 600mg fixed dose (flat dose).

33. The method according to any one of embodiments 28 to 32, wherein the PD-1 antagonist is an antibody that binds PD-1.

34. The method according to embodiment 33, wherein the antibody is a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody or a chimeric antibody.

35. The method according to embodiment 33 or 34, wherein the antibody is selected from the group consisting of nivolumab, pembrolizumab, carpriclizumab, cimiralizumab, sendilimab (sintilimab), and PDR 001.

36. The method according to any one of embodiments 28 to 35, wherein the PD-1 antagonist is nivolumab.

37. The method according to any one of embodiments 28 to 32, wherein the PD-1 antagonist is an antibody that binds PD-L1.

38. The method according to embodiment 37, wherein the antibody is a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody or a chimeric antibody.

39. The method according to embodiment 37 or 38, wherein the antibody is selected from the group consisting of atilizumab, avizumab, dewaluzumab and BMS-936559.

40. The method according to any one of embodiments 1 to 39, further comprising administering to the subject an effective dose of one or more chemotherapeutic agents.

41. The method according to embodiment 40, wherein the one or more chemotherapeutic agents are selected from the group consisting of: acimexin, aclarubicin, alcogazole hydrochloride, aclopram, doxorubicin, adolesin, aldesleukin, einadine, altretamine, ambroxycin, aceracene acetate to a subject is administered a quinone, aminoglutethimide, amsacrine, anastrozole, anthranilamide, asparaginase, clindamin, azacitidine, azatepa, azomycin, batimastat, benzotepa, bicalutamide, bisabolol hydrochloride, bisnefad dimesylate, bevacizumab, bizelesin, bleomycin sulfate, brequina sodium, bripirimid, busulfan, actinomycin, carulone, cameramide, carbitemm, carboplatin, carmustine (BiCNU), carubin hydrochloride, cableteprazole, sildengo, chlorambucil, cerubicin, cisplatin, linaabine, methamphetamine mesylate, clathrazidone, clathrazidovudine, clathrone, clathrazine, carbapenem, acarbozine acetate, and acerbazine acetate, Actinomycin D, daunorubicin hydrochloride, decitabine, dexomalatinum, dizagutanine, dizagutaline mesylate, diazaquinone, docetaxel, doxorubicin hydrochloride, droloxifene citrate, drostandrosterone propionate, daptomycin, idatroxate, eflornithine hydrochloride, elsamicin, enloplatin, emprine, epipipidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine sodium phosphate, etanidazole, etoposide phosphate, etoposide, fazole hydrochloride, fazarabine, fentrexone A amine, floxuridine, fludarabine phosphate, fluorouracil, flusitabine, fosquidone, fostrexin sodium, gemcitabine hydrochloride, gemcitabine, doxycycline hydrochloride, doxoraline hydrochloride, doxorubicin hydrochloride, doxorabicine hydrochloride, nalin hydrochloride, droxib hydrochloride, droxydine hydrochloride, fluvudine hydrochloride, fossilidone, fostrexin hydrochloride, fostrexin sodium, fostrexadone, gemcitabine, fostrexin, fosfomycin hydrochloride, foscamine, fosfomycin hydrochloride, and other,Tablet, hydroxyurea, idarubicin hydrochloride, ifosfamide, imofovir and interferonAlpha-2 a, interferon alpha-2 b, interferon alpha-n 1, interferon alpha-n 3, interferon beta-1 a, interferon gamma-1 b, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, linazole hydrochloride, lometrexol sodium, lomustine (CCNU), losoxanone hydrochloride, maxol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, minoriter, mercaptopurine, methotrexate sodium, chlorphenamine, metoclopramide, meltepa, mitodomide, mitokacin, mitorubicin, mitomycin, mitotane, mitoxantrone hydrochloride, mycophenolate, nocodazole, noradriamycin, olpamatin, oxsulam, zolpidem (paclitaxel), paclitaxel, pezox, paclitaxel, pexib, paclitaxel, etc, Pellicin, pentomustard, pellomycin sulfate, perphosphamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, profometant, porfimer sodium, pofimycin, poitemustine, procarbazine hydrochloride, puromycin hydrochloride, pyrazolofuranin, liboin, luodimine, safrog, saffipronol hydrochloride, semustine, octreozine, sorafenib, sodium spartamate, spicamycin, germanospiramine hydrochloride, spiromustine, spiroplatin, puromycin, streptozocin, sulfobenclamide, sunitinib, talimox, taxol, tegolan sodium (tecogalan sodium), tegafur, tilloxanone hydrochloride, temoporfin, temozolomide, teniposide, tirulone, testolactone, thioprimine, thioguanine, tiazofurin, tiazofurazone hydrochloride, topotecan hydrochloride, Toremifene citrate, triptolone acetate, triciribine phosphate, trimetrexate, tritrexate glucuronate, triptorelin, tobrozole hydrochloride, uracil mustard, urotropine, vapreotide, verteporfin, vinpocetine sulfate, vincristine sulfate, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinoxidine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, Zeniplatin (Zeniplatin), setastine and zorubicin hydrochloride.

42. The method according to any one of embodiments 1 to 41, wherein the vector comprises, consists of, or consists essentially of SEQ ID NO 19.

43. The method according to any one of embodiments 1 to 42, wherein the vector is an isolated virus having European Collection of Animal Cell Cultures (ECACC) accession number 13021201.

Detailed Description

I. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present disclosure, including definitions, will control. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other documents mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the disclosure will be apparent from the detailed description, and from the claims.

To further define the disclosure, the following terms and definitions are provided.

In this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a polynucleotide" is understood to represent one or more polynucleotides. Thus, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

Further, as used herein, "and/or" will be considered to specifically disclose each of the two particular features or components, with or without the other. Thus, the term "and/or" as used herein in phrases such as "a and/or B" is intended to include "a and B", "a or B", "a" (alone) and "B" (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass the following: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

The term "about" is used herein to mean about, approximately, about, or within the range of … …. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variation of 10% (higher or lower).

As used herein, "antibody" refers to an intact immunoglobulin, an antigen-binding fragment thereof, or an antigen-binding molecule. The antibodies of the present disclosure may be of any isotype or class (e.g., M, D, G, E and a) or of any subclass (e.g., G1-4, a1-2), and may have kappa (κ) or lambda (λ) light chains.

As used herein, the term "effective amount" refers to an amount effective to achieve the desired effect, in the necessary dosage and period of time. The desired result may be, for example, a reduction or inhibition of neovascularization or angiogenesis in vitro or in vivo; reducing or inhibiting tumor size; or inducing or improving T cell activation. An effective amount need not "cure" or completely remove neovascularization or angiogenesis. In some embodiments, an effective amount can reduce tumor size or volume. In some embodiments, the effective amount may reduce or ameliorate one or more symptoms of cancer.

As used herein, the phrase "treating a tumor" refers to inhibiting tumor growth, reducing tumor size, preventing tumor recurrence, and combinations thereof.

The term "polynucleotide" or "nucleotide" is intended to include a single nucleic acid as well as a plurality of nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger rna (mrna) or plasmid dna (pdna). In certain embodiments, the polynucleotide comprises a conventional phosphodiester bond or an unconventional bond (e.g., an amide bond, such as found in Peptide Nucleic Acids (PNAs)).

As used herein, "polynucleotide," "nucleotide," or "nucleic acid" are used interchangeably and encompass nucleotide sequences of full-length cDNA sequences, including untranslated 5 'and 3' sequences, coding sequences, as well as fragments, epitopes, domains, and variants of nucleic acid sequences. The polynucleotide may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide may be composed of single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is a mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded regions or, more commonly, double-stranded regions, or a mixture of single-and double-stranded regions. In addition, a polynucleotide may be composed of a triple-stranded region comprising RNA or DNA or both RNA and DNA. Polynucleotides may also comprise one or more modified bases or DNA or RNA backbones that are modified for stability or other reasons. "modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically or metabolically modified forms.

In the present disclosure, a polypeptide may consist of amino acids linked to each other by peptide bonds or modified peptide bonds (i.e., peptide isosteres) and may comprise amino acids other than the 20 gene-encoded amino acids (e.g., non-naturally occurring amino acids). The polypeptides of the present disclosure may be modified by natural processes such as post-translational processing or by chemical modification techniques well known in the art. Such modifications are well described in the basic text and in more detailed monographs, as well as in a number of research documents. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. It is understood that the same type of modification may be present to the same or different extent in several sites of a given polypeptide. In addition, a given polypeptide may comprise many types of modifications. Polypeptides may be branched, for example, due to ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processes, or may also be prepared by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenization, sulfation, transfer RNA mediated addition of amino acids to proteins, such as arginination and ubiquitination. (see, e.g., protein-Structure And Molecular Properties,2nd Ed., T.E.Creighton, W.H.Freeman And Company, New York (1993); Posttranslation compatibility Modification of Proteins, B.C.Johnson, Ed., Academic Press, New York, pgs.1-12 (1983); Seifter et al, Meth Enzymol 182:626-646 (1990); Rattan et al, Ann NY Acad Sci 663 48-62 (1992)).

The terms "fragment," "variant," "derivative," and "analog" when referring to any polypeptide or polynucleotide of the present disclosure include any polypeptide or polynucleotide that retains at least some activity, i.e., the ability to function as any naturally occurring function of the polypeptide or polynucleotide. For example, "fragments," "variants," "derivatives," and "analogs" of tumor necrosis factor receptor 1(TNFR1) have some of the activity of naturally occurring full-length TNFR1, e.g., the ability to bind to TNFR1 ligand, i.e., TNF- α or lymphotoxin. In another example, the "fragments," "variants," "derivatives," and "analogs" of a Fas polypeptide have some of the activity of a naturally occurring full-length Fas polypeptide, e.g., the ability to induce apoptosis. In other examples, a "fragment," "variant," "derivative," and "analog" of an endothelial cell-specific promoter can induce endothelial cell-specific expression of a gene operably linked to the promoter. Additional non-limiting examples of various fragments, variants, analogs or derivatives of TNFR1, Fas polypeptide, and endothelial cell specific promoters are described below.

The term "percent sequence identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences in a comparison window, which takes into account the additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target and reference sequences. Since the gaps are not nucleotides or amino acids, the gaps present in the target sequence are not counted. Likewise, gaps that occur in the reference sequence are not counted because the nucleotides or amino acids of the target sequence are counted rather than the nucleotides or amino acids from the reference sequence.

The percentage of sequence identity was calculated by: the number of positions at which the identical amino acid residue or nucleic acid base occurs in both sequences is determined to yield the number of matched positions, the number of matched positions is divided by the total number of positions in the window of comparison, and the result is multiplied by 100 to yield the percentage of sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using off-the-shelf software used and downloaded on-line. Suitable software programs are available from a variety of sources and are used for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is b12seq, which is part of the BLAST program suite and is available from the national center for biotechnology information BLAST website (BLAST. ncbi. nlm. nih. gov.) of the united states government. B12seq A comparison between two sequences is performed using the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher (part of the EMBOSS suite of bioinformatics programs), also available at www.ebi.ac.uk/Tools/psa from the European Bioinformatics Institute (EBI).

Different regions in a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence may each have their own percentage of sequence identity. Note that the percentage sequence identity values are rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It should also be noted that the length value will always be an integer.

One skilled in the art will appreciate that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-to-sequence comparisons driven only by primary sequence data. A sequence alignment may result from multiple sequence alignments. One suitable program for generating multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com/MUSCLE/Inc. ClustalW2 and MUSCLE may optionally be obtained, for example, from EBI.

It is also understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystal protein structure), functional data (e.g., mutation positions), or phylogenetic data. A suitable program for integrating the isomeric data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, or available from, for example, EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity may be performed automatically or manually.

As used herein, the terms "linked," "fused," "fusion," "chimeric," and "chimera" are used interchangeably. These terms refer to the joining together of two or more elements or components by any means including chemical conjugation or recombinant means. By "in-frame fusion" is meant the joining of two or more Open Reading Frames (ORFs) in a manner that preserves the correct reading frame of the original ORF, forming a continuous longer ORF. Thus, the resulting recombinant fusion protein or chimeric protein is a single protein comprising two or more segments corresponding to the polypeptide encoded by the original ORF (which segments are not normally so linked in nature). Although the reading frames are contiguous throughout the fusion segment, the segments may be physically or spatially separated by, for example, in-frame linker sequences.

The term "heterologous nucleotide sequence" means a different entity of a polynucleotide from which the polynucleotide is derived from the entity with which it is compared. For example, heterologous polynucleotides may be synthetic, or derived from different species, different cell types of an individual, or the same or different types of cells of different individuals. In one aspect, a heterologous nucleotide sequence can be a polynucleotide operably linked to another polynucleotide to produce a fusion polynucleotide. In some aspects, the heterologous nucleotide sequence can encode a polypeptide. For example, the heterologous nucleotide sequence can be a promoter element operably linked to a gene encoding a polypeptide. The heterologous nucleotide sequence may also include other cis-regulatory elements operably linked to the gene encoding the polypeptide. In other aspects, the heterologous nucleotide sequence may not encode a polypeptide.

As used herein, the term "expression" refers to the process by which a gene is produced to produce a biochemical, such as an RNA or polypeptide. The process includes any manifestation of the functional presence of genes within the cell, including but not limited to gene knockdown as well as transient and stable expression. Including, but not limited to, transcription of genes into messenger RNA (mRNA), transfer RNA (trna), small hairpin RNA (shrna), small interfering RNA (sirna), or any other RNA product, and translation of such mrnas into one or more polypeptides. If the final desired product is a biochemical, expression includes the production of the biochemical and any precursors.

As used herein, the term "complementarity determining region" (CDR) refers to the amino acid residues of an antibody that are responsible for binding to an antigen. The CDR regions of an antibody are present within the hypervariable regions of the heavy and light chains of an antibody. A full-length antibody comprises three CDR regions in the heavy chain variable domain and three CDR regions in the light chain variable domain.

As used herein, the term "anti-tumor response" refers to the physical response of a subject to the presence of a tumor. For example, in some aspects, an anti-tumor response in the present disclosure can be an anti-tumor immune response. In some aspects, the anti-tumor immune response is characterized by the presence of tumor-infiltrating CD8+ lymphocytes within the tumor bed. In some aspects, the anti-tumor immune response is characterized by a particular cytokine profile of the subject. In some aspects, the anti-tumor immune response is characterized by the presence of circulating anti-tumor antibodies against a tumor marker or tumor tissue in the subject.

As used herein, the term "cold tumor" refers to a tumor with little or no immune cells present within the tumor. For example, a cold tumor may have little or no tumor infiltrating lymphocytes (e.g., T cells and B cells), Natural Killer (NK) cells, or macrophages present in the tumor microenvironment. Tumors need not be completely devoid of immune cells to become cold tumors.

As used herein, the term "thermal tumor" refers to: tumors in which the presence of immune cells within the tumor is increased compared to cold tumors. For example, a hot tumor may increase the presence of tumor infiltrating lymphocytes (e.g., T cells and B cells), Natural Killer (NK) cells, or macrophages in the tumor microenvironment compared to a cold tumor.

As used herein, the term "immune checkpoint" refers to a biomolecule that acts as a positive or negative regulator of the immune system. Immune checkpoints play a role in maintaining self-tolerance, preventing autoimmunity, and protecting tissues from immune collateral damage. Immune checkpoint molecules may include, but are not limited to, CD27, CD28, CD40, CD122, CD137, OX40, glucocorticoid-induced TNFR family-related Gene (GITR), induced T-cell co-stimulator (ICOS), A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, and SIGLEC 7.

As used herein, the term "administered repeatedly" refers to the administration of a therapeutic agent on a repeated basis at defined fixed intervals. The time interval between each administration may vary over the course of repeated administrations, and may be as long as1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more.

The term "combination therapy" as used herein refers to the administration of two or more treatment modalities to treat a disease or condition. In one aspect of the disclosure, the combination therapy is directed to administering the vector and an immune checkpoint inhibitor to a subject or population of subjects in need thereof. In some embodiments, the combination therapy comprises administering an immune checkpoint inhibitor prior to administration of the vector. In another embodiment, the combination therapy comprises administration of an immune checkpoint inhibitor concurrently with administration of the vector. In another embodiment, the combination therapy comprises administration of an immune checkpoint inhibitor after administration of the vector.

As used herein, the term "adenovirus" refers to a human adenovirus of the family adenoviridae. The adenoviruses of the present disclosure can include, for example, adenoviruses from any of seven and 57 serotypes, including a (serotypes 12, 18, and 31), B (serotypes 3,7, 11, 14, 16, 21, 34, 35, 50, and 55), C (serotypes 1, 2, 5, 6, and 57), D (serotypes 8,9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51, 53, 54, and 56), E (serotype 4), F (serotypes 40 and 41), or G (serotype 52). As used herein, the term "adenoviral vector" refers to an adenovirus that has been genetically modified to behave differently than the native wild-type virus. For example, an adenoviral vector can be modified so that it cannot replicate outside of a particular packaging cell line. In some aspects, the adenoviral vector is genetically modified to carry one or more genes encoding non-adenoviral proteins.

II. Methods of treatment of the present disclosure

Although a variety of chemotherapeutic drugs are available for the treatment of different types of tumors, cancer remains one of the leading causes of death worldwide. Chemotherapeutic drugs often have undesirable toxicity because they indiscriminately target rapidly dividing cells, kill tumor cells, and rapidly dividing healthy cells. Other drugs are limited to treating specific tumor types with specific genetic mutations. These problems become more complicated when the primary tumor is transferred to other parts of the body, making effective treatment more difficult.

Immune checkpoints play a role in tumor growth and development. By naturally stimulating immune checkpoints through receptor/ligand interactions, tumor cells are able to evade the host immune system. Thus, molecules that block immune checkpoints (e.g., immune checkpoint inhibitors) have been tested for the treatment of cancer. However, these inhibitors only work on a small fraction of subjects with only a few types of tumors. In addition, the subject's response to immune checkpoint therapy is often accompanied by relapse and disease progression.

The present disclosure provides a method of reducing or inhibiting tumor size or eliminating a tumor in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. In particular aspects, the subject has a tumor that is reduced in size or inhibited, or a tumor that is eliminated, as compared to a tumor of a subject that has not been administered a vector.

The present disclosure also provides a method of treating a tumor or metastasis thereof in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. In particular aspects, a tumor or metastasis thereof in a subject is treated as compared to a tumor in the subject without administration of the vector. By stimulating immune checkpoints naturally, tumor cells can down-regulate anti-tumor T cell activity and evade the anti-tumor immune response of the host. This results in tumor-induced T cell tolerance and allows the tumor to continue to grow out of the control of the host immune system. Therefore, immune checkpoint inhibitors are being investigated as cancer therapeutics. However, immune checkpoint inhibitors are not effective therapeutics against "cold tumors" (tumors with little or no immune cells within the tumor). Cold tumors may have little or no tumor infiltrating lymphocytes (e.g., T cells and B cells), Natural Killer (NK) cells, or macrophages present in the tumor microenvironment. In contrast, a hot tumor is a tumor in which the presence of immune cells within the tumor is increased compared to a cold tumor. For example, a hot tumor may increase the presence of tumor infiltrating lymphocytes (e.g., T cells and B cells), Natural Killer (NK) cells, or macrophages in the tumor microenvironment compared to a cold tumor.

The present disclosure also provides a method of inducing or improving T cell activation in a subject having a tumor, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. In particular aspects, T cell activation is induced or improved in a subject compared to T cell activation in a subject not administered a vector.

The present disclosure also provides a method of inducing or increasing the therapeutic efficacy of an immune checkpoint inhibitor comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. In particular aspects, the therapeutic effect of the immune checkpoint inhibitor is induced or improved in a subject in which the vector is not administered as compared to the therapeutic effect of the immune checkpoint inhibitor in the subject.

The present disclosure also provides a method of converting a cold tumor to a hot tumor in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of an immune checkpoint inhibitor. In particular aspects, a cold tumor in the subject is converted to a hot tumor as compared to a cold tumor in a subject not administered the vector.

Tumor growth can be measured by techniques known in the art, including but not limited to Magnetic Resonance Imaging (MRI) scans, functional MRI (fmri) scans, Computed Tomography (CT) scans, or Positron Emission Tomography (PET) scans. In a particular aspect, tumor growth is measured by MRI. In some aspects, the tumor of the subject is a recurrent tumor that occurs during treatment with the vector. In yet other embodiments, the tumor of the subject is a metastatic tumor that occurs during treatment with the vector.

In some aspects, the methods of the present disclosure increase the overall survival rate of the subject. In some aspects, the methods of the present disclosure increase progression-free survival in a subject.

The term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, that has a positive response or is expected to have a positive response to the methods of the present disclosure. In some aspects, the subject is a human. In some aspects, the subject is a cancer patient.

A. Immune checkpoint inhibitors

The methods of the present disclosure comprise administering to a subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell specific promoter, and also administering to the subject an effective dose of an immune checkpoint inhibitor.

Immune checkpoints are biomolecules that are involved in stimulating or suppressing an immune response. The immune system naturally attempts to eliminate tumor cells by activating an anti-tumor immune response against cells containing tumor antigens. Anti-tumor immune responses may include tumor-specific CD8+ lymphocytes (cytotoxic T cells), Natural Killer (NK) cells, macrophages, and other immune cells, which migrate to the tumor site, infiltrate the tumor, and kill the tumor cells. Throughout the course of the immune response, various signaling checkpoints exist to stimulate or inhibit T cell activation, thereby modulating the extent and duration of the anti-tumor response.

Some immune checkpoints help stimulate immune responses (e.g., stimulate T cell activation). Stimulatory immune checkpoints include, but are not limited to, CD27, CD28, CD40, CD40L (CD154), CD58, CD80, CD86, CD122, CD137(4-1BB), CD134(OX40), CD252(OX40L), and CD278 (ICO). Other immunodetection sites have an inhibitory effect on immune responses (e.g., inhibition of T cell activation). Inhibitory immune checkpoints include, but are not limited to, adenosine A2A receptor (A2AR), CD152(CTLA-4), CD272(BTLA), CD276(B7-H3), IDO, TDO, killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3(LAG3), NOX2, VTCN1(B7-H4), PD-1, PD-L1, PD-L2, T cell immunoglobulin and mucin domain-3 (TIM3), CD328(SIGLEC7), CD329(SIGLEC9), and T cell immune receptor with Ig and ITIM domains (TIGIT).

Tumors can evade the host's anti-tumor response by participating in inhibitory immune checkpoints and down-regulating anti-tumor responses. Thus, molecules that block immune checkpoints (e.g., immune checkpoint inhibitors) are being investigated for the treatment of cancer. However, these inhibitors work only in a small fraction of patients with only a few types of tumors. In addition, patient response to immune checkpoint inhibitor therapy is often accompanied by relapse and disease progression.

In some aspects, the immune checkpoint inhibitor useful in the methods of the present disclosure is a molecule that binds to an immune checkpoint receptor or an immune checkpoint receptor ligand. In some aspects, the immune checkpoint inhibitor is an antibody. In some aspects, the antibody is a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody, or a chimeric antibody. In another aspect, the immune checkpoint inhibitor comprises Fab, F (ab)₂Fv or scFv.

In some aspects, the immune checkpoint inhibitor binds to an immune checkpoint receptor or an immune checkpoint receptor ligand involved in inhibiting T cell activation. In particular aspects, the immune checkpoint inhibitor is a molecule that inhibits the T cell stimulatory activity of the A2A receptor (A2AR), CD152(CTLA-4), CD272(BTLA), CD276(B7-H3), IDO, TDO, killer cell immunoglobulin-like receptor (KIR), lymphocyte activator gene 3(LAG3), NOX2, VTCN1(B7-H4), PD-1, PD-L1, PD-L2, T cell immunoglobulin and mucin domain-3 (TIM3), CD328(SIGLEC7), CD329(SIGLEC9), and T cell immune receptor with Ig and ITIM domains (TIGIT).

PD-1 antagonists

In a particular aspect of the disclosure, the immune checkpoint inhibitor is a PD-1 antagonist. PD-1 (programmed cell death protein 1) is a cell surface receptor that plays a role in inhibiting T cell responses in vivo. The B7 homolog, known as programmed death ligand 1(PD-L1), is a natural ligand of PD-1, which transmits its T cell inhibitory signal by binding to the PD-1 receptor. Most normal human tissues do not express PD-L1 on the cell surface. However, human cancers express large amounts of PD-L1 on the cell surface. Binding of PD-L1 expressed on the surface of tumor cells to PD-1 on the surface of T cells can lead to T cell apoptosis, T cell depletion, T cell disability, T cell IL-10 production, and dendritic cell suppression. These signals inhibit the activity of anti-tumor T cells and act as an immune barrier, helping tumor cells to evade anti-tumor immune responses.

PD-1 antagonists may prevent PD-1 signaling by binding directly to PD-1 and inhibiting the interaction of PD-1 with PD-L1. This reduces signaling from the PD-1 receptor and blocks PD-1 mediated T cell suppression. In one embodiment, a PD-1 antagonist useful in the present disclosure is an anti-PD-1 antibody. In another embodiment, the anti-PD-1 antibody is a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody, or a chimeric antibody. In another embodiment, the anti-PD-1 antibody for treatment comprises Fab, F (ab)₂Fv or scFv.

In some aspects, the PD-1 antagonist is an anti-PD-1 antibody selected from the group consisting of: nivolumab (a) (b)See, e.g., U.S. Pat. No. 8,008,449 and Wang et al 2014, Cancer Immunol Res.2(9):846-56), pembrolizumab (R)See, e.g., U.S. patent nos. 8,354,509 and 8,900,587), carpriclizumab (SHR-1210; see, e.g., Huang et al, Clin Cancer Res.2018, PMID: 29358502), cimetipril mab (See, e.g., U.S. patent application publication No. 2015/0203579A1), Cedilizumab (C.B.)See, e.g., Liu et al, J Hematol oncol, 2017; 10(1):136) and PDR001 (see, e.g., Naing et al, J Clin Oncol.2016).

In some embodiments, the immune checkpoint inhibitor is a composition comprising nivolumab V_HThe anti-PD-1 antibody of 3 CDRs. In some embodiments, the immune checkpoint inhibitor is a composition comprising nivolumab V_LThe anti-PD-1 antibody of 3 CDRs. In other embodiments, the immune checkpoint inhibitor is a packageContaining nivolumab V_H3 CDRs and nivolumab V_L3 CDR (V)_HCDR1、V_H CDR2、V_H CDR3、V_L CDR1、V_LCDR2 and V_LCDR 3). In other embodiments, the immune checkpoint inhibitor is a peptide comprising V_HAnd V_LThe anti-PD-1 antibody of (1), the V_HComprising nivolumab V_HThe amino acid sequence of (A), the_LComprising nivolumab V_LThe amino acid sequence of (a). In some embodiments, the immune checkpoint inhibitor is nivolumab.

Another PD-1 antagonist that reduces or inhibits PD-1 signaling is a molecule that binds to PD-L1 and blocks the interaction of PD-L1 with PD-1. This interference of receptor/ligand binding reduces signaling from the PD-1 receptor and blocks PD-1 mediated T cell suppression. In one embodiment, a PD-1 antagonist useful in the present disclosure is an anti-PD-L1 antibody. In another embodiment, the anti-PD-L1 antibody is a monoclonal antibody, a humanized antibody, a human antibody, a single chain antibody, or a chimeric antibody. In another embodiment, the anti-PD-L1 antibody comprises a Fab, F (ab)₂Fv or scFv.

In some aspects, the PD-1 antagonist is an anti-PD-L1 antibody selected from the group consisting of: amilizumab (a)See, e.g., U.S. Pat. No. 8,217,149), Abamectin: (See, e.g., US 2014/0341917a1), de Waluzumab (c)See, e.g., US 2013/0034559 a1) and BMS-936559 (see, e.g., US patent nos. 7,943,743; WO 2013/173223).

C. Combination therapy comprising a Fas-chimera vector and a PD-1 antagonist

The present disclosure provides a method of reducing or inhibiting tumor size or eliminating a tumor in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of a PD-1 antagonist. In some aspects, the subject's tumor size is reduced or inhibited, or the subject's tumor is eliminated, as compared to a tumor of a subject to which the vector is not administered.

The present disclosure also provides a method of treating a tumor or metastasis thereof in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of a PD-1 antagonist. In some aspects, the tumor or metastasis thereof in the subject is treated as compared to the tumor or metastasis thereof in a subject not administered the vector.

The present disclosure also provides a method of inducing or improving T cell activation in a subject having a tumor, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of a PD-1 antagonist. In some aspects, T cell activation is induced or improved in a subject compared to T cell activation in a subject not administered a vector.

The present disclosure also provides a method of inducing or increasing the therapeutic efficacy of a PD-1 antagonist in a subject having a tumor, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of a PD-1 antagonist. In some aspects, the therapeutic effect of the PD-1 antagonist is induced or improved in a subject in which the vector is not administered, as compared to the therapeutic effect of the PD-1 antagonist in the subject.

The present disclosure also provides a method of converting a cold tumor to a hot tumor in a subject in need thereof, comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of a PD-1 antagonist. In some aspects, the cold tumor in the subject is converted to a hot tumor as compared to a cold tumor in a subject not administered the vector.

For the methods of the present disclosure, an effective dose of the carrier is 0.5 x 10 administered every six weeks¹³-2×10¹³An effective dose of each viral particle, a PD-1 antagonist (e.g., an anti-PD-1 antibody (e.g., nivolumab)) is about 240mg every two weeks or about 480mg every four weeks. In some aspects, the anti-PD-1 antibody (e.g., pembrolizumab) is administered at a dose of about 200mg every three weeks.

Tumor growth may be measured by techniques known in the art, including, but not limited to, Magnetic Resonance Imaging (MRI) scans, functional MRI (fmri) scans, Computed Tomography (CT) scans, or Positron Emission Tomography (PET) scans. In a particular aspect, tumor growth is measured by MRI. In some aspects, the tumor of the subject is a recurrent tumor that occurs during treatment with the vector. In other embodiments, the tumor of the subject is a metastatic tumor that occurs during treatment with the vector.

The term "subject" or "individual" or "animal" or "subject" or "mammal" refers to any subject, particularly a mammalian subject, that has, or is expected to have, an increased or improved anti-tumor response as a result of the present disclosure. In some aspects, the term "subject" or "individual" or "animal" or "subject" or "mammal" refers to any subject, particularly a mammalian subject, and a PD-1 antagonist, that has been administered a combination regimen comprising a vector expressing a Fas chimera protein. In one embodiment, the subject is a human. In another embodiment, the subject is a cancer patient.

In certain aspects, the PD-1 antagonist is administered prior to, concurrently with, or after administration of the vector. In other aspects, the vector is administered at least 1 day ahead, at least 2 days ahead, at least 3 days ahead, at least 4 days ahead, at least 5 days ahead, at least 6 days ahead, at least 7 days ahead, at least 9 days ahead, at least 10 days ahead, at least 2 weeks ahead, at least 3 weeks ahead, at least 4 weeks ahead, at least 1 month ahead, at least 2 months ahead, or more prior to the PD-1 antagonist. In other aspects, the PD-1 antagonist is administered at least 1 day ahead, at least 2 days ahead, at least 3 days ahead, at least 4 days ahead, at least 5 days ahead, at least 6 days ahead, at least 7 days ahead, at least 9 days ahead, at least 10 days ahead, at least 2 weeks ahead, at least 3 weeks ahead, at least 4 weeks ahead, at least 1 month ahead, at least 2 months ahead, or more prior to the vector.

In some aspects, the tumor of the subject is a recurrent tumor that occurs during treatment with the vector. In other embodiments, the tumor of the subject is a metastatic tumor that occurs during treatment with the vector. In some aspects, the vector is administered prior to administration of the PD-1 antagonist, and the PD-1 antagonist is administered as the tumor progresses. In some aspects, the vector is administered prior to administration of the PD-1 antagonist, and the PD-1 antagonist is administered upon tumor recurrence.

An effective dose of a vector administered as part of the present disclosure can be measured as a Viral Particle (VP). In some embodiments, an effective dose of the carrier includes, but is not limited to, equal to or less than about 1 × 10¹⁶、1×10¹⁵、1×10¹⁴、5×10¹³、4×10¹³、3×10¹³、2×10¹³、1×10¹³、9×10¹²、8×10¹²、7×10¹²、6×10¹²、5×10¹²、4×10¹²、3×10¹²、2×10¹²、1×10¹²、9×10¹¹、8×10¹¹、7×10¹¹、6×10¹¹、5×10¹¹、4×10¹¹、3×10¹¹、2×10¹¹、1×10¹¹、9×10¹⁰、8×10¹⁰、7×10¹⁰、6×10¹⁰、5×10¹⁰、4×10¹⁰、3×10¹⁰、2×10¹⁰Or 1X 10¹⁰And (c) viral particles. In other embodiments, the effective dose of the carrier is about 1X 10¹⁰To about 1X 10¹⁶About 1X 10¹¹To about 1X 10¹⁵About 1X 10¹¹To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁵About 1X 10¹²To about 1X 10¹⁶About 1X 10¹²To about 1X 10¹⁴About 5X 10¹²To about 1X 10¹⁶About 5X 10¹²To about 1X 10¹⁵About 5X 10¹²To about 1X 10¹⁴About 1X 10¹²To about 1X 10¹³About 1X 10¹³To about 1X 10¹⁴And (c) viral particles.

In some aspects, at least about 1 × 10¹¹An effective dose of each viral particle is administered with the vector. In some aspects, at least about 1 × 10¹²An effective dose of each viral particle is administered with the vector. In some aspects, at least about 1 × 10¹³An effective dose of each viral particle is administered with the vector. In some aspects, at least about 1 × 10¹⁴An effective dose of each viral particle is administered with the vector. In some aspects, at least about 1 × 10¹⁵An effective dose of each viral particle is administered with the vector. In some aspects, at least about 1 × 10¹⁶An effective dose of each viral particle is administered with the vector. In some aspects, at least about 1 × 10⁷、1×10⁸、1×10⁹、1×10¹⁰Or 5X 10¹⁰An effective dose of each viral particle is administered with the vector.

In some aspects of the disclosure, an effective dose of a PD-1 antagonist is administered as a fixed dose. For the purposes of this disclosure, the use of the term "fixed dose" means a dose that is administered to a patient without regard to the patient's weight or Body Surface Area (BSA). Thus, the fixed dose is not provided as a mg/kg dose, but as an absolute amount of drug (e.g., anti-PD-1 antibody). For example, a 60kg human and a 100kg human will receive the same dose of the composition (e.g., 240mg of anti-PD-1 antibody).

In some aspects, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody. In some aspects, an effective dose of an anti-PD-1 or anti-PD-L1 antibody is a dose between about 100mg to about 600mg (e.g., a fixed dose). In some aspects, an effective dose of anti-PD-1 or PD-L1 antibody is a fixed dose of about 100-300mg, such as about 200-300mg, about 220-260mg, about 230-250mg, or about 240 mg. In some aspects, an effective dose of anti-PD-1 or anti-PD-L1 antibody is a fixed dose of about 400-600mg, such as about 450-520mg, about 460-510mg, about 470-500mg, or about 480 mg. In some aspects, an effective dose of an anti-PD-1 or PD-L1 antibody is one dose (e.g., a fixed dose), such as about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 470mg, about 480mg, about 490mg, about 500mg, about 510mg, about 530mg, about 540mg, about 550mg, about 560mg, about 570mg, about 580mg, about 590mg or about 600 mg.

In some aspects, an effective dose of an anti-PD-1 antibody or an anti-PD-L1 antibody is a dose (e.g., a fixed dose) of between about 60-100mg, about 60-200mg, about 60-300mg, about 60-400mg, about 60-500mg, or about 60-600 mg. In some aspects, an effective dose of the anti-PD-1 antibody or anti-PD-L1 antibody is a dose (e.g., a fixed dose) of between about 100-200mg, about 100-300mg, about 100-400mg, or about 100-500 mg. In some aspects, an effective dose of the anti-PD-1 antibody or anti-PD-L1 antibody is a dose (e.g., a fixed dose) of between about 300-400mg or about 300-500 mg. In some aspects, an effective dose of an anti-PD-1 antibody or an anti-PD-L1 antibody is a dose (e.g., a fixed dose) of between about 400 and 500 mg. In a particular aspect, an effective dose of the anti-PD-1 antibody or anti-PD-L1 antibody is a dose (e.g., fixed dose) of about 480 mg.

The term "weight-based dose" as referred to herein refers to a dose that is administered to a patient based on the calculation of the patient's weight. For example, when a patient weighing 60kg requires 3mg/kg of anti-PD-1 antibody, an appropriate amount of anti-PD-1 antibody (i.e., 180mg) can be taken.

In some aspects, an effective dose of a PD-1 antagonist is equal to or less than about 15mg/kg, 14mg/kg, 13mg/kg, 12mg/kg, 11mg/kg, 10mg/kg, 9mg/kg, 8mg/kg, 7mg/kg, 6mg/kg, 5mg/kg, 4mg/kg, 3mg/kg, 2mg/kg, or 1 mg/kg. In a specific embodiment, the effective dose of the PD-1 antagonist is about 3 mg/kg.

In some aspects, the PD-1 antagonist is nivolumab. In some aspects, at 3 × 10¹²To 3X 10¹³An effective dose of each viral particle is administered with the vector, and nivolumab is administered at an effective dose (weight-based dose) of 2mg/kg to 12 mg/kg. In other aspects at 3 × 10¹²To 3X 10¹³An effective dose of each viral particle is administered with the vector, and nivolumab is administered at an effective dose (fixed dose) of 460mg to 500 mg.

In particular aspects at 1 × 10¹³The amount of each viral particle is administered with an effective dose of the vector, and an effective dose of nivolumab is administered in an amount of 3mg/kg (weight-based dose). In another aspect, at 3 × 10¹²To 1X 10¹³The amount of each viral particle is administered with an effective dose of the vector, and an effective dose of nivolumab is administered in an amount of 200mg to 260mg (fixed dose). In particular aspects at 1 × 10¹³The amount of each viral particle is administered with an effective dose of the vector and nivolumab is administered in an effective amount (fixed dose) of 240 mg.

In another aspect, at 1 × 10¹³The amount of each viral particle is administered with an effective dose of the vector, and an effective dose of nivolumab is administered in an amount of 3mg/kg to 12mg/kg (weight based dose). In another aspect, at 3 × 10¹²To 1X 10¹³The amount of each viral particle is administered with an effective dose of the vector, and an effective dose of nivolumab is administered in an amount of 460mg to 500mg (fixed dose). In particular aspects at 1 × 10¹³The amount of each viral particle is administered with an effective dose of the vector, and an effective dose of nivolumab is administered in an amount of 480mg (fixed dose).

In some aspects, the PD-1 antagonist is pembrolizumab. In some aspects, at 3 × 10¹²To 3X 10¹³An effective dose of each viral particle is administered with the vehicle and pembrolizumab is administered at an effective dose (fixed dose) of 150mg to 250 mg.

In another aspect, at 3 × 10¹²To 1X 10¹³Administering an effective dose of the vector in an amount of 180mg toAn effective dose of pembrolizumab is administered in an amount of 220mg (fixed dose). In particular aspects at 1 × 10¹³The amount of each viral particle is administered in an effective dose of the carrier and pembrolizumab is administered in an effective amount of 200mg (fixed dose).

The methods of the present disclosure comprise administering at least one effective dose of a carrier and a PD-1 antagonist. The regimen for administering the vector and the PD-1 antagonist includes repeated administrations of the vector and the PD-1 antagonist. In one embodiment, the vehicle is repeatedly administered daily, once per about 2 days, once per about 3 days, once per about 4 days, once per about 5 days, once per about 6 days, once per about 7 days, once per about 2 weeks, once per about 3 weeks, once per about 4 weeks, once per about 5 weeks, once per about 6 weeks, once per about 7 weeks, once per about 2 months, or once per about 6 months. In another embodiment, the PD-1 antagonist is administered repeatedly once about 7 days, once about 2 weeks, once about 3 weeks, once about 4 weeks, once about 2 months, once about 3 months, once about 4 months, once about 5 months, or once about 6 months. In a particular embodiment, the vector is administered every 2 months and the PD-1 antagonist is administered every 2 weeks. In a specific embodiment, the PD-1 antagonist is nivolumab.

D. Diseases and disorders

The methods of the present disclosure can be used to reduce or inhibit tumor size or eliminate tumors in a subject in need thereof. In some aspects of the disclosure, the tumor is derived from or associated with: metastatic colorectal cancer (mCRC), advanced non-squamous non-small cell lung cancer (NSCLC), metastatic renal cell carcinoma (mRCC), glioblastoma multiforme (GBM), muller's carcinoma, ovarian cancer, peritoneal cancer, fallopian tube cancer, or papillary serous carcinoma of the uterus. In some aspects, the methods of the present disclosure reduce the volume of malignant peritoneal fluid (e.g., ascites), reduce pain in the subject, prolong the survival of the subject, or any combination thereof. The combination of the vector and the PD-1 antagonist can reduce, inhibit or treat tumors. The tumor may be a solid tumor, a primary tumor, a metastatic tumor, or any combination thereof. The term "metastatic" or "metastasis" refers to a tumor cell capable of establishing a secondary neoplastic lesion in another site or organ.

In the methods of the present disclosure, a "solid tumor" includes, but is not limited to, sarcoma, melanoma, carcinoma, or other solid tumor cancers. "sarcoma" refers to a tumor that consists of material like embryonic connective tissue and is usually composed of tightly packed cells embedded in fibrous or homologous material. Sarcomas include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanoma, myxosarcoma, osteosarcoma, Ebeneset's sarcoma, liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms ' tumor sarcoma, endometrial sarcoma, interstitial sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblast sarcoma, giant cell sarcoma, granulocyte sarcoma, Hodgkin's sarcoma, idiopathic multicopy-synergic sarcoma, immunoblastic sarcoma of B-cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer's cell sarcoma, angiosarcoma, white sarcoma, malignant interstitial sarcoma, paraosteosarcoma, reticulosarcoma, Rous sarcoma, Goniose's sarcoma, and Goniose's sarcoma, Serous cystic sarcoma, synovial sarcoma, and capillary dilated sarcoma.

The term "melanoma" refers to tumors produced by the melanocytic system of the skin and other organs. Melanoma includes, for example, acral sparrow melanoma, melanotic melanoma, benign juvenile melanoma, claudman' S melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo malignant melanoma, metastatic melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

The term "carcinoma (carcinoma)" refers to a malignant neoplasm consisting of epithelial cells that tend to infiltrate the surrounding tissue and cause metastasis. Exemplary cancers include, for example, acinar cancer (acinar carcinosoma), cystic adenoid cancer (adenocystic carcinosoma), adenoid cystic cancer (adenoid cystic cancer), adenocarcinoma (carcinosoma), adrenocortical carcinoma, alveolar cell carcinoma, basal cell carcinoma (carcinosoma basocellulare), basal cell carcinoma (basoloid carcinosoma), basal squamous cell carcinoma (basolous cell carcinosoma), bronchioloalveolar carcinoma (broncheolar carcinosoma), bronchioloid cancer, medullary cancer, cholangiocellular carcinoma, choriocarcinoma, jelly-like carcinoma, acne carcinoma, dendrima, papillary carcinoma (carcinose), cutaneous carcinoma, columnar carcinoma, squamous cell carcinoma (carcinosis), choriocarcinoma, ductal carcinoma, papillary carcinoma (carcinosoma), papillary carcinoma (carcinosis), carcinosis, ductal carcinoma (carcinosis), adenoid carcinosis, ductal carcinoma (carcinosis), adenoid carcinosis, ductal carcinoma (carcinosis), adenoid carcinosis, ductal carcinoma, adenoid (carcinosis), adenoid carcinosis, ductal carcinoma, carcinosis, ductal carcinoma, carcinosis, and is, Ulcerative carcinoma (carcinosoma ex ulcerous), fibrous carcinoma (carcinosum), colloidal carcinoma (gelatinforme carcinosoma), colloidal carcinoma (gelatinous carcinosoma), giant cell carcinoma, adenocarcinoma (glaandular carcinosoma), granular cell carcinoma, mother-of-hair carcinoma (hair-matrix carcinosoma), leukemia, hepatocellular carcinoma, schorlike cell adenocarcinoma (hurthlehler cell carcinoma), vitreous carcinoma (hyaline carcinosoma), renal cell carcinoma (hypephrocardia), juvenile embryonal carcinoma (embryonic carcinoma), carcinoma in situ, intraepidermal carcinoma (intraepithelial carcinoma), intraepithelial carcinoma (intraepithelial carcinosoma), guillierocele's carcinoma (krinosis), squamous cell carcinoma (carcinosoma), mammary carcinoma (carcinosis), mammary carcinoma (cystic carcinoma's carcinoma), mammary carcinoma (cystic carcinoma), mammary carcinoma (carcinosis), mammary carcinoma (mammary carcinoma), mammary carcinoma (carcinosis), melanoma), mammary carcinoma (mammary carcinoma), mammary carcinoma (mammary carcinoma), mammary carcinoma (carcinosis), or carcinosis), or carcinosis), carcinosis), or carcinosis, and is, Melanoma (melanotic carcinoma), carcinoma of the soft (carcinoma molle), mucous cancer (mucous carcinoma), mucous cancer (mucoma mucosum), mucous cell carcinoma (carcinoma mucocele), mucoepidermoid carcinoma (mucoepidermoid carcinoma), mucous cancer (mucoma mucosum), mucomatoid carcinoma (mucoma myxomatodes), nasopharyngeal carcinoma (nanopharmaceutical carcinoma), oat cell carcinoma (oat cell carcinoma), ossified carcinoma (carcinosiliciformis), osteoid carcinoma (osteoplastic carcinoma), papillary carcinoma (palatinoma), periportal carcinoma (periportal carcinoma), precancerous carcinoma (precancerous carcinoma), papillary carcinoma (cancer), sarcomatoid carcinoma (cancer of the kidney), sarcomatoid carcinoma of the kidney (cancer of the kidney), cancer of the liver (cancer of the kidney, cancer of the liver, cancer of the kidney (cancer of the kidney, kidney (cancer of the liver, kidney cancer of the kidney, kidney, Simple cancer (carcinosoma simplex), small cell carcinoma, potato-like (solanaceous carcinosa), globular cell carcinoma (sphenoid cell carcinosa), spindle cell carcinoma (spindle cell carcinosa), medullary carcinoma (carcinosa spongisum), squamous carcinoma (squamous carcinosa), squamous cell carcinoma (stringcarcinoma), dilated carcinoma (carcinosa), transitional cell carcinoma (metastatic cell carcinosa), nodular carcinoma (carcinosa), nodular skin carcinoma (tuberous carcinosa), verrucous carcinoma (verrucous carcinosa), and choriocarcinoma (carcinosum).

Additional cancers (cancer) that may be inhibited or treated according to the present methods include, for example, leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer (including non-small cell lung cancer (NSCLC)), rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung cancer, primary brain tumors, gliomas (including glioblastoma multiforme (GBM) and recurrent GBM), Gastrointestinal (GI) cancers (including but not limited to esophageal, gall bladder, biliary, liver, pancreas, stomach, small intestine, large intestine, colon, rectum, and anal cancers), malignant pancreatic islets, malignant carcinoids, bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, genitourinary tract cancer, Malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortex cancer, prostate cancer, Muller's cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, or papillary serous carcinoma of uterus.

In some aspects, the tumor is a recurrent tumor. In some aspects, the tumor is a metastatic tumor.

III nucleic acid construct comprising a Fas-chimera gene and an endothelial cell-specific promoter

The present disclosure provides a method of anti-tumor therapy comprising: (a) administering to the subject an effective dose of a vector comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter; and (b) administering to the subject an effective dose of a checkpoint inhibitor.

In the present disclosure, the gene encoding the Fas-chimera protein (or gene product) can be linked to an endothelial cell-specific promoter that directs expression of the Fas-chimera gene product in endothelial cells. Expression of such cytotoxic gene products is useful in situations where excessive neovascularization or vascular growth is undesirable (e.g., in tumors).

Fas-chimeras

The Fas-chimera proteins expressed from the nucleic acid constructs of the present disclosure comprise at least two "death receptor" polypeptides, each of which is derived from a different protein. The first polypeptide of the Fas-chimera protein comprises the ligand binding domain of tumor necrosis factor receptor 1(TNFR 1). The second polypeptide of the Fas-chimera protein comprises the effector domain of the Fas polypeptide.

The ligand binding domain of TNFR1 can be any domain that binds to TNFR1 ligand. In one embodiment, the TNFR1 ligand is TNF- α. In another embodiment, the TNFR1 ligand is lymphotoxin- α. The ligand binding domain of TNFR1 may be the extracellular domain of TNFR1 or any fragment, variant, derivative or analog thereof. Non-limiting examples of TNFR1 ligand binding domains are described below.

Effector domains useful for Fas polypeptides of the present disclosure include any Fas domain that forms a death-inducing signaling complex (DISC) to induce apoptosis. In one embodiment, the effector domain of the Fas polypeptide comprises an intracellular domain, a transmembrane domain, or both. Non-limiting examples of Fas polypeptide effector domains are described below.

TNFR1 and Fas polypeptide can be linked by a peptide bond or by a linker. The linker linking the TNFR1 ligand binding domain to the Fas effector domain may be a polypeptide linker or a non-peptide linker. For example, the linker of the Fas-chimera protein may comprise one or more glycines, serines, leucines, or any combination thereof. In one embodiment, the linker for use in the present disclosure comprises Ser-Leu. In another embodiment, linkers useful in the present disclosure include (GGGS) n, (Denise et al.J.biol.chem.277: 35035-.

1. Tumor necrosis factor receptor 1

Full-length human TNFR1 polypeptide is 455 amino acids in length and is also known as TNF-R1, tumor necrosis factor receptor type I (TNFRI), TNFR-I, TNFRSF1A, tnpar, P55, P60, or CD120 a. The naturally occurring human TNFR1 polypeptide is known to bind to TNF- α or homotrimeric lymphotoxin- α. Binding of TNF- α to the extracellular domain results in homotrimerization of TNFRI, which then interacts specifically with the death domain of the tumor necrosis factor receptor type 1 associated death domain protein (TRADD). Various TRADD interacting proteins, such as TNF receptor-related factor (TRAFS), receptor interacting serine/threonine protein kinase 1(RIPK1), and Fas associated protein with death domain (FADD), are recruited into the complex by their association with the TRADD. The complex activates at least two distinct signaling cascades, apoptosis and NF- κ -B signaling.

The 455aa polypeptide sequence (reported as the human TNFR1 polypeptide sequence) has the identification number P19438-1 in the UniProtKB database. This human TNFR1 polypeptide sequence is designated herein as isoform A and SEQ ID NO 2. SEQ ID NO 1 is a nucleotide sequence encoding SEQ ID NO 2. The 108aa polypeptide sequence was reported as an isoform of the human TNFR1 polypeptide sequence and has the identification number P19438-2 in the UniProtKB database. The 108aa polypeptide corresponds to amino acids 1 to 108 of isoform A (SEQ ID NO:2) and is designated herein as isoform B. Another variant of the human TNFR1 polypeptide with 232aa is reported in the UniProtKB database as identification number P19438-3. The 232aa polypeptide corresponds to amino acids 1 to 232 of isoform A (SEQ ID NO:2) and is referred to herein as isoform C. Additional natural variants of human TNFR1 include, but are not limited to, TNFR1 polypeptides of isoform A, B and C comprising one or more mutations selected from the group consisting of: H51Q, C59R, C59S, C62G, C62Y, P75L, T79M, C81F, C99S, S115G, C117R, C117Y, R121P, R121Q, P305T and any combination thereof. Other known TNFR1 variants include TNFR1 polypeptides of isoforms A, B and C, which comprise L13LILPQ, K255E, S286G, R394L, 412: deletion, GPAA443-446APP, or any combination thereof.

Table 1 shows the amino acid sequence of human wild-type TNFR1 and the nucleotide sequence encoding wild-type TNFR 1.

TABLE 1 TNFR1 sequences

The mouse TNFR1 polypeptide sequence and variants thereof are also reported. The 454aa mouse TNFR1 polypeptide has the identification number P25118 in the UniProtKB database. TNFR1 polypeptides known in other animals include, but are not limited to, rat (e.g., P22934 in the UniProtKB database), bovine (e.g., O19131 in the UniProtKB database), porcine (e.g., P50555 in the UniProtKB database), or equine (e.g., D1MH71 in the UniProtKB database).

Full-length TNFR1 can be cleaved into two chains, (1) TNF receptor superfamily member 1A, membrane form (i.e., corresponding to amino acids 22 to 455 of full-length TNFR1) and (2) TNF binding protein 1(TBPI) (i.e., corresponding to amino acids 41 to 291 of full-length TNFR 1). Full-length human TNFR1 polypeptide consists of a signal sequence (amino acids 1 to 21 of SEQ ID NO:2), an extracellular domain (amino acids 22 to 211 of SEQ ID NO:2), a transmembrane domain (amino acids 212 to 234 of SEQ ID NO:2), and a cytoplasmic domain (amino acids 235 to 455 of SEQ ID NO: 2). The extracellular domain of TNFR1 comprises four cysteine repeats, TNFR-Cys1 (corresponding to amino acids 43 to 82 of SEQ ID NO:2), TNFR-Cys2 (corresponding to amino acids 83 to 125 of SEQ ID NO:2), TNFR-Cys3 (corresponding to amino acids 126 to 166 of SEQ ID NO:2), and TNFR-Cys4 (corresponding to amino acids 167 to 196 of SEQ ID NO: 2).

As will be appreciated by those skilled in the art, the starting and terminating residues of the domains listed above may vary depending on the computer modeling program used or the method used to determine the domain. Thus, the various functional domains of TNFR1 may differ from those defined above.

In one embodiment, the ligand binding domain of TNFR1 useful for Fas-chimera protein comprises, consists essentially of, or consists of the extracellular domain of TNFR1 or any fragment, variant, derivative or analog thereof, wherein the extracellular domain of TNFR1 or any fragment, variant, derivative or analog thereof binds TNF- α. In another embodiment, the ligand binding domain of TNFR1 includes TNFR-Cys 1; TNFR-Cys 2; TNFR-Cys 3; TNFR-Cys 4; TNFR-Cysl and TNFR-Cys 2; TNFR-Cysl and TNFR-Cys 3; TNFR-Cysl and TNFR-Cys 4; TNFR-Cys2 and TNFR-Cys 3; TNFR-Cys2 and TNFR-Cys 4; TNFR-Cys3 and TNFR-Cys 4; TNFR-Cysl, TNFR-Cys2 and TNFR-Cys 3; TNFR-Cysl, TNFR-Cys2 and TNFR-Cys 4; TNFR-Cys2, TNFR-Cys3 and TNFR-Cys 4; or TNFR-Cys1, TNFR-Cys2, TNFR-Cys3 and TNFR-Cys 4. In other embodiments, the ligand binding domain of TNFR1 in the Fas-chimera protein comprises TNF binding protein I. In other embodiments, the TNFR1 ligand binding domain of the Fas-chimera protein comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 22 to 190, amino acids 22 to 191, amino acids 22 to 192, amino acids 22 to 193, amino acids 22 to 197, amino acids 22 to 198, amino acids 22 to 199, amino acids 22 to 200, amino acids 22 to 201, amino acids 22 to 202, amino acids 22 to 203, amino acids 22 to 204, amino acids 22 to 205, amino acids 22 to 206, amino acids 22 to 207, amino acids 22 to 208, amino acids 22 to 209, amino acids 22 to 210, or amino acids 22 to 211 of SEQ ID No. 2, wherein the ligand binding domain binds to a TNFR1 ligand (e.g., TNF- α).

In other embodiments, the ligand binding domain of TNFR1 further comprises a signal peptide. An example of a suitable signal peptide is the signal peptide of TNFR1, e.g., amino acids 1 to 21 of SEQ ID NO. 2. In yet other embodiments, the ligand binding domain of the Fas-chimera gene product comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 1 to 190, amino acids 1 to 191, amino acids 1 to 192, amino acids 1 to 193, amino acids 1 to 194, amino acids 1 to 198, amino acids 1 to 199, amino acids 1 to 200, amino acids 1 to 201, amino acids 1 to 202, amino acids 1 to 203, amino acids 1 to 204, amino acids 1 to 205, amino acids 1 to 206, amino acids 1 to 207, amino acids 1 to 208, amino acids 1 to 209, amino acids 1 to 210 or amino acids 1 to 211 of SEQ ID NO 2, wherein the ligand binding domain binds to a TNFR1 ligand, such as TNF- α. In certain embodiments, the TNFR1 ligand binding domain of the Fas-chimera protein comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 4, wherein the ligand binding domain binds to a TNFR1 ligand (e.g., TNF- α).

In other embodiments, the ligand binding domain of TNFR1 is encoded by a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 3.

In yet other embodiments, the TNFR1 ligand binding domain of the Fas-chimera protein comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 22 to 108 of SEQ ID NO: 2(TNFR1 isoform B), amino acids 22 to 232 of SEQ ID NO: 2(TNFR1 isoform C), or amino acids 44 to 291 of SEQ ID NO:2 (TBP1), wherein the ligand binding domain binds to a TNFR1 ligand (e.g., TNF- α).

Fas Polypeptides

The full-length human Fas polypeptide is 335 amino acids in length and is also known as tumor necrosis factor receptor superfamily member 6, Apo-1 antigen, apoptosis-mediating surface antigen Fas, FasLG receptor or CD 95. Naturally occurring Fas polypeptide is a receptor for TNFSF 6/FasLG. When the Fas polypeptide is bound to a Fas ligand (FasL), the interaction between Fas and FasL results in the formation of a Death Inducing Signaling Complex (DISC) comprising FADD, caspase-8 and caspase-10. In some types of cells (type I), the treated caspase-8 directly activates other members of the caspase family and triggers the execution of apoptosis. In other types of cells (type II), Fas-DISC initiates a feedback loop that screws into the progressive release of pro-apoptotic factors from mitochondria and the amplified activation of caspase-8. Fas-mediated apoptosis can have a role in inducing peripheral tolerance, antigen-stimulated mature cell suicide, or both.

The 335aa polypeptide sequence reported as the human Fas polypeptide sequence has the identification number P25445-1 in the UniProtKB database. The human Fas polypeptide sequence is designated herein as SEQ ID NO 6. SEQ ID NO 5 is a nucleotide sequence encoding SEQ ID NO 6. The nucleotide sequence encoding the Fas polypeptide is also referred to as APT1, Fas1 or TNFRSF 6. The full-length Fas polypeptide comprises a signal peptide (corresponding to amino acids 1 to 25 of SEQ ID NO: 6), an extracellular domain (corresponding to amino acids 26 to 173 of SEQ ID NO: 6), a transmembrane domain (corresponding to amino acids 174 to 190 of SEQ ID NO: 6) and an intracellular (or cytoplasmic) domain (corresponding to amino acids 191 to 335 of SEQ ID NO: 6). The intracellular domain comprises a death domain (e.g., corresponding to amino acids 230 to 314 of SEQ ID NO: 6).

As will be understood by those skilled in the art, the above listed domain start and end residues can be varied according to the computer modeling program used or the method used to determine the domain. Thus, the various functional domains of Fas can be different from those defined above. Table 2 shows the amino acid sequence of wild-type human Fas and the nucleotide sequence encoding the Fas protein.

TABLE 2 Fas sequence

Mouse Fas polypeptide sequences and variants thereof are also reported. The mouse Fas polypeptide of 327aa has the identification number P25446 in the UniProtKB database. Fas polypeptides known in other animals include, but are not limited to, old world monkeys (e.g., Q9BDN4 in the UniProtKB database), rhesus monkeys (e.g., Q9BDP2 in the UniProtKB database), rats (e.g., Q63199 in the UniProtKB database), or cows (e.g., P51867 in the UniProtKB database).

Based on sequence variations in the Fas polypeptide, one of ordinary skill in the art can identify sequence variations in the effector domain of the Fas polypeptide. For example, a natural variant of a Fas effector domain may include one or more substitutions or mutations of C178R, L180F, P183L, I184V, T198I, Y232C, T241K, T241P, V249L, R250P, R250Q, G253D, G253S, N255D, a257D, I259R, D260G, D260V, D260Y, I262S, N264K, T270I, T270K, E63272 272G, E272K, L278F, K299N, T305I, I310S, or any combination thereof.

In one embodiment, the effector domain useful for a Fas polypeptide of the present disclosure comprises a death domain of a Fas polypeptide. In another embodiment, the effector domain of the Fas polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 230 to 314 of SEQ ID No. 6. In other embodiments, the effector domain of the Fas polypeptide comprises an intracellular domain of the Fas polypeptide. In yet other embodiments, the effector domain of the Fas polypeptide comprises an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 185 to 335, amino acids 186 to 335, amino acids 187 to 335, amino acids 188 to 335, amino acids 189 to 335, amino acids 190 to 335, amino acids 191 to 335, amino acids 192 to 335, amino acids 193 to 335, amino acids 194 to 335, amino acids 195 to 335, amino acids 196 to 335, amino acids 197 to 335, amino acids 198 to 335 or amino acids 199 to 335 of SEQ ID No. 6.

In yet other embodiments, the effector domain of the Fas polypeptide further comprises a transmembrane domain of a Fas polypeptide. In yet other embodiments, the effector domain of the Fas polypeptide comprises an amino acid sequence having at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to amino acids 174 to 335 of SEQ ID NO 6. In some embodiments, the effector domain of the Fas polypeptide further comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 amino acid from the C-terminal portion of the Fas extracellular domain. In certain embodiments, the effector domain of the Fas polypeptide comprises an amino acid sequence that is at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 179 to 335, amino acids 178 to 335, amino acids 177 to 335, amino acids 176 to 335, amino acids 175 to 335, amino acids 174 to 335, amino acids 173 to 335, amino acids 168 to 335, amino acids 167 to 335, amino acids 166 to 335, amino acids 165 to 335, amino acids 164 to 335 or amino acids 163 to 335 of SEQ ID No. 6, wherein the effector domain forms a death-inducing signal complex (DISC), activates caspase 8, or induces apoptosis.

In some embodiments, the effector domain of the Fas polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 8, wherein the effector domain forms a death-inducing signal complex (DISC), activates caspase 8, or induces apoptosis.

In other embodiments, the effector domain of the Fas polypeptide is encoded by a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 7.

In one embodiment, a Fas-chimera gene product of the present disclosure comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 10, wherein the Fas-chimera gene product induces apoptosis. In another embodiment, the Fas-chimera gene product consists of a sequence identical to EQ ID NO: 9, wherein the Fas-chimera gene product induces apoptosis, a nucleotide sequence encoding at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity.

B. Endothelial cell specific promoters

The nucleic acid construct comprising the Fas-chimera gene also comprises one or more expression control elements for regulating the expression of the operatively linked Fas-chimera gene. Such expression control elements include, but are not limited to, promoters, secretion signals, and other regulatory elements.

The nucleic acid constructs useful in the present disclosure utilize endothelial cell specific promoters to direct the expression of Fas-chimera protein in endothelial cells, thereby inducing endothelial apoptosis.

For the purposes of this disclosure, an endothelial cell-specific promoter may comprise one or more cis-regulatory elements, which increase the endothelial cell specificity of the promoter compared to a promoter not comprising cis-regulatory elements. In one example, the cis-regulatory element comprises an enhancer. In another aspect, the cis regulatory element comprises an hypoxia response element. In other examples, cis-regulatory elements include enhancers and hypoxia response elements.

In one embodiment, cis-regulatory elements useful in the present disclosure comprise a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 11 or SEQ ID NO 12 (the complement of SEQ ID NO 11), wherein the cis-regulatory element increases endothelial cell specificity of the promoter compared to a promoter that does not comprise the cis-regulatory element. The cis-regulatory element may also comprise other nucleotide sequences having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 13 or SEQ ID NO 14 (the complement of SEQ ID NO 13).

In another embodiment, a cis-regulatory element of the present disclosure comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 13 or SEQ ID NO 14 (the complement of SEQ ID NO 13), wherein the cis-regulatory element increases endothelial cell specificity of the promoter compared to a promoter that does not comprise the cis-regulatory element. The cis-regulatory element may also comprise other nucleotide sequences having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 11 or SEQ ID NO 12 (the complement of SEQ ID NO 11).

In other embodiments, the cis-regulatory elements of the present disclosure comprise a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO 15 or SEQ ID NO 16 (the complement of SEQ ID NO 15), wherein the cis-regulatory element increases endothelial cell specificity of the promoter compared to a promoter that does not comprise the cis-regulatory element. In other embodiments, the cis-regulatory element of the nucleic acid construct comprises SEQ ID NO 15 or SEQ ID NO 16 or any fragment, variant, derivative or analog thereof, wherein the fragment, variant, derivative or analog increases endothelial cell specificity of the promoter compared to a promoter not comprising the cis-regulatory element.

In some embodiments, a cis-regulatory element of the present disclosure comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 20 or SEQ ID No. 21, wherein the cis-regulatory element increases endothelial cell specificity of the promoter compared to a promoter that does not comprise the cis-regulatory element. In other embodiments, the cis-regulatory element of the nucleic acid construct comprises SEQ ID NO 20 or SEQ ID NO 21 or any fragment, variant, derivative or analog thereof, wherein the fragment, variant, derivative or analog increases endothelial cell specificity of the promoter compared to a promoter not comprising the cis-regulatory element.

In some embodiments, a cis-regulatory element of the present disclosure comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 22 or SEQ ID No. 23, wherein the cis-regulatory element increases endothelial cell specificity of the promoter compared to a promoter that does not comprise the cis-regulatory element. In other embodiments, the cis-regulatory element of the nucleic acid construct comprises SEQ ID NO 22 or SEQ ID NO 23 or any fragment, variant, derivative or analog thereof, wherein the fragment, variant, derivative or analog increases endothelial cell specificity of the promoter compared to a promoter not comprising the cis-regulatory element.

Table 3 shows various cis-regulatory element sequences useful for the present disclosure.

TABLE 3 endothelial cell-specific cis-regulatory elements and promoters

The cis-regulatory element of the present disclosure may be linked to the promoter upstream or downstream of the promoter or inserted between two nucleotides in the promoter. The endothelial cell specific promoters of the present disclosure may utilize any promoter known in the art. For example, suitable promoters useful in the present disclosure include endothelial-specific promoters: proendothelin-1 (PPE-1 promoter), US2010/0282634 (published 11/2010; and WO2011/083464 published 7/14/2011); the PPE-1-3X promoter (U.S. Pat. No. 7,579,327, U.S. Pat. No. 8,071,740, U.S. Pat. No. 8,039,261, U.S. Pat. No. 2010/0282634, U.S. Pat. No. 2007/0286845, WO2011/083464, and WO 2011/083466); TIE-1(S79347, S79346) and TIE-2(U53603) promoters [ Sato TN, Proc Natl Acad Sci U S A1993 Oct 15; 90(20) 9355-8), endoglin promoter [ Y11653; rius C, Blood 1998Dec 15; 92(12) 4677-90), von Willerbrandd factor [ AF 152417; collins CJ Proc Natl Acad Sci U A1987 Jul; 4393-7, KDR/flk-1 promoter [ X89777, X89776; ronicke V, Circ Res 1996 Aug; 79(2), 277-85), FLT-1 promoter [ D64016 AJ 224863; morishita K, J Biol Chem 1995Nov 17; 270(46), 27948-53], the Egr-1 promoter [ AJ 24926; sukhaltm VP, Oncogene Res 1987 Sep-Oct; l (4): 343-55), the E-selectin promoter [ Y12462; collins T J Biol Chem 1991Feb 5; 266(4) 2466-73], endothelial adhesion molecule promoter: ICAM-1[ X84737; horley KJ EMBO J10 months 1989; 2889-96% of 8(10), VCAM-1[ M92431; iadeecrco MF, J Biol Chem, 8/15, 1992; 267(23) 16323-9), PECAM-1[ AJ313330X 96849; CD31, Newman PJ, Science 1990Mar 9; 247(4947) 1219-22], vascular smooth muscle specific elements: the calg box X53154 and aortic carboxypeptidase-like protein (ACLP) promoter [ AF 332596; layne MD, Circ Res.2002; 90: 728-.

In one embodiment, the promoter linked to the endothelial cell specific element comprises a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID No. 17, wherein the promoter linked to the element induces endothelial cell specificity of a gene operably linked to the promoter. In another embodiment, the promoter linked to an endothelial cell specific element comprises a fragment, variant, derivative or analog of the wild-type PPE-1 promoter, wherein the fragment, variant, derivative or analog induces endothelial cell specificity of a gene operably linked to the promoter. In one example, an endothelial cell specific element can be inserted between nucleotide residues 442 and 449, which correspond to SEQ ID NO 17.

In further embodiments, the endothelial cell specific promoter comprises an hypoxia responsive element. Hypoxia Response Element (HRE) is located on the antisense strand of the endothelin-1 promoter. This element is the binding site for hypoxia inducible factor-1, which is required for the positive regulation of the endothelin-1 promoter (of the human, rat and murine genes) by hypoxia. Hypoxia is a potent signal that induces the expression of several genes including erythropoietin (Epo), VEGF, and various glycolytic enzymes. The core sequence (8 base pairs) is conserved in all genes that respond to hypoxic conditions, and the flanking regions differ from the other genes. The ET-I hypoxia responsive element is located between GAT-A-2 and the AP-1 binding site. In one example, the hypoxia response element comprises SEQ ID NO 24, a fragment, variant, derivative or analog thereof.

In other embodiments, endothelial cell specific promoters useful in the present disclosure comprise, consist essentially of, or consist of a nucleotide sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID No. 18, wherein a promoter linked to a cis regulatory element induces endothelial cell specificity of a gene operably linked to the promoter. In another embodiment, the endothelial cell specific promoter comprises a fragment, variant, derivative or analog of SEQ ID No. 18, wherein said fragment, variant, derivative or analog thereof induces endothelial cell specificity of a gene operably linked to said promoter.

Other variations of endothelial cell specific promoters can be found in WO2011/083464, WO2011/083466 and WO2012/052423, which are incorporated herein by reference in their entirety.

The present disclosure also provides a novel promoter sequence comprising the nucleotide sequence of SEQ ID NO 17. In one example, the promoter further comprises an endothelial cell-specific cis regulatory element. In one example, the endothelial cell-specific cis regulatory element comprises SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24 or any fragment, derivative, variant or analog thereof, wherein the fragment, derivative, variant or analog thereof increases endothelial cell specificity of the promoter as compared to a promoter that does not comprise the cis regulatory element. In another example, the promoter comprises the nucleotide sequence of SEQ ID NO. 18. The present disclosure includes nucleic acid constructs comprising a novel promoter and a heterologous nucleotide sequence. In one embodiment, the heterologous nucleic acid sequence comprises a nucleotide sequence encoding a Fas-chimera protein as described herein. In another embodiment, the heterologous nucleotide sequence comprises an adenoviral sequence.

C. Carrier

The present disclosure also provides a vector comprising a nucleic acid construct comprising a Fas-chimera gene operably linked to an endothelial cell-specific promoter. For purposes of this disclosure, many carrier systems may be employed. For example, various viral gene delivery systems that can be used to practice this aspect of the disclosure include, but are not limited to, adenoviral vectors, alphaviral vectors, enteroviral vectors, pestiviral vectors, lentiviral vectors, baculoviral vectors, herpesvirus vectors, epstein barr virus vectors, papovaviral vectors, poxvirus vectors, vaccinia virus vectors, adeno-associated virus vectors, and herpes simplex virus vectors.

In another embodiment, the vector comprising the Fas-chimera gene operably linked to an endothelial cell specific promoter is an adenovirus. For example, the adenovirus may be any one or more of human adenovirus species a (serotypes 12, 18 and 31), B (serotypes 3,7, 11, 14, 16, 21, 34, 35, 50 and 55), C (serotypes 1, 2, 5, 6 and 57), D (8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, 42-49, 51, 53, 54 and 56), E (serotype 4), F (serotypes 40 and 41) or G (serotype 52). In a particular embodiment, the adenovirus of the present disclosure is a human adenovirus serotype 5. In some embodiments, the adenovirus used for gene therapy is a recombinant non-replicative adenovirus that does not contain the E1 region and the E3 region.

In a particular embodiment, the vector is the Ad5-PPE-1-3X-Fas-c vector. In a more specific embodiment, the vector is an Ad5-PPE-1-3X-Fas-c vector comprising, consisting essentially of, or consisting of SEQ ID NO 19. In another embodiment, the adenoviral vector is an isolated virus having european animal cell collection (ECACC) accession number 13021201.

IV, the treatment further comprises one or more chemotherapeutic agents

In some aspects, the methods of the present disclosure further comprise administering to the subject one or more chemotherapeutic agents.

One or more chemotherapeutic agents that may be co-administered with the adenoviruses of the present disclosure include, but are not limited to, acivicin, doxorubicin, aristodazole hydrochloride, aclopram, doxorubicin, adolescent, aldesleukin, elargol, ambroxycin, amimalthrone acetate, aminoglutethimide, amsacrine, anastrozole, amthomycin, asparaginase, clindamycin, azacitidine, azatepa, azomycin, batistat, benzoxastat, bicalutamide hydrochloride, bisnefad dimesylate, bevacizumab, bizelesin, bleomycin sulfate, brequinar sodium, briepimine, busulfan, actinomycin, carroterone, cametamide, cabetimer, carboplatin, carmustine (BiCNU), carvacizumab, carvaquone hydrochloride, cisapride, cisagotaro, chlorambucil, sirolimus, cisapride, meclizine, amil, amikava, meclizine, amicin, and a, Cladribine, clinostat mesylate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, daunorubicin hydrochloride, decitabine, dexomaplatin, dizagutanine mesylate, diazaquinone, docetaxel, doxorubicin hydrochloride, droloxifene citrate, drotaandrosterone propionate, dactinomycin, edatrexate, eflornithine hydrochloride, elsamicin, enloplatin, entiperidine, epipipidine, epirubicin hydrochloride, erbulozole, esorubicin hydrochloride, estramustine sodium phosphate, etanidazole, etoposide phosphate, etoposide, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flucitabine, fosquizalone, fossiltricin sodium, gemcitabine hydrochloride, gemcitabine,Tablets, hydroxyurea, idarubicin hydrochloride, ifosfamide, imofovir, interferon alpha-2 a, interferon alpha-2 b, interferon alpha-nl, interferon alpha-n 3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprorelin acetate, liazole hydrochloride, lometrexol sodium, lomustine (CCNU), loxinoxantrone hydrochloride, maxol, maytansine, meclizine hydrochloride, megestrol acetate, melphalan, melnolium, mercaptopurine, methotrexate sodium, chlorphenidine, metoteracil, mitodomide, mitoxantrone hydrochloride, mycophenolate mofetil, norgestimatinib, mitomycin, mitoxantrone, mitomycin, mitotane hydrochloride, mitoxantrone, medroxy, norgestrel, norgestimate hydrochloride, ipratropium, Ormaplatin, Oxysulfman, Pazopani (pazotinib), paclitaxel, pemetrexed, pellitorin, penthemum, pellomycin sulfate, perphosphamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, Prolometan, porfimer sodium, Pofilomycin, Pennimustine, procarbazine hydrochloride, puromycin hydrochloride, pyrazolofuranidin, Riboadenosine, Rogomine, Safengo hydrochloride, Semustine, octreozine, Sorafenib, sodium spartinate, Spafenib, Geospiramine hydrochloride, Spiromustine, Spiroplatin, puromycin, streptozocin, Sulfolbenzone, sunitinib, Taliroxol, taxol, Teogalan sodium (tecalan sodium), Gatifen, Tilapinium, Tiloxanone hydrochloride, Temoprofen, Temoxon, Tedizocinolone, testolactone, Thiocladib, Thiocladin, Thiocladinone, guanylne, Gekkonidine, and Thiaclonidine, Thiotepa, tiazofurin (tiazofurin), tirapazamine, topotecan hydrochloride, toremifene citrate, tritolone acetate, triciribine phosphate, trimetrexate glucal ester, triptorelin, tobrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinoxirane sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorozole, zeniplane (zeiplane)tin), neat stastatin, or zorubicin hydrochloride. Additional Antineoplastic Agents include those disclosed in chapter 52 of Antineoplastic Agents (Antineoplastic Agents) (Paul calcium and Bruce A. Chabner) and The introduction thereto, page 1202-1263 of The Pharmacological Basis of therapy (The Pharmacological Basis of Therapeutics) by Goodman and Gilman (eighth edition, 1990, McGraw-Hill corporation).

In some aspects of the disclosure, the one or more chemotherapeutic agents are selected from the group consisting of altretamine, raltitrexed, topotecan, paclitaxel, docetaxel, cisplatin, carboplatin, oxaliplatin, liposomal doxorubicin, gemcitabine, cyclophosphamide, vinorelbine, ifosfamide, etoposide, altretamine, capecitabine, irinotecan, melphalan, pemetrexed, bevacizumab, and albumin-bound paclitaxel.

In some aspects, the subject has previously received up to three, up to two, or up to one line of chemotherapy. In other aspects, the subject has not previously received more than 3 lines of chemotherapy for recurrent cancer.

Effective doses of chemotherapeutic agents are available in the art.

In some aspects, the administration of one or more chemotherapeutic agents is repeated. In particular aspects, the one or more chemotherapeutic agents are administered repeatedly about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.

V, pharmaceutical composition

The present disclosure also provides pharmaceutical compositions comprising vectors expressing Fas-chimera proteins for use in the methods of the present disclosure. The pharmaceutical composition may be formulated for administration to a mammal, including a human. Pharmaceutical compositions for use in the methods of the present disclosure comprise a pharmaceutically acceptable carrier, including, for example, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts, or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate), polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin. In one embodiment, the composition is formulated by the addition of physiological saline.

The compositions of the present disclosure may be administered by any suitable method, for example, parenterally (e.g., including subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques), intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implanted depot. As previously described, the compositions comprise a nucleic acid construct comprising a Fas-chimera gene in endothelial cells, thereby inducing apoptosis of the endothelial cells. Thus, the composition can inhibit, reduce, or reduce the size of a tumor or a metastasis thereof by inhibiting neovascularization and/or angiogenesis of tumor endothelial cells. Likewise, a VEGF antagonist used in conjunction with the nucleic acid construct inhibits neovascularization and/or angiogenesis by directly inhibiting VEGF activity. Thus, in one embodiment, the combination therapy is delivered systemically or locally. For systemic or local delivery, the pharmaceutical formulation containing the nucleic acid construct, adenovirus or a homogeneous population of adenoviruses may utilize a mechanical device such as a needle, cannula or surgical instrument.

The sterile injectable form of the compositions for use in the methods of the present disclosure may be an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a suspension in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween (Tween), Span (Span) and other emulsifiers or bioavailability enhancers, commonly used to prepare pharmaceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

Parenteral formulations may be single bolus doses, infusion or loading bolus doses, followed by a maintenance dose. These compositions may be administered at specific fixed or variable intervals (e.g., once a day or on an "as needed" basis).

Certain pharmaceutical compositions for use in the methods of the present disclosure may be administered orally in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions may also be administered by nasal aerosol or inhalation. Such compositions may be prepared as a salt solution using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

Examples

Example 1

Evaluation of efficacy and safety of anti-PD-L1 antibody in combination with VB-111 in treatment of metastatic Lewis lung cancer mice

The purpose is as follows: the purpose of this study was to investigate the efficacy and safety of the combination of Ad5-PPE-1-3X-Fas-c and anti-PD-L1 monoclonal antibody for the treatment of mouse model of metastatic Lewis lung cancer.

Materials: the following materials were used in this study:

purified anti-mouse CD274(B7-H1, PD-L1)

The cargo number: BLG-124328, BLG-124329 hamster IgG

Concentration: 2.03mg/ml

The provider: BIOLEGEND (supplied by ENCO)

Physical state: liquid, method for producing the same and use thereof

Batch number: 8210782

Size: one bottle is 25mg, and the other bottle is 5mg

The medium: PBS (Poly Butylene succinate)

Preparation: the anti-PD-L1 antibody was dissolved in PBS to a concentration of 1mg/ml (200. mu.l per mouse at a dose of 200. mu.g/mouse).

Ad5-PPE-1-3X-Fas-c (VB-111) per mouse 10⁹Viral particles

Concentration: 1X 10¹²Individual viral particle (vp)/ml

Physical state: liquid, method for producing the same and use thereof

And storing at the temperature of less than or equal to-65 ℃ in a low-temperature bottle.

The medium: physiological saline

Preparation: the vials were thawed and mixed by inversion on the day of treatment. VB-111 (1X 10)¹²/ml) was diluted 100-fold. (by collecting 10. mu.l of VB-111 (1X 10)¹²/ml) and 990. mu.l of physiological saline was added to obtain VB-1111X 10¹⁰Concentration of/ml), the mouse has been injected with 100. mu.l/mouse to reach (1X 10)⁹Individual virus particles/mouse).

Ad5-PPE-1-3X-Fas-c (VB-111) per mouse 10¹¹Viral particles

Concentration: 1X 10¹²Viral Particles (VP)/ml

Physical state: liquid, method for producing the same and use thereof

Storage conditions: at the temperature of less than or equal to-65 ℃, and is placed in a low-temperature bottle.

The medium: physiological saline

Preparation: the vials were thawed and mixed by inversion on the day of treatment. VB-111 (1X 10) was administered at 100. mu.l/mouse¹²/ml) to achieve 1X 10¹¹Individual virus particles/mouse.

Negative control/vehicle

Name: physiological saline

The provider: LIFE

Physical state: liquid, method for producing the same and use thereof

Batch number: g172446

Size: each bottle contains 5ml

Storage conditions: at room temperature

Animal(s) production

Male C57BL/6 mice of 12-14 weeks of age were used in this study. The care and handling procedures for the mice were performed according to the laboratory animal care and use guidelines printed by the national academy of sciences Press, Washington, D.C..

The method comprises the following steps:

in addition to the F group (healthy untreated group), male C57BL/6 mice (12-14 weeks) were injected with 50. mu.l of 5X 10 in the left footpad⁵D122 cells.

Mice were monitored for tumor diameter every 5 days. When the tumor diameter reached 5mm, the mice were closely attended each day until the tumor diameter reached 7 mm. The day when the tumor diameter reached 7mm was defined as day 0, and the primary tumor was excised by resection. After the amputation, mice were randomized into different treatment groups (fig. 1 and table 4). Treatment was started 5 days after the amputation. Each animal received:

A. 100 mul of physiological saline;

B.VB-111(l×10¹¹VP/mouse, i.v. 100 μ l), once on day 5;

C.VB-111(1×10⁹VP/mouse, i.v. 100 μ l), once on day 5;

D.VB-111(1×10⁹VP/mouse, i.v. 100 μ l), once on day 5; and anti-PD-L1 antibody (200 μ g/mouse, i.p.), once every 3 days on days 5, 8, 11;

E. anti-PD-L1 antibody (200 μ g/mouse, IP), once every 3 days on days 5, 8, 11; or

F. Treatment-free group (healthy group).

The average number of days from amputation to death of the first three consecutive deaths determines the number of days the remaining animals are amputated.

The treatment groups are summarized in table 4 below.

Table 4: test compositions and dosage levels

And (3) evaluating the curative effect: lung weights were recorded, photographed and collected in 4% formaldehyde. Tumor burden was calculated by subtracting normal lung weight (0.1526 g). Each parameter detected was analyzed by T-test.

As a result:

weight:

there was no significant difference in mean body weight between all treatment groups. A significant difference was observed between the saline group and the healthy group (p < 0.05).

Lung weight

The average lung weight was increased 6-fold in the negative control, saline-treated group compared to the healthy group.

Using VB-111 (1X 10)¹¹VP/mouse) treatment showed the strongest effect, with a significant (p ≦ 0.001) reduction in average lung weight of 62%. VB-111 at 1X 10⁹A significant 36% reduction in lung weight (p) at lower doses in VP/mice<0.005) similar to the 44% reduction observed after anti-PD-L1 treatment (p ≦ 0.001). However, VB-111 (1X 10)⁹VP/mouse) in combination with anti-PD-L1 resulted in a mean lung weight reduction of 58% (p.ltoreq.0.001), similar to that associated with high dose VB-111 (1X 10)¹¹VP/mouse) observed after treatment. VB-111 (1X 10)⁹VP/mouse) and anti-PD-L1 in combination with VB-111 (1X 10)⁹VP/mouse) monotherapy is more effective (p)<0.05) (fig. 2).

Tumor burden

Using VB-111 (1X 10)¹¹VP/mouse) showed the strongest effect, reducing the average tumor burden significantly (p.ltoreq.0.00 l) by 72%. VB-111 at 1X 10⁹Lower doses of VP/mice significantly reduced the average tumor burden by 42% (p)<0.005), and a 51% reduction (p.ltoreq.0.001) was observed after anti-PD-L1 treatment. However, VB-111 (1X 10)⁹VP/mouse) in combination with anti-PD-Ll resulted in a significant changeA significant reduction of 67% (p.ltoreq.0.001), similar to that obtained with a 1X 10¹¹Reduction observed after high dose VB-111 treatment in VP/mice. VB-111 (1X 10)⁹VP/mouse) and anti-PD-L1 were significantly better than VB-111 (1X 10)⁹VP/mouse) monotherapy is more effective (p)<0.05S) (fig. 3).

Conclusion

In this study, high dose VB-111 (1X 10)¹¹VP/mouse) treatment significantly reduced the average lung weight and tumor burden of LLC mouse models. With anti-PD-L1 or VB-111 (1X 10)⁹VP/mouse) lower dose of VB-111 (1X 10) compared to monotherapy⁹VP/mouse) in combination with anti-PD-L1 showed an advantage in reducing mean tumor lung burden and an effect similar to 1 x 10¹¹High dose VB-111 monotherapy in VP/mice.

Example 2

Evaluation of efficacy and safety of anti-PD-L1 antibody in combination with VB-111 in B16F10 melanoma mice

The purpose is as follows: the purpose of this study was to investigate the efficacy and safety of the combination treatment of Ad5-PPE-1-3X-Fas-c and anti-PD-L1 monoclonal antibodies in a mouse model of melanoma.

Materials: the following materials were used in this study:

purified anti-mouse CD274(B7-H1, PD-L1)

The cargo number: BLG-124329, hamster IgG

Concentration: 2.0mg/ml

The provider: BIOLEGEND (supplied by ENCO)

Physical state: liquid, method for producing the same and use thereof

Batch number: b214210

Size: one bottle of 25mg

The medium: PBS (Poly Butylene succinate)

Preparation: the anti-PD-L1 antibody was dissolved in PBS to a concentration of 1mg/ml (200. mu.l per mouse at a dose of 200. mu.g/mouse).

¹¹10 virus particles of Ad5-PPE-1-3X-Fas-c (VB-111) per mouse

Concentration: 1X 10¹²Individual virus particleGranule (vp)/ml

Physical state: liquid, method for producing the same and use thereof

Storage conditions: at a temperature of less than or equal to-65 ℃ in a low-temperature bottle

The medium: physiological saline

Preparation: the vials were thawed and mixed by inversion on the day of treatment. VB-111 (1X 10) was administered at 100. mu.l/mouse¹²/ml) to achieve 1X 10¹¹Individual virus particles/mouse.

Negative control/vehicle

Name: physiological saline

The provider: LIFE

Physical state: liquid, method for producing the same and use thereof

Batch number: g172446

Size: each bottle contains 5ml

Storage conditions: at room temperature

Animal(s) production

Male C57BL/6 mice of 12-14 weeks of age were used in this study. Animals were 12-14 weeks old at the start of the study. The Care and handling procedures for mice were performed according to the Laboratory animal Care and Use guidelines printed by the national academy of sciences Press, Washington, D.C..

The method comprises the following steps:

50 μ l PBS + 2X 10 in 50 μ l MATRIGEL⁵B16F10 cells were injected subcutaneously into the left side of male C57BL/6 mice (12-14 weeks).

Mice were monitored three to six times per week for tumor size. Treatment began on day 9 ("the day of distribution"), when the animals had developed tumors to about 100mm³Mice were randomly assigned to different groups according to tumor size and weight (mice that did not show any measurable tumor or mice with liquid tumors were excluded at that time point). Body weight data were recorded 3 times per week and 3-6 clinical signs were recorded weekly.

Each animal received:

A. normal saline, intravenous injection;

B.VB-111(l×10¹¹VP/mouse, i.v. 100 μ Ι), once on day 9;

C. anti-PD-L1 antibody (200 μ g/mouse, i.p.) once every 2-3 days on days 9, 12 and 14; or

D.VB-111(1×10¹¹VP/mouse, i.v. 100 μ L), once on day 9, anti-PD-L1 antibody (200 μ g/mouse, i.p.), once every 2-3 days on days 9, 12 and 14.

The treatment groups are summarized in table 5 below.

Table 5: test group and dose level

And (3) evaluating the curative effect: individual tumor sizes were recorded, photographed and collected in 4% formaldehyde.

As a result:

body weight

There was no significant difference in mean body weight between all treatment groups.

Tumor size (mm)³)

VB-111 (1X 10) used alone¹¹VP/mouse) or treatment with anti-PD-L1 alone can moderately reduce tumor size; however, the most potent effect was after the combination therapy of VB-111 and anti-PD-L1. From day 15 to death, combination treatment significantly reduced the mean tumor volume. The value of the combination treatment was most significant on day 17 (figure 4).

Conclusion

VB-111(1×10¹¹VP/mouse) in combination with anti-PD-L1 showed an improvement in reducing mean tumor volume over the use of anti-PD-L1 or VB-111(1 x 10)¹¹VP/mouse).

Example 3

Phase I/II randomized, open-label, multicenter study of previous treated patients with advanced or metastatic non-squamous cell non-small cell lung carcinoma (NSCLC) with VB-111 in combination with nivolumab

Study design and treatment planning

This open label study was conducted to evaluate the safety and efficacy of VB-111 (intravenous (IV) every two months) in combination with nivolumab (infused at 3mg/kg every two weeks as standard of care) compared to nivolumab alone in patients with advanced or metastatic non-squamous cell NSCLC. The study will begin with a single arm, multicenter, dose escalation phase I portion, where the combination will be administered to up to 12 patients using a 3+3 dose escalation model and, if successful, will proceed to further include randomized phase II, as detailed below.

Phase I part: dose level 1 (cohort 1): VB-1113X 10¹²Individual Viral Particles (VPs) + nivolumab 3 mg/kg.

The phase I portion is depicted in fig. 5. At least 3 subjects will receive intravenous infusion of nivolumab (3mg/kg) followed by intravenous infusion of VB-111 (3X 10)¹²Viral Particles (VPs)) and observed for the development of dose-limiting toxicities (DLTs) for 28 days. Initially, only one subject was enrolled and treatment was initiated, while two additional subjects were enrolled at least 5 days after the 1 st subject began treatment. Cohort 2 will be enrolled open at the same time if no DLT was recorded in the first group of 3 subjects during the 28 day period. However, if two DLTs were recorded in the first group of 3 subjects, the trial would be terminated. If only one DLT is observed, 3 additional subjects will be administered the drug at the same dose level 1 and will evaluate DLT for up to 28 days. If a DLT is observed once in the second group of subjects (i.e., 2/6 subjects experienced a DLT), the trial will terminate. Otherwise, group 2 will be open recruited.

Phase I part: dose level 2 (cohort 2): VB-1111 x 10¹³VPs + nivolumab 3 mg/kg.

Initiation of cohort 2-cohort is only approved after all patients of cohort 1 completed a 28-day observation period and reported DLTs of less than 2. See fig. 5. In this cohort 2, at least 3 patients will receive intravenous infusion of nivolumab (3mg/kg) followed by intravenous infusion of VB-111 (1X 10)¹³VPs) and observed for the occurrence of DLT for 28 days. The grouping schedule for cohort 2 will be similar to that of cohort 1: first, only 1 patient was enrolled and treatment was initiated,while two other patients will be enrolled at least 5 days after the day patient 1 started treatment. If no DLT was recorded in the first group of 3 patients during the 28 day period, this dose would be determined as a safe dose for combination therapy and used as the recommended phase II dose (RP 2D). However, if two DLTs were recorded in the first group of 3 patients, the trial would be terminated. If only 1 DLT is observed, three additional patients will be administered the drug at the same dose level 2 and will evaluate DLT for up to 28 days. If a DLT is observed once in the second group of patients (i.e., 2/6 patients experienced a DLT), the trial will terminate. Otherwise, enrollment of the phase II portion of the study will be authorized.

At that time, the intra-patient dose escalation was allowed: that is, a patient treated at dosage level 1 may be escalated to receive subsequent treatment at dosage level 2. All patients entering phase I will be evaluated for efficacy in a phase II trial analysis.

Definition of DLT: any drug-related (VB-111 or nivolumab) ≧ 3 toxicity occurring within the first 28 days of treatment, excluding the following:

grade 3 liver or hematologic toxicity.

Drug-controlled ≧ 3 grade nausea or vomiting (DLT if nausea and/or vomiting cannot be controlled by the drug and occurs within a 28-day observation period).

Not less than grade 3 hypokalemia, hyponatremia, hypophosphatemia, hypomagnesemia and hypocalcemia, if easily corrected, are clinically asymptomatic and are not accompanied by medically significant complications (e.g. electrocardiographic changes).

Grade 3-4 febrile events occurring within 24 hours after VB-111 administration should not be considered DLT if they respond to symptomatic treatment.

Nivolumab treats the generally expected grade 3-4 events (e.g., rash, thyroiditis, diarrhea, hepatitis, nephritis, pneumonia) and is considered by researchers that nivolumab-related events will not be considered DLTs unless they are determined by the researchers to be more severe in grade, extent, duration or time of onset than nivolumab monotherapy toxicity.

Patients who complete the 28-day observation period without DLT will continue to receive VB-111 treatment on day 1 of every fourth 14-day cycle (every 56 ± 5 days) and receive nivolumab treatment on day 1 of every 14-day cycle. On the days of administration of both drugs, nivolumab will be administered first. These interviewees will continue with all assessments and evaluations according to the schedule of events. It is not allowed to reduce the dose of VB-111 and/or nivolumab (delayed or discontinued medication only according to the treatment delay or discontinuation guidelines given in this protocol).

If a DLT is reported in this study, the patient reporting a DLT will discontinue study treatment. Therapeutic FU should be performed on patients with DLT and other patients who have stopped treatment (FU is continued for further anticancer therapy and CT scan acquisition). These patients will be subjected to further anti-cancer treatment at the discretion of the investigator.

Patients who quit for any reason other than DLT during the 28 day observation period will be replaced by patients who received the same dose regimen treatment. AEs that occurred after the 28-day observation period will be recorded as AEs even though they met the DLT standard. If an AE meeting the DLT criteria occurs after a 28 day observation period, the investigator will consult a medical supervisor to obtain specific safety assessment guidelines.

Phase II:

phase II portion is depicted in fig. 6. If less than 2 DLTs were reported in cohort 2 patients, phase II will be initiated and new patients will be enrolled in the open. In this part of the study, patients will be randomized to one of two treatment groups (study or control) using a centralized randomization procedure at a 1:1 ratio, receiving the following treatments:

arm 1 (VB-111 in combination with nivolumab):

VB-111 dose 1X 10¹³VPs (RP2D) were administered as intravenous infusions on day 1 and every 4 th 14-day cycle (every 56 days ± 5 days).

Nivolumab administered as a standard of care at 3mg/kg intravenous infusion on day 1 of each 14 day cycle.

Arm 2 (nivolumab only):

nivolumab administered as a standard of care at 3mg/kg intravenous infusion on day 1 of each 14 day cycle.

In this study, the length of one cycle will be 14 days. The first dose of study drug should be administered within 48 hours after randomization. On the day of administration of both drugs, nivolumab will be administered first. It is not allowed to reduce the dose of VB-111 and/or nivolumab (delayed or discontinued medication only according to the treatment delay or discontinuation guidelines given in this protocol). In addition, there was no crossover from the control group to the combination group. See fig. 6.

Randomization will be layered with the following layering factors:

expression level of PD-L1 < 1% vs. > 1%

Smoking status: former light smoker or non-smoker smokers

O non-smokers, defined as patients with less than or equal to 100 cigarettes for a lifetime;

o a conventional light smoker, defined as a subject who has a smoking amount of more than 100 and less than or equal to 10 years later and gives up smoking for more than or equal to 1 year before entering the group;

sex: male to female.

Study duration of treatment

In both phases of the study (phases I and II), treatment will continue until the patient develops unacceptable treatment-related toxicity until disease Progression (PD) is confirmed (as defined by irRECIST) or other causes (e.g., withdrawal of consent, judgment of the investigator, failure of disease progression to investigator-judged withdrawal criteria). Study treatment will be considered complete if the patient stops treatment due to confirmed PD. Discontinuing treatment for any other reason will be considered incomplete treatment and recorded as "discontinuing treatment".

After completion or cessation of study treatment, the patient will be treated at the discretion of the physician. All effort will be expended in collecting post-treatment scans (performed according to standard of care), information on subsequent anti-cancer treatments and patient reported outcome measures until death, withdrawal of consent or loss of visit, with time intervals defined by standard specifications. For patients who discontinue study treatment for reasons other than PD, follow-up scans should be performed every 8 weeks (+ -7 days) until PD, withdrawal of consent, death, missed visits.

Recruitment and withdrawal

Deviations from any inclusion or exclusion criteria are not allowed, as deviations may compromise scientific integrity, regulatory acceptability, or subject safety of the study. Therefore, the standards specified in the protocol must be adhered to. Any questions regarding subject eligibility should be discussed with the sponsor prior to enrollment.

Subjects included criteria. To qualify for the study, the subject must meet all of the following criteria:

signed informed consent was obtained prior to initiating any study-specific procedures and treatments to confirm the patient's awareness and willingness to comply with the study requirements.

The female or male patients aged 18 years or more.

Patients have stage IV or stage III histologically documented non-squamous cell non-small cell lung cancer (NSCLC), or have recurrent disease and are not candidates for curative treatment, but second line nivolumab is a candidate for advanced disease as a standard of treatment.

Disease recurrence or progression during/after a previous platinum-dual drug-based chemotherapy regimen for advanced or metastatic NSCLC. If the disease recurs within 6 months after the last platinum-based treatment, previous treatment (adjuvant or neoadjuvant) of the early disease can be counted as first line treatment of stage IV.

Disease measurable by Computed Tomography (CT) according to response evaluation criteria for solid tumors (RECIST)1.1 was performed within 28 days prior to the first administration of the study drug. The target lesion may be located in a previously irradiated area if there is a recorded progression of the disease at that location.

The physical ability status of the Eastern Cooperative Oncology Group (ECOG) is less than or equal to 1.

Adequate kidney, liver and bone marrow function was achieved according to the following criteria:

absolute neutrophil count of 1500 cells/. mu.l or more

Hemoglobin. gtoreq.9.0 g/dL

Platelets ≥ 100,000 cells/. mu.l

Total bilirubin < 1.5 times the Upper Limit of Normal (ULN), with the exception of Gilbert syndrome patients, which must be <3.0mg/dL,

aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT) are less than or equal to 2.5 × ULN.

Serum creatinine levels ≦ 1.5ULN or creatinine clearance ≧ 40ml/min for patients with creatinine levels above the normal range (creatinine clearance calculated by the Cockcroft-Gault equation).

Prothrombin Time (PT), Partial Thromboplastin Time (PTT) (in seconds) must not extend more than 20% of ULN unless due to the use of anticoagulants;

the expected life is more than or equal to 12 weeks.

Radiotherapy, including significant lung volume (e.g., 10% or greater V20), must be completed at least 4 weeks prior to the first administration of the study drug. Radiation therapy, which does not include significant lung volume, must be completed at least 2 weeks prior to the first administration of study drug.

The previous chemotherapy and/or study drug must be used at least 4 weeks prior to the first administration of the study drug.

Sexually active women of reproductive age (WOCBP) or men sexually active with WOCBP must use the effective birth control method defined in section 10.6.3 during the study in a manner that minimizes the risk of failure. Before inclusion in the study, fertile women must be informed of the importance of avoiding pregnancy during participation in the trial and the potential risk factors for accidental pregnancy. All women with fertility must be tested for negative pregnancy within 7 days before the first administration.

Subject exclusion criteria: subjects who meet any of the following criteria will be excluded from the study:

patients with active or recent history of known or suspected autoimmune disease and in need of systemic treatment within 12 months prior to the first administration of the study treatment. Patients with type 1 diabetes, residual hypothyroidism caused only by autoimmune thyroiditis, skin disorders (vitiligo, psoriasis or alopecia) requiring hormone replacement or not requiring systemic treatment are allowed to be enrolled.

Patients with diseases requiring systemic treatment with corticosteroids (> 10mg prednisone equivalent per day) or other immunosuppressive drugs within 14 days prior to the first administration of study treatment. In the absence of active autoimmune disease, inhalation or topical use of steroids is permitted.

There is an activated EGFR mutation or ALK gene rearrangement according to the subject's medical record.

NSCLC is pathologically mixed with small cell lung cancer.

Treatment with anti-programmed death protein 1(PD-1), anti-programmed cell death ligand 1(PD-L1), anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4) antibodies, or any other antibody or drug, has previously been used, targeting the T cell co-stimulatory or checkpoint pathway.

All toxicities attributed to prior anti-cancer treatments (except alopecia, fatigue and grade 2 peripheral neuropathy) had to be resolved to grade 1 (NCT CTCAE version 4) or baseline prior to study drug administration.

Patients diagnosed with clinically relevant interstitial lung disease.

The known Human Immunodeficiency Virus (HIV) tests have a positive history or are known to have acquired immunodeficiency syndrome (AIDS).

Hepatitis B virus surface antigen (HBV sAg) or hepatitis C virus ribonucleic acid (HCV RNA) detection is positive within 6 months before screening, which indicates the existence of acute or chronic active infection. Note that: seropositive HBV indicates that a patient who has been exposed but has no evidence of active infection (e.g., negative PCR) is eligible.

History of severe hypersensitivity to other monoclonal antibodies.

There was a history of other clinically active malignancies within the first 5 years of enrollment, except for those with negligible risk of metastasis or death, such as basal cell carcinoma or squamous cell carcinoma of the skin of superficial resection or carcinoma of the cervix or breast in situ of local ablation/resection.

Major surgery (including open biopsy) was performed within 4 weeks prior to study initiation, or was expected to be required during study treatment. Patients must recover from the effects of major surgery or major trauma at least 14 days prior to the first administration of study treatment.

Minor surgery was performed within 24 hours prior to the first study treatment.

Women who are breast-fed.

New York Heart Association (NYHA) secondary or more severe congestive heart failure.

The study treatment had a history of myocardial infarction or unstable angina within 6 months prior to the first administration.

A history of stroke or transient ischemic attack within 6 months prior to the first administration of the study treatment.

Study treatment there was a history of hemoptysis (greater than or equal to 1/2 teaspoons of bright red blood per episode) within 6 months prior to the first administration.

Patients with known proliferative and/or vascular retinopathy (e.g., diabetic patients).

Known CNS diseases, post-treatment brain metastases are in addition: post-treatment brain metastases were defined as evidence of no progression or bleeding ≦ grade 1 for at least 4 weeks post-treatment (NCT CTCAE 4 th edition), with the period determined by clinical examination and brain MRI during screening. CNS metastases must be asymptomatic and patients return neurologically to baseline at least 2 weeks prior to the initiation of study treatment. In addition, the patient must discontinue corticosteroids, or use a steady or decreasing dose of <10 mg prednisone (or equivalent) per day. Patients with CNS metastases treated by neurosurgical resection or brain biopsy within 3 months prior to enrollment will be excluded.

Significant vascular disease (e.g., aortic aneurysm, need for surgical repair, or recent peripheral arterial thrombosis) occurred within 6 months prior to the first administration of the study treatment. Note that: allowing for stable peripheral vascular disease.

Clinical evidence of hemorrhagic diathesis or significant coagulation dysfunction (in the absence of therapeutic anticoagulation therapy).

Study drug there was a history of abdominal fistulas or gastrointestinal perforations within one year prior to first administration.

Severe, non-healing wounds, active ulcers or untreated fractures.

Subjects receiving Tyrosine Kinase Inhibitors (TKIs) within the last 4 weeks or antibody-based therapy, anti-angiogenic therapy within 6 weeks. Note that: after an elution period of 4 weeks bevacizumab treatment was allowed to proceed beforehand.

The tumor invades the great vessels (aorta, vena cava, major pulmonary vessels, etc.) or the pericardium or heart.

Treatment prohibition and/or treatment restriction:

anti-cancer therapies other than those specified in this study are being performed or planned for use.

A strong CYP3a4 inhibitor.

Pretreatment with VB-111

Any other serious or uncontrolled physical disease, active infection, physical examination results, laboratory examination results, mental state change, or mental disorder, in the opinion of the researcher, would limit the subject's ability to comply with the requirements, significantly increase the patient's risk, or affect the interpretability of the study results.

Failure to follow the study and/or follow-up procedures.

Treatment distribution program

The investigator must screen all subjects likely to participate in the study to determine subject eligibility. Written informed consent must be obtained prior to any screening procedure. Subjects signed an Informed Consent Form (ICF) and assigned a unique number. All subjects will be identified by this assigned number and their initials. The unique subject identification number is comprised of four digits, the first and second digits representing the study site, and the third and fourth digits representing the subject's number at that site. The first subject to be enrolled will be subject number 01, the second 02, and so on.

Subjects who failed to meet the recruitment criteria at any stage during the screening period were defined as failed screening. All screening failures will be recorded in the screening log, including the reason for the screening failure. The filter log will be saved in the investigator's site file according to the ICH GCP guidelines. The failure rate of the screen was estimated to be 10% for the phase II portion of the study and the withdrawal rate was estimated to be 2%. Thus, it is expected that up to 112 subjects will be screened to achieve a target number of 100 enrolled subjects, ensuring at least 50 evaluable subjects per group.

The phase II portion of this open label trial will include randomization (1:1) to treatment, group 1 or group 2. A centralized randomization procedure will be used.

Randomization will be layered with the following layering factors:

PD-L1 expression level: less than 1% to more than or equal to 1%

Smoking status: former light smoker or non-smoker smokers

Omicron non-smokers, defined as patients with less than or equal to 100 cigarettes for a lifetime

O' a conventional light smoker, defined as a patient who has a smoking amount of more than 100 cigarettes and less than or equal to 10 years old and gives up smoking for more than or equal to 1 year before entering the group

Sex: male to female.

Study intervention

VB-111 product description

Ofranergene Obandenovec VB-111 formula: VB-111 was formulated as a sterile carrier solution. The solution was provided in frozen (below 65 ℃) form for single use, in 10ml glass vials. Each vial contained 5mL of virus with a titer of 10¹²VP/ml vehicle and vehicle (10% glycerol in phosphate buffered saline). The carrier solution should be thawed and maintained at 2-8 ℃ until dilution, and maintained at room temperature until administration.

Ofranergene Obandenovec VB-111 supplies: study drugs were packaged in a small sealed carton: 6 bottles per box. The study site will provide a sufficient number of VB-111 to treat the subject. The study medication will be delivered to the intended recipient (pharmacist or other designated personnel, according to the rules of the study center) under appropriate storage conditions. Each delivery must be confirmed by the recipient. The pharmacist or its prescribing personnel will dispense the medication to the researcher in the relevant dosage.

The pharmacist or prescribing person will maintain a dispensing log in which he/she will record the date and quantity of study product dispensed for each patient. The manifest file will be provided to the study supervisor who will verify responsibility for dose identification and verification during the study. During the study, all used and unused containers will be inventoried and, if the sponsor agrees, will be returned to the sponsor for destruction or destroyed in the field. Written confirmation of destruction will be provided.

Ofranergene Obadenovec VB-111 storage and stability: VB-111 stability studies are ongoing and so far support 48 months of storage at temperatures below 65 ℃. Shelf life will be described in the documentation shipped with each batch of medication prepared. VB-111 vials should be stored in frozen closed vials (below 65 ℃).

Ofranergene Obandenovec VB-111 preparation: VB-111 was prepared as shown in the following table:

table 6: preparation of Ofranergene Obadenovec VB-111

The pharmacist can use a sterile empty bag, or can separately add 40ml of Normal Saline (NS) +10ml of VB-111 into the bag; or a 50ml bag of NS was used and the excess volume was removed, followed by addition of VB-111.

For <50kg subjects, 35/11.5ml indicated a 30% reduction in VB-111.

The entire process of drug preparation should be performed in a Biological Safety Cabinet (BSC) type II at room temperature. After thawing, the drug should be diluted as soon as possible in normal saline at room temperature. Note that the drug can be stored on ice for up to 3 hours before dilution, if desired. Once the drug is in the final formulation in physiological saline, it is kept at room temperature.

VB-111 dose and administration: VB-111 will be administered intravenously at a rate of 3ml/min on day 1 (56. + -. 5 days) of each of the four treatment cycles. It is not necessary to fast before taking VB-111. An infusion pump may be used. In the day and cohort where both nivolumab and VB-111 were used, nivolumab will be used first.

The maximum time of the drug in physiological saline was 60 minutes at room temperature (plus a 30 minute window). Patients weighing less than 50kg will receive a reduced dose of VB-111 as shown in Table 6.

On the day of administration when the patient is concurrently receiving VB-111 and nivolumab therapy, nivolumab should be prepared and administered prior to VB-111. This is based on the paradigm that investigators should be given last as a safety precaution. No order-dependent changes in the pharmacology of these two drugs are expected. Although it is expected that this will occur immediately (within 1 hour) after nivolumab, it can also be administered later (within 24 hours) if clinically indicated, if longer, as discussed with the sponsor's medical supervisor.

Nawu monoclonal antibody product statement

Nivolumab formulation, dosage and administration: nivolumab is a fully human monoclonal antibody suitable for treating metastatic non-small cell lung cancer, patients with progression during or after platinum chemotherapy. The antibody blocks apoptosis receptor-1 (PD-1) activity, resulting in reduced tumor growth.

Nivolumab is a sterile, preservative-free, pyrogen-free, transparent to milky-white, colorless to pale yellow liquid, possibly containing small amounts of (few) particles. The injection of nivolumab for intravenous infusion is provided in the form of a disposable vial (40mg/4mL or 100mg/10mL solution). Each ml of the solution of nivolumab contains 10mg of nivolumab, mannitol (30mg), pentetic acid (0.008mg), polysorbate 80(0.2mg), sodium chloride (2.92mg), sodium citrate dihydrate (5.88mg) and water for injection, United states Pharmacopeia. Hydrochloric acid and/or sodium hydroxide may be included to adjust the pH to 6.

On day 1 of each 14 day cycle, a 3mg/kg dose will be administered intravenously over 60 minutes. Infusion will be via an intravenous line containing a sterile, pyrogen-free, low protein, in-line filter (pore size 0.2 microns to 1.2 microns). Other drugs are not co-administered through the same intravenous line. The venous line is flushed at the end of infusion.

Obtaining the nivolumab: nivolumab will be prescribed as part of the standard of care treatment for the patient.

Nivolumab storage and stability: the product contains no preservative. After preparation, the nivolumab infusion is stored:

nivolumab infusion was stored at room temperature for no more than 4 hours from the time of preparation. This includes the time of room temperature storage of the infusion in an IV container and administration of the infusion, or

Under refrigerated conditions of 2 ℃ to 8 ℃ (36 ° f-46 ° f), no more than 24 hours from the preparation of the infusion.

Do not freeze.

VB-111 preoperative drug

And (3) fever abatement treatment: acetaminophen (900-1000mg) will be administered 1-2 hours prior to VB-111 administration, followed by 450-500mg of acetaminophen up to 36 hours after administration as needed.

Corticosteroid treatment: for subjects who develop grade 3 fever after taking VB-111, or at the discretion of the investigator, dexamethasone can be given at 10mg 30 minutes prior to administration (up to 3 hours prior to treatment, but not earlier than 20 minutes), followed by a VB-111 dose. Further corticosteroid treatment will be performed at the discretion of the investigator.

The nivolumab does not need preoperative medication.

The crowd: up to 112 previously treated advanced or metastatic non-squamous cell NSCLC subjects (age. gtoreq.18) meeting eligibility criteria will be included in the study. And (3) stage I: a minimum of 6 and a maximum of 12 patients will be enrolled. And (2) in a stage II: 100 patients will be enrolled and randomly assigned (1:1) to one of two treatment groups (50 patients per group).

Number of sites: and (3) stage I: two sites in israel; and (2) in a stage II: sites are added as needed.

The research objective is as follows: safety: the safety and tolerability of the combination of intravenous VB-111 and nivolumab was examined in patients with advanced or metastatic non-squamous cell NSCLC as compared to nivolumab alone. The curative effect is as follows: evaluation of the efficacy of the combination of intravenous VB-111 and nivolumab in advanced or metastatic non-squamous cell NSCLC patients compared to nivolumab alone.

Study endpoint:

safety endpoint: treatment safety and tolerability will be evaluated based on DLT, AE, Severe Adverse Events (SAE), subject clinical status and standard laboratory test results taken periodically during the treatment period and up to 60 days after cessation of treatment. The security evaluation will include:

medical interview

Monitoring and assessment of adverse events

Physical examination

Vital signs

An electrocardiogram

Laboratory measurements-clinical chemistry, hematology, urinalysis.

The severity/intensity of adverse events will be graded using the national cancer institute's general term criteria for adverse events.

The therapeutic end point is as follows:

primary end point:

subject response Rate (ORR) of RECIST1.1

Secondary endpoint:

total survival time (OS)

OS Rate (12 months after randomization)

ORR defined by irRECIST

Duration of subject response (DOR)

Reaction time (TTR)

Progression Free Survival (PFS)

PFS Rate (12 months after randomization)

Exploratory and sub-study endpoints:

OS (monthly) as a function of the expression of PD-L1 before treatment

PFS (monthly) as a function of the expression of PD-L1 before treatment

ORR as a function of the expression of PD-L1 before treatment

Patient reported results: lung Cancer Symptom Scale (LCSS)

Comparison of primary and secondary efficacy endpoints for a subgroup of patients who developed fever after at least one treatment with a subgroup of patients who did not develop fever.

The following samples will be taken from all patients for exploratory analysis:

archival tumor tissue for histopathology

If the investigator determines that a biopsy is clinically needed as part of a standard of care treatment during study patient participation or within 3 months after study drug withdrawal, the biopsy samples can be used for further examination of histopathology to obtain evidence of anti-tumor activity, immunotherapeutic activity, and viral transgenesis.

Study procedure/evaluation

Medical history and subject status: relevant medical and medication histories will be obtained by interview or from medical records at screening visit and day 1 of each 14 day cycle from cycle 2 start. Data collection will confirm histological diagnosis and PD-L1 protein expression (the latest test before study enrollment) and will focus on previously relevant medical conditions and treatments, concomitant medications and co-morbidities. Medical records will be reviewed to record contraindications. In addition, the subject will be asked to provide a list of current or planned medications (prescription and over-the-counter) and procedures. At the screening visit, archived tumor tissue will be collected from all eligible patients.

Physical examination: physical examination will be performed within 7 days prior to day 1 of the first treatment cycle, on day 1 of each 14 day treatment cycle, and 30 ± 7 days after the last study drug administration. Weight is measured for each physical examination, while height is only measured during the screening physical examination.

Vital signs and oxygen saturation: vital signs and oxygen saturation will be measured 7 days prior to day 1 of the first treatment cycle, then on days 1 and 8 of cycle 1. Starting from cycle 2, these parameters will be measured on day 1 of each 14 day treatment cycle and 30 ± 7 days after the last study drug administration.

Blood pressure, body temperature, respiration and heart rate, pulse oximetry (and monitoring of supplemental oxygen amounts, if applicable) will be recorded 30 minutes before each administration (+/-5 minutes), 30 minutes after each administration (+/-5 minutes) and at only the first dose VB-1114 hours (+/-5 minutes) and 6 hours (+/-60 minutes). These parameters will also be recorded when the subject develops any new or worsening respiratory symptoms.

Archiving tumor tissue: all effort will be expended to obtain and submit tissue from prior surgery for relevant research. Archived tissues or slides were submitted within 30 days after randomization (see research laboratory reference manual). The samples will be immunohistochemically stained using antibodies against CD4 and CD8T cells. VBL retains the option of expanding the analysis for additional testing to support further elucidation of the mechanism of action and to determine a subpopulation of patients that are likely to respond to VB-111.

Optional fresh biopsy samples: if the investigator determines that a biopsy is clinically needed as part of a standard of care treatment during study patient participation or within 3 months after study drug withdrawal, these biopsy samples can be used for further testing of VBL (evidence for anti-tumor and immunotherapeutic activity and viral transgenes). If one tissue sample is taken, the remaining tissue will be prepared into 3 samples:

a portion of the tissue (-60 mL) will be placed immediately in 30mL of 10% formalin and transported to the VBL for processing at ambient conditions within one week after collection, preferably after block preparation. Samples will be stored at ambient conditions in the VBL or VBL designated central laboratory.

Two parts of the tissue were prepared as fresh pieces, immediately snap frozen in liquid nitrogen in two 2ml cryovials, and then cryogenically transported to the VBL. Frozen samples will be shipped to the VBL at ambient conditions every three months along with dry ice and will be stored in a frost-free refrigerator at-70 ℃ or below in a central laboratory.

In other assays (anti-tumor activity and immunotherapeutic activity), VBL will also explore and verify the presence and expression of viral transgenes in tumor tissues. DNA and/or RNA will be extracted from fresh frozen tissue samples using DNA and/or RNA isolation kits. The DNA sample will be tested by PCR for the presence of the inserted viral transgene sequence in the tissue. The RNA samples will be tested by PCR for viral transgene expression in the tissue.

ECOG performance status: evaluation will be performed within 7 days of day 1 of the first treatment cycle, on day 1 of each 14 day treatment cycle and 30 ± 7 days after the last study drug administration.

Electrocardiogram: a 12 lead ECG will be performed within 7 days of cycle 1 day 1 and within 30 ± 7 days after the last administration. Researchers will report whether the electrocardiogram is normal or abnormal and its clinical significance. All clinically significant abnormalities found at screening should be recorded as a medical history in CRF.

Clinical laboratory evaluation: laboratory tests for eligibility, safety and treatment impact will be performed at the local laboratory according to local standards of care and clinical indications and the results will be recorded in the study database. The CRO should be provided with local laboratory reference ranges and associated laboratory certificates for all test parameters prior to receiving the study drug shipment.

Hematology: hematological assessments will be made within 7 days after the start of cycle 1, day 1 and day 8 of cycle 1, day 1 of each subsequent 14-day cycle, and 30 ± 7 days after the last administration (all the time before administration). Complete blood cell count (CBC) will include an assessment of hemoglobin, hematocrit, White Blood Cells (WBC) and by complete manual or automatic classification (total neutrophils, lymphocytes, monocytes, eosinophils, basophils; both absolute and percent), Red Blood Cells (RBC), platelet count and Erythrocyte Sedimentation Rate (ESR)

And (3) biochemical treatment: biochemical assessments will be made within 7 days after the start of cycle 1, day 1 and day 8 of cycle 1, day 1 of each subsequent 14-day cycle, and 30 ± 7 days after the last administration (all before). Assessment will include evaluation of the integrated metabolome (alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), albumin, calcium, magnesium, sodium, potassium, gamma-glutamyltransferase (GGT), total bilirubin, creatinine), creatinine clearance, differential total protein, uric acid, urea (BUN), glucose, and International Normalized Ratios (INR). Liver function test results must be obtained within 72 hours prior to administration (which may be done on the day of administration, but must not exceed 3 days prior to administration).

Thyroid function: thyroid function will be assessed on day 1 of cycle 1, on day 1 of every second treatment cycle (i.e. every 4 weeks) and 30 ± 7 days after the last administration. The TSH will be evaluated and if any abnormalities are noted, the reflection for T3 and free T4 will also be measured.

And (3) urine analysis: general urinalysis will be assessed within 7 days after the first treatment, on day 1 of each treatment cycle, starting from treatment cycle 2 and 30 ± 7 days after the last administration. The assay will include a proteinuria assay performed according to local standards. If new or increased proteinuria occurs, it may take 24 hours for urine to be collected. The +2 dipstick results required a 24 hour collection, while the +3 dipstick results required retention of study drug and a 24 hour collection. The presence of increased creatinine in pus urine requires the assessment of possible nephritis.

Blood coagulation function: prothrombin Time (PT) and Partial Thromboplastin Time (PTT) (in seconds) will be evaluated within 7 days of the first therapeutic dose, on day 1 of each treatment cycle, starting from treatment cycle 2, and 30 ± 7 days after the last dose. Extending beyond 20% of the ULN would require pausing VB-111 administration unless the investigator believes the increase is due to the use of anticoagulants.

And (3) pregnancy test: a serum or urine hCG pregnancy test will be performed on women with fertility potential within 7 days before the first treatment is started. Subsequent testing will be performed on day 1 of every second treatment cycle (every 28 days) prior to administration. Before VB-111 treatment is given, a negative result must be obtained. Women who are unable to become pregnant for one of the following reasons do not need to undergo pregnancy tests:

confirmation of menopause by healthcare provider

The female has had either the uterus or both ovaries or fallopian tubes removed

Computed Tomography (CT): CT scans of the chest, abdomen and pelvis, as well as any areas being monitored during screening and treatment, and other areas of known or suspected disease (including the CNS) will be taken within 28 days of the first study dose and on day 1 of every four treatment cycles (every 8 weeks ± 5 days). After discontinuation of study drug treatment, all efforts will be undertaken to collect post-treatment scans until death, withdrawal of consent, or loss of visit. For patients who discontinue study treatment for reasons other than PD, follow-up scans will be performed every 8 weeks (+ -7 days) according to standard of care until PD, withdrawal of consent, death, missed visits. Tumor assessment at screening will be performed by institutional standard CT. The tumor assessment method should remain consistent throughout all visits and be performed until disease progression. Throughout the course of the study, researchers will evaluate patients for disease response or progression based on RECIST1.1 and irRECIST. CT scans will be acquired for central laboratory review, but are only applicable to patients in phase II part of the study, and are only performed on an "acquisition and storage" basis. Each CT requires the collection of CDs and their storage in the patient's archive for later analysis by the sponsor. At the same time and during the course of an ongoing study, researchers will read and analyze CT. The responsibility for determining the response from a real-time scan during the study will not be at the core reader.

Results reported by the patient: patients will complete the Lung Cancer Symptom Scale (LCSS) on day 1 of cycle 1, day 1 of every fourth cycle (every 8 weeks ± 5 days), and day 30 ± 7 after the last administration. Thereafter, all efforts will be done to complete post-treatment lcs every 8 weeks ± 7 days until death, withdrawal of consent or loss of visit. In general, patients will be required to complete the LCSS prior to any study procedure and study treatment.

Study timetable

Screening (days-1 to-28)

Once the subjects signed an Informed Consent Form (ICF), the screening period for a particular subject began. Written informed consent must be obtained before any protocol-specific test or procedure can be performed. After informed consent, screening evaluations will be performed within 28 days after the planned start of treatment, except for tests performed within 7 days after the start of treatment as shown below. Standards of care testing conducted prior to ICF, including physical and blood tests, can be used for screening.

Screening will assign a unique subject number for identifying the subject throughout the clinical study, and this number must be used in all study documents associated with that subject.

At the screening visit the following assessments will be made:

obtaining and recording consent from potential subjects on the ICF

Obtaining medical history by interviewing and/or medical record review to determine eligibility

Review of medication history by interview and/or medical record to determine eligibility

Obtaining archived tumor tissue from patients meeting eligibility criteria.

Assessment of tumor status according to RECIST1.1 guidelines

The following tests must be performed no more than 7 days before the first application:

performing a whole body physical examination including weight, height, vital signs and oxygen saturation

Carry out a 12-lead electrocardiogram

Assessment of ECOG Performance status

Hematology, biochemistry, coagulation, thyroid function and urinalysis tests were performed.

Urine pregnancy tests were performed on fertile women, where applicable.

Research visit

Cycle 1, day 1-baseline

Review and record concomitant medication to confirm eligibility

Measuring vital signs and oxygen saturation

Performing hematology, biochemistry and thyroid function tests

Let the patient complete the LCSS questionnaire

If necessary, a tumor biopsy is performed.

Confirmation of qualification according to inclusion/exclusion criteria

The first dose of study treatment was administered. Patients who received both nivolumab and VB-111 were first infused with nivolumab.

Recording adverse events

Cycle 1, day 8 (. + -. 1 day)

Review and record concomitant medication

Recording adverse events

Measuring vital signs and oxygen saturation

Hematological and biochemical, coagulation, thyroid function and urinalysis tests are performed-if necessary, tumor biopsies are performed.

Cycle 2 and above, day 1 (. + -. 5 days)

Assessment of adverse events reported by the subject or observed by the investigator

Review and record concomitant medication

Performing a whole body physical examination including weight, vital signs and oxygen saturation

Assessment of ECOG Performance status

Performing hematology, biochemistry, thyroid function, coagulation, urinalysis tests.

Serum or urine pregnancy tests (every second cycle) were performed on fertile women.

If necessary, a tumor biopsy is performed.

Study treatment was administered.

Only every fourth cycle (cycles 4, 8, 12, etc.):

let the patient complete the LCSS questionnaire before performing any other tests or procedures

CT scans were acquired and tumor response assessed according to RECIST1.1 and irRECIST guidelines

Recording adverse events reported by the subject or observed by the investigator

Record concomitant medication

Study completion visit-30 days after last administration (+ -7 days)

Adverse events reported by the subjects or observed by the investigators were recorded. AE recordings will continue until 60 days after the last application.

Record concomitant medication

Let the patient complete the LCSS questionnaire before performing any other tests or procedures

Performing a physical examination including weight, vital signs and oxygen saturation

Assessment of ECOG Performance status

Carrying out an electrocardiogram

Blood samples were collected for hematology, biochemistry, thyroid function, coagulation tests.

Collecting urine for urine analysis

Post-study monitoring-every 8 weeks (± 7 days) following discontinuation of drug

Follow-up will continue on all patients receiving treatment as long as all efforts are done until death, withdrawal of consent, or loss of visit.

Adverse events reported by the subjects or observed by the investigator (up to 60 days only after the last administration)

Record any further anti-cancer therapies

Let the patient complete the LCSS questionnaire

CT scans were acquired and tumor response assessed according to RECIST1.1 and irRECIST guidelines

Vital signs can be collected over the phone.

Evaluation criteria for efficacy

Reaction parameter-RECIST 1.1:

Measurable disease is defined as at least one lesion that can be accurately measured in at least one dimension (the longest diameter to be recorded). The longest diameter of each lesion must be 10mm, as measured by CT scanning using a contrast agent. The CT scan layer thickness should be no more than 5 mm. Thoracic, abdominal and pelvic CT should be performed at each time point. The same approach should be used for tumor assessment throughout the study.

Malignant lymph nodes should be considered as measurable disease if their minor axis is >15 mm.

All measurable lesions of up to 2 lesions per organ and 5 lesions in total representing all affected organs should be identified as target lesions and will be recorded and measured at baseline.

The target lesion should be selected based on its size (the lesion with the longest diameter), representative of all involved organs, and whether they are suitable for accurate and repeatable repeated measurements by a consistent assessment method (by imaging techniques or clinically). The sum of the Longest Diameters (LD) of all target lesions will be calculated and reported as the baseline sum LD.

All other lesions (or disease sites) should be identified as non-target lesions and should also be recorded at baseline. These lesions should be tracked as "present", "absent" or "clearly progressing", without measurement.

The baseline assessment of all disease states should be made as close to the start of treatment as possible and not more than 4 weeks before the start of treatment.

Reaction standard: follow-up requires measuring the longest diameter of each target lesion. For lymph nodes, the minimum diameter needs to be measured for follow-up. The change in the sum of these diameters provides some estimate of the change in tumor size and thus the change in therapeutic efficacy. All lesions must be evaluated using the same technique as baseline.

Complete Reaction (CR): is evidence that all target and non-target lesions have disappeared and that there are no new lesions. The minor axis of any pathological lymph node must be reduced to <10 mm. CR must be recorded by two disease assessments, at least 4 weeks apart.

Partial Reaction (PR): the sum of the Longest Diameters (LD) of all target measurable lesions is reduced by at least 30% with reference to the baseline sum of LD. There is no clear progression of non-target lesions, nor new lesions. At least two disease assessment documents separated by 4 weeks are required. If the only target lesion is an isolated pelvic mass measured by physical examination and the irradiation radiation cannot be measured, a 50% reduction in LD is required.

Progressive Disease (PD): the sum of LD of the target lesions is increased by at least 20% with reference to the minimum total LD. The sum must demonstrate an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered an increased disease. The treating physician believes that within 12 weeks of the study, there is clear progression of non-target lesions, apart from pleural effusion, with no cytological evidence of tumor origin, also thought to be disease-increasing (in this case, an explanation must be provided). If the only target lesion is an isolated pelvic mass measured by physical examination and the irradiation radiation cannot be measured, a 50% increase in LD is required.

Stable disease condition: any case that does not meet the above criteria is meant.

The reaction could not be evaluated: defined as no tumor assessments repeated after initiation of study treatment for reasons unrelated to disease symptoms or signs.

Reaction parameters-irRECIST criteria:

in addition to the evaluation using RECIST1.1 criteria, RECIST's immune response adaptation will be applied to this assay. In this study, each CT should evaluate RECIST1.1 and irRECIST. irRECIST will be used for decisions regarding treatment continuation/discontinuation. The essential differences between irRECIST and RECIST standards are as follows:

new measurable lesions do not necessarily constitute progressive disease, they should be added to the total tumor burden. New unmeasurable lesions do not contribute to disease progression, but would prevent the determination of irCR.

In the absence of symptoms consistent with clinical exacerbations, significant disease progression should be confirmed after 4 weeks.

At baseline, the sum of the longest diameters (SumD) of all target lesions (maximum 2 lesions per organ, maximum 5 lesions in total) was measured. In each subsequent Tumor Assessment (TA), SumD (longest diameter ≧ 10mm [ shortest diameter of lymph node ≧ 15mm ]; maximum 2 new lesions per organ, total 5 new lesions) of the target lesion and the new measurable lesion were added to yield a Total Measurable Tumor Burden (TMTB): TMTB ═ SumD target lesion + SumD new measurable lesion.

The percent change in TMTB at each evaluation time point describes the size and growth kinetics at the time of the appearance of the old and new measurable lesions. In each tumor assessment, the response of the target and new measurable lesions was defined as the change in TMTB (excluding irPD) as follows:

complete reaction (irCR): all targets and new, measurable lesions disappeared completely, but lymph nodes had to be reduced to a short axis <10mm

Partial reaction (irPR): TMTB decreased by ≧ 30% (see below) relative to baseline

Stable disease (irSD): not meeting the criteria of irCR or irPR, without irPD

Progressive disease (irPD): relative to the lowest point, TMTB increased by ≥ 20%. Unless rapid clinical exacerbations occur, irPD should be confirmed by a second continuous scan obtained ≧ 4 weeks from the initial irPD record. Once confirmed, the irPD date will be considered the date of the initial irPD record.

The overall response according to irRECIST comes from the response in measurable lesions (based on TMTB) and the presence of any non-measurable lesions.

Additional parameters of the reaction

Overall survival is the length of life observed from the first use of VB-111 to death or last exposure.

Progression-free survival (measurable disease study) is the date from the first use of VB-111 to disease progression, death or last exposure.

The Overall Reaction Rate (ORR) is the ratio of the complete reaction [ CR ] to the partial reaction [ PR ]

Duration of reaction (DOR) is the time from the first evidence of PR or better to the confirmation of PD or death for any reason. The DOR of a subject who achieves CR or PR will be calculated.

The Time To Response (TTR) is the time from the start of treatment to the recorded PR or better.

Statistical method

All data collected will be aggregated and presented. Continuous variables will be described as the mean, median, standard deviation, and range of n observations. The classification data will be described using a tabulation, including frequency and percentage. A list of individual subjects for all data will be generated and presented. Statistical tests comparing treatment groups will be performed at 5% level on both sides. Statistical description and analysis will be performed using R version 3.4.3(R development core team. vienna, austria).

The study population is as follows: a safe population would include all subjects who received at least one dose of study medication. All safety analyses will be performed in the safety population. The improved treatment intent (mITT) population will include all subjects from the safety population who have had at least one post-baseline efficacy measurement (RECIST 1.1). Efficacy analysis will be performed on the mITT population. In addition, separate analyses will include mltt subjects and subjects from study part I.

Demographic and baseline parameters: demographic and baseline parameters will be summarized overall and by treatment group. All continuous variables will be summarized by descriptive statistics. All discrete variables will be summarized in terms of frequency and percentage.

Study duration and compliance: all study drug administration and compliance data will be summarized.

Prior and concomitant medication: all relevant prior medications and all concomitant medications will be summarized in terms of frequency and percentage. All drugs will be coded using the World Health Organization (WHO) drug dictionary.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such modifications and adaptations are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

Example 4

Stage II trial of VB-111 in combination with nivolumab for treatment of metastatic colorectal cancer (mCRC).

Background:

unfortunately, immune-based approaches in gastrointestinal cancer, with the notable exception of immune checkpoint suppression in microsatellite instability (MSI-H) disease and gastric cancer, have been largely unsuccessful. The reason for this is not clear, but is undoubtedly related to the fact that: in advanced disease, gastrointestinal cancer appears to be less immunogenic, as evidenced by the lack of infiltrating lymphocytes for T-phase progression and an immunosuppressive tumor microenvironment.

VB-111 is an antiangiogenic agent comprising a non-replicating E1-deleted adenovirus type 5 containing a modified murine pro-endothelin (PPE) promoter and a Fas chimera transgene

VB-111 has been tested and shows promise in glioblastoma, ovarian and thyroid tumors

Nivolumab is a human monoclonal antibody to PD-1.

The aim of this study was to investigate the role of VB-111 in colorectal cancer (CRC) and to evaluate whether it is possible to enhance the anti-tumor immunity induced by VB-111 treatment by PD-1 inhibition.

The purpose is as follows:

determine the safety and tolerability of VB-111 in combination with nivolumab in refractory metastatic CRC patients.

The optimal overall response (BOR) (partial response (PR) + Complete Response (CR)) was determined according to the response evaluation criteria (RECIST v1.1) for VB-111 and nivolumab in combination therapy refractory metastatic CRC patients.

Qualified conditions are as follows:

histopathological confirmation of colorectal cancer metastasis to liver.

Patients must progress in the standard chemotherapy regimen for colorectal cancer above 2 lines, or be intolerant to chemotherapy or refuse previous chemotherapy.

The patient's tumor must be proven to be microsatellite stable (MSS).

The patient must have at least 1 metastasis, can undergo pre-and mid-treatment biopsies, and is willing to accept this.

All patients enrolled must have measurable disease according to RECIST v1.1 criteria.

Designing:

the proposed study was a phase II study of VB-111 in combination with immune checkpoint inhibition (nivolumab) to treat patients with metastatic CRC

Treatment will be carried out on a periodic basis, including 2 weeks, VB-111 every 6 weeks, and nivolumab every 2 weeks, until progression or unacceptable toxicity occurs.

Disease status assessments will be made every 8(+/-1) weeks after study treatment initiation.

Inclusion criteria

The patient must obtain histopathological confirmation of colorectal cancer at the NCI pathology laboratory. The patient must have radiologically confirmed liver metastases.

The patient must:

has been in>Progress was made in 2 standard-of-care chemotherapy lines for colorectal cancer

Or

Intolerance of standard-of-care chemotherapy for colorectal cancer

Or

Previous standard of care chemotherapy for colorectal cancer was rejected.

Patients with known KRAS wild-type tumors must have progressed, not tolerate, or reject anti-EGFR based treatments.

The patient's tumor must be proven to be microsatellite stable (MSS).

The patient must have at least 1 metastasis, can undergo pre-and mid-treatment biopsies, and is willing to accept this. Ideally, the biopsy lesion should not be one of the target measurable lesions, although this depends on the judgment of the researcher

Patients must have measurable disease according to RECIST v1.1 criteria.

Age ≥ 18 years. Since there is currently no dose or adverse event data for nivolumab in combination with VB-111 for subjects under 18 years of age, children were excluded from the study but would be eligible for future pediatric trials.

ECOG Performance status.

Sufficient hematological function is defined as:

omicron leukocyte (WBC) count ≥ 3 × 10⁹/L

Omicron absolute neutrophil count (ANC)≥1.5×10⁹/L

The lymphocyte count is more than or equal to 0.5 multiplied by 10⁹/L

O. platelet count ≥ 100 × 10⁹/L

Omicron Hgb is more than or equal to 9g/dL (more than 48 hours after the transfusion)

PT and PTT (seconds) <1.2 × ULN. Patients receiving anticoagulant therapy need not meet the criteria for PT and PTT

INR, fibrinogen <1.2 × ULN. Patients receiving anticoagulant therapy need not meet INR criteria.

Sufficient liver function is defined as:

the total bilirubin level is less than or equal to 1.5 × ULN,

omicron AST levels ≦ 2.5 × ULN without liver metastasis; or ≦ 5 × ULN in the presence of liver metastases,

in the absence of liver metastasis, ALT levels are ≦ 2.5 × ULN; or ≦ 5 × ULN in the presence of liver metastases

Adequate kidney function is defined as:

the effect of nivolumab and VB-111 on the developing human fetus is not known. For this reason, fertile women and men must agree to take appropriate contraceptive measures for up to 5 months (female) and 7 months (male) or 2 months after the last dose of nivolumab, for longer periods of time, before entering the study and during study participation. If a woman is pregnant or suspects that she is pregnant while her or her partner is participating in the study, she should immediately notify her attending physician.

Troponin levels were within the normal range when administered to the group.

The patient must be able to understand and be willing to sign a written informed consent document.

Body weight >35kg

The patient must participate in the tissue acquisition protocol.

Exclusion criteria

Patients who received standard anti-cancer therapy or study medication (e.g., chemotherapy, immunotherapy, endocrine therapy, targeted therapy, biotherapy, tumor embolization, monoclonal antibodies or other study medication), extensive radiation therapy or major surgery within 4 weeks prior to enrollment.

Subjects receiving anti-VEGF treatment within 4 weeks prior to enrollment.

Subjects with a current corticosteroid dose greater than the physiological replacement dose, defined as 10mg cortisone or its equivalent per day.

Patients with known brain metastases, because of their poor prognosis and frequent progressive neurological dysfunction, which can confound the evaluation of the nervous system and other adverse events.

Patients with signs of liver failure, e.g. ascites, encephalopathy or variceal bleeding, which have clinical significance within 6 months prior to enrollment.

Previous major hepatectomy: residual liver < 50% of the initial liver volume. A biliary stent patient may be included.

Patients with a history of active autoimmune disease or potentially recurrent autoimmune disease, which may affect vital organ function or require immunosuppressive therapy, including systemic corticosteroids. These include, but are not limited to, patients with: a history of immune-related neurological diseases, multiple sclerosis, autoimmune (demyelinating) neuropathy, Guillain-Barre syndrome or CIDP, myasthenia gravis; systemic autoimmune diseases such as SLE, connective tissue disease, scleroderma, Inflammatory Bowel Disease (IBD), crohn's disease, ulcerative colitis, hepatitis; and patients with a history of Toxic Epidermal Necrolysis (TEN), Steiner-Johnson syndrome, or Phospholipids syndrome. Such diseases should be excluded due to the risk of recurrence or exacerbation of the disease.

Notably, patients with vitiligo, endocrine deficiency (including thyroiditis) use alternative hormones (including hormone replacement therapy)Rational corticosteroids) are eligible for treatment. For patients with rheumatoid arthritis and other arthropathies, sjogren's syndrome and psoriasis controlled by topical drugs, as well as seropositive (such as antinuclear antibodies (ANA), antithyroid antibodies), it should be assessed whether there is target organ involvement and whether systemic treatment is required but should be qualified.

History of idiopathic pulmonary fibrosis (including bronchiolitis obliterans with organizing pneumonia) or chest CT screening showed evidence of active pneumonia.

Uncontrolled complications, including but not limited to persistent or active infection, symptomatic congestive heart failure, unstable angina, arrhythmia or psychiatric/social events (within the timeframe identified in the following bulletins), which may limit compliance with research requirements.

History of severe or unstable cerebrovascular disease.

Indoor air pulse oximetry < 92%.

Myocardial infarction occurred within 6 months prior to group entry.

History of myocarditis.

Persistent hypotension (<90/50mmHg) or uncontrolled hypertension (>160/100mmHg)

Stroke within 6 months prior to group entry.

Proliferative and/or vascular retinopathy.

Severe vascular disease (e.g. aortic aneurysm, need for surgical repair or recent peripheral arterial thrombosis) occurred within 6 months prior to enrollment.

There was a history of hemoptysis (> 1/2 teaspoons of bright red blood per episode) or active gastrointestinal bleeding within 6 months prior to group entry.

Evidence of bleeding diathesis or overt coagulation dysfunction (in the absence of therapeutic anticoagulant therapy)

History of abdominal fistulas or gastrointestinal perforations

HIV-positive patients were excluded because HIV causes a complex immunodeficiency, and study treatment may pose more risk to this patient.

Autologous or allogeneic hematopoietic stem cell transplantation.

A subject with ascites.

Subjects with surgical wounds that did not heal for more than 30 days.

History of anaphylaxis due to compounds with similar chemical or biological composition to nivolumab or VB-111.

A history of severe hypersensitivity to any monoclonal antibody.

Pre-existing aggressive malignancies (except for non-melanoma skin cancers) were disease-free for at least 3 years prior to group entry.

Pregnant women were excluded from this study because the potential teratogenic or abortive effects of nivolumab and VB-111 were unclear. Since the risk of adverse events in nursing infants after receiving nivolumab and VB-111 treatment is unknown but potentially, breast feeding should be discontinued if the mother receives nivolumab and/or VB-111 treatment.

Study design and treatment planning

This study will be an open label, one arm phase II study of VB-111 in combination with the anti-PD 1 antibody nivolumab in patients with advanced refractory CRC.

Treatment will be performed on a cycle consisting of 2 weeks (+/-3 days).

VB-111 was administered every 6 weeks starting on day 1 of cycle 1 and nivolumab was administered every 2 weeks starting on day 1 of cycle 2 (fig. 7 and table 7).

Treatment will continue until non-treatment criteria are met.

Patients were monitored for imaging every 8(+/-1) weeks.

Table 7: treatment regimens

VB-111 administration

VB-111 will be administered on day 1 of cycle 1 and at a rate of 1X 10¹³Or 0.7X 10¹³The fixed dose of VP continues to be administered every 3 cycles (4 th, 7 th, 10 th, etc.). Will be provided withVB-11 is administered by intravenous infusion over about 60-90 minutes.

At room temperature, the maximum time from the dilution of VB-111 in 0.9% sodium chloride solution to the start of infusion should be less than 60 minutes.

Acetaminophen 500-1000mg will be administered orally 1-2 hours prior to VB-111 infusion and then 500mg orally every 4-6 hours following treatment as needed up to 36 hours. For patients who develop > grade 3 fever after taking VB-111, or at the discretion of the investigator, dexamethasone IV 10mg may be administered 20 minutes to 3 hours (but not earlier than 20 minutes) prior to treatment with the subsequent VB-111 dose.

Administration of nivolumab

Nivolumab will be administered at a fixed dose of 240mg on day 1 of each cycle starting with cycle 2. Nivolumab will be administered by intravenous infusion over about 30-60 minutes.

Nivolumab will be administered through a low protein binding in-line filter of 0.2 micron to 1.2 micron pore size.

On days of administration of both drugs, VB-111 will be administered first. Nivolumab infusion will begin about 1 hour after the VB-111 infusion is complete.

Vital signs will be collected within 1 hour prior to VB-111 and nivolumab infusion, at least once during each infusion, and within 30 minutes after completion of the infusion.

For nivolumab, if grade 2 or less infusion-related reactions occurred, the infusion rate of study drug could be reduced by 50% or discontinued until the event resolved and restarted at 50% of the initial rate until the infusion was completed. Acetaminophen and/or an antihistamine (e.g., diphenhydramine) or an equivalent meeting institutional standards may be used at the discretion of the researcher. If the severity of infusion-related reactions is grade 3 or greater, study medication will be discontinued.

TABLE 8 study calendar

¹If the baseline assessment is performed at screening or within a specified time frame, the baseline and C1D1 assessments need not be repeated. All assessments will be completed within 72 hours prior to the start of treatment on day 1 of each cycle. If treatment is not initiated within 28 days after enrollment, the screening assessment will be repeated. The cycle was 14(+/-3) days.

²240mg of nivolumab was infused intravenously on day 1 of each cycle starting at cycle 2.

³Intravenous injection 1 × 10 on day 1 of cycle 1 and every +3 cycles (4, 7, 10, etc.)¹³VB-111 for VP. For body weight>35kg of and<50kg of subjects, a reduction of 0.7X 10¹³The dose of VP.

⁴Confirmed by gene analysis or immunohistochemistry.

⁵If clinically indicated.

⁶Vital signs will be collected within 1 hour prior to VB-111 and nivolumab infusion, at least once during each infusion, and within 30 minutes after completion of the infusion.

⁷Biochemical profile: electrolytes, BUN, creatinine, AST, ALT, total and direct bilirubin, calcium, phosphorus, albumin, magnesium.

⁸CT scans or MRI were performed on the chest, abdomen and pelvis at screening, baseline and every 8(+/-1) weeks after study treatment initiation. If treatment is continued after the initial estimate of PD, a conformational scan will be performed after 4 weeks (+/-1 week). If the patient stops treatment for reasons other than disease progression, imaging will continue during the follow-up until disease progression.

⁹Forced tumor biopsies will be performed at baseline and on day 1 of cycle 2 or cycle 4. If the patient's disease progresses before the biopsy is planned, a post-treatment biopsy may be taken as appropriate according to PI at the time of progression.

¹⁰1 week +/-1

¹¹Follow-up was planned at 60(+/-14 days) and 90(+/-14 days) after cessation of treatment to assess patient safety. After this visit, the patient's survival will be tracked by phone or email every 6 months (+ -1 month). Note that: if the patient stops treatment for reasons other than disease progression, we will continue to invite the patient to an imaging exam every 8(+/-1) weeks. External scanning may be accepted.

¹²If the subject is unwilling to do an FU visit with the NIH, they will be contacted by phone or email to learn about survival and adverse events.

Reaction standard

For the purposes of this study, patients should be re-evaluated for response every 8 weeks (+/-1 week). The study will evaluate response and progression using revised response evaluation criteria for solid tumors (RECIST) guidelines (version 1.1) and new international criteria proposed for modified immune-related responses.

While immune-related RECIST criteria will be considered in growth situations to continue treatment, standard RECIST criteria will be the primary method for evaluating primary endpoints.

According to RECIST1.1, if the disease progresses, the study treatment can be continued at the discretion of the investigator. For this case, modified immune-related response criteria (irRC) based on RECIST1.1 will be used in all subjects without worsening existing symptoms or developing new tumor-related symptoms as they progress.

Response criteria-evaluation of target lesions

Complete Reaction (CR): all target lesions disappeared. The minor axis of any pathological lymph node (whether targeted or non-targeted) must be reduced to<10mm。

Partial Reaction (PR): the target lesion sum diameter is reduced by at least 30% with reference to the baseline sum diameter.

Progressive Disease (PD): the sum of the diameters of the lesions of interest was increased by at least 20% with reference to the minimum sum in the study (including the baseline sum if present in the study). In addition to the relative increase of 20%,the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered to be progressing).

Stable disease condition (SD): taking the minimum sum of diameters at the time of study as a reference, there was neither sufficient shrinkage to meet the requirements of PR nor sufficient increase to meet the requirements of PD.

Assessment of non-target lesions

Complete Reaction (CR):all non-target lesions disappeared and tumor marker levels normalized. All lymph node sizes must be non-pathological (<10mm minor axis). Note that: if tumor markers are initially above the upper normal limit, they must normalize before the patient can be considered a complete clinical response.

non-CR/non-PD:one or more non-target lesions persist and/or tumor marker levels remain above the normal range.

Progressive Disease (PD):a clear progression of one or more new lesions and/or existing non-target lesions occurs. The clear progression should generally not exceed the target lesion state. It must represent a change in the overall disease state rather than an increase in individual lesions. Although the clear progression of "non-target" lesions is an exception, in this case the opinion of the attending physician should be dominant and the status of the progression should be confirmed later by the review group (or the lead investigator).

Evaluation of optimal Overall response

The best overall response is the best response recorded from the start of treatment to disease progression/recurrence (the smallest measure recorded since the start of treatment is taken as a reference for progressive disease). The optimal response assignment for a patient will depend on the implementation of both the measurement and confirmation criteria.

For patients with measurable disease (i.e., target disease), see table 9:

table 9.

For subjects with unmeasurable disease (i.e., non-target disease), see table 10.

Table 10.

Duration of reaction

Overall reaction duration:the overall duration of response is from the time the CR or PR criteria are met (whichever is recorded first) until the first date that relapse or progressive disease is objectively recorded (the smallest measurement recorded since the start of treatment serves as a reference for progressive disease).

The duration of overall CR is from the time the measurement criteria for CR are first met to the first date the disease progression is objectively documented.

Duration of stable disease: from the start of treatment until the progression criteria are reached, the minimum measurements recorded since the start of treatment are taken as a reference, including baseline measurements.

Toxicity criteria

The following adverse event management guidelines aim to ensure the safety of each patient during the study. The descriptions and rating scale in the revised NCI adverse event generic terminology standard (CTCAE) version 5.0 will be used for adverse event reporting.

·CorrelationThe reasonable probability of the study product causing an adverse event. Reasonable likelihood means that there is evidence of a causal relationship between the product of interest and the adverse event.

·Is not relatedThere is no reasonable possibility that administration of the study product will result in this event.

Statistical analysis

General methods

After determining safety and tolerability based on reported toxicity, the proportion and confidence interval for all evaluable subjects experiencing a response will be reported.

Primary endpoint analysis

The toxicity rating and type of each patient will be tabulated and reported.

The proportion of evaluable subjects who experience a response (PR + CR) will be reported with a 95% bilateral confidence interval.

Minor endpoint analysis

PFS and OS will be determined using the Kaplan-Meier method (survival analysis) and median PFS and OS and 95% confidence intervals will be reported.

Security analysis

The proportion of patients experiencing toxicity will be listed by toxicity rating and type.

Baseline descriptive statistics

Baseline demographics will be reported.

Middle term analysis

As shown in the two-stage design, the number of responses in 9 evaluable patients after treatment was recorded and used to determine whether phase II enrollment could be performed.

Further analysis: VB-111 glandular vector levels will be measured by RT-PCR in blood and tumor samples from patients receiving VB-111 treatment. Descriptive statistical analysis results, including confidence intervals, will be used where appropriate. Any statistical tests done for the purpose of evaluating exploratory properties will be performed not in the case of formal adjustment of multiple comparisons, but in the case of the number of tests performed.

Sequence listing

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<120> method of antitumor therapy

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atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttggtg 60

ggaatatacc cctcaggggt tattggactg gtccctcacc taggggacag ggagaagaga 120

gatagtgtgt gtccccaagg aaaatatatc caccctcaaa ataattcgat ttgctgtacc 180

aagtgccaca aaggaaccta cttgtacaat gactgtccag gcccggggca ggatacggac 240

tgcagggagt gtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc 300

agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg cacagtggac 360

cgggacaccg tgtgtggctg caggaagaac cagtaccggc attattggag tgaaaacctt 420

ttccagtgct tcaattgcag cctctgcctc aatgggaccg tgcacctctc ctgccaggag 480

aaacagaaca ccgtgtgcac ctgccatgca ggtttctttc taagagaaaa cgagtgtgtc 540

tcctgtagta actgtaagaa aagcctggag tgcacgaagt tgtgcctacc a 591

<210> 4

<211> 197

<212> PRT

<213> Artificial sequence

<220>

<223> amino acid sequence of ligand binding domain of TNFR1

<400> 4

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1 5 10 15

Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

20 25 30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

35 40 45

Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

50 55 60

Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp

65 70 75 80

Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu

85 90 95

Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

100 105 110

Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

115 120 125

Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

130 135 140

Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu

145 150 155 160

Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

165 170 175

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

180 185 190

Lys Leu Cys Leu Pro

195

<210> 5

<211> 1008

<212> DNA

<213> Artificial sequence

<220>

<223> full-Length FAS

<400> 5

atgctgggca tctggaccct cctacctctg gttcttacgt ctgttgctag attatcgtcc 60

aaaagtgtta atgcccaagt gactgacatc aactccaagg gattggaatt gaggaagact 120

gttactacag ttgagactca gaacttggaa ggcctgcatc atgatggcca attctgccat 180

aagccctgtc ctccaggtga aaggaaagct agggactgca cagtcaatgg ggatgaacca 240

gactgcgtgc cctgccaaga agggaaggag tacacagaca aagcccattt ttcttccaaa 300

tgcagaagat gtagattgtg tgatgaagga catggcttag aagtggaaat aaactgcacc 360

cggacccaga ataccaagtg cagatgtaaa ccaaactttt tttgtaactc tactgtatgt 420

gaacactgtg acccttgcac caaatgtgaa catggaatca tcaaggaatg cacactcacc 480

agcaacacca agtgcaaaga ggaaggatcc agatctaact tggggtggct ttgtcttctt 540

cttttgccaa ttccactaat tgtttgggtg aagagaaagg aagtacagaa aacatgcaga 600

aagcacagaa aggaaaacca aggttctcat gaatctccaa ctttaaatcc tgaaacagtg 660

gcaataaatt tatctgatgt tgacttgagt aaatatatca ccactattgc tggagtcatg 720

acactaagtc aagttaaagg ctttgttcga aagaatggtg tcaatgaagc caaaatagat 780

gagatcaaga atgacaatgt ccaagacaca gcagaacaga aagttcaact gcttcgtaat 840

tggcatcaac ttcatggaaa gaaagaagcg tatgacacat tgattaaaga tctcaaaaaa 900

gccaatcttt gtactcttgc agagaaaatt cagactatca tcctcaagga cattactagt 960

gactcagaaa attcaaactt cagaaatgaa atccaaagct tggtctag 1008

<210> 6

<211> 335

<212> PRT

<213> Artificial sequence

<220>

<223> full-Length FAS

<400> 6

Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala

1 5 10 15

Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser

20 25 30

Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn

35 40 45

Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro

50 55 60

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

65 70 75 80

Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His

85 90 95

Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly

100 105 110

Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg

115 120 125

Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp

130 135 140

Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr

145 150 155 160

Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn Leu Gly Trp

165 170 175

Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg

180 185 190

Lys Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly

195 200 205

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

210 215 220

Ser Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met

225 230 235 240

Thr Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu

245 250 255

Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu

260 265 270

Gln Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys

275 280 285

Glu Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys

290 295 300

Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser

305 310 315 320

Asp Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val

325 330 335

<210> 7

<211> 505

<212> DNA

<213> Artificial sequence

<220>

<223> Effector Domain of FAS

<400> 7

aggatccaga tctaacttgg ggtggctttg tcttcttctt ttgccaattc cactaattgt 60

ttgggtgaag agaaaggaag tacagaaaac atgcagaaag cacagaaagg aaaaccaagg 120

ttctcatgaa tctccaacct taaatcctga aacagtggca ataaatttat ctgatgttga 180

cttgagtaaa tatatcacca ctattgctgg agtcatgaca ctaagtcaag ttaaaggctt 240

tgttcgaaag aatggtgtca atgaagccaa aatagatgag atcaagaatg acaatgtcca 300

agacacagca gaacagaaag ttcaactgct tcgtaattgg catcaacttc atggaaagaa 360

agaagcgtat gacacattga ttaaagatct caaaaaagcc aatctttgta ctcttgcaga 420

gaaaattcag actatcatcc tcaaggacat tactagtgac tcagaaaatt caaacttcag 480

aaatgaaatc caaagcttgg tctag 505

<210> 8

<211> 167

<212> PRT

<213> Artificial sequence

<220>

<223> Effector Domain of FAS

<400> 8

Gly Ser Arg Ser Asn Leu Gly Trp Leu Cys Leu Leu Leu Leu Pro Ile

1 5 10 15

Pro Leu Ile Val Trp Val Lys Arg Lys Glu Val Gln Lys Thr Cys Arg

20 25 30

Lys His Arg Lys Glu Asn Gln Gly Ser His Glu Ser Pro Thr Leu Asn

35 40 45

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

50 55 60

Ile Thr Thr Ile Ala Gly Val Met Thr Leu Ser Gln Val Lys Gly Phe

65 70 75 80

Val Arg Lys Asn Gly Val Asn Glu Ala Lys Ile Asp Glu Ile Lys Asn

85 90 95

Asp Asn Val Gln Asp Thr Ala Glu Gln Lys Val Gln Leu Leu Arg Asn

100 105 110

Trp His Gln Leu His Gly Lys Lys Glu Ala Tyr Asp Thr Leu Ile Lys

115 120 125

Asp Leu Lys Lys Ala Asn Leu Cys Thr Leu Ala Glu Lys Ile Gln Thr

130 135 140

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

145 150 155 160

Asn Glu Ile Gln Ser Leu Val

165

<210> 9

<211> 1101

<212> DNA

<213> Artificial sequence

<220>

<223> FAS-chimera

<400> 9

atgggcctct ccaccgtgcc tgacctgctg ctgccgctgg tgctcctgga gctgttggtg 60

ggaatatacc cctcaggggt tattggactg gtccctcacc taggggacag ggagaagaga 120

gatagtgtgt gtccccaagg aaaatatatc caccctcaaa ataattcgat ttgctgtacc 180

aagtgccaca aaggaaccta cttgtacaat gactgtccag gcccggggca ggatacggac 240

tgcagggagt gtgagagcgg ctccttcacc gcttcagaaa accacctcag acactgcctc 300

agctgctcca aatgccgaaa ggaaatgggt caggtggaga tctcttcttg cacagtggac 360

cgggacaccg tgtgtggctg caggaagaac cagtaccggc attattggag tgaaaacctt 420

ttccagtgct tcaattgcag cctctgcctc aatgggaccg tgcacctctc ctgccaggag 480

aaacagaaca ccgtgtgcac ctgccatgca ggtttctttc taagagaaaa cgagtgtgtc 540

tcctgtagta actgtaagaa aagcctggag tgcacgaagt tgtgcctacc aagcttagga 600

tccagatcta acttggggtg gctttgtctt cttcttttgc caattccact aattgtttgg 660

gtgaagagaa aggaagtaca gaaaacatgc agaaagcaca gaaaggaaaa ccaaggttct 720

catgaatctc caaccttaaa tcctgaaaca gtggcaataa atttatctga tgttgacttg 780

agtaaatata tcaccactat tgctggagtc atgacactaa gtcaagttaa aggctttgtt 840

cgaaagaatg gtgtcaatga agccaaaata gatgagatca agaatgacaa tgtccaagac 900

acagcagaac agaaagttca actgcttcgt aattggcatc aacttcatgg aaagaaagaa 960

gcgtatgaca cattgattaa agatctcaaa aaagccaatc tttgtactct tgcagagaaa 1020

attcagacta tcatcctcaa ggacattact agtgactcag aaaattcaaa cttcagaaat 1080

gaaatccaaa gcttggtcta g 1101

<210> 10

<211> 366

<212> PRT

<213> Artificial sequence

<220>

<223> FAS-chimera

<400> 10

Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu

1 5 10 15

Glu Leu Leu Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Leu Val Pro

20 25 30

His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro Gln Gly Lys

35 40 45

Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys

50 55 60

Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp

65 70 75 80

Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu

85 90 95

Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val

100 105 110

Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg

115 120 125

Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe

130 135 140

Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu

145 150 155 160

Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu

165 170 175

Asn Glu Cys Val Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr

180 185 190

Lys Leu Cys Leu Pro Ser Leu Gly Ser Arg Ser Asn Leu Gly Trp Leu

195 200 205

Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg Lys

210 215 220

Glu Val Gln Lys Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly Ser

225 230 235 240

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

245 250 255

Asp Val Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met Thr

260 265 270

Leu Ser Gln Val Lys Gly Phe Val Arg Lys Asn Gly Val Asn Glu Ala

275 280 285

Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln Asp Thr Ala Glu Gln

290 295 300

Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys Glu

305 310 315 320

Ala Tyr Asp Thr Leu Ile Lys Asp Leu Lys Lys Ala Asn Leu Cys Thr

325 330 335

Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser Asp

340 345 350

Ser Glu Asn Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val

355 360 365

<210> 11

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 11

ctggagggtg actttgcttc tggagccagt acttcatact tttcatt 47

<210> 12

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 12

aatgaaaagt atgaagtact ggctccagaa gcaaagtcac cctccag 47

<210> 13

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 13

gtacttcata cttttcattc caatggggtg actttgcttc tgga 44

<210> 14

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 14

tccagaagca aagtcacccc attggaatga aaagtatgaa gtac 44

<210> 15

<211> 131

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 15

ctccagaagc aaagtcaccc cattggaatg aaaagtatga agtacaatga aaagtatgaa 60

gtactggctc cagaagcaaa gtcaccctcc agaagcaaag tcaccccatt ggaatgaaaa 120

gtatgaagta c 131

<210> 16

<211> 131

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 16

gtacttcata cttttcattc caatggggtg actttgcttc tggagggtga ctttgcttct 60

ggagccagta cttcatactt ttcattgtac ttcatacttt tcattccaat ggggtgactt 120

tgcttctgga g 131

<210> 17

<211> 850

<212> DNA

<213> Artificial sequence

<220>

<223> endothelial cell-specific PPE-1 promoter

<400> 17

gtacgtgtac ttctgatcgg cgatactagg gagataagga tgtgcctgac aaaaccacat 60

tgttgttgtt atcattatta tttagttttc cttccttgct aactcctgac ggaatctttc 120

tcacctcaaa tgcgaagtac tttagtttag aaaagacttg gtggaagggg tggtggtgga 180

aaagtagggt gatcttccaa actaatctgg ttccccgccc gccccagtag ctgggattca 240

agagcgaaga gtggggatcg tccccttgtt tgatcagaaa gacataaaag gaaaatcaag 300

tgaacaatga tcagccccac ctccacccca cccccctgcg cgcgcacaat acaatctatt 360

taattgtact tcatactttt cattccaatg gggtgacttt gcttctggag aaactcttga 420

ttcttgaact ctggggctgg cagctagcaa aaggggaagc gggctgctgc tctctgcagg 480

ttctgcagcg gtctctgtct agtgggtgtt ttctttttct tagccctgcc cctggattgt 540

cagacggcgg gcgtctgcct ctgaagttag ccgtgatttc ctctagagcc gggtcttatc 600

tctggctgca cgttgcctgt gggtgactaa tcacacaata acattgttta gggctggaat 660

gaagtcagag ctgtttaccc ccactctata ggggttcaat ataaaaaggc ggcggagaac 720

tgtccgagtc agaagcgttc ctgcaccggc gctgagagcc tgacccggtc tgctccgctg 780

tccttgcgcg ctgcctcccg gctgcccgcg acgctttcgc cccagtggaa gggccacttg 840

ctgcggccgc 850

<210> 18

<211> 987

<212> DNA

<213> Artificial sequence

<220>

<223> endothelial cell-specific PPE-13X promoter

<400> 18

gtacgtgtac ttctgatcgg cgatactagg gagataagga tgtgcctgac aaaaccacat 60

tgttgttgtt atcattatta tttagttttc cttccttgct aactcctgac ggaatctttc 120

tcacctcaaa tgcgaagtac tttagtttag aaaagacttg gtggaagggg tggtggtgga 180

aaagtagggt gatcttccaa actaatctgg ttccccgccc gccccagtag ctgggattca 240

agagcgaaga gtggggatcg tccccttgtt tgatcagaaa gacataaaag gaaaatcaag 300

tgaacaatga tcagccccac ctccacccca cccccctgcg cgcgcacaat acaatctatt 360

taattgtact tcatactttt cattccaatg gggtgacttt gcttctggag aaactcttga 420

ttcttgaact ctggggctgg cagctagcct ccagaagcaa agtcacccca ttggaatgaa 480

aagtatgaag tacaatgaaa agtatgaagt actggctcca gaagcaaagt caccctccag 540

aagcaaagtc accccattgg aatgaaaagt atgaagtacg ctagcaaaag gggaagcggg 600

ctgctgctct ctgcaggttc tgcagcggtc tctgtctagt gggtgttttc tttttcttag 660

ccctgcccct ggattgtcag acggcgggcg tctgcctctg aagttagccg tgatttcctc 720

tagagccggg tcttatctct ggctgcacgt tgcctgtggg tgactaatca cacaataaca 780

ttgtttaggg ctggaatgaa gtcagagctg tttaccccca ctctataggg gttcaatata 840

aaaaggcggc ggagaactgt ccgagtcaga agcgttcctg caccggcgct gagagcctga 900

cccggtctgc tccgctgtcc ttgcgcgctg cctcccggct gcccgcgacg ctttcgcccc 960

agtggaaggg ccacttgctg cggccgc 987

<210> 19

<211> 35207

<212> DNA

<213> Artificial sequence

<220>

<223> VB-111 complete construct

<400> 19

catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60

ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120

gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180

gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240

taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300

agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360

gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420

cgggtcaaag ttggcgtttt attattatag tcagtacgta cgtgtacttc tgatcggcga 480

tactagggag ataaggatgt gcctgacaaa accacattgt tgttgttatc attattattt 540

agttttcctt ccttgctaac tcctgacgga atctttctca cctcaaatgc gaagtacttt 600

agtttagaaa agacttggtg gaaggggtgg tggtggaaaa gtagggtgat cttccaaact 660

aatctggttc cccgcccgcc ccagtagctg ggattcaaga gcgaagagtg gggatcgtcc 720

ccttgtttga tcagaaagac ataaaaggaa aatcaagtga acaatgatca gccccacctc 780

caccccaccc ccctgcgcgc gcacaataca atctatttaa ttgtacttca tacttttcat 840

tccaatgggg tgactttgct tctggagaaa ctcttgattc ttgaactctg gggctggcag 900

ctagcctcca gaagcaaagt caccccattg gaatgaaaag tatgaagtac aatgaaaagt 960

atgaagtact ggctccagaa gcaaagtcac cctccagaag caaagtcacc ccattggaat 1020

gaaaagtatg aagtacgcta gcaaaagggg aagcgggctg ctgctctctg caggttctgc 1080

agcggtctct gtctagtggg tgttttcttt ttcttagccc tgcccctgga ttgtcagacg 1140

gcgggcgtct gcctctgaag ttagccgtga tttcctctag agccgggtct tatctctggc 1200

tgcacgttgc ctgtgggtga ctaatcacac aataacattg tttagggctg gaatgaagtc 1260

agagctgttt acccccactc tataggggtt caatataaaa aggcggcgga gaactgtccg 1320

agtcagaagc gttcctgcac cggcgctgag agcctgaccc ggtctgctcc gctgtccttg 1380

cgcgctgcct cccggctgcc cgcgacgctt tcgccccagt ggaagggcca cttgctgcgg 1440

ccgctaattc tgcagatcgg gatccggcat gggcctctcc accgtgcctg acctgctgct 1500

gccgctggtg ctcctggagc tgttggtggg aatatacccc tcaggggtta ttggactggt 1560

ccctcaccta ggggacaggg agaagagaga tagtgtgtgt ccccaaggaa aatatatcca 1620

ccctcaaaat aattcgattt gctgtaccaa gtgccacaaa ggaacctact tgtacaatga 1680

ctgtccaggc ccggggcagg atacggactg cagggagtgt gagagcggct ccttcaccgc 1740

ttcagaaaac cacctcagac actgcctcag ctgctccaaa tgccgaaagg aaatgggtca 1800

ggtggagatc tcttcttgca cagtggaccg ggacaccgtg tgtggctgca ggaagaacca 1860

gtaccggcat tattggagtg aaaacctttt ccagtgcttc aattgcagcc tctgcctcaa 1920

tgggaccgtg cacctctcct gccaggagaa acagaacacc gtgtgcacct gccatgcagg 1980

tttctttcta agagaaaacg agtgtgtctc ctgtagtaac tgtaagaaaa gcctggagtg 2040

cacgaagttg tgcctaccaa gcttaggatc cagatctaac ttggggtggc tttgtcttct 2100

tcttttgcca attccactaa ttgtttgggt gaagagaaag gaagtacaga aaacatgcag 2160

aaagcacaga aaggaaaacc aaggttctca tgaatctcca accttaaatc ctgaaacagt 2220

ggcaataaat ttatctgatg ttgacttgag taaatatatc accactattg ctggagtcat 2280

gacactaagt caagttaaag gctttgttcg aaagaatggt gtcaatgaag ccaaaataga 2340

tgagatcaag aatgacaatg tccaagacac agcagaacag aaagttcaac tgcttcgtaa 2400

ttggcatcaa cttcatggaa agaaagaagc gtatgacaca ttgattaaag atctcaaaaa 2460

agccaatctt tgtactcttg cagagaaaat tcagactatc atcctcaagg acattactag 2520

tgactcagaa aattcaaact tcagaaatga aatccaaagc ttggtctagc tcgagcatgc 2580

atctaggcgg ccgcatggca gaaattcgcg aattcgctag cgttaacgga tcctctagac 2640

gagatccgaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 2700

atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 2760

atgtatctta tcatgtctag atctgtactg aaatgtgtgg gcgtggctta agggtgggaa 2820

agaatatata aggtgggggt cttatgtagt tttgtatctg ttttgcagca gccgccgccg 2880

ccatgagcac caactcgttt gatggaagca ttgtgagctc atatttgaca acgcgcatgc 2940

ccccatgggc cggggtgcgt cagaatgtga tgggctccag cattgatggt cgccccgtcc 3000

tgcccgcaaa ctctactacc ttgacctacg agaccgtgtc tggaacgccg ttggagactg 3060

cagcctccgc cgccgcttca gccgctgcag ccaccgcccg cgggattgtg actgactttg 3120

ctttcctgag cccgcttgca agcagtgcag cttcccgttc atccgcccgc gatgacaagt 3180

tgacggctct tttggcacaa ttggattctt tgacccggga acttaatgtc gtttctcagc 3240

agctgttgga tctgcgccag caggtttctg ccctgaaggc ttcctcccct cccaatgcgg 3300

tttaaaacat aaataaaaaa ccagactctg tttggatttg gatcaagcaa gtgtcttgct 3360

gtctttattt aggggttttg cgcgcgcggt aggcccggga ccagcggtct cggtcgttga 3420

gggtcctgtg tattttttcc aggacgtggt aaaggtgact ctggatgttc agatacatgg 3480

gcataagccc gtctctgggg tggaggtagc accactgcag agcttcatgc tgcggggtgg 3540

tgttgtagat gatccagtcg tagcaggagc gctgggcgtg gtgcctaaaa atgtctttca 3600

gtagcaagct gattgccagg ggcaggccct tggtgtaagt gtttacaaag cggttaagct 3660

gggatgggtg catacgtggg gatatgagat gcatcttgga ctgtattttt aggttggcta 3720

tgttcccagc catatccctc cggggattca tgttgtgcag aaccaccagc acagtgtatc 3780

cggtgcactt gggaaatttg tcatgtagct tagaaggaaa tgcgtggaag aacttggaga 3840

cgcccttgtg acctccaaga ttttccatgc attcgtccat aatgatggca atgggcccac 3900

gggcggcggc ctgggcgaag atatttctgg gatcactaac gtcatagttg tgttccagga 3960

tgagatcgtc ataggccatt tttacaaagc gcgggcggag ggtgccagac tgcggtataa 4020

tggttccatc cggcccaggg gcgtagttac cctcacagat ttgcatttcc cacgctttga 4080

gttcagatgg ggggatcatg tctacctgcg gggcgatgaa gaaaacggtt tccggggtag 4140

gggagatcag ctgggaagaa agcaggttcc tgagcagctg cgacttaccg cagccggtgg 4200

gcccgtaaat cacacctatt accggctgca actggtagtt aagagagctg cagctgccgt 4260

catccctgag caggggggcc acttcgttaa gcatgtccct gactcgcatg ttttccctga 4320

ccaaatccgc cagaaggcgc tcgccgccca gcgatagcag ttcttgcaag gaagcaaagt 4380

ttttcaacgg tttgagaccg tccgccgtag gcatgctttt gagcgtttga ccaagcagtt 4440

ccaggcggtc ccacagctcg gtcacctgct ctacggcatc tcgatccagc atatctcctc 4500

gtttcgcggg ttggggcggc tttcgctgta cggcagtagt cggtgctcgt ccagacgggc 4560

cagggtcatg tctttccacg ggcgcagggt cctcgtcagc gtagtctggg tcacggtgaa 4620

ggggtgcgct ccgggctgcg cgctggccag ggtgcgcttg aggctggtcc tgctggtgct 4680

gaagcgctgc cggtcttcgc cctgcgcgtc ggccaggtag catttgacca tggtgtcata 4740

gtccagcccc tccgcggcgt ggcccttggc gcgcagcttg cccttggagg aggcgccgca 4800

cgaggggcag tgcagacttt tgagggcgta gagcttgggc gcgagaaata ccgattccgg 4860

ggagtaggca tccgcgccgc aggccccgca gacggtctcg cattccacga gccaggtgag 4920

ctctggccgt tcggggtcaa aaaccaggtt tcccccatgc tttttgatgc gtttcttacc 4980

tctggtttcc atgagccggt gtccacgctc ggtgacgaaa aggctgtccg tgtccccgta 5040

tacagacttg agaggcctgt cctcgagcgg tgttccgcgg tcctcctcgt atagaaactc 5100

ggaccactct gagacaaagg ctcgcgtcca ggccagcacg aaggaggcta agtgggaggg 5160

gtagcggtcg ttgtccacta gggggtccac tcgctccagg gtgtgaagac acatgtcgcc 5220

ctcttcggca tcaaggaagg tgattggttt gtaggtgtag gccacgtgac cgggtgttcc 5280

tgaagggggg ctataaaagg gggtgggggc gcgttcgtcc tcactctctt ccgcatcgct 5340

gtctgcgagg gccagctgtt ggggtgagta ctccctctga aaagcgggca tgacttctgc 5400

gctaagattg tcagtttcca aaaacgagga ggatttgata ttcacctggc ccgcggtgat 5460

gcctttgagg gtggccgcat ccatctggtc agaaaagaca atctttttgt tgtcaagctt 5520

ggtggcaaac gacccgtaga gggcgttgga cagcaacttg gcgatggagc gcagggtttg 5580

gtttttgtcg cgatcggcgc gctccttggc cgcgatgttt agctgcacgt attcgcgcgc 5640

aacgcaccgc cattcgggaa agacggtggt gcgctcgtcg ggcaccaggt gcacgcgcca 5700

accgcggttg tgcagggtga caaggtcaac gctggtggct acctctccgc gtaggcgctc 5760

gttggtccag cagaggcggc cgcccttgcg cgagcagaat ggcggtaggg ggtctagctg 5820

cgtctcgtcc ggggggtctg cgtccacggt aaagaccccg ggcagcaggc gcgcgtcgaa 5880

gtagtctatc ttgcatcctt gcaagtctag cgcctgctgc catgcgcggg cggcaagcgc 5940

gcgctcgtat gggttgagtg ggggacccca tggcatgggg tgggtgagcg cggaggcgta 6000

catgccgcaa atgtcgtaaa cgtagagggg ctctctgagt attccaagat atgtagggta 6060

gcatcttcca ccgcggatgc tggcgcgcac gtaatcgtat agttcgtgcg agggagcgag 6120

gaggtcggga ccgaggttgc tacgggcggg ctgctctgct cggaagacta tctgcctgaa 6180

gatggcatgt gagttggatg atatggttgg acgctggaag acgttgaagc tggcgtctgt 6240

gagacctacc gcgtcacgca cgaaggaggc gtaggagtcg cgcagcttgt tgaccagctc 6300

ggcggtgacc tgcacgtcta gggcgcagta gtccagggtt tccttgatga tgtcatactt 6360

atcctgtccc ttttttttcc acagctcgcg gttgaggaca aactcttcgc ggtctttcca 6420

gtactcttgg atcggaaacc cgtcggcctc cgaacggtaa gagcctagca tgtagaactg 6480

gttgacggcc tggtaggcgc agcatccctt ttctacgggt agcgcgtatg cctgcgcggc 6540

cttccggagc gaggtgtggg tgagcgcaaa ggtgtccctg accatgactt tgaggtactg 6600

gtatttgaag tcagtgtcgt cgcatccgcc ctgctcccag agcaaaaagt ccgtgcgctt 6660

tttggaacgc ggatttggca gggcgaaggt gacatcgttg aagagtatct ttcccgcgcg 6720

aggcataaag ttgcgtgtga tgcggaaggg tcccggcacc tcggaacggt tgttaattac 6780

ctgggcggcg agcacgatct cgtcaaagcc gttgatgttg tggcccacaa tgtaaagttc 6840

caagaagcgc gggatgccct tgatggaagg caatttttta agttcctcgt aggtgagctc 6900

ttcaggggag ctgagcccgt gctctgaaag ggcccagtct gcaagatgag ggttggaagc 6960

gacgaatgag ctccacaggt cacgggccat tagcatttgc aggtggtcgc gaaaggtcct 7020

aaactggcga cctatggcca ttttttctgg ggtgatgcag tagaaggtaa gcgggtcttg 7080

ttcccagcgg tcccatccaa ggttcgcggc taggtctcgc gcggcagtca ctagaggctc 7140

atctccgccg aacttcatga ccagcatgaa gggcacgagc tgcttcccaa aggcccccat 7200

ccaagtatag gtctctacat cgtaggtgac aaagagacgc tcggtgcgag gatgcgagcc 7260

gatcgggaag aactggatct cccgccacca attggaggag tggctattga tgtggtgaaa 7320

gtagaagtcc ctgcgacggg ccgaacactc gtgctggctt ttgtaaaaac gtgcgcagta 7380

ctggcagcgg tgcacgggct gtacatcctg cacgaggttg acctgacgac cgcgcacaag 7440

gaagcagagt gggaatttga gcccctcgcc tggcgggttt ggctggtggt cttctacttc 7500

ggctgcttgt ccttgaccgt ctggctgctc gaggggagtt acggtggatc ggaccaccac 7560

gccgcgcgag cccaaagtcc agatgtccgc gcgcggcggt cggagcttga tgacaacatc 7620

gcgcagatgg gagctgtcca tggtctggag ctcccgcggc gtcaggtcag gcgggagctc 7680

ctgcaggttt acctcgcata gacgggtcag ggcgcgggct agatccaggt gatacctaat 7740

ttccaggggc tggttggtgg cggcgtcgat ggcttgcaag aggccgcatc cccgcggcgc 7800

gactacggta ccgcgcggcg ggcggtgggc cgcgggggtg tccttggatg atgcatctaa 7860

aagcggtgac gcgggcgagc ccccggaggt agggggggct ccggacccgc cgggagaggg 7920

ggcaggggca cgtcggcgcc gcgcgcgggc aggagctggt gctgcgcgcg taggttgctg 7980

gcgaacgcga cgacgcggcg gttgatctcc tgaatctggc gcctctgcgt gaagacgacg 8040

ggcccggtga gcttgaacct gaaagagagt tcgacagaat caatttcggt gtcgttgacg 8100

gcggcctggc gcaaaatctc ctgcacgtct cctgagttgt cttgataggc gatctcggcc 8160

atgaactgct cgatctcttc ctcctggaga tctccgcgtc cggctcgctc cacggtggcg 8220

gcgaggtcgt tggaaatgcg ggccatgagc tgcgagaagg cgttgaggcc tccctcgttc 8280

cagacgcggc tgtagaccac gcccccttcg gcatcgcggg cgcgcatgac cacctgcgcg 8340

agattgagct ccacgtgccg ggcgaagacg gcgtagtttc gcaggcgctg aaagaggtag 8400

ttgagggtgg tggcggtgtg ttctgccacg aagaagtaca taacccagcg tcgcaacgtg 8460

gattcgttga tatcccccaa ggcctcaagg cgctccatgg cctcgtagaa gtccacggcg 8520

aagttgaaaa actgggagtt gcgcgccgac acggttaact cctcctccag aagacggatg 8580

agctcggcga cagtgtcgcg cacctcgcgc tcaaaggcta caggggcctc ttcttcttct 8640

tcaatctcct cttccataag ggcctcccct tcttcttctt ctggcggcgg tgggggaggg 8700

gggacacggc ggcgacgacg gcgcaccggg aggcggtcga caaagcgctc gatcatctcc 8760

ccgcggcgac ggcgcatggt ctcggtgacg gcgcggccgt tctcgcgggg gcgcagttgg 8820

aagacgccgc ccgtcatgtc ccggttatgg gttggcgggg ggctgccatg cggcagggat 8880

acggcgctaa cgatgcatct caacaattgt tgtgtaggta ctccgccgcc gagggacctg 8940

agcgagtccg catcgaccgg atcggaaaac ctctcgagaa aggcgtctaa ccagtcacag 9000

tcgcaaggta ggctgagcac cgtggcgggc ggcagcgggc ggcggtcggg gttgtttctg 9060

gcggaggtgc tgctgatgat gtaattaaag taggcggtct tgagacggcg gatggtcgac 9120

agaagcacca tgtccttggg tccggcctgc tgaatgcgca ggcggtcggc catgccccag 9180

gcttcgtttt gacatcggcg caggtctttg tagtagtctt gcatgagcct ttctaccggc 9240

acttcttctt ctccttcctc ttgtcctgca tctcttgcat ctatcgctgc ggcggcggcg 9300

gagtttggcc gtaggtggcg ccctcttcct cccatgcgtg tgaccccgaa gcccctcatc 9360

ggctgaagca gggctaggtc ggcgacaacg cgctcggcta atatggcctg ctgcacctgc 9420

gtgagggtag actggaagtc atccatgtcc acaaagcggt ggtatgcgcc cgtgttgatg 9480

gtgtaagtgc agttggccat aacggaccag ttaacggtct ggtgacccgg ctgcgagagc 9540

tcggtgtacc tgagacgcga gtaagccctc gagtcaaata cgtagtcgtt gcaagtccgc 9600

accaggtact ggtatcccac caaaaagtgc ggcggcggct ggcggtagag gggccagcgt 9660

agggtggccg gggctccggg ggcgagatct tccaacataa ggcgatgata tccgtagatg 9720

tacctggaca tccaggtgat gccggcggcg gtggtggagg cgcgcggaaa gtcgcggacg 9780

cggttccaga tgttgcgcag cggcaaaaag tgctccatgg tcgggacgct ctggccggtc 9840

aggcgcgcgc aatcgttgac gctctagacc gtgcaaaagg agagcctgta agcgggcact 9900

cttccgtggt ctggtggata aattcgcaag ggtatcatgg cggacgaccg gggttcgagc 9960

cccgtatccg gccgtccgcc gtgatccatg cggttaccgc ccgcgtgtcg aacccaggtg 10020

tgcgacgtca gacaacgggg gagtgctcct tttggcttcc ttccaggcgc ggcggctgct 10080

gcgctagctt ttttggccac tggccgcgcg cagcgtaagc ggttaggctg gaaagcgaaa 10140

gcattaagtg gctcgctccc tgtagccgga gggttatttt ccaagggttg agtcgcggga 10200

cccccggttc gagtctcgga ccggccggac tgcggcgaac gggggtttgc ctccccgtca 10260

tgcaagaccc cgcttgcaaa ttcctccgga aacagggacg agcccctttt ttgcttttcc 10320

cagatgcatc cggtgctgcg gcagatgcgc ccccctcctc agcagcggca agagcaagag 10380

cagcggcaga catgcagggc accctcccct cctcctaccg cgtcaggagg ggcgacatcc 10440

gcggttgacg cggcagcaga tggtgattac gaacccccgc ggcgccgggc ccggcactac 10500

ctggacttgg aggagggcga gggcctggcg cggctaggag cgccctctcc tgagcggcac 10560

ccaagggtgc agctgaagcg tgatacgcgt gaggcgtacg tgccgcggca gaacctgttt 10620

cgcgaccgcg agggagagga gcccgaggag atgcgggatc gaaagttcca cgcagggcgc 10680

gagctgcggc atggcctgaa tcgcgagcgg ttgctgcgcg aggaggactt tgagcccgac 10740

gcgcgaaccg ggattagtcc cgcgcgcgca cacgtggcgg ccgccgacct ggtaaccgca 10800

tacgagcaga cggtgaacca ggagattaac tttcaaaaaa gctttaacaa ccacgtgcgt 10860

acgcttgtgg cgcgcgagga ggtggctata ggactgatgc atctgtggga ctttgtaagc 10920

gcgctggagc aaaacccaaa tagcaagccg ctcatggcgc agctgttcct tatagtgcag 10980

cacagcaggg acaacgaggc attcagggat gcgctgctaa acatagtaga gcccgagggc 11040

cgctggctgc tcgatttgat aaacatcctg cagagcatag tggtgcagga gcgcagcttg 11100

agcctggctg acaaggtggc cgccatcaac tattccatgc ttagcctggg caagttttac 11160

gcccgcaaga tataccatac cccttacgtt cccatagaca aggaggtaaa gatcgagggg 11220

ttctacatgc gcatggcgct gaaggtgctt accttgagcg acgacctggg cgtttatcgc 11280

aacgagcgca tccacaaggc cgtgagcgtg agccggcggc gcgagctcag cgaccgcgag 11340

ctgatgcaca gcctgcaaag ggccctggct ggcacgggca gcggcgatag agaggccgag 11400

tcctactttg acgcgggcgc tgacctgcgc tgggccccaa gccgacgcgc cctggaggca 11460

gctggggccg gacctgggct ggcggtggca cccgcgcgcg ctggcaacgt cggcggcgtg 11520

gaggaatatg acgaggacga tgagtacgag ccagaggacg gcgagtacta agcggtgatg 11580

tttctgatca gatgatgcaa gacgcaacgg acccggcggt gcgggcggcg ctgcagagcc 11640

agccgtccgg ccttaactcc acggacgact ggcgccaggt catggaccgc atcatgtcgc 11700

tgactgcgcg caatcctgac gcgttccggc agcagccgca ggccaaccgg ctctccgcaa 11760

ttctggaagc ggtggtcccg gcgcgcgcaa accccacgca cgagaaggtg ctggcgatcg 11820

taaacgcgct ggccgaaaac agggccatcc ggcccgacga ggccggcctg gtctacgacg 11880

cgctgcttca gcgcgtggct cgttacaaca gcggcaacgt gcagaccaac ctggaccggc 11940

tggtggggga tgtgcgcgag gccgtggcgc agcgtgagcg cgcgcagcag cagggcaacc 12000

tgggctccat ggttgcacta aacgccttcc tgagtacaca gcccgccaac gtgccgcggg 12060

gacaggagga ctacaccaac tttgtgagcg cactgcggct aatggtgact gagacaccgc 12120

aaagtgaggt gtaccagtct gggccagact attttttcca gaccagtaga caaggcctgc 12180

agaccgtaaa cctgagccag gctttcaaaa acttgcaggg gctgtggggg gtgcgggctc 12240

ccacaggcga ccgcgcgacc gtgtctagct tgctgacgcc caactcgcgc ctgttgctgc 12300

tgctaatagc gcccttcacg gacagtggca gcgtgtcccg ggacacatac ctaggtcact 12360

tgctgacact gtaccgcgag gccataggtc aggcgcatgt ggacgagcat actttccagg 12420

agattacaag tgtcagccgc gcgctggggc aggaggacac gggcagcctg gaggcaaccc 12480

taaactacct gctgaccaac cggcggcaga agatcccctc gttgcacagt ttaaacagcg 12540

aggaggagcg cattttgcgc tacgtgcagc agagcgtgag ccttaacctg atgcgcgacg 12600

gggtaacgcc cagcgtggcg ctggacatga ccgcgcgcaa catggaaccg ggcatgtatg 12660

cctcaaaccg gccgtttatc aaccgcctaa tggactactt gcatcgcgcg gccgccgtga 12720

accccgagta tttcaccaat gccatcttga acccgcactg gctaccgccc cctggtttct 12780

acaccggggg attcgaggtg cccgagggta acgatggatt cctctgggac gacatagacg 12840

acagcgtgtt ttccccgcaa ccgcagaccc tgctagagtt gcaacagcgc gagcaggcag 12900

aggcggcgct gcgaaaggaa agcttccgca ggccaagcag cttgtccgat ctaggcgctg 12960

cggccccgcg gtcagatgct agtagcccat ttccaagctt gatagggtct cttaccagca 13020

ctcgcaccac ccgcccgcgc ctgctgggcg aggaggagta cctaaacaac tcgctgctgc 13080

agccgcagcg cgaaaaaaac ctgcctccgg catttcccaa caacgggata gagagcctag 13140

tggacaagat gagtagatgg aagacgtacg cgcaggagca cagggacgtg ccaggcccgc 13200

gcccgcccac ccgtcgtcaa aggcacgacc gtcagcgggg tctggtgtgg gaggacgatg 13260

actcggcaga cgacagcagc gtcctggatt tgggagggag tggcaacccg tttgcgcacc 13320

ttcgccccag gctggggaga atgttttaaa aaaaaaaaaa gcatgatgca aaataaaaaa 13380

ctcaccaagg ccatggcacc gagcgttggt tttcttgtat tccccttagt atgcggcgcg 13440

cggcgatgta tgaggaaggt cctcctccct cctacgagag tgtggtgagc gcggcgccag 13500

tggcggcggc gctgggttct cccttcgatg ctcccctgga cccgccgttt gtgcctccgc 13560

ggtacctgcg gcctaccggg gggagaaaca gcatccgtta ctctgagttg gcacccctat 13620

tcgacaccac ccgtgtgtac ctggtggaca acaagtcaac ggatgtggca tccctgaact 13680

accagaacga ccacagcaac tttctgacca cggtcattca aaacaatgac tacagcccgg 13740

gggaggcaag cacacagacc atcaatcttg acgaccggtc gcactggggc ggcgacctga 13800

aaaccatcct gcataccaac atgccaaatg tgaacgagtt catgtttacc aataagttta 13860

aggcgcgggt gatggtgtcg cgcttgccta ctaaggacaa tcaggtggag ctgaaatacg 13920

agtgggtgga gttcacgctg cccgagggca actactccga gaccatgacc atagacctta 13980

tgaacaacgc gatcgtggag cactacttga aagtgggcag acagaacggg gttctggaaa 14040

gcgacatcgg ggtaaagttt gacacccgca acttcagact ggggtttgac cccgtcactg 14100

gtcttgtcat gcctggggta tatacaaacg aagccttcca tccagacatc attttgctgc 14160

caggatgcgg ggtggacttc acccacagcc gcctgagcaa cttgttgggc atccgcaagc 14220

ggcaaccctt ccaggagggc tttaggatca cctacgatga tctggagggt ggtaacattc 14280

ccgcactgtt ggatgtggac gcctaccagg cgagcttgaa agatgacacc gaacagggcg 14340

ggggtggcgc aggcggcagc aacagcagtg gcagcggcgc ggaagagaac tccaacgcgg 14400

cagccgcggc aatgcagccg gtggaggaca tgaacgatca tgccattcgc ggcgacacct 14460

ttgccacacg ggctgaggag aagcgcgctg aggccgaagc agcggccgaa gctgccgccc 14520

ccgctgcgca acccgaggtc gagaagcctc agaagaaacc ggtgatcaaa cccctgacag 14580

aggacagcaa gaaacgcagt tacaacctaa taagcaatga cagcaccttc acccagtacc 14640

gcagctggta ccttgcatac aactacggcg accctcagac cggaatccgc tcatggaccc 14700

tgctttgcac tcctgacgta acctgcggct cggagcaggt ctactggtcg ttgccagaca 14760

tgatgcaaga ccccgtgacc ttccgctcca cgcgccagat cagcaacttt ccggtggtgg 14820

gcgccgagct gttgcccgtg cactccaaga gcttctacaa cgaccaggcc gtctactccc 14880

aactcatccg ccagtttacc tctctgaccc acgtgttcaa tcgctttccc gagaaccaga 14940

ttttggcgcg cccgccagcc cccaccatca ccaccgtcag tgaaaacgtt cctgctctca 15000

cagatcacgg gacgctaccg ctgcgcaaca gcatcggagg agtccagcga gtgaccatta 15060

ctgacgccag acgccgcacc tgcccctacg tttacaaggc cctgggcata gtctcgccgc 15120

gcgtcctatc gagccgcact ttttgagcaa gcatgtccat ccttatatcg cccagcaata 15180

acacaggctg gggcctgcgc ttcccaagca agatgtttgg cggggccaag aagcgctccg 15240

accaacaccc agtgcgcgtg cgcgggcact accgcgcgcc ctggggcgcg cacaaacgcg 15300

gccgcactgg gcgcaccacc gtcgatgacg ccatcgacgc ggtggtggag gaggcgcgca 15360

actacacgcc cacgccgcca ccagtgtcca cagtggacgc ggccattcag accgtggtgc 15420

gcggagcccg gcgctatgct aaaatgaaga gacggcggag gcgcgtagca cgtcgccacc 15480

gccgccgacc cggcactgcc gcccaacgcg cggcggcggc cctgcttaac cgcgcacgtc 15540

gcaccggccg acgggcggcc atgcgggccg ctcgaaggct ggccgcgggt attgtcactg 15600

tgccccccag gtccaggcga cgagcggccg ccgcagcagc cgcggccatt agtgctatga 15660

ctcagggtcg caggggcaac gtgtattggg tgcgcgactc ggttagcggc ctgcgcgtgc 15720

ccgtgcgcac ccgccccccg cgcaactaga ttgcaagaaa aaactactta gactcgtact 15780

gttgtatgta tccagcggcg gcggcgcgca acgaagctat gtccaagcgc aaaatcaaag 15840

aagagatgct ccaggtcatc gcgccggaga tctatggccc cccgaagaag gaagagcagg 15900

attacaagcc ccgaaagcta aagcgggtca aaaagaaaaa gaaagatgat gatgatgaac 15960

ttgacgacga ggtggaactg ctgcacgcta ccgcgcccag gcgacgggta cagtggaaag 16020

gtcgacgcgt aaaacgtgtt ttgcgacccg gcaccaccgt agtctttacg cccggtgagc 16080

gctccacccg cacctacaag cgcgtgtatg atgaggtgta cggcgacgag gacctgcttg 16140

agcaggccaa cgagcgcctc ggggagtttg cctacggaaa gcggcataag gacatgctgg 16200

cgttgccgct ggacgagggc aacccaacac ctagcctaaa gcccgtaaca ctgcagcagg 16260

tgctgcccgc gcttgcaccg tccgaagaaa agcgcggcct aaagcgcgag tctggtgact 16320

tggcacccac cgtgcagctg atggtaccca agcgccagcg actggaagat gtcttggaaa 16380

aaatgaccgt ggaacctggg ctggagcccg aggtccgcgt gcggccaatc aagcaggtgg 16440

cgccgggact gggcgtgcag accgtggacg ttcagatacc cactaccagt agcaccagta 16500

ttgccaccgc cacagagggc atggagacac aaacgtcccc ggttgcctca gcggtggcgg 16560

atgccgcggt gcaggcggtc gctgcggccg cgtccaagac ctctacggag gtgcaaacgg 16620

acccgtggat gtttcgcgtt tcagcccccc ggcgcccgcg ccgttcgagg aagtacggcg 16680

ccgccagcgc gctactgccc gaatatgccc tacatccttc cattgcgcct acccccggct 16740

atcgtggcta cacctaccgc cccagaagac gagcaactac ccgacgccga accaccactg 16800

gaacccgccg ccgccgtcgc cgtcgccagc ccgtgctggc cccgatttcc gtgcgcaggg 16860

tggctcgcga aggaggcagg accctggtgc tgccaacagc gcgctaccac cccagcatcg 16920

tttaaaagcc ggtctttgtg gttcttgcag atatggccct cacctgccgc ctccgtttcc 16980

cggtgccggg attccgagga agaatgcacc gtaggagggg catggccggc cacggcctga 17040

cgggcggcat gcgtcgtgcg caccaccggc ggcggcgcgc gtcgcaccgt cgcatgcgcg 17100

gcggtatcct gcccctcctt attccactga tcgccgcggc gattggcgcc gtgcccggaa 17160

ttgcatccgt ggccttgcag gcgcagagac actgattaaa aacaagttgc atgtggaaaa 17220

atcaaaataa aaagtctgga ctctcacgct cgcttggtcc tgtaactatt ttgtagaatg 17280

gaagacatca actttgcgtc tctggccccg cgacacggct cgcgcccgtt catgggaaac 17340

tggcaagata tcggcaccag caatatgagc ggtggcgcct tcagctgggg ctcgctgtgg 17400

agcggcatta aaaatttcgg ttccaccgtt aagaactatg gcagcaaggc ctggaacagc 17460

agcacaggcc agatgctgag ggataagttg aaagagcaaa atttccaaca aaaggtggta 17520

gatggcctgg cctctggcat tagcggggtg gtggacctgg ccaaccaggc agtgcaaaat 17580

aagattaaca gtaagcttga tccccgccct cccgtagagg agcctccacc ggccgtggag 17640

acagtgtctc cagaggggcg tggcgaaaag cgtccgcgcc ccgacaggga agaaactctg 17700

gtgacgcaaa tagacgagcc tccctcgtac gaggaggcac taaagcaagg cctgcccacc 17760

acccgtccca tcgcgcccat ggctaccgga gtgctgggcc agcacacacc cgtaacgctg 17820

gacctgcctc cccccgccga cacccagcag aaacctgtgc tgccaggccc gaccgccgtt 17880

gttgtaaccc gtcctagccg cgcgtccctg cgccgcgccg ccagcggtcc gcgatcgttg 17940

cggcccgtag ccagtggcaa ctggcaaagc acactgaaca gcatcgtggg tctgggggtg 18000

caatccctga agcgccgacg atgcttctga tagctaacgt gtcgtatgtg tgtcatgtat 18060

gcgtccatgt cgccgccaga ggagctgctg agccgccgcg cgcccgcttt ccaagatggc 18120

taccccttcg atgatgccgc agtggtctta catgcacatc tcgggccagg acgcctcgga 18180

gtacctgagc cccgggctgg tgcagtttgc ccgcgccacc gagacgtact tcagcctgaa 18240

taacaagttt agaaacccca cggtggcgcc tacgcacgac gtgaccacag accggtccca 18300

gcgtttgacg ctgcggttca tccctgtgga ccgtgaggat actgcgtact cgtacaaggc 18360

gcggttcacc ctagctgtgg gtgataaccg tgtgctggac atggcttcca cgtactttga 18420

catccgcggc gtgctggaca ggggccctac ttttaagccc tactctggca ctgcctacaa 18480

cgccctggct cccaagggtg ccccaaatcc ttgcgaatgg gatgaagctg ctactgctct 18540

tgaaataaac ctagaagaag aggacgatga caacgaagac gaagtagacg agcaagctga 18600

gcagcaaaaa actcacgtat ttgggcaggc gccttattct ggtataaata ttacaaagga 18660

gggtattcaa ataggtgtcg aaggtcaaac acctaaatat gccgataaaa catttcaacc 18720

tgaacctcaa ataggagaat ctcagtggta cgaaacagaa attaatcatg cagctgggag 18780

agtcctaaaa aagactaccc caatgaaacc atgttacggt tcatatgcaa aacccacaaa 18840

tgaaaatgga gggcaaggca ttcttgtaaa gcaacaaaat ggaaagctag aaagtcaagt 18900

ggaaatgcaa tttttctcaa ctactgaggc agccgcaggc aatggtgata acttgactcc 18960

taaagtggta ttgtacagtg aagatgtaga tatagaaacc ccagacactc atatttctta 19020

catgcccact attaaggaag gtaactcacg agaactaatg ggccaacaat ctatgcccaa 19080

caggcctaat tacattgctt ttagggacaa ttttattggt ctaatgtatt acaacagcac 19140

gggtaatatg ggtgttctgg cgggccaagc atcgcagttg aatgctgttg tagatttgca 19200

agacagaaac acagagcttt cataccagct tttgcttgat tccattggtg atagaaccag 19260

gtacttttct atgtggaatc aggctgttga cagctatgat ccagatgtta gaattattga 19320

aaatcatgga actgaagatg aacttccaaa ttactgcttt ccactgggag gtgtgattaa 19380

tacagagact cttaccaagg taaaacctaa aacaggtcag gaaaatggat gggaaaaaga 19440

tgctacagaa ttttcagata aaaatgaaat aagagttgga aataattttg ccatggaaat 19500

caatctaaat gccaacctgt ggagaaattt cctgtactcc aacatagcgc tgtatttgcc 19560

cgacaagcta aagtacagtc cttccaacgt aaaaatttct gataacccaa acacctacga 19620

ctacatgaac aagcgagtgg tggctcccgg gctagtggac tgctacatta accttggagc 19680

acgctggtcc cttgactata tggacaacgt caacccattt aaccaccacc gcaatgctgg 19740

cctgcgctac cgctcaatgt tgctgggcaa tggtcgctat gtgcccttcc acatccaggt 19800

gcctcagaag ttctttgcca ttaaaaacct ccttctcctg ccgggctcat acacctacga 19860

gtggaacttc aggaaggatg ttaacatggt tctgcagagc tccctaggaa atgacctaag 19920

ggttgacgga gccagcatta agtttgatag catttgcctt tacgccacct tcttccccat 19980

ggcccacaac accgcctcca cgcttgaggc catgcttaga aacgacacca acgaccagtc 20040

ctttaacgac tatctctccg ccgccaacat gctctaccct atacccgcca acgctaccaa 20100

cgtgcccata tccatcccct cccgcaactg ggcggctttc cgcggctggg ccttcacgcg 20160

ccttaagact aaggaaaccc catcactggg ctcgggctac gacccttatt acacctactc 20220

tggctctata ccctacctag atggaacctt ttacctcaac cacaccttta agaaggtggc 20280

cattaccttt gactcttctg tcagctggcc tggcaatgac cgcctgctta cccccaacga 20340

gtttgaaatt aagcgctcag ttgacgggga gggttacaac gttgcccagt gtaacatgac 20400

caaagactgg ttcctggtac aaatgctagc taactataac attggctacc agggcttcta 20460

tatcccagag agctacaagg accgcatgta ctccttcttt agaaacttcc agcccatgag 20520

ccgtcaggtg gtggatgata ctaaatacaa ggactaccaa caggtgggca tcctacacca 20580

acacaacaac tctggatttg ttggctacct tgcccccacc atgcgcgaag gacaggccta 20640

ccctgctaac ttcccctatc cgcttatagg caagaccgca gttgacagca ttacccagaa 20700

aaagtttctt tgcgatcgca ccctttggcg catcccattc tccagtaact ttatgtccat 20760

gggcgcactc acagacctgg gccaaaacct tctctacgcc aactccgccc acgcgctaga 20820

catgactttt gaggtggatc ccatggacga gcccaccctt ctttatgttt tgtttgaagt 20880

ctttgacgtg gtccgtgtgc accagccgca ccgcggcgtc atcgaaaccg tgtacctgcg 20940

cacgcccttc tcggccggca acgccacaac ataaagaagc aagcaacatc aacaacagct 21000

gccgccatgg gctccagtga gcaggaactg aaagccattg tcaaagatct tggttgtggg 21060

ccatattttt tgggcaccta tgacaagcgc tttccaggct ttgtttctcc acacaagctc 21120

gcctgcgcca tagtcaatac ggccggtcgc gagactgggg gcgtacactg gatggccttt 21180

gcctggaacc cgcactcaaa aacatgctac ctctttgagc cctttggctt ttctgaccag 21240

cgactcaagc aggtttacca gtttgagtac gagtcactcc tgcgccgtag cgccattgct 21300

tcttcccccg accgctgtat aacgctggaa aagtccaccc aaagcgtaca ggggcccaac 21360

tcggccgcct gtggactatt ctgctgcatg tttctccacg cctttgccaa ctggccccaa 21420

actcccatgg atcacaaccc caccatgaac cttattaccg gggtacccaa ctccatgctc 21480

aacagtcccc aggtacagcc caccctgcgt cgcaaccagg aacagctcta cagcttcctg 21540

gagcgccact cgccctactt ccgcagccac agtgcgcaga ttaggagcgc cacttctttt 21600

tgtcacttga aaaacatgta aaaataatgt actagagaca ctttcaataa aggcaaatgc 21660

ttttatttgt acactctcgg gtgattattt acccccaccc ttgccgtctg cgccgtttaa 21720

aaatcaaagg ggttctgccg cgcatcgcta tgcgccactg gcagggacac gttgcgatac 21780

tggtgtttag tgctccactt aaactcaggc acaaccatcc gcggcagctc ggtgaagttt 21840

tcactccaca ggctgcgcac catcaccaac gcgtttagca ggtcgggcgc cgatatcttg 21900

aagtcgcagt tggggcctcc gccctgcgcg cgcgagttgc gatacacagg gttgcagcac 21960

tggaacacta tcagcgccgg gtggtgcacg ctggccagca cgctcttgtc ggagatcaga 22020

tccgcgtcca ggtcctccgc gttgctcagg gcgaacggag tcaactttgg tagctgcctt 22080

cccaaaaagg gcgcgtgccc aggctttgag ttgcactcgc accgtagtgg catcaaaagg 22140

tgaccgtgcc cggtctgggc gttaggatac agcgcctgca taaaagcctt gatctgctta 22200

aaagccacct gagcctttgc gccttcagag aagaacatgc cgcaagactt gccggaaaac 22260

tgattggccg gacaggccgc gtcgtgcacg cagcaccttg cgtcggtgtt ggagatctgc 22320

accacatttc ggccccaccg gttcttcacg atcttggcct tgctagactg ctccttcagc 22380

gcgcgctgcc cgttttcgct cgtcacatcc atttcaatca cgtgctcctt atttatcata 22440

atgcttccgt gtagacactt aagctcgcct tcgatctcag cgcagcggtg cagccacaac 22500

gcgcagcccg tgggctcgtg atgcttgtag gtcacctctg caaacgactg caggtacgcc 22560

tgcaggaatc gccccatcat cgtcacaaag gtcttgttgc tggtgaaggt cagctgcaac 22620

ccgcggtgct cctcgttcag ccaggtcttg catacggccg ccagagcttc cacttggtca 22680

ggcagtagtt tgaagttcgc ctttagatcg ttatccacgt ggtacttgtc catcagcgcg 22740

cgcgcagcct ccatgccctt ctcccacgca gacacgatcg gcacactcag cgggttcatc 22800

accgtaattt cactttccgc ttcgctgggc tcttcctctt cctcttgcgt ccgcatacca 22860

cgcgccactg ggtcgtcttc attcagccgc cgcactgtgc gcttacctcc tttgccatgc 22920

ttgattagca ccggtgggtt gctgaaaccc accatttgta gcgccacatc ttctctttct 22980

tcctcgctgt ccacgattac ctctggtgat ggcgggcgct cgggcttggg agaagggcgc 23040

ttctttttct tcttgggcgc aatggccaaa tccgccgccg aggtcgatgg ccgcgggctg 23100

ggtgtgcgcg gcaccagcgc gtcttgtgat gagtcttcct cgtcctcgga ctcgatacgc 23160

cgcctcatcc gcttttttgg gggcgcccgg ggaggcggcg gcgacgggga cggggacgac 23220

acgtcctcca tggttggggg acgtcgcgcc gcaccgcgtc cgcgctcggg ggtggtttcg 23280

cgctgctcct cttcccgact ggccatttcc ttctcctata ggcagaaaaa gatcatggag 23340

tcagtcgaga agaaggacag cctaaccgcc ccctctgagt tcgccaccac cgcctccacc 23400

gatgccgcca acgcgcctac caccttcccc gtcgaggcac ccccgcttga ggaggaggaa 23460

gtgattatcg agcaggaccc aggttttgta agcgaagacg acgaggaccg ctcagtacca 23520

acagaggata aaaagcaaga ccaggacaac gcagaggcaa acgaggaaca agtcgggcgg 23580

ggggacgaaa ggcatggcga ctacctagat gtgggagacg acgtgctgtt gaagcatctg 23640

cagcgccagt gcgccattat ctgcgacgcg ttgcaagagc gcagcgatgt gcccctcgcc 23700

atagcggatg tcagccttgc ctacgaacgc cacctattct caccgcgcgt accccccaaa 23760

cgccaagaaa acggcacatg cgagcccaac ccgcgcctca acttctaccc cgtatttgcc 23820

gtgccagagg tgcttgccac ctatcacatc tttttccaaa actgcaagat acccctatcc 23880

tgccgtgcca accgcagccg agcggacaag cagctggcct tgcggcaggg cgctgtcata 23940

cctgatatcg cctcgctcaa cgaagtgcca aaaatctttg agggtcttgg acgcgacgag 24000

aagcgcgcgg caaacgctct gcaacaggaa aacagcgaaa atgaaagtca ctctggagtg 24060

ttggtggaac tcgagggtga caacgcgcgc ctagccgtac taaaacgcag catcgaggtc 24120

acccactttg cctacccggc acttaaccta ccccccaagg tcatgagcac agtcatgagt 24180

gagctgatcg tgcgccgtgc gcagcccctg gagagggatg caaatttgca agaacaaaca 24240

gaggagggcc tacccgcagt tggcgacgag cagctagcgc gctggcttca aacgcgcgag 24300

cctgccgact tggaggagcg acgcaaacta atgatggccg cagtgctcgt taccgtggag 24360

cttgagtgca tgcagcggtt ctttgctgac ccggagatgc agcgcaagct agaggaaaca 24420

ttgcactaca cctttcgaca gggctacgta cgccaggcct gcaagatctc caacgtggag 24480

ctctgcaacc tggtctccta ccttggaatt ttgcacgaaa accgccttgg gcaaaacgtg 24540

cttcattcca cgctcaaggg cgaggcgcgc cgcgactacg tccgcgactg cgtttactta 24600

tttctatgct acacctggca gacggccatg ggcgtttggc agcagtgctt ggaggagtgc 24660

aacctcaagg agctgcagaa actgctaaag caaaacttga aggacctatg gacggccttc 24720

aacgagcgct ccgtggccgc gcacctggcg gacatcattt tccccgaacg cctgcttaaa 24780

accctgcaac agggtctgcc agacttcacc agtcaaagca tgttgcagaa ctttaggaac 24840

tttatcctag agcgctcagg aatcttgccc gccacctgct gtgcacttcc tagcgacttt 24900

gtgcccatta agtaccgcga atgccctccg ccgctttggg gccactgcta ccttctgcag 24960

ctagccaact accttgccta ccactctgac ataatggaag acgtgagcgg tgacggtcta 25020

ctggagtgtc actgtcgctg caacctatgc accccgcacc gctccctggt ttgcaattcg 25080

cagctgctta acgaaagtca aattatcggt acctttgagc tgcagggtcc ctcgcctgac 25140

gaaaagtccg cggctccggg gttgaaactc actccggggc tgtggacgtc ggcttacctt 25200

cgcaaatttg tacctgagga ctaccacgcc cacgagatta ggttctacga agaccaatcc 25260

cgcccgccta atgcggagct taccgcctgc gtcattaccc agggccacat tcttggccaa 25320

ttgcaagcca tcaacaaagc ccgccaagag tttctgctac gaaagggacg gggggtttac 25380

ttggaccccc agtccggcga ggagctcaac ccaatccccc cgccgccgca gccctatcag 25440

cagcagccgc gggcccttgc ttcccaggat ggcacccaaa aagaagctgc agctgccgcc 25500

gccacccacg gacgaggagg aatactggga cagtcaggca gaggaggttt tggacgagga 25560

ggaggaggac atgatggaag actgggagag cctagacgag gaagcttccg aggtcgaaga 25620

ggtgtcagac gaaacaccgt caccctcggt cgcattcccc tcgccggcgc cccagaaatc 25680

ggcaaccggt tccagcatgg ctacaacctc cgctcctcag gcgccgccgg cactgcccgt 25740

tcgccgaccc aaccgtagat gggacaccac tggaaccagg gccggtaagt ccaagcagcc 25800

gccgccgtta gcccaagagc aacaacagcg ccaaggctac cgctcatggc gcgggcacaa 25860

gaacgccata gttgcttgct tgcaagactg tgggggcaac atctccttcg cccgccgctt 25920

tcttctctac catcacggcg tggccttccc ccgtaacatc ctgcattact accgtcatct 25980

ctacagccca tactgcaccg gcggcagcgg cagcaacagc agcggccaca cagaagcaaa 26040

ggcgaccgga tagcaagact ctgacaaagc ccaagaaatc cacagcggcg gcagcagcag 26100

gaggaggagc gctgcgtctg gcgcccaacg aacccgtatc gacccgcgag cttagaaaca 26160

ggatttttcc cactctgtat gctatatttc aacagagcag gggccaagaa caagagctga 26220

aaataaaaaa caggtctctg cgatccctca cccgcagctg cctgtatcac aaaagcgaag 26280

atcagcttcg gcgcacgctg gaagacgcgg aggctctctt cagtaaatac tgcgcgctga 26340

ctcttaagga ctagtttcgc gccctttctc aaatttaagc gcgaaaacta cgtcatctcc 26400

agcggccaca cccggcgcca gcacctgttg tcagcgccat tatgagcaag gaaattccca 26460

cgccctacat gtggagttac cagccacaaa tgggacttgc ggctggagct gcccaagact 26520

actcaacccg aataaactac atgagcgcgg gaccccacat gatatcccgg gtcaacggaa 26580

tacgcgccca ccgaaaccga attctcctgg aacaggcggc tattaccacc acacctcgta 26640

ataaccttaa tccccgtagt tggcccgctg ccctggtgta ccaggaaagt cccgctccca 26700

ccactgtggt acttcccaga gacgcccagg ccgaagttca gatgactaac tcaggggcgc 26760

agcttgcggg cggctttcgt cacagggtgc ggtcgcccgg gcagggtata actcacctga 26820

caatcagagg gcgaggtatt cagctcaacg acgagtcggt gagctcctcg cttggtctcc 26880

gtccggacgg gacatttcag atcggcggcg ccggccgctc ttcattcacg cctcgtcagg 26940

caatcctaac tctgcagacc tcgtcctctg agccgcgctc tggaggcatt ggaactctgc 27000

aatttattga ggagtttgtg ccatcggtct actttaaccc cttctcggga cctcccggcc 27060

actatccgga tcaatttatt cctaactttg acgcggtaaa ggactcggcg gacggctacg 27120

actgaatgtt aagtggagag gcagagcaac tgcgcctgaa acacctggtc cactgtcgcc 27180

gccacaagtg ctttgcccgc gactccggtg agttttgcta ctttgaattg cccgaggatc 27240

atatcgaggg cccggcgcac ggcgtccggc ttaccgccca gggagagctt gcccgtagcc 27300

tgattcggga gtttacccag cgccccctgc tagttgagcg ggacagggga ccctgtgttc 27360

tcactgtgat ttgcaactgt cctaaccctg gattacatca agatctttgt tgccatctct 27420

gtgctgagta taataaatac agaaattaaa atatactggg gctcctatcg ccatcctgta 27480

aacgccaccg tcttcacccg cccaagcaaa ccaaggcgaa ccttacctgg tacttttaac 27540

atctctccct ctgtgattta caacagtttc aacccagacg gagtgagtct acgagagaac 27600

ctctccgagc tcagctactc catcagaaaa aacaccaccc tccttacctg ccgggaacgt 27660

acgagtgcgt caccggccgc tgcaccacac ctaccgcctg accgtaaacc agactttttc 27720

cggacagacc tcaataactc tgtttaccag aacaggaggt gagcttagaa aacccttagg 27780

gtattaggcc aaaggcgcag ctactgtggg gtttatgaac aattcaagca actctacggg 27840

ctattctaat tcaggtttct ctagaatcgg ggttggggtt attctctgtc ttgtgattct 27900

ctttattctt atactaacgc ttctctgcct aaggctcgcc gcctgctgtg tgcacatttg 27960

catttattgt cagcttttta aacgctgggg tcgccaccca agatgattag gtacataatc 28020

ctaggtttac tcacccttgc gtcagcccac ggtaccaccc aaaaggtgga ttttaaggag 28080

ccagcctgta atgttacatt cgcagctgaa gctaatgagt gcaccactct tataaaatgc 28140

accacagaac atgaaaagct gcttattcgc cacaaaaaca aaattggcaa gtatgctgtt 28200

tatgctattt ggcagccagg tgacactaca gagtataatg ttacagtttt ccagggtaaa 28260

agtcataaaa cttttatgta tacttttcca ttttatgaaa tgtgcgacat taccatgtac 28320

atgagcaaac agtataagtt gtggccccca caaaattgtg tggaaaacac tggcactttc 28380

tgctgcactg ctatgctaat tacagtgctc gctttggtct gtaccctact ctatattaaa 28440

tacaaaagca gacgcagctt tattgaggaa aagaaaatgc cttaatttac taagttacaa 28500

agctaatgtc accactaact gctttactcg ctgcttgcaa aacaaattca aaaagttagc 28560

attataatta gaataggatt taaacccccc ggtcatttcc tgctcaatac cattcccctg 28620

aacaattgac tctatgtggg atatgctcca gcgctacaac cttgaagtca ggcttcctgg 28680

atgtcagcat ctgactttgg ccagcacctg tcccgcggat ttgttccagt ccaactacag 28740

cgacccaccc taacagagat gaccaacaca accaacgcgg ccgccgctac cggacttaca 28800

tctaccacaa atacacccca agtttctgcc tttgtcaata actgggataa cttgggcatg 28860

tggtggttct ccatagcgct tatgtttgta tgccttatta ttatgtggct catctgctgc 28920

ctaaagcgca aacgcgcccg accacccatc tatagtccca tcattgtgct acacccaaac 28980

aatgatggaa tccatagatt ggacggactg aaacacatgt tcttttctct tacagtatga 29040

ttaaatgaga catgattcct cgagttttta tattactgac ccttgttgcg ctttttttgt 29100

gcgtgctcca cattggctgc ggtttctcac atcgaagtag actgcattcc agccttcaca 29160

gtctatttgc tttacggatt tgtcaccctc acgctcatct gcagcctcat cactgtggtc 29220

atcgccttta tccagtgcat tgactgggtc tgtgtgcgct ttgcatatct cagacaccat 29280

ccccagtaca gggacaggac tatagctgag cttcttagaa ttctttaatt atgaaattta 29340

ctgtgacttt tctgctgatt atttgcaccc tatctgcgtt ttgttccccg acctccaagc 29400

ctcaaagaca tatatcatgc agattcactc gtatatggaa tattccaagt tgctacaatg 29460

aaaaaagcga tctttccgaa gcctggttat atgcaatcat ctctgttatg gtgttctgca 29520

gtaccatctt agccctagct atatatccct accttgacat tggctggaac gcaatagatg 29580

ccatgaacca cccaactttc cccgcgcccg ctatgcttcc actgcaacaa gttgttgccg 29640

gcggctttgt cccagccaat cagcctcgcc caccttctcc cacccccact gaaatcagct 29700

actttaatct aacaggagga gatgactgac accctagatc tagaaatgga cggaattatt 29760

acagagcagc gcctgctaga aagacgcagg gcagcggccg agcaacagcg catgaatcaa 29820

gagctccaag acatggttaa cttgcaccag tgcaaaaggg gtatcttttg tctggtaaag 29880

caggccaaag tcacctacga cagtaatacc accggacacc gccttagcta caagttgcca 29940

accaagcgtc agaaattggt ggtcatggtg ggagaaaagc ccattaccat aactcagcac 30000

tcggtagaaa ccgaaggctg cattcactca ccttgtcaag gacctgagga tctctgcacc 30060

cttattaaga ccctgtgcgg tctcaaagat cttattccct ttaactaata aaaaaaaata 30120

ataaagcatc acttacttaa aatcagttag caaatttctg tccagtttat tcagcagcac 30180

ctccttgccc tcctcccagc tctggtattg cagcttcctc ctggctgcaa actttctcca 30240

caatctaaat ggaatgtcag tttcctcctg ttcctgtcca tccgcaccca ctatcttcat 30300

gttgttgcag atgaagcgcg caagaccgtc tgaagatacc ttcaaccccg tgtatccata 30360

tgacacggaa accggtcctc caactgtgcc ttttcttact cctccctttg tatcccccaa 30420

tgggtttcaa gagagtcccc ctggggtact ctctttgcgc ctatccgaac ctctagttac 30480

ctccaatggc atgcttgcgc tcaaaatggg caacggcctc tctctggacg aggccggcaa 30540

ccttacctcc caaaatgtaa ccactgtgag cccacctctc aaaaaaacca agtcaaacat 30600

aaacctggaa atatctgcac ccctcacagt tacctcagaa gccctaactg tggctgccgc 30660

cgcacctcta atggtcgcgg gcaacacact caccatgcaa tcacaggccc cgctaaccgt 30720

gcacgactcc aaacttagca ttgccaccca aggacccctc acagtgtcag aaggaaagct 30780

agccctgcaa acatcaggcc ccctcaccac caccgatagc agtaccctta ctatcactgc 30840

ctcaccccct ctaactactg ccactggtag cttgggcatt gacttgaaag agcccattta 30900

tacacaaaat ggaaaactag gactaaagta cggggctcct ttgcatgtaa cagacgacct 30960

aaacactttg accgtagcaa ctggtccagg tgtgactatt aataatactt ccttgcaaac 31020

taaagttact ggagccttgg gttttgattc acaaggcaat atgcaactta atgtagcagg 31080

aggactaagg attgattctc aaaacagacg ccttatactt gatgttagtt atccgtttga 31140

tgctcaaaac caactaaatc taagactagg acagggccct ctttttataa actcagccca 31200

caacttggat attaactaca acaaaggcct ttacttgttt acagcttcaa acaattccaa 31260

aaagcttgag gttaacctaa gcactgccaa ggggttgatg tttgacgcta cagccatagc 31320

cattaatgca ggagatgggc ttgaatttgg ttcacctaat gcaccaaaca caaatcccct 31380

caaaacaaaa attggccatg gcctagaatt tgattcaaac aaggctatgg ttcctaaact 31440

aggaactggc cttagttttg acagcacagg tgccattaca gtaggaaaca aaaataatga 31500

taagctaact ttgtggacca caccagctcc atctcctaac tgtagactaa atgcagagaa 31560

agatgctaaa ctcactttgg tcttaacaaa atgtggcagt caaatacttg ctacagtttc 31620

agttttggct gttaaaggca gtttggctcc aatatctgga acagttcaaa gtgctcatct 31680

tattataaga tttgacgaaa atggagtgct actaaacaat tccttcctgg acccagaata 31740

ttggaacttt agaaatggag atcttactga aggcacagcc tatacaaacg ctgttggatt 31800

tatgcctaac ctatcagctt atccaaaatc tcacggtaaa actgccaaaa gtaacattgt 31860

cagtcaagtt tacttaaacg gagacaaaac taaacctgta acactaacca ttacactaaa 31920

cggtacacag gaaacaggag acacaactcc aagtgcatac tctatgtcat tttcatggga 31980

ctggtctggc cacaactaca ttaatgaaat atttgccaca tcctcttaca ctttttcata 32040

cattgcccaa gaataaagaa tcgtttgtgt tatgtttcaa cgtgtttatt tttcaattgc 32100

agaaaatttc aagtcatttt tcattcagta gtatagcccc accaccacat agcttataca 32160

gatcaccgta ccttaatcaa actcacagaa ccctagtatt caacctgcca cctccctccc 32220

aacacacaga gtacacagtc ctttctcccc ggctggcctt aaaaagcatc atatcatggg 32280

taacagacat attcttaggt gttatattcc acacggtttc ctgtcgagcc aaacgctcat 32340

cagtgatatt aataaactcc ccgggcagct cacttaagtt catgtcgctg tccagctgct 32400

gagccacagg ctgctgtcca acttgcggtt gcttaacggg cggcgaagga gaagtccacg 32460

cctacatggg ggtagagtca taatcgtgca tcaggatagg gcggtggtgc tgcagcagcg 32520

cgcgaataaa ctgctgccgc cgccgctccg tcctgcagga atacaacatg gcagtggtct 32580

cctcagcgat gattcgcacc gcccgcagca taaggcgcct tgtcctccgg gcacagcagc 32640

gcaccctgat ctcacttaaa tcagcacagt aactgcagca cagcaccaca atattgttca 32700

aaatcccaca gtgcaaggcg ctgtatccaa agctcatggc ggggaccaca gaacccacgt 32760

ggccatcata ccacaagcgc aggtagatta agtggcgacc cctcataaac acgctggaca 32820

taaacattac ctcttttggc atgttgtaat tcaccacctc ccggtaccat ataaacctct 32880

gattaaacat ggcgccatcc accaccatcc taaaccagct ggccaaaacc tgcccgccgg 32940

ctatacactg cagggaaccg ggactggaac aatgacagtg gagagcccag gactcgtaac 33000

catggatcat catgctcgtc atgatatcaa tgttggcaca acacaggcac acgtgcatac 33060

acttcctcag gattacaagc tcctcccgcg ttagaaccat atcccaggga acaacccatt 33120

cctgaatcag cgtaaatccc acactgcagg gaagacctcg cacgtaactc acgttgtgca 33180

ttgtcaaagt gttacattcg ggcagcagcg gatgatcctc cagtatggta gcgcgggttt 33240

ctgtctcaaa aggaggtaga cgatccctac tgtacggagt gcgccgagac aaccgagatc 33300

gtgttggtcg tagtgtcatg ccaaatggaa cgccggacgt agtcatattt cctgaagcaa 33360

aaccaggtgc gggcgtgaca aacagatctg cgtctccggt ctcgccgctt agatcgctct 33420

gtgtagtagt tgtagtatat ccactctctc aaagcatcca ggcgccccct ggcttcgggt 33480

tctatgtaaa ctccttcatg cgccgctgcc ctgataacat ccaccaccgc agaataagcc 33540

acacccagcc aacctacaca ttcgttctgc gagtcacaca cgggaggagc gggaagagct 33600

ggaagaacca tgtttttttt tttattccaa aagattatcc aaaacctcaa aatgaagatc 33660

tattaagtga acgcgctccc ctccggtggc gtggtcaaac tctacagcca aagaacagat 33720

aatggcattt gtaagatgtt gcacaatggc ttccaaaagg caaacggccc tcacgtccaa 33780

gtggacgtaa aggctaaacc cttcagggtg aatctcctct ataaacattc cagcaccttc 33840

aaccatgccc aaataattct catctcgcca ccttctcaat atatctctaa gcaaatcccg 33900

aatattaagt ccggccattg taaaaatctg ctccagagcg ccctccacct tcagcctcaa 33960

gcagcgaatc atgattgcaa aaattcaggt tcctcacaga cctgtataag attcaaaagc 34020

ggaacattaa caaaaatacc gcgatcccgt aggtcccttc gcagggccag ctgaacataa 34080

tcgtgcaggt ctgcacggac cagcgcggcc acttccccgc caggaaccat gacaaaagaa 34140

cccacactga ttatgacacg catactcgga gctatgctaa ccagcgtagc cccgatgtaa 34200

gcttgttgca tgggcggcga tataaaatgc aaggtgctgc tcaaaaaatc aggcaaagcc 34260

tcgcgcaaaa aagaaagcac atcgtagtca tgctcatgca gataaaggca ggtaagctcc 34320

ggaaccacca cagaaaaaga caccattttt ctctcaaaca tgtctgcggg tttctgcata 34380

aacacaaaat aaaataacaa aaaaacattt aaacattaga agcctgtctt acaacaggaa 34440

aaacaaccct tataagcata agacggacta cggccatgcc ggcgtgaccg taaaaaaact 34500

ggtcaccgtg attaaaaagc accaccgaca gctcctcggt catgtccgga gtcataatgt 34560

aagactcggt aaacacatca ggttgattca catcggtcag tgctaaaaag cgaccgaaat 34620

agcccggggg aatacatacc cgcaggcgta gagacaacat tacagccccc ataggaggta 34680

taacaaaatt aataggagag aaaaacacat aaacacctga aaaaccctcc tgcctaggca 34740

aaatagcacc ctcccgctcc agaacaacat acagcgcttc cacagcggca gccataacag 34800

tcagccttac cagtaaaaaa gaaaacctat taaaaaaaca ccactcgaca cggcaccagc 34860

tcaatcagtc acagtgtaaa aaagggccaa gtgcagagcg agtatatata ggactaaaaa 34920

atgacgtaac ggttaaagtc cacaaaaaac acccagaaaa ccgcacgcga acctacgccc 34980

agaaacgaaa gccaaaaaac ccacaacttc ctcaaatcgt cacttccgtt ttcccacgtt 35040

acgtcacttc ccattttaag aaaactacaa ttcccaacac atacaagtta ctccgcccta 35100

aaacctacgt cacccgcccc gttcccacgc cccgcgccac gtcacaaact ccaccccctc 35160

attatcatat tggcttcaat ccaaaataag gtatattatt gatgatg 35207

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 20

ggtgactttg cttctggag 19

<210> 21

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 21

ctccagaagc aaagtcacc 19

<210> 22

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 22

gtacttcata cttttcatt 19

<210> 23

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> cis regulatory element

<400> 23

aatgaaaagt atgaagtac 19

<210> 24

<211> 6

<212> DNA

<213> Artificial sequence

<220>

<223> anoxic reaction element

<400> 24

gcacgt 6

<210> 25

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 25

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser Gly Gly Gly Ser

35 40

PCT/RO/134 Table