Compositions and methods for treating cancer

Abstract

One aspect of the present disclosure is directed to a method for treating cancer in a subject in need thereof, by administering at least a first compound and a second compound to the subject, in any order, together or separately.The first compound is an effective amount of a checkpoint inhibitor, which may be accompanied by at least one pharmaceutically acceptable carrier.The second compound is an effective amount of a therapeutic double-stranded RNA (tdsRNA), which may be accompanied by at least one pharmaceutically acceptable carrier.The compounds may be administered together or separately.Compositions for carrying out the method are also described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 885,143, filed August 9, 2019, and entitled "Compositions For Cancer Therapy And Methods" (Attorney Docket No. 500051-000849); U.S. Provisional Patent Application No. 62 / 869,909, filed July 2, 2019, and entitled "Synergistic Cancer Compositions and Methods Involving Same" (Attorney Docket No. 500051-000820); U.S. Provisional Patent Application No. 62 / 792,760, filed January 15, 2019, and entitled "Cancer Treatment Compositions and Methods" (Attorney Docket No. 500051-000766); U.S. Provisional Patent Application No. 62 / 792,760, filed January 15, 2019, and entitled "Cancer Treatment Compositions and Methods" (Attorney Docket No. 500051-000766); The present disclosure claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 792,765 (Attorney Docket No. 500051-000765), filed December 21, 2018, entitled "Methods for Promoting Cancer," and U.S. Provisional Patent Application No. 62 / 783,834 (Attorney Docket No. 500051-000753), filed December 21, 2018, entitled "Cancer Treatment." All publications, patent applications, and patents mentioned in this disclosure are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present application, including any definitions, will control. [Background technology]

[0002] Immunotherapy is a rapidly developing field for the treatment of cancer, but unfortunately has faced limited success. An ever-growing collection of new drugs that target the body's immune system against tumors is gaining attention in cancer treatment. Immunotherapy has been successful in achieving survival or symptom-free survival in a minority of patients. Unfortunately, immunotherapy only helps a minority of patients with a given type of cancer, and has had little or no success in some types of cancer. Summary of the Invention [Problem to be solved by the invention]

[0003] There is a need to develop methods and combination therapies that induce or enhance the effectiveness of checkpoint inhibitors in both non-responsive and responsive subject populations. There is a long-felt need to discover why immunotherapy is ineffective against some types of cancer and how it can be improved to work against more types of cancer. [Means for solving the problem]

[0004] In this disclosure, the term "in any embodiment of the disclosure" is understood to include at least the meaning of "in any of the methods and compositions of the disclosure."

[0005] One embodiment is directed to a method for treating cancer in a subject in need thereof, comprising administering to the subject at least a first compound and a second compound, in any order, together or separately. In the method, the first compound comprises an effective amount of a checkpoint inhibitor, optionally accompanied by at least one pharmaceutically acceptable carrier, and the second compound is an effective amount of a therapeutic double-stranded RNA (tdsRNA), optionally accompanied by at least one pharmaceutically acceptable carrier. The present disclosure further provides a checkpoint inhibitor and a therapeutic double-stranded RNA (tdsRNA) for use in a cancer treatment method or for use in preparing a medicament for cancer treatment. The checkpoint inhibitor and the tdsRNA may be administered simultaneously or separately.

[0006] Treating cancer can include at least one selected from the group consisting of inhibiting tumor growth in a subject; eliciting the effect of a checkpoint inhibitor in a subject; enhancing the effect of a checkpoint inhibitor in a subject; prolonging the effect of a checkpoint inhibitor in a subject; and activating a response to a checkpoint inhibitor in a subject.

[0007] Any cancer can be treated by the method and composition of the present disclosure.In one embodiment, cancer is at least one selected from the group consisting of pancreatic cancer, skin cancer, colorectal cancer, ovarian cancer, melanoma, breast cancer, triple-negative breast cancer, head and neck tumor, bladder cancer, renal cell carcinoma, and lung cancer.Preferably, cancer is pancreatic cancer, colorectal cancer, melanoma, bladder cancer, or renal cell carcinoma.

[0008] In any embodiment of the present disclosure, the tdsRNA is n ribo(C 4-29 U) n or rI n ribo(C 11-14 U) n ; preferably, rI n ribo(C 11 U)n ;rI n ribo(C 13 U) n ; or rI n ribo(C 14 U) n and most preferably rI n ribo(C 12 U) n It is possible.

[0009] In any embodiment of the present disclosure, the tdsRNA can be a Rugged dsRNA. Rugged dsRNA is a hybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strand (rI) of the same or similar length (e.g., the value of n is the same or similar). n ·rC n ) is resistant to denaturation under conditions that allow separation of the

[0010] In any embodiment of the present disclosure, the Rugged dsRNA as a weight percentage of total RNA in a method or composition may exceed a value selected from the group consisting of 1 weight percent; 5 weight percent; 10 weight percent; 20 weight percent; 30 weight percent; 40 weight percent; 50 weight percent; 60 weight percent; 70 weight percent; 80 weight percent; and 90 weight percent.

[0011] In any embodiment of the present disclosure, a tdsRNA can have a lower length limit of 40; 50; 60; 70; 80; or 380, and the same tdsRNA can have an upper length limit of 50,000; 10,000; 9000; 8000; 7000; or 450. As described above, any lower length limit can be combined with any upper length limit. For example, a tdsRNA in any embodiment of the present disclosure can have a length or value of "n" between 40 and 50,000 bases or base pairs, depending on whether one or both strands are measured. In preferred embodiments of any embodiment of the present disclosure, the length or value of "n" can be 50 to 10,000; 60 to 9000; 70 to 8000; 80 to 7000; or 380 to 450. Preferably, n is 40 to 50,000; 50 to 10,000; 60 to 9000; 70 to 8000; 80 to 7000; or 380 to 450.

[0012] In any embodiment of the present disclosure, the tdsRNA can have an RNA duplex between 4 and about 5000 helical turns, preferably between 30 and 38 helical turns.

[0013] In any embodiment of the present disclosure, the tdsRNA can have a molecular weight of about 2 kilodaltons to about 30,000 kilodaltons, preferably 250 kilodaltons to 320 kilodaltons.

[0014] In any embodiment of the present disclosure, the tdsRNA can have a linear structure without branched RNA structures.

[0015] In any embodiment of the present disclosure, the second compound comprises tdsRNA, and at least 30 weight percent of the total dsRNA is linear; at least 40 weight percent of the total dsRNA is linear; at least 50 weight percent of the total dsRNA is linear; at least 60 weight percent of the total dsRNA is linear; at least 70 weight percent of the total dsRNA is linear; at least 80 weight percent of the total dsRNA is linear; or at least 90 weight percent of the total dsRNA is linear.In any embodiment of the present disclosure, tdsRNA forms a complex with stabilizing polymer.For example, stabilizing polymer can be selected from the group consisting of polylysine; polylysine + carboxymethylcellulose; polyarginine; polyarginine + carboxymethylcellulose; and combinations thereof.

[0016] In any embodiment of the present disclosure, the tdsRNA is n ribo(C 11-14 U) n ;rI n ribo(C4U) n ;rI n ribo(C5U) n ;rI n ribo(C6U) n ;rI n ribo(C7U) n ;rI n ribo(C8U) n ;rI n ribo(C9U) n ;rI n ribo(C 10 U) n ;rI n ribo(C 11 U) n ;rI n ribo(C 13 U) n ;rI n ribo(C 14 U) n ;rI n ribo(C 15HE) n ;country n ·ribo(C 16 HE) n ;country n ·ribo(C 17 HE) n ;country n ·ribo(C 18 HE) n ;country n ·ribo(C 19 HE) n ;country n ·ribo(C 20 HE) n ;country n ·ribo(C 21 HE) n ;country n ·ribo(C 22 HE) n ;country n ·ribo(C 23 HE) n ;country n ·ribo(C 24 HE) n ;country n ·ribo(C 25 HE) n ;country n ·ribo(C 26 HE) n ;country n ·ribo(C 27 HE) n ;country n ·ribo(C 28 HE) n ;country n ·ribo(C 29 HE) n ;country n ·ribo(C 30 HE) n ;country n ·ribo(C 31 HE) n ;country n ·ribo(C 32 HE) n ;country n ·ribo(C 33 HE) n ;country n ·ribo(C 34 HE) n ;country nribo(C 35 U) n ;rI n ribo(C 4-30 U) n ;rI n ribo(C 14-30 U) n ;rI n ribo(C 11-14 G) n ;rI n ribo(C 4-29 G) n ;rI n ribo(C 30-35 U) n ;r(Poly I·Poly C) n ; and r(polyA·polyU) n As disclosed above, n can have numerous upper and lower limits, such as 40 to 50,000; 50 to 10,000; 60 to 9000; 70 to 8000; 80 to 7000; and 380 to 450.

[0017] In any embodiment of the present disclosure, the effective amount of tdsRNA is a synergistic therapeutically effective amount.

[0018] In any embodiment of the present disclosure, the combination of administered tdsRNA and checkpoint inhibitor produces a synergistic effect in treating cancer or inhibiting tumor cell proliferation. This synergistic effect can be selected from the group consisting of: extending the subject's survival; extending the subject's time to progression; inhibiting tumor growth; inducing tumor cell death; increasing tumor regression; preventing tumor recurrence; preventing tumor growth; preventing tumor expansion; delaying tumor recurrence; delaying tumor growth; delaying tumor expansion; and promoting tumor disappearance. In any embodiment of the present disclosure, the effective amount of checkpoint inhibitor is a synergistic therapeutically effective amount. In other words, the administered checkpoint inhibitor produces an additive or synergistic effect in treating cancer or an additive or synergistic effect in inhibiting tumor cell proliferation.

[0019] In any embodiment of the present disclosure, one additional step, which may be performed in any order with one or more of the previously disclosed steps, further comprises administering a third compound to a subject. The composition of the present disclosure may also include this third compound. The third compound may be one or more selected from the group consisting of chemotherapeutic drugs (anticancer drugs); targeted anticancer drugs; and targeted anticancer drugs including antibodies. A targeted anticancer drug is any drug designed to bind to cancer cells. For example, the drug may include an antibody, a ligand, or a receptor, a hormone, a nutrient, a biochemical, or a mimic thereof, or a binding portion thereof. In a preferred embodiment, the effective amount of the third compound is synergistic with the tdsRNA and the checkpoint inhibitor, a therapeutically effective amount, or both. In another preferred embodiment, the third compound is administered at a subtherapeutic dose and has no effect on cancer without the combination of the first compound (i.e., the checkpoint inhibitor) and the second compound (tdsRNA).

[0020] In any embodiment of the present disclosure, the method may further comprise administering to the subject a compound selected from the group consisting of interferon; interferon mixture; Alferon; and alpha-interferon molecular species. The interferon may be an interferon molecular species produced by human leukocytes and purified as a mixture of at least seven molecular species of alpha-interferon. The seven molecular species may be, for example, interferon alpha 2, interferon alpha 4, interferon alpha 7, interferon alpha 8, interferon alpha 10, interferon alpha 16, and interferon alpha 17.

[0021] In one embodiment, the first compound, the second compound, optionally the third compound, and optionally the fourth compound are each distinct or chemically significantly different from each other, i.e., for example, one compound cannot be both the first compound and the second compound.

[0022] Any method of administration is suitable, but in any embodiment of the present disclosure, administration may be intravenous; intradermal; subcutaneous; intramuscular; intranasal; intraperitoneal; intracranial; intravesical; orally; or topical.

[0023] In any embodiment of the present disclosure, tdsRNA and checkpoint inhibitor can be administered simultaneously or separately.For example, tdsRNA and checkpoint inhibitor can be administered separately at different time intervals, but tdsRNA (for example, in the second compound) is administered at a frequency selected from the group consisting of once a month, once every 3 weeks, once every 2 weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, and daily.As another example, tdsRNA and checkpoint inhibitor can be administered separately, but can be administered within a time period selected from the group consisting of 2 months; 1 month; 3 weeks; 2 weeks; 1 week; 3 days; 1 day; 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, and 30 minutes. In any embodiment of the present disclosure, the second compound comprising tdsRNA can be administered intravenously to a subject 1 to 5 times per week at a dose that averages about 25 to 700 milligrams of tdsRNA per day for up to one month or for periods greater than one month. For example, the second compound comprising tdsRNA can be administered to a subject 1 to 5 times per week at a dose that averages about 25 to 700 milligrams of tdsRNA per day for at least one month, continuously.

[0024] In any embodiment of the present disclosure, the tdsRNA and checkpoint inhibitor together may provide a synergistic effect in treating cancer or inhibiting tumor cell growth that is greater than the use of the tdsRNA alone, the checkpoint inhibitor alone, or the combined effects of the tdsRNA alone and the checkpoint inhibitor alone.

[0025] In any embodiment of the present disclosure, the checkpoint inhibitor may have at least one feature selected from the group consisting of an antibody; a monoclonal antibody; a humanized antibody; a human antibody; a fusion protein; a pegylated antibody; a multimeric antibody; an antibody fragment comprising an epitope-binding region; and combinations thereof.

[0026] In any embodiment of the disclosure, the checkpoint inhibitor is selected from the group consisting of 2B4; A2aR; B7 family ligand; B7 H3; B7 H4; B and T lymphocyte attenuator (BTLA); BMA; CD112; CD137; CD160; CD2; CD20; CD226; CD27; CD276; CD28; CD30; CD33; CD40; CD47; CD52; CD70; CD80; CD86; CGEN The antibody may inhibit, interact with, or bind to a checkpoint protein, a ligand for a checkpoint protein, or a receptor for a checkpoint protein selected from the group consisting of: 15049; CHK1; CHK2; cytotoxic T-lymphocyte antigen 4 (CTLA-4); DR3; galectin 9 (GAL9); GITR; herpes virus entry mediator (HVEM); ICOS; IDO1; IDO2; killer cell immunoglobulin-like receptor (KIR); LAG3; LAIR; LAIR1; LAIR2; LIGHT; lymphocyte activation gene 3 (LAG-3); MARCO; OX-40; PD-1; PD-L1; PD-L2; PS; SIRP alpha; SLAM; T cell immunoreceptor with Ig and ITIM domains (TIGIT); T cell membrane protein 3 (TIM3); V-domain immunoglobulin (Ig)-containing suppressor of T cell activation (VISTA); VTCN1; and combinations thereof.

[0027] In any aspect of the present disclosure, the checkpoint inhibitor may inhibit, interact with, or bind to a checkpoint protein, a checkpoint protein ligand, or a checkpoint protein receptor. For example, the checkpoint protein, checkpoint protein ligand, or checkpoint protein receptor may be selected from the group consisting of PD-1; PD-L1; cytotoxic T-lymphocyte antigen 4 (CTLA-4); CD80; CD86; and combinations thereof. In a preferred embodiment, the checkpoint inhibitor inhibits PD-1 or PD-L1. Additional members of this checkpoint inhibitor / receptor group are further listed elsewhere in this disclosure. In one embodiment, the checkpoint inhibitor may comprise an antibody. For example, the checkpoint inhibitor may comprise an antibody that binds to one or more checkpoint proteins, checkpoint protein ligands, or checkpoint protein receptors.

[0028] In any embodiment of the disclosure, the checkpoint inhibitor is alemtuzumab [CAMPATH-1H®]; AMP-224 (GlaxoSmithKline / Amplimmune); AMP-514 (Amplimmune / AZ); alelumab (Merck Serono); atezolizumab [TECENTRIQ®; Roche / Genentech] [targets PD-L1]; AUNP 12 (Aurigene and Pierre Fabre); avelumab [BAVENCIO®] [targets PD-L1]; BMS-936559, BMS-986016 (Bristol-Meyers Squibb); BMS-986016 (Bristol-Meyers Squibb). Squibb); cemiplimab [LIBTAYO®] [targets PD-1]; CP-870,893 (Genentech); CT-011; durvalumab [IMFINZI®]; durvalumab [IMFINZI®] [targets PD-L1]; galiximab (Biogen Idec); IMP321 (Immutep SA); INCB024360 (Incyte); indoximod (NewLink Genetics); IPH2101 (Innate Pharma / Bristol-Myers Squibb); ipilimumab [YERVOY® Bristol-Myers Squibb]; Libtayo (semiplimab-rwlc); lambrolizumab; lirilumab (Bristol-Myers Squibb); MDX-1105 (Medarex, Inc. / Bristol Myer Squibb; MEDI-4736 (Medimmune / AstraZeneca); MEDI-6469 (MedImmune / AZ); MGA271 (Macrogenics); MIHI; mogamulizumab (Kyowa Kirin Co., Ltd.); MPDL3280A (Roche); nivolumab [OPDIVO® Bristol-Myers Squibb] [targets PD-1]; NLG-919 (NewLink Genetics); ofatumumab [ARZERRA®]; pembrolizumab [KEYTRUDA®; Merck] [targets PD-1]; PF-05082566 (Pfizer); pidilizumab (Curetech); rituximab [RITUXAN®]; tremelimumab; urelumab (Bristol-Meyers Squibb); valilumab (CelIDex Therapeutics); and combinations thereof.

[0029] In any embodiment of the present disclosure, the subject to be treated can be a mammal. The mammal can be, for example, a human.

[0030] In any embodiment of the present disclosure, the cancer can be one that is unresponsive to treatment with a checkpoint inhibitor alone, and / or is unresponsive to a chemotherapeutic agent alone, and / or is unresponsive to a combination of a checkpoint inhibitor and a chemotherapeutic agent.

[0031] In another aspect, the present disclosure is directed to a method for treating cancer in a subject in need thereof, comprising exposing or contacting the cancer to or with a first compound and a second compound, in any order, either together or separately, wherein the first compound comprises an effective amount of a checkpoint inhibitor, optionally associated with at least one pharmaceutically acceptable carrier, and the second compound is an effective amount of a therapeutic double-stranded RNA (tdsRNA), optionally associated with at least one pharmaceutically acceptable carrier.

[0032] In another embodiment, the present disclosure is directed to a composition for treating cancer, comprising a checkpoint inhibitor and a tdsRNA. The composition may be a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. The composition may improve progression-free survival or overall survival of a subject administered the composition. In one embodiment, the checkpoint inhibitor may be selected from the group consisting of a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, and a combination thereof. In one embodiment, the checkpoint inhibitor is selected from the group consisting of 2B4; A2aR; B7 family ligand; B7 H3; B7 H4; B and T lymphocyte attenuator (BTLA); BMA; CD112; CD137; CD160; CD2; CD20; CD226; CD27; CD276; CD28; CD30; CD33; CD40; CD47; CD52; CD70; CD80; CD86; CGEN The compound may inhibit, bind to, or interact with a checkpoint protein, a ligand of a checkpoint protein, or a receptor of a checkpoint protein selected from the group consisting of: 15049; CHK1; CHK2; cytotoxic T-lymphocyte antigen 4 (CTLA-4); DR3; galectin 9 (GAL9); GITR; herpes virus entry mediator (HVEM); ICOS; IDO1; IDO2; killer cell immunoglobulin-like receptor (KIR); LAG3; LAIR; LAIR1; LAIR2; LIGHT; lymphocyte activation gene 3 (LAG-3); MARCO; OX-40; PD-1; PD-L1; PD-L2; PS; SIRP alpha; SLAM; T cell immunoreceptor with Ig and ITIM domains (TIGIT); T cell membrane protein 3 (TIM3); V-domain immunoglobulin (Ig)-containing suppressor of T cell activation (VISTA); VTCN1; their ligands; their receptors; and combinations thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of PD-1, PD-L1, cytotoxic T-lymphocyte antigen 4 (CTLA-4), CD80, CD86, their ligands, and their receptors.and combinations thereof. For example, the checkpoint inhibitor is selected from the group consisting of ipilimumab (YERVOY®, Bristol-Myers Squibb); nivolumab (OPDIVO®, Bristol-Myers Squibb); pembrolizumab (KEYTRUDA®; Merck); and combinations thereof. As another example, checkpoint inhibitors include alemtuzumab [CAMPATH-1H®]; AMP-224 (GlaxoSmithKline / Amplimmune); AMP-514 (Amplimmune / AZ); allerumab (Merck Serono); atezolizumab [TECENTRIQ®; Roche / Genentech] [targets PD-L1]; AUNP 12 (Aurigene and Pierre Fabre); avelumab [BAVENCIO®] [targets PD-L1]; BMS-936559, BMS-986016 (Bristol-Meyers Squibb); BMS-986016 (Bristol-Meyers Squibb); cemiplimab [LIBTAYO®] [targets PD-1]; CP-870,893 (Genentech); CT-011; durvalumab [IMFINZI®]; durvalumab [IMFINZI®] [targets PD-L1]; galiximab (Biogen Idec); IMP321 (Immutep SA); INCB024360 (Incyte); indoximod (NewLink Genetics); IPH2101 (Innate Pharma / Bristol-Myers Squibb); ipilimumab [YERVOY®, Bristol-Myers Squibb]; Libtayo (cemiplimab-rwlc); lambrolizumab; lirilumab (Bristol-Myers Squibb); MDX-1105 (Medarex, Inc. / Bristol Myer Squibb);The compound may be selected from the group consisting of MEDI-4736 (Medimmune / AstraZeneca); MEDI-6469 (MedImmune / AZ); MGA271 (Macrogenics); MIHI; mogamulizumab (Kyowa Kirin Co., Ltd.); MPDL3280A (Roche); nivolumab [OPDIVO®, Bristol-Myers Squibb] [targets PD-1]; NLG-919 (NewLink Genetics); ofatumumab [ARZERRA®]; pembrolizumab [KEYTRUDA®; Merck] [targets PD-1]; PF-05082566 (Pfizer); pidilizumab (Curetech); rituximab [RITUXAN®]; tremelimumab; urelumab (Bristol-Meyers Squibb); valilumab (CelIDex Therapeutics); and combinations thereof.

[0033] In any embodiment of the disclosure, the anticancer or chemotherapy drug is selected from the group consisting of ABVD; AC; ACE; abiraterone (Zytiga); Abraxane; Abstral; actinomycin D; Actiq; adriamycin; afatinib (Giotrif); Afinitor; aflibercept (Zaltrap); Aldara; aldesleukin (IL-2, proleukin or interleukin 2); alemtuzumab (MabCampath); Alkeran; amsacrine (Amcidin, m-AMSA); Amcidin; anastrozole (Arimidex); Ara C; Aredia; Arimidex; Aromasin; Arsenic trioxide (Trisenox, ATO); Asparaginase (Crisantaspase, Erwinase); Axitinib (Inlyta); Azacitidine (Vidaza); BEACOPP; BEAM; Bendamustine (Levact); Bevacizumab (Avastin); Bexarotene (Targretin); Bicalutamide (Casodex); Bleomycin; Bleomycin, etoposide and platinum (BEP); Bortezomib (Velcade) ;Bosulif;bosutinib (Bosulif);brentuximab (Adcetris);Brufen;buserelin (Suprefact);Busilvex;busulfan (Myleran, Busilvex);CAPE-OX;CAPOX;CAV;CAVE;CCNU;CHOP;CMF;CMV;CVP;cabazitaxel (Jevtana);cabozantinib (Cometriq);Caelyx;Calpol;Campto;capecitabine (Xeloda);Caprelsa;Carbo MV; CarboTaxol; carboplatin; carboplatin and etoposide; carboplatin and paclitaxel; carmustine (BCNU, Gliadel); Casodex; ceritinib (Zykadia); Cerubidin; cetuximab (Erbitux); ChlVPP; chlorambucil (Leukeran); cisplatin; cisplatin and Teysuno; cisplatin and capecitabine (CX); cisplatin, etoposide, and ifosfamide (PEI); cisplatin, fluorouracil (5-FU), and trastuzumab; cladribine (Leustat, LITAK); Clasteon;Clofarabine (Evoltra); Co-codamol (Kapake, Solpadol, Tylex); Cometriq; Cosmegen; Crisantaspase; Crizotinib (Xalkori); Cyclophosphamide; Cyclophosphamide, thalidomide, and dexamethasone (CTD); Cyprostat; Cyproterone acetate (Cyprostat); Cytarabine (Ara C, cytosine arabinoside); Cytarabine into spinal fluid; Cytosine arabinoside; DHAP; DTIC; Dabrafenib (Tafinlar); Dacarbazine (DTIC); Dacogen; Dactinomycin (Actinomycin D, Cosmegen); Dasatinib (Sprycel); Daunorubicin; De Gramont; Decapeptyl SR; decitabine (Dacogen); degarelix (Firmagon); denosumab (Prolia, Xgeva); Depocyt (Depocyte); dexamethasone; diamorphine; pamidronate disodium; Disprol; docetaxel (Taxotere); docetaxel, cisplatin and fluorouracil (TPF); Doxifos; Doxil; doxorubicin (Adriamycin); doxorubicin and ifosfamide (Doxifos); Drogenil; Durogesic; EC; ECF; EOF; EOX; EP (etoposide and cisplatin); ESHAP; Effentora; Efudix; Eldisine; Eloxatin; enzalutamide; epirubicin [Fa rumorubicin (Pharmorubicin)]; epirubicin, cisplatin, and capecitabine (ECX); epirubicin, carboplatin, and capecitabine (ECarboX); Eposin; Erbitux; eribulin (Halaven); erlotinib (Tarceva); Erwinase; Estracyt; Etopophos; etoposide (Eposin, Etopophos, Vepesid); everolimus (Afinitor); Evoltra; exemestane (Aromasin); FAD; FEC; FEC-T chemotherapy; FMD; Folfirinox; FOLFOX; Faslodex; Femara; fentanyl; Firmagon; Fludara; fludarabine (Fludara);Fludarabine, cyclophosphamide, and rituximab (FCR); fluorouracil (5FU); flutamide; folinic acid, fluorouracil, and irinotecan (FOLFIRI); fulvestrant (faslodex); G-CSF; gefitinib (Iressa); GemCarbo (gemcitabine and carboplatin); GemTaxol; gemcitabine (Gemzar); gemcitabine and capecitabine (GemCap); gemcitabine and cisplatin (GC); gemcitabine and paclitaxel (GemTaxol); Gemzar; Giotrif; Gliadel; Glivec; Gonapeptyl Depot; Goserelin (Zoladex); Goserelin (Zoladex, Novgos); Granulocyte colony-stimulating factor (G-CSF); Halaven; Herceptin; Hycamtin; Hydrea; Hydroxycarbamide (Hydrea); Hydroxyurea; I-DEX; ICE; IL-2; IPE; Ibandronate; Ibritumomab (Zevalin); Ibrutinib (Imbruvica); Ibuprofen (Brufen, Nurofen); Iclusig; Idarubicin (Zavedos); Idarubicin and dexamethasone; Idelalisib (Zydelig); Ifosfamide (Mitoxana); Imatinib (Glivec); Imiquimod cream (Aldara); Imnovid; Instanyl; Interferon (Intron A); Interleukin; Intron A; ipilimumab (Yervoy); Iressa; irinotecan (Campto); irinotecan and capecitabine (Xeliri); irinotecan de Gramont; irinotecan modified de Gramont; Javlor; Jevtana; Kadcyla; Kapake; Keytruda; lanreotide (Somatuline); Lanvis; lapatinib (Tyverb); lenalidomide (Revlimid); letrozole (Femara); Leukeran; leuprorelin (Prostap, Lutrate); Leustat; Levact; liposomal doxorubicin; Litak; lomustine (CCNU); Lynparza; Lysodren; MIC; MMM; MPT; MST Continus; MVAC; MVP;MabCampath; Mabthera; Maxtrex; Medroxyprogesterone acetate (Provera); Megace; Megestrol acetate (Megace); Melphalan (Alkeran); Mepact; Mercaptopurine (Xaluprine); Methotrexate; Methylprednisolone; Mifamurtide (Mepact); Mitomycin C; Mitotane; Mitoxana; Mitoxantrone (Mitozantrone); Morphgesic SR; morphine; Myleran; Myocet; Nab-paclitaxel; Nab-paclitaxel (Abraxane); Navelbine; Nelarabine (Atriance); Nexavar; Nilotinib (Tasigna); Nintedanib (Vargatef); Nipent; Nivolumab (Opdivo); Novgos; Nurofen; Obinutuzumab (Gazyvaro); Octreotide; Ofatumumab (Arzerra); Olaparib (Lynparza); Oncovin; Onkotrone; Opdivo; Oramorph; Oxaliplatin (Eloxatin); Oxaliplatin and capecitabine (Xelox); PAD; PC (paclitaxel and carboplatin, CarboTaxol); PE; PMitCEBO; POMB / ACE; Paclitaxel (Taxol); Paclitaxel and carboplatin; Pamidronate; Pana dol; panitumumab (Vectibix); paracetamol; pazopanib (Votrient); pembrolizumab (Keytruda); pemetrexed (Alimta); pemetrexed and carboplatin; pemetrexed and cisplatin; pentostatin (Nipent); Perjeta; pertuzumab (Perjeta); pixantrone (Pixuvri); Pixuvri; pomalidomide (Imnovid); Ponatinib; Potactasol; Prednisolone; Procarbazine; Procarbazine, lomustine, and vincristine (PCV); Proleukin; Prolia; Prostap; Provera; Purinethol; R-CHOP; R-CVP; R-DHAP; R-ESHAP; R-GCVP; RICE; Raloxifene; Raltitrexed (Tomudex); Regorafenib (Stivarga); Revlimid;Rituximab (Mabthera); Sevredol; Clodronate sodium (Bonefos, Clasteon, Loron); Solpadol; Sorafenib (Nexavar); Steroids (dexamethasone, prednisolone, methylprednisolone); Streptozocin (Zanosar); Sunitinib (Sutent); Sutent; TAC; TIP; Tafinlar; Tamoxifen; Tarceva; Targretin; Tasigna; Taxol; Taxotere; Taxotere and cyclophosphamide Famid (TC); Temodal; Temozolomide (Temodal); Temsirolimus; Tepadina; Teysuno; Thalidomide; Thiotepa (Tepadina); Thioguanine (thioguanine, 6-TG, 6-thioguanine); Tomudex; Topotecan (Hycamtin, Potactasol); Torisel; Trabectedin (Yondelis); Trastuzumab (Herceptin); Trastuzumab emtansine (Kadcyla); Treosulfan; Tretinoin (Vesanoid, ATRA) ; Triptorelin; Trizenox; Tyrex; Tyverb; VIDE; Vandetanib (Caprelsa); Valgatef; VeIP; Vectibix; Velbe; Velcade; Vemurafenib (Zelboraf); Bepecid; Vesanoid; Vidaza; Vinblastine (Velbe); Vincristine; Vincristine, actinomycin D (dactinomycin) and cyclophosphamide (VAC); Vincristine, actinomycin and ifosfamide (VAI); Vincristine, doxorubicin, and dexamethasone (V AD); Vindesine (Eldisine); Vinflunine (Javlor); Vinorelbine (Navelbine); Vismodegib (Erivedge); Votrient; XELOX; Xalkori; Xeloda; Xgeva; Xtandi; Yervoy; Yondelis; Z-DEX; Zaltrap; Zanosar; Zavedos; Zelboraf; Zevalin; Zoladex (breast cancer); Zoladex (prostate cancer); Zoledronic acid (Zometa); Zometa; Zomorph; Zydelig; Zytiga;and combinations thereof.

[0034] The following are non-limiting preferred embodiments of the present disclosure. 1. Checkpoint inhibitors and therapeutic double-stranded RNA (tdsRNA) for use in the treatment of cancer. 2. The checkpoint inhibitor and tdsRNA for use according to embodiment 1, wherein the tdsRNA and the checkpoint inhibitor are administered simultaneously or separately. 3. The method further comprising administering to the subject a third compound, wherein the third compound is: chemotherapy drugs; targeted anti-cancer drugs; and Targeted anti-cancer drugs, including antibodies 3. The checkpoint inhibitor and tdsRNA for use according to embodiment 1 or 2, wherein the checkpoint inhibitor and tdsRNA are one or more selected from the group consisting of: 4. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 3, further comprising administering to the subject one or more selected from the group consisting of interferon; interferon cocktail; Alferon; and alpha-interferon species. 5. A composition for treating cancer comprising a checkpoint inhibitor and a therapeutic double-stranded RNA (tdsRNA). 6. Checkpoint inhibitors Antibodies; monoclonal antibodies; humanized antibodies; human antibodies; fusion proteins; pegylated antibodies; multimeric antibodies; antibody fragments containing epitope-binding regions; and combinations thereof. 6. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 5, or the composition according to any one of embodiments 1 to 5, selected from: 7. Checkpoint inhibitors 2B4; A2aR; B7 family ligand; B7 H3; B7 H4; B and T lymphocyte attenuator (BTLA); BMA; CD112; CD137; CD160; CD2; CD20; CD226; CD27; CD276; CD28; CD30; CD33; CD40; CD47; CD52; CD70; CD80; CD86; CGEN 15049; CHK1; CHK2; cytotoxic T-lymphocyte antigen 4 (CTLA-4); DR3; galectin 9 (GAL9); GITR; herpesvirus entry mediator (HVEM); ICOS; IDO1; IDO2; killer cell immunoglobulin-like receptor (KIR); LAG3; LAIR; LAIR1; LAIR2; LIGHT; lymphocyte activation gene 3 (LAG-3); MARCO; OX-40; PD-1; PD-L1; PD-L2; PS; SIRP alpha; SLAM; T cell immunoreceptor with Ig and ITIM domains (TIGIT); T cell membrane protein 3 (TIM3); V domain immunoglobulin (Ig)-containing suppressor of T cell activation (VISTA); VTCN1; and combinations thereof 7. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 6, or the composition according to any one of embodiments 1 to 6, which inhibits, interacts with, or binds to a checkpoint protein, a ligand of a checkpoint protein, or a receptor for a checkpoint protein selected from the group consisting of: 8. Checkpoint inhibitors PD-1; PD-L1; cytotoxic T-lymphocyte antigen 4 (CTLA-4); CD80; CD86; and combinations thereof 8. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 7, or the composition according to any one of embodiments 1 to 7, which inhibits, interacts with, or binds to a checkpoint protein, a ligand of a checkpoint protein, or a receptor for a checkpoint protein selected from the group consisting of: 9. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 8, or the composition according to any one of embodiments 1 to 8, wherein the checkpoint inhibitor inhibits PD-1 or PD-L1. 10. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 9, or the composition according to any one of embodiments 1 to 9, wherein the cancer is pancreatic cancer; skin cancer; colorectal cancer; ovarian cancer; melanoma; breast cancer; triple-negative breast cancer; head and neck tumor; bladder cancer; renal cell carcinoma; and lung cancer. 11. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 10, or the composition according to any one of embodiments 1 to 10, wherein the cancer is pancreatic cancer, colorectal cancer, melanoma, bladder cancer, or renal cell carcinoma. 12.tdsRNA is rI n ribo(C 11-14 U) n ;rI n ribo(C4U) n ;rI n ribo(C5U) n ;rI n ribo(C6U) n ;rI n ribo(C7U) n ;rI n ribo(C8U) n ;rI n ribo(C9U) n ;rI n ribo(C 10 U) n ;rI n ribo(C 11 U) n ;rI n ribo(C 13 U) n ;rI n ribo(C 14 U) n ;rI n ribo(C 15 U) n ;rI n ribo(C 16HE) n ;country n ·ribo(C 17 HE) n ;country n ·ribo(C 18 HE) n ;country n ·ribo(C 19 HE) n ;country n ·ribo(C 20 HE) n ;country n ·ribo(C 21 HE) n ;country n ·ribo(C 22 HE) n ;country n ·ribo(C 23 HE) n ;country n ·ribo(C 24 HE) n ;country n ·ribo(C 25 HE) n ;country n ·ribo(C 26 HE) n ;country n ·ribo(C 27 HE) n ;country n ·ribo(C 28 HE) n ;country n ·ribo(C 29 HE) n ;country n ·ribo(C 30 HE) n ;country n ·ribo(C 31 HE) n ;country n ·ribo(C 32 HE) n ;country n ·ribo(C 33 HE) n ;country n ·ribo(C 34 HE) n ;country n ·ribo(C 35 HE) n ;country nribo(C 4-30 U) n ;rI n ribo(C 14-30 U) n ;rI n ribo(C 11-14 G) n ;rI n ribo(C 4-29 G) n ;rI n ribo(C 30-35 U) n ;r(Poly I·Poly C) n ;r(Poly A·Poly U) n ; and Rugged dsRNA 12. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 11, or the composition according to any one of embodiments 1 to 11, selected from: 13. tdsRNA is rI n ribo(C 4-29 U) n or rI n ribo(C 30-35 U) n , preferably rI n ribo(C 4-29 U) n 13. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 12, or the composition according to any one of embodiments 1 to 12, wherein 14. tdsRNA is rI n ribo(C 11-14 U) n 14. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 13, or the composition according to any one of embodiments 1 to 13, wherein 15. tdsRNA is r(I n )·ribo(C 12 U) n or r(I n )·ribo(C 30 U) n 13. The checkpoint inhibitor and tdsRNA for use according to embodiment 12, wherein the checkpoint inhibitor and tdsRNA are 16. tdsRNA is r(I n )·ribo(C 12 U) n 16. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 15, or the composition according to any one of embodiments 1 to 15, wherein 17. tdsRNA is Rugged dsRNA, and Rugged dsRNA is a hybridized poly(riboinosinic acid) strand and poly(ribocytosinic acid) strand (rI n ·rC n 17. The checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 16, or the composition according to any one of embodiments 1 to 16, wherein the checkpoint inhibitor and tdsRNA are resistant to denaturation under conditions that allow separation of the tdsRNA from the checkpoint inhibitor and tdsRNA. 18. A checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 17, or a composition according to any one of embodiments 1 to 17, wherein n is an integer selected from 40 to 50,000; 50 to 10,000; 60 to 9000; 70 to 8000; 80 to 7000; or 380 to 450. 19. A checkpoint inhibitor and tdsRNA for use according to any one of embodiments 1 to 18, or a composition according to any one of embodiments 1 to 18, wherein the tdsRNA and the checkpoint inhibitor together provide a synergistic effect in treating cancer or inhibiting tumor cell proliferation that is greater than the use of the tdsRNA alone, the checkpoint inhibitor alone, or the combined use of the tdsRNA alone and the checkpoint inhibitor alone. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 depicts the synergy of tdsRNA with checkpoint blockers in an animal model for pancreatic cancer, showing a synergistic extension of time to progression and overall survival. [Figure 2] FIG. 2 depicts survival of patients with pancreatic cancer who have low or high SIII. [Figure 3]FIG. 3 depicts data showing a decline in SIII over 18 weeks for nine patients whose metastatic pancreatic cancer stabilized after treatment with AMPLIGEN®. [Figure 4] Figure 4 depicts a significant improvement (p=0.0015) in the ratio of CXCL10 chemokine (the "benign" C-X-C motif chemokine 10):CCL22 chemokine (the "malignant" C-C motif chemokine ligand 22) in tumor samples from colorectal cancer patients treated with tdsRNA compared to similarly collected historical data. [Figure 5] Figure 5 depicts improvement in the ratio of CXCL10 chemokine ("benign" C-X-C motif chemokine 10) / CCL22 chemokine ("malignant" C-C motif chemokine ligand 22) and the ratio of T cell markers (Teff to Treg ratio) within excised tumors after tdsRNA treatment (patients versus historical controls). [Figure 6] FIG. 6 depicts a greater than 250% extension of survival using the combination of tdsRNA plus anti-PD-1 compared to anti-PD-1 alone. [Figure 7] Figure 7 shows the inhibition of renal cell carcinoma (786-0) xenograft growth in nude mice treated with AMPLIGEN. The inhibition of renal cell carcinoma (786-0) xenograft growth by tdsRNA (lower curve) is depicted compared to the untreated control (upper curve). [Figure 8] Figure 8 shows survival from renal cell carcinoma (7860) in nude mice treated with AMPLIGEN. 100% survival (top line) of nude mice bearing renal cell carcinoma (786-0) xenografts treated with tdsRNA is depicted compared to 100% mortality for untreated controls. [Figure 9] FIG. 9 depicts a CT scan of the chest showing a dramatic clinical response of triple-negative breast cancer. [Figure 10]FIG. 10 depicts a CT scan of the abdominal cavity showing a partial clinical response of ovarian cancer to a complete response (CR). DETAILED DESCRIPTION OF THE INVENTION

[0036] Immunotherapies based on checkpoint inhibitors (monoclonal antibodies that block immune-depleting inhibitors in T cells or tumor cells) are currently being rapidly approved by the FDA for a variety of specific indications.

[0037] Non-limiting examples of specific cancer types requiring improved immunotherapy As used herein, "tumor" and "cancer" are used interchangeably. Tumors can be benign or malignant.

[0038] Pancreatic cancer Pancreatic cancer is the fourth most common cause of cancer-related death in the United States and the eighth most common cause worldwide. Pancreatic cancer has one of the highest mortality rates of all cancers and is the fourth leading cause of cancer deaths in both men and women. The 1-year and 5-year relative survival rates for all stages combined are shockingly low, at 25% and 6%, respectively. For localized disease, the 5-year survival rate is approximately 20%. The median survival for locally advanced disease and metastatic disease, which together represent more than 80% of individuals, is approximately 10 and 6 months, respectively.

[0039] Treatment for pancreatic cancer depends on the stage of the cancer. Only localized cancers are currently considered suitable for surgery with curative intent, but only about 20% of cases present with localized disease at the time of diagnosis. Palliative surgery may also be performed if the malignancy is invasive or compressing the duodenum or colon. In such cases, bypass surgery may overcome the obstruction and improve quality of life, but is not intended as a cure. For disease not considered suitable for resection, palliative chemotherapy may be used to improve quality of life and provide a modest survival benefit for the patient.

[0040] There is a need for the improvement of the method for treating pancreatic cancer, especially locally advanced pancreatic cancer and metastatic pancreatic cancer.Metastasis is the leading cause of death in cancer patients.However, there is no effective therapy that targets the development and progression of metastasis in pancreatic cancer.In one of the preferred embodiments of the present disclosure, cancer is pancreatic cancer.

[0041] melanoma Globally, melanoma is diagnosed at an incidence rate of 3.0 cases per 100,000 people, representing 1.7% of all cancer cases. In 2012, 232,000 women were diagnosed with melanoma. The mortality rate of 0.7 per 100,000 women is substantially lower than the incidence rate (Ferlay et al., 2013). The lifetime risk of developing melanoma is approximately 2.4% (1 in 40 cases) for Caucasians, 0.1% (1 in 1,000 cases) for African Americans, and 0.5% (1 in 200 cases) for Hispanics. The average age at melanoma diagnosis is 62 years, but it is one of the most common cancers in young adults, particularly young women (American Cancer Society, 2015).

[0042] For patients with localized melanoma, the prognosis is good with adequate surgical resection, reflected in a relatively low mortality rate (World Cancer Report, 2014). Five-year survival rates are greater than 90% and 80% for stage I and II disease, respectively (Kaufman et al., 2013).

[0043] However, most metastatic melanoma is resistant to current therapies (World Cancer Report, 2014). Five-year survival rates are 78-40% for stages IIIA-C and 15-20% for stage IV (American Cancer Society, 2015).

[0044] In addition to exposure to sunlight, the risk of developing melanoma is influenced by other environmental factors, such as age and sex, as well as anatomical location and individual susceptibility. UV-emitting tanning devices also increase the risk of malignant melanoma. CDKN2A mutations have been found in 20-40% of people with a family history of melanoma (World Cancer Report, 2014).

[0045] Melanoma primarily arises in the skin (more than 95% of cases), but is also found in the mucosa of the oral cavity, nasal cavity, anus, and vagina, and, to a lesser extent, the mucosa of the small intestine. Furthermore, melanocytes are present in the conjunctiva, retina, and meninges. Melanoma can be histologically subtyped into superficial spreading melanoma, nodular melanoma, acral lentiginous melanoma, and lentigo maligna melanoma. Melanoma is classified according to the TNM classification. As recommended by the American Joint Committee on Cancer Staging Manual, melanoma patients are classified into three groups: localized disease without evidence of metastasis (stage I-II), regional disease (stage III), and distant metastatic disease (stage IV) (World Cancer Report, 2014).

[0046] The standard treatment for melanoma is complete surgical excision with surrounding healthy tissue. If excision is incomplete or not possible at all, patients receive primary radiation therapy, which may be combined with interferon-alpha administration in advanced stages (stages IIB / C and IIIA-C). Treatment options include single-agent chemotherapy, polychemotherapy, and targeted therapy with specific inhibitors. Dacarbazine, temozolamide, and fotemustin are currently being used in single-agent chemotherapy trials. Different combinations of chemotherapy agents are also being explored in multi-drug chemotherapy studies: CarboTax regimen (carboplatin + paclitaxel), GemTreo regimen (gemcitabine + treosulfan), DVP regimen (dacarbazine + vindesine + cisplatin), BHD regimen (carmustine + hydroxyurea + dacarbazine), and BOLD regimen (bleomycin + vincristine + lomustine + dacarbazine). Furthermore, chemotherapy in combination with ipilimumab and the administration of specific inhibitors of BRAF, c-kit, and N-RAS to patients with mutations in the respective genes is also being evaluated in clinical trials (S3-Leitlinie Melanom, 2013). In one preferred embodiment of the present disclosure, the cancer is melanoma.

[0047] Colorectal cancer (CRC) Colorectal cancer (CRC) is one of the most common cancers worldwide. Early detection and surgery with tumor resection are currently crucial for successful treatment. For localized tumors, i.e., tumors that have not progressed to metastatic disease, surgical intervention is performed, involving radical resection of the tumor and surrounding bowel and tissue. Colorectal tumors are graded into several stages according to Duke stages A-D or higher, but more recently according to the TNM classification. Early-stage tumors (Duke stages A and B) are generally associated with a relatively favorable outcome, while later-stage tumors that present with metastasis (Duke stages C and D) have poor survival rates. Unfortunately, metastasis often progresses undetected until the tumor has grown to a significant size. Tumors typically metastasize to regional lymph nodes, but distant metastases to the liver and lungs are also common.

[0048] Patients with early-stage CRC (stages I and II or Duke stages A and B) undergo surgical resection alone and are not treated with chemotherapy. However, nearly one-quarter of early-stage patients with nonmetastatic disease subsequently relapse with metastasis, and the 5-year survival rates for patients diagnosed with metastatic forms of CRC, i.e., Duke stage C with lymph node metastasis and Duke stage D with blood-borne dissemination, are 37% and 11%, respectively. Patients diagnosed with early-stage CRC (Duke stages A and B) without evidence of metastatic disease at surgery have a significantly better prognosis, with 5-year survival rates of 85% and 67%, respectively (Cancer Research UK, 2004). However, a significant proportion (10%-45%) of these patients relapse with metastatic disease.

[0049] Chemotherapy has been demonstrated to be effective against Duke stage C tumors. New research also indicates the value of chemotherapy for some patients with early stage colorectal cancer who are at risk of metastatic recurrence. However, although chemotherapy intervention is implemented for some patients with early stage colon cancer, its implementation as a routine treatment can be cost-effective and counterproductive. Side effects associated with treatment make it desirable to avoid the use of chemotherapy except in cases with a high risk of recurrence. In one of the preferred embodiments of the present disclosure, the cancer is colorectal cancer.

[0050] Ovarian / endometrial cancer Ovarian cancer is among the deadliest gynecological malignancies in developed countries. In the United States, approximately 23,000 women are diagnosed with ovarian cancer, and nearly 14,000 women die from the disease each year. There are three major types of ovarian cancer: epithelial cancer, germ cell cancer, and sex cord-stromal cancer. Approximately 90% of ovarian cancers begin within the epithelial tissue (the outer lining of the ovaries). These types of ovarian cancer are divided into serous, mucinous, endometrioid, clear cell, transitional, and undifferentiated types. The risk of ovarian epithelial cancer increases with age, particularly after age 50. Germ cell follicle tumors account for approximately 5% of ovarian cancers. Germ cell follicle tumors begin in egg-producing cells. This type of ovarian cancer can also occur in women of any age, but approximately 80% are found in women under the age of 30. The major subtypes are teratomas, dysgerminomas, endodermal sinus tumors, and choriocarcinomas. Sex cord-stromal tumors, which account for approximately 5% of ovarian cancers, grow within the connective tissue of the ovary. The majority are found in older women. Despite advances in cancer treatment, the mortality rate from ovarian cancer has remained virtually unchanged over the past 20 years. Given the steep survival gradient relative to the stage at which the disease is diagnosed, early detection remains the most important factor in improving the long-term survival of ovarian cancer patients.

[0051] Endometrial cancer is the most common gynecological malignancy, accounting for approximately 13% of all malignancies occurring in women. Approximately 34,000 cases of endometrial cancer are diagnosed annually in the United States. All endometrial cancers arise from the glands of the uterine lining. Adenocarcinomas account for 75% of all endometrial cancers. Endometrial adenocarcinomas, which contain benign or malignant squamous epithelium, are known as adenocanthomas and adenosquamous carcinomas, respectively, and account for 30% of endometrial cancers. The remaining types of endometrial cancer have a more poor prognosis. Approximately 3% have the clear cell carcinoma form, and approximately 1% have the papillary carcinoma form.

[0052] Ovarian cancer refers to at least one or more cancers selected from the group consisting of serous ovarian cancer, mucinous ovarian cancer, endometrioid ovarian cancer, clear cell ovarian cancer, transitional ovarian cancer and / or undifferentiated ovarian cancer, teratoma, dysgerminoma, endodermal sinus tumor, and choriocarcinoma, and endometrial cancer includes endometrial carcinoma, adenocarcinoma, endometrial adenocarcinoma, adenosquamous carcinoma, clear cell carcinoma, and papillary carcinoma. In one of the preferred embodiments of the present disclosure, the cancer is ovarian cancer.

[0053] Breast cancer Breast cancer is a heterogeneous malignant disease with diverse biological characteristics and clinical responses. Gene expression profiling has defined gene signatures corresponding to at least five distinct molecular subtypes of breast cancer, including an aggressive form known as triple-negative (TN) breast cancer.

[0054] Three endogenous molecules have been identified that drive many breast cancers: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). By definition, triple-negative (TN) breast cancers do not express these three molecules. TN breast cancers represent a relatively small proportion (approximately 10%) of all breast cancers, but they are typically high-grade (poorly differentiated), rapidly progressive, have a high risk of recurrence, and have lower survival rates than other subtypes of breast cancer. Therefore, TN breast cancers are associated with a disproportionate number of deaths. Additionally, for unknown reasons, TN breast cancers are more likely to be diagnosed at younger ages and in women of African-American descent. Women who carry mutant germline BRCA1 or BRCA2 genes are at increased risk for developing both breast and ovarian cancer.

[0055] Current clinical treatments for breast cancer typically involve drugs targeting three molecules identified to promote many breast cancers, such as endocrine therapy and trastuzumab, a monoclonal antibody targeting HER2.TN breast cancer is defined as the absence of these targets, so conventional cytotoxic chemotherapy is currently the mainstay systemic treatment for patients with TN breast cancer.However, conventional systemic treatment is limited by poor therapeutic response, high toxicity, and the development of resistance.Although new approaches have emerged in the treatment of TN breast cancer, such as targeting DNA repair with PARP inhibitors, compared with other subtypes of breast cancer, there have been relatively few therapeutic advances in TN breast cancer.Therefore, there is a strong need for targeted approaches to the treatment of TN breast cancer.In one of the preferred embodiments of the present disclosure, the cancer is breast cancer.

[0056] Bladder cancer Bladder cancer, also known as urothelial carcinoma (transitional cell carcinoma), is a type of cancer found in the lining of the urinary tract, including the renal pelvis, ureter, bladder, and part of the urethra. The most common form of bladder cancer is urothelial carcinoma. Bladder cancer occurs in people of all races and can affect people of any age. Bladder cancer is the fourth most common type of cancer in men and the ninth most common cancer in women. Bladder cancer causes approximately 170,000 deaths in the United States each year.

[0057] Researchers do not know the exact cause(s) of bladder cancer, but smoking is thought to be a major known contributing factor. Occupational exposure to carcinogens such as benzidine (i.e., aromatic amines) in the workplace can also result in bladder tumors. Occupations at risk for benzidine exposure include bus drivers, rubber processors, auto mechanics, leather workers, blacksmiths, machine mechanics, mechanics, and hairdressers (due to frequent exposure to hair dyes during permanent hair application). Another modifiable factor less strongly associated with bladder cancer is obesity.

[0058] Bladder cancer or urothelial carcinoma is often described based on how far it has invaded the bladder wall. Papillary or non-invasive bladder cancer grows by thin, finger-like projections from the inner surface of the bladder into the hollow part. Papillary tumors often grow into the center of the bladder without growing into the deeper layers of the bladder. Low-grade (slow-growing) non-invasive papillary carcinoma in situ tends to have a good outcome. Squamous carcinoma is another example of non-invasive bladder cancer. Squamous carcinoma does not grow into the hollow part of the bladder. When a papillary or flat tumor grows into the deep layers of the bladder, it is called invasive urothelial carcinoma. Invasive bladder cancer is more likely to spread and is much more difficult to treat.

[0059] Other cancers of the bladder are squamous cell carcinoma, adenocarcinoma, small cell carcinoma, and sarcoma.

[0060] Current bladder cancer treatment involves invasive surgery, radical cystectomy, intravesical therapy, chemotherapy, radiotherapy and / or immunotherapy.However, these treatments have many drawbacks, such as flu-like symptoms, extreme fatigue, hair loss, DNA damage, the development of secondary cancer, cell migration into bloodstream and complications from surgery.In one of the preferred embodiments of the present disclosure, cancer is bladder cancer.

[0061] Kidney cancer Kidney cancer (also called renal cancer or renal cell carcinoma) most often affects adults between the ages of 50 and 70. If detected early, kidney cancer is curable. However, symptoms do not appear until the tumor grows to a large size or metastasizes to other organs, at which point treatment becomes palliative.

[0062] In this disclosure, renal cancer and kidney cancer refer to renal cell carcinoma.

[0063] The 5-year survival rate for individuals diagnosed with kidney cancer is approximately 90% for those whose tumors are localized to the kidney, approximately 60% for those whose tumors have spread limitedly to nearby tissues, and approximately 9% for those whose tumors have spread to distant sites [American Cancer Society, Detailed Guide: Kidney Cancer. "What Are the Key Statistics for Kidney Cancer (Renal Cell Carcinoma)?"].

[0064] The majority of kidney cancers are renal cell carcinomas, also known as renal adenocarcinomas or clear cell carcinomas (accounting for over 90% of malignant kidney tumors). There are five major types of renal cell carcinomas, identified based on microscopic examination for cell type: clear cell carcinoma, papillary carcinoma, chromophobe carcinoma, collecting duct carcinoma, and "unclassifiable" carcinoma. Renal cancers are also typically graded on a scale of 1 to 4, which indicates how similar the cancer cell nuclei are to those of normal kidney cells. (Grade 1 renal cell carcinomas have nuclei that are virtually identical to normal kidney cell nuclei and generally have a favorable prognosis, while grade 4 renal cell carcinomas have nuclei that appear undifferentiated, as distinguished from differentiated normal kidney cell nuclei, and have a poor prognosis.) In addition to grade, kidney cancers are also characterized by stage, which describes the size of the cancer and the extent of metastasis. The most commonly used staging system is the American Joint Committee on Cancer (AJCC) staging system (also called the TNM system), although the Robson system is also an older system that is sometimes used.

[0065] Risk factors for kidney cancer include: age over 50 years; male gender (men are twice as likely to develop kidney cancer as women); smoking; exposure to asbestos, cadmium, or organic solvents; obesity; a high-fat diet; and von Hippel-Lindau disease (a genetic condition that increases the incidence of kidney cancer).

[0066] Symptoms of kidney cancer include hematuria (blood in the urine), pain in the abdomen or lower back, weight loss, fatigue, anemia, fever, high blood pressure, and swelling in the legs or ankles.

[0067] In addition to a detailed medical history, physical examination, and laboratory blood tests, diagnosing kidney cancer may typically include a computed tomography (CT) scan, ultrasound, magnetic resonance imaging (MRI), intravenous pyelography (a kidney test that uses dye and x-rays), or arteriography (a test in which dye is applied to the blood vessels that supply the kidneys). Chest x-rays and bone scans are commonly performed to detect metastatic disease.

[0068] In individuals whose tumors are confined to the kidney, the treatment of renal cancer involves surgical removal of the kidney and surrounding tissue (nephrectomy).Radiation therapy can be applied to treat pain and advanced or metastatic renal cancer, or to cause the regression of tumors that are causing obstruction.Immunotherapy, such as interferon and interleukin 2, can be used to boost the immune system in patients with advanced renal cancer (Journal of the American Medical Association, JAMA Patient Page: Kidney Cancer).In one of the preferred embodiments of the present disclosure, the cancer is renal cancer.

[0069] lung cancer Lung cancer is the leading cause of cancer death in the United States. Lung cancer is classified as non-small cell lung cancer (NSCLC) or small cell lung cancer, with NSCLC representing more than 80% of cases. For NSCLC, the most common type of lung cancer, the 5-year survival rate is 70-80% for stage I disease without lymph node or distant metastasis, but only 5-15% for stage IV advanced (distant) disease.

[0070] Current treatments for lung cancer include surgery, radiation, classical chemotherapy agents (platinum compounds, taxanes), and targeted therapy (VEGFR, EGFR, IGFR, HDACS, and proteasome inhibitors). However, despite advances in treatment, the 5-year survival rate is about 16%. Numerous clinical trials evaluating classical chemotherapy agents for lung cancer indicate that current drugs have reached a therapeutic plateau. Therefore, new drugs for the treatment of lung cancer with different mechanisms of action are needed. In one of the preferred embodiments of the present disclosure, the cancer is lung cancer.

[0071] Checkpoint inhibitors One area of ​​research into expanding the effects of immunotherapeutic drugs is the class of checkpoint inhibitors. As used herein, the term "immune checkpoint inhibitor" refers to a substance that blocks the activity of molecules involved in attenuating immune responses. Examples of immune checkpoint inhibitors are described in the present disclosure. In one embodiment, checkpoint inhibitors are antibody-based drugs that mobilize T cell-mediated immune responses. Checkpoint inhibitors block cancer cells from using molecular switches known as checkpoints that normally prevent T cells from attacking healthy tissues. When these checkpoints, such as PD-1 (programmed death 1) and CTLA4 (cytotoxic T lymphocyte-associated protein 4), are hijacked by cancer cells, the immune system's T cell response is turned off, allowing cells to multiply and tumors to grow. Checkpoint inhibitors [e.g., anti-PD-1, anti-CTL4, anti-PDL-1 (programmed death ligand 1, expressed on the surface of tumor cells), and anti-PDL-2] flip the switch back on, unleashing the immune response so that T cells become activated and destroy cancer cells.

[0072] Checkpoint inhibitors work best against so-called hot tumors, which are cancers infiltrated by T cells and macrophages, creating inflamed tumors. This response by the immune system does not kill the tumor, but because T cells are present within the tumor, they are more easily recruited to the cancer. Checkpoint inhibitors release the stranglehold that the tumor places on T cells. Once freed from inhibition, the T cells are free to kill cancer cells.

[0073] Tumors can be classified as "hot" or "cold" depending on the functional ability of cells within the tumor microenvironment to mount a cytotoxic immune response against the tumor. Hot tumors are often densely populated with cytotoxic T cells and have a high mutational burden. That is, hot tumors have many changes in their DNA code that cause cancer cells to produce new, distinctive proteins, called "neoantigens," that are expressed on their cell surface. These neoantigens make the tumor more susceptible to recognition by the immune system and therefore more likely to provoke a strong immune response.

[0074] In contrast, "cold" tumors are cancers that, for a variety of reasons, have not been recognized by the immune system or have not elicited a strong tumor cytotoxic response by the immune system. Immune T cells are unable to invade the tumor microenvironment. The microenvironment within and around tumor cells contains blood vessels, structural elements, and specialized immune cells, the latter including myeloid-derived suppressor cells and regulatory T cells (abbreviated as Tregs). These Tregs are capable of cytotoxic T cells (T eff They secrete immunosuppressive chemical messengers, such as cytokines, that prevent effector T cells (abbreviated as T cells) from migrating to tumors, thereby reducing the strength of normal immune responses by creating an "immune desert" that contains cold tumors.

[0075] This inability to suppress cold tumors is one of the limitations of current immunotherapies. There is a long-standing need to effectively apply immunotherapy to immunologically cold cancers—in other words, how to make immunologically cold cancers immune-responsive.

[0076] However, current checkpoint inhibitor therapies are effective in treating cancer within a relatively small subset of cancer subjects, due in part to the presence of pre-existing immune activation and inhibitory receptors. Thus, there is a need to develop methods and combination therapies that induce or enhance the effectiveness of checkpoint inhibitors in both non-responsive and responsive subject populations.

[0077] Therapeutic double-stranded RNA (tdsRNA; formerly known as antitumor immune enhancer or ATIE) This disclosure is directed, in part, to tdsRNA, formerly known as antitumor immune enhancers (ATIEs). Specific embodiments of tdsRNA include AMPLIGEN® (also known as lintatolimod), rugged dsRNA, mismatched dsRNA, or dsRNA. The term therapeutic double-stranded RNA or tdsRNA is a new term that replaces the former term antitumor immune enhancer or ATIE. In this disclosure, ATIE and tdsRNA have exactly the same meaning and can be used interchangeably. tdsRNA (formerly known as ATIEs) is described in more detail below.

[0078] For purposes of this disclosure, tdsRNA or ATIE may refer to any dsRNA discussed in this disclosure, particularly any dsRNA disclosed in this section.

[0079] One preferred embodiment for tdsRNA is AMPLIGEN®, which is a poly(I:poly(C)) 12Poly(I):poly(C) is a synthetic double-stranded ribonucleic acid in which uridine acid (U) substitutions in the cytidyl chain create non-hydrogen-bonded regions in the molecular configuration. Its chemical name is polyriboinosinic acid:polyribocytidyl (12:1) uridine acid, or poly(I):poly(C). 12 The USAN name (United States Adopted Name) of AMPLIGEN® is lintatolimod. Therefore, in this disclosure, AMPLIGEN® and lintatolimod have the same meaning.

[0080] Poly I:Poly C 12 U is a structural analogue of the polyribonucleotide complex, polyI:polyC, which is a hydrogen-bonded polyriboinosinic acid with polyribocytidylic acid. Within the polyC chain, uridine acid substitutions occur on average every 12–13 bases, and polyI:polyC contains unique structural regions interspersed with continuous regions. 12 Poly I and poly C are the raw materials for single-stranded RNA (ssRNA), resulting in a U duplex. 12 U is a double-stranded RNA (dsRNA) molecule, lintatorimod (poly I:poly C). 12 The polymers are annealed under controlled conditions to form a β-block-α (β-block-α) molecule.

[0081] In one embodiment, the tdsRNA is RNA strands containing riboinosinic acid and RNA strands containing ribocytidylic acid and ribouracilic acid, or RNA strands containing riboinosinic acid and RNA strands containing ribocytosinic acid and guanine, mismatched dsRNA, such as RNA strands containing adenine and ribouracillic acid and the like.

[0082] Another embodiment(s) of the tdsRNA is a specific type of mismatched dsRNA. In one aspect, the mismatched dsRNA is preferably rI n ·r(C 11 U) n ;rI n·r(C 13 U) n ;rI n ·r(C 14 U) n and most preferably rI n ·r(C 12 U) n of the general formula rI n ·r(C 4-35 U) n or rI n ·r(C 11-14 U) n The mismatched dsRNA may be of the formula rI n ·r(C 11-14 U) n This means that one chain is rI n and the other chain is represented by (C 11-14 U) n where the dot symbol "" represents the two strands hybridizing to form the double-stranded RNA structure. Note that although reference is made to two hybridized strands, not 100% of the bases are base-paired as mismatches exist.

[0083] rI n represents polyriboinosine of n bases. The "r" represents riboinosine, the RNA-like form of inosine. This is in contrast to 2'-deoxyinosine. The n represents the total length of this single-stranded inosine molecule (single-stranded RNA).

[0084] For example, r(C 11-14 U) n "n" refers to a single-stranded RNA that contains C and U bases, with a ratio of C to U bases of 11 to 14 Cs to 1 U. "n" refers to the total length of the single-stranded RNA in base units.

[0085] Therefore, rI n ·r(C 11-14 U) n What is rI n But r(C 11-14 U) nn represents the length for both strands, so that both ssRNA strands are the same length, resulting in a dsRNA with no significant single-stranded regions in the middle or at the ends of the double-stranded structure.

[0086] In this disclosure, unless otherwise indicated, all polynucleotides administered to a patient are dsRNA or chemical analogs thereof, such as riboinosine (i.e., RNA, not DNA, unless otherwise indicated). "n" is the length of the dsRNA (in bases), and n is an integer having a value of 40 to 50,000; 10 to 40,000; 10 to 500; 10 to 50; or 40 to 500 (rugged dsRNA). In this and other formulas that follow, r = riboinosine and r = riboinosine.

[0087] Rugged dsRNA is a hybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strand (rI). n ·rC n tdsRNA that is resistant to denaturation under conditions that allow separation of the Rugged dsRNA. For further description of Rugged dsRNA and exemplary methods for preparing such molecules, see U.S. Patent Nos. 8,722,874 and 9,315,538. In a preferred embodiment of Rugged dsRNA, the Rugged dsRNA is rI n ribo(C 4-29 U) n ; rI n ribo(C 11-14 U) n ; rI n ribo(C 12 U) n ; and rI n ribo(C 30-35 U) n Preferably, the Rugged dsRNA has a formula selected from the group consisting of rI n ribo(C 30-35 U) n It has the following structure.

[0088] In a preferred embodiment, the Rugged dsRNA has the following characteristics: double-stranded RNA between 30 and 38 helical turns; Molecular weight of 250 kilodaltons to 320 kilodaltons; Each strand of the rugged dsRNA is approximately 380–450 bases in length (or approximately 380–450 double-stranded base pairs in length). The present invention has one or more of the following:

[0089] Under analytical or preparative high performance liquid chromatography, the Rugged dsRNA exhibits an HPLC peak of approximately 4.5 to 6.5 minutes, preferably between 4.5 to 6 minutes, and most preferably 5 minutes, and is a substantially purified, pharmaceutically active molecule of poly(I):poly(C). 12 U) n [For example, poly(I):poly(C 11-14 U) n In some embodiments, the molecular weight is about 30 kilodaltons to 300 kilodaltons, and the length is about 50 to 500 base pairs, with about 4.7 to 46.7 complete turns of the RNA helix. Rugged dsRNA refers to a molecular species that is inherently resistant to denaturation and unfolding. Rugged dsRNA is a molecular species that is inherently resistant to denaturation and unfolding. Rugged dsRNA is a molecular species that is inherently resistant to denaturation and unfolding, with the same length of r(polyI·polyC) n It may be characterized as a dsRNA that is more resistant to denaturation, and has an HPLC peak at approximately 5 minutes, poly(I):poly(C x U) n It may also be characterized as.

[0090] Other mismatched dsRNAs for use in the present invention include poly(C m ,U) or poly(Cm , G) [where m is an integer having a value of 4 to 29], and an unpaired base [uracil (U) or guanine (G)] is substituted with polyribocytidylic acid (rC m ) chain, n ·rC n Alternatively, dsRNA may be a mismatched analog of the complex formed by modifying polyriboinosinic acid (rI) with polyribocytidylic acid. n r(I)·r(C) dsRNA can also be derived from r(I)·r(C) dsRNA by modifying the ribosyl backbone, for example, by incorporating 2'-O-methylribosyl residues. Mismatched dsRNAs can be complexed with RNA-stabilizing polymers, such as lysine carboxymethylcellulose or poly ICLC, as described in the next paragraph. These r(I)·r(C) dsRNAs can be complexed with RNA-stabilizing polymers, such as lysine carboxymethylcellulose or poly ICLC, as described in the next paragraph. n ·rC n Of the mismatch analogs of the formula rI, preferred analogs are of the formula rI n ·r(C 11-14 ,U) n and are described by Ts'o & Carter in U.S. Patent Nos. 4,024,222 and 4,130,641, the disclosures of which are incorporated herein by reference. The dsRNAs described therein are generally suitable for use in accordance with the present invention.

[0091] Another embodiment is poly(riboinosinic acid), ribo(I nThis specification relates to specifically structured dsRNAs derived from ribo(I)·ribo(C) dsRNA by modifying the ribosyl backbone of ribo(I)·ribo(C), for example, by incorporating 2'-O-methylribosyl residues. Specific structured dsRNAs may also be modified at the ends of the molecule to add hinge(s) to prevent base pair mismatch and thereby confer specific biological activity in solvents or in the aqueous environments present in human body fluids. Specific structured dsRNAs described in U.S. Pat. Nos. 4,024,222; 4,130,641; and 5,258,369 (incorporated by reference) are generally suitable as starting materials after selection for rugged dsRNAs. Although the present disclosure describes Rugged dsRNA, other dsRNAs (including tdsRNAs) that are not Rugged dsRNAs described in the present disclosure are also suitable as starting materials for producing Rugged dsRNAs. In any embodiment, tdsRNAs, including Rugged dsRNAs, can be complexed with stabilizing polymers such as polylysine, polylysine + carboxymethylcellulose, polyarginine, polyarginine + carboxymethylcellulose, or any combination thereof.

[0092] Other examples of mismatched dsRNAs for use as tdsRNA include: The ratio of C to U in one chain is 4:1, rI n ribo(C4U) n ; The ratio of C to U in one chain is 5:1, rI n ribo(C5U) n ; The ratio of C to U in one chain is 6:1, rI n ribo(C6U) n ; The ratio of C to U in one chain is 7:1, rI n ribo(C7U) n ; The ratio of C to U in one chain is 8:1, rI n ribo(C8U) n ; The ratio of C to U in one chain is 9:1, rI n ribo(C9U) n ; The ratio of C to U in one chain is 10:1, rI n ribo(C 10 U) n ; The ratio of C to U in one chain is 11:1, rI n ribo(C 11 U) n ; The ratio of C to U in one chain is 12:1, rI n ribo(C 12 U) n ; The ratio of C to U in one chain is 13:1, rI n ribo(C 13 U) n ; The ratio of C to U in one chain is 14:1, rI n ribo(C 14 U) n ; The ratio of C to U in one chain is 15:1, rI n ribo(C 15 U) n ; The ratio of C to U in one chain is 16:1, rI n ribo(C 16 U) n ; The ratio of C to U in one chain is 17:1, rI n ribo(C 17 U) n ; The ratio of C to U in one chain is 18:1, rI n ribo(C 18 U) n ; The ratio of C to U in one chain is 19:1, rI n ribo(C 19 U) n ; The ratio of C to U in one chain is 20:1, rIn ribo(C 20 U) n ; The ratio of C to U in one chain is 21:1, rI n ribo(C 21 U) n ; The ratio of C to U in one chain is 22:1, rI n ribo(C 22 U) n ; The ratio of C to U in one chain is 23:1, rI n ribo(C 23 U) n ; The ratio of C to U in one chain is 24:1, rI n ribo(C 24 U) n ; The ratio of C to U in one chain is 25:1, rI n ribo(C 25 U) n ; The ratio of C to U in one chain is 26:1, rI n ribo(C 26 U) n ; The ratio of C to U in one chain is 27:1, rI n ribo(C 27 U) n ; The ratio of C to U in one chain is 28:1, rI n ribo(C 28 U) n ; The ratio of C to U in one chain is 29:1, rI n ribo(C 29 U) n ; The ratio of C to U in one chain is 4 to 29:1, rI n ribo(C 4-29 U) n ; The ratio of C to G in one strand is 4 to 29:1, rI n ribo(C 4-29G) n ; The ratio of C to U in one chain is 11-14:1, rI n ·r(C 11-14 U) n ; The ratio of C to U in one chain is 12:1, rI n ribo(C 12 U) n ; The ratio of C to U in one chain is 30:1, rI n ribo(C 30 U) n ; The ratio of C to U in one chain is 30-35:1, rI n ribo(C 30-35 U) n ; and r(Poly A·Poly U) n Includes.

[0093] Briefly, tdsRNA is the type of dsRNA described below. If one strand is n long, it is understood that the other strand is also n long, even if not stated otherwise. Also, when a range is claimed, each intermediate ratio value is also claimed.

[0094] For example, rI n ribo(C 4-29 U) n are individually, rI n ribo(C4U) n , rI n ribo(C5U) n , rI n ribo(C6U) n , rI n ribo(C7U) n , rI n ribo(C8U) n , rI n ribo(C9U) n , rI n ribo(C 10 U) n , rI n ribo(C11 HE) n ,country n ·ribo(C 12 HE) n ,country n ·ribo(C 13 HE) n ,country n ·ribo(C 14 HE) n ,country n ·ribo(C 15 HE) n ,country n ·ribo(C 16 HE) n ,country n ·ribo(C 17 HE) n ,country n ·ribo(C 18 HE) n ,country n ·ribo(C 19 HE) n ,country n ·ribo(C 20 HE) n ,country n ·ribo(C 21 HE) n ,country n ·ribo(C 22 HE) n ,country n ·ribo(C 23 HE) n ,country n ·ribo(C 24 HE) n ,country n ·ribo(C 25 HE) n ,country n ·ribo(C 26 HE) n ,country n ·ribo(C 27 HE) n ,country n ·ribo(C 28 HE) n 、およびrI n ·ribo(C 29 HE) n Someone who cannot remember the rI n ·ribo(C 30-35U) n are individually, rI n ribo(C 30 U) n , rI n ribo(C 31 U) n , rI n ribo(C 32 U) n , rI n ribo(C 33 U) n , rI n ribo(C 34 U) n , and rI n ribo(C 35 U) n It will encompass

[0095] That is, each of the above molecules is also individually claimed and considered as an embodiment as part of the present invention.

[0096] The specific constitutive tdsRNA has the general formula: ribo(I n )·ribo(C 4-29 U) n , ribo(I n )·ribo(C 11-14 U) n , or ribo(I n )·ribo(C 12 U) n [wherein the chain is composed of ribonucleotides (ribo), and n is an integer between about 40 and about 40,000]. For example, poly(ribocytosine acid 4-29 Ribouracilic acid), poly(ribocytosinic acid) 11-14 Ribouracilic acid), or poly(ribocytosinic acid) 12 A strand composed of poly(riboinosinic acid) can partially hybridize with an opposing strand composed of poly(riboinosinic acid) such that the two strands form an RNA double helix (dsRNA) that is unpaired (i.e., mismatched) at the uracil bases.

[0097] For a subject (eg, a 150 lb or 70 Kg human), the dose of dsRNA can range from 0.1 to 1,000,000 μg, preferably from 0.4 to 400,000 μg.

[0098] Alternatively, the tdsRNA can be matched (i.e., not in a mismatched form). Thus, polyadenylic acid (polyA·polyU) complexed with polyuridine acid (i.e., r(polyA·polyU) n ) can be used. The matched dsRNA can be administered in the same manner as any of the mismatched tdsRNA.

[0099] The tdsRNA can be administered by any known administration method (eg, see the detailed description under "Administration Methods" for a more detailed list).

[0100] Formulations for administration include aqueous solutions, syrups, elixirs, powders, granules, tablets, and capsules, which typically contain conventional excipients such as binders, fillers, lubricants, disintegrants, humectants, suspending agents, emulsifiers, preservatives, buffer salts, flavoring agents, coloring agents, and / or sweeteners. Formulations can also be applied intranasally by spray or nebulizer. It is appreciated that the preferred route will vary with the condition and age of the recipient, the nature of the infection or condition, and the active ingredient selected.

[0101] In another embodiment, the mismatched dsRNA can be a rugged dsRNA (see, e.g., U.S. Patent Nos. 8,722,874 and 9,315,538). In one embodiment, the rugged dsRNA can be an isolated double-stranded ribonucleic acid (dsRNA) that is resistant to denaturation under conditions that allow separation of hybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strands, where only one strand of the isolated dsRNA contains one or more uracil or guanine bases that are not base-paired with the opposite strand, and the one strand is a poly(riboinosinic acid) strand. 30-35In another embodiment, the rugged dsRNA can be an isolated double-stranded ribonucleic acid (dsRNA) that is resistant to denaturation under conditions that allow separation of the hybridized poly(riboinosinic acid) and poly(ribocytosinic acid) strands, and the isolated dsRNA can be a ribo(I) strand. n )·ribo(C 30-35 U) n [wherein ribo is a ribonucleotide, and n is an integer of 40 to 500 or 40 to about 40,000].

[0102] In another embodiment, the tdsRNA is an isolated double-stranded ribonucleic acid (dsRNA) that is enzymatically active under heat stress, wherein each strand has a molecular weight of about 250 kDa to about 320 kDa. 4-29 In another embodiment, the rugged dsRNA is an isolated double-stranded ribonucleic acid (dsRNA) that is enzymatically active under heat stress, wherein each strand is about 380 bases to about 450 bases long and each strand is composed of poly(riboinosinic acid), wherein the two strands do not base pair at the uracil base positions and the two strands base pair at the cytosine base positions, and the strands are partially hybridized. 4-29 In another embodiment, the rugged dsRNA is an isolated double-stranded ribonucleic acid (dsRNA) that is enzymatically active under heat stress, comprising one strand composed of poly(riboinosinic acid) and one strand composed of poly(uracyl) and one strand composed of poly(riboinosinic acid), wherein the two strands do not base pair at the uracil base positions and the two strands base pair at the cytosine base positions, and the strands are partially hybridized. In another embodiment, the rugged dsRNA is an isolated double-stranded ribonucleic acid (dsRNA) that is enzymatically active under heat stress, wherein each strand is composed of poly(riboinosinic acid), with the RNA duplex (dsRNA) spanning from about 4 to about 5,000 helical turns, preferably 30 to 38 helical turns. 4-29The dsRNA can be an isolated dsRNA comprising one strand composed of poly(uracil acid) and an opposite strand composed of poly(riboinosinic acid), wherein the two strands do not base pair at the positions of the uracil bases and the two strands base pair at the positions of the cytosine bases, and the strands are partially hybridized.

[0103] After synthesis, rugged dsRNA can be isolated by placing it under conditions that denature at least the majority of dsRNA in the population (more than 10 wt% or mol%, more than 20 wt% or mol%, more than 30 wt% or mol%, more than 40 wt% or mol%, more than 50 wt% or mol%, more than 60 wt% or mol%, more than 70 wt% or mol%, more than 80 wt% or mol%, more than 90 wt% or mol%, more than 95 wt% or mol%, or more than 98 wt% or mol%), and then subjecting it to negative or positive selection (or both) for dsRNA that is still partially hybridized. The denaturing conditions that unfold at least partially hybridized dsRNA strands can include the appropriate selection of buffer salt, pH, solvent, temperature, or any combination thereof. The conditions can be empirically determined by observing the unfolding or melting of ribonucleic acid duplexes. The yield of rugged dsRNA can be improved by partial hydrolysis of long ribonucleic acid strands followed by selection of (partially) hybridized strands that are of appropriate size and resistant to denaturation.

[0104] Thus, the purity of rugged dsRNA that functions as tdsRNA can increase after synthesis from less than about 0.1-10 mol% relative to the total RNA in the population (e.g., the rugged dsRNA is present at at least 0.1 mol% or 0.1 wt% but less than about 10 mol% or 10 wt%) to high purity. High purity can be greater than 20 wt% or mol%, greater than 30 wt% or mol%, greater than 40 wt% or mol%, greater than 50 wt% or mol%, greater than 60 wt% or mol%, greater than 70 wt% or mol%, greater than 80 wt% or mol%, greater than 90 wt% or mol%, and greater than 98 wt% or mol%. All wt% or mol% are wt% or mol% relative to the total RNA present in the same composition.

[0105] The molecular weight of the rugged dsRNA can be about 250 kDa to about 320 kDa, or about 270 kDa to about 300 kDa. The length of one or both strands of the rugged dsRNA can be about 380 bases to about 450 bases, or about 400 bases to about 430 bases. The number of helix turns provided by the RNA duplex of the rugged dsRNA can be about 30 to about 38, or about 32 to about 36.

[0106] In another embodiment, at least one or more different rugged dsRNAs can be administered to a subject (eg, a human patient or an animal) in need of such treatment.

[0107] The recommended dosage of tdsRNA depends on the subject's clinical condition and the physician's or veterinarian's experience treating the disease or other pathological condition. While the dosage and / or frequency of administration may vary depending on the physician or veterinarian's condition, mismatched dsRNA may be administered to a subject (e.g., a human patient weighing approximately 70-80 kg) at about 0.5 mg to about 60 mg per day, about 5 mg to about 400 mg per day, 25 mg to about 700 mg per day, or about 10 mg to about 800 mg per day, on a schedule of once daily to seven days per week, or once weekly to three times weekly (preferably twice weekly). That is, for example, administration may be 50 to 1400 mg every other day, resulting in an average daily dose of 25 to 700 milligrams per day.

[0108] The solid form of the nucleic acid can be dissolved using a known diluent for administration, such as physiological phosphate-buffered saline, and then injected intravenously. It is understood that the preferred dosage may vary depending on the age, condition, sex, or health of the subject; the nature of the disease, or other pathological conditions, including the number and severity of symptoms; and the active ingredient selected.

[0109] Immune checkpoints and checkpoint inhibitors (also called immune checkpoint inhibitors) Immune checkpoints, which act as an off-switch for T cells in the immune system, are being explored to indirectly treat cancer by restoring immune responses with targeted drugs and thus activating the body's immune system. As used herein, the terms "checkpoint inhibitor" and "immune checkpoint inhibitor" are interchangeable and refer to molecules that (1) reduce, (2) inhibit, (3) interfere with, (4) modulate, or (5) any combination of (1) through (4) one or more checkpoint proteins, either in whole or in part. Immune checkpoint proteins (checkpoint proteins) are proteins that regulate the activation or function of T cells. These proteins contribute to costimulatory or inhibitory interactions in T cell responses. These checkpoint proteins include, for example, checkpoint inhibitors such as PD-1 and checkpoint inhibitor receptors such as PD-L1. Other checkpoint proteins are also listed in this disclosure.

[0110] Immune checkpoint proteins regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses. The immune checkpoint inhibitor comprises or is derived from an antibody. In preferred aspects of this and other embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD80 antibody; an anti-CD86 antibody; and combinations thereof. In more preferred aspects, the immune checkpoint inhibitor is at least one selected from the group consisting of ipilimumab (YERVOY®, Bristol-Myers Squibb); nivolumab (OPDIVO®, Bristol-Myers Squibb); and pembrolizumab (KEYTRUDA®; Merck).

[0111] Preferably, the immune checkpoint inhibitor is alemtuzumab [CAMPATH-1H®]; AMP-224 (GlaxoSmithKline / Amplimmune); AMP-514 (Amplimmune / AZ); allerumab (Merck Serono); atezolizumab [TECENTRIQ®; Roche / Genentech] [targets PD-L1]; AUNP 12 (Aurigene and Pierre Fabre); avelumab [BAVENCIO®] [targets PD-L1]; BMS-936559, BMS-986016 (Bristol-Meyers Squibb); BMS-986016 (Bristol-Meyers Squibb) Squibb); cemiplimab [LIBTAYO®] [targets PD-1]; CP-870,893 (Genentech); CT-011; durvalumab [IMFINZI®]; durvalumab [IMFINZI®] [targets PD-L1]; galiximab (Biogen Idec); IMP321 (Immutep SA); INCB024360 (Incyte); indoximod (NewLink Genetics); IPH2101 (Innate Pharma / Bristol-Myers Squibb); ipilimumab [YERVOY®, Bristol-Myers Squibb]; Libtayo (semiplimab-rwlc); lambrolizumab; lirilumab (Bristol-Myers Squibb); MDX-1105 (Medarex, Inc. / Bristol Myer Squibb; MEDI-4736 (Medimmune / AstraZeneca); MEDI-6469 (MedImmune / AZ); MGA271 (Macrogenics); MIHI; mogamulizumab (Kyowa Kirin Co., Ltd.); MPDL3280A (Roche); nivolumab [OPDIVO®, Bristol-Myers Squibb] [targets PD-1]; NLG-919 (NewLink Genetics); ofatumumab [ARZERRA®]; pembrolizumab [KEYTRUDA®; Merck] [targets PD-1]; PF-05082566 (Pfizer); pidilizumab (Curetech); rituximab [RITUXAN®]; tremelimumab; urelumab (Bristol-Meyers Squibb); valilumab (CelIDex Therapeutics); and combinations thereof. The combination may be an FDA-approved combination, such as, for example, Opdivo plus Yervoy for certain forms of colorectal cancer; Keytruda with Lenvima for advanced endometrial cancer; or Tecentriq plus certain chemotherapy drugs for small cell lung cancer.

[0112] Aspects of immune checkpoints are known and include those described in US 8,168,757; US 8,735,553; WO2002086083; WO2004004771; WO2004056875; WO2006121168; WO2008156712; WO2010077634; WO2011066389; WO2011161699; W WO2012168944; WO2013132317; WO2013144704; WO2014055897; WO2014100079; WO2016044900; WO2016142833; WO2016142835; WO2016142852; WO2016142886; and WO2016142894.

[0113] Ipilimumab (YERVOY), a monoclonal antibody that targets cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) on the surface of T cells, and nivolumab (Opdivo), a monoclonal antibody that targets programmed cell death protein 1 (PD-1), are approved by the U.S. Food and Drug Administration for the treatment of advanced melanoma, advanced renal cell carcinoma, and non-small cell lung cancer.

[0114] Examples of immune checkpoint inhibitors include reagents that inhibit, bind to, or interact with ligands of checkpoint proteins. A partial list of checkpoint proteins is as follows: 2B4; A2aR; B-7 family ligands; B7-H3; B7-H4; B and T lymphocyte attenuator (BTLA); BMA; CD112; CD137; CD160; CD2; CD20; CD226; CD27; CD276; CD28; CD30; CD33; CD40; CD47; CD52; CD70; CD80; CD86; CGEN-15049; CHK1; CHK2; cytotoxic T lymphocyte antigen 4 (CTLA-4); DR3; galectin 9 (GAL9); GITR; herpesvirus entry mediator ( HVEM); HVEM; ICOS; IDO1; IDO2; killer cell immunoglobulin-like receptor (KIR); LAG3; LAIR; LAIR1; LAIR2; LIGHT; lymphocyte activation gene 3 (LAG-3); MARCO; OX-40; PD-1; PD-L1; PD-L2; PS; SIRP alpha; SLAM; T cell immunoreceptor with Ig and ITIM domains (TIGIT); T cell membrane protein 3 (TIM3); V domain immunoglobulin (Ig)-containing suppressor of T cell activation (VISTA); VTCN1; and any combination thereof.

[0115] PD-L1 and PD-L2 PD-L1 and PD-L2 are receptors and negative regulators of immune activation through the inhibition of effective T cell function. PD-L1 and PD-L2 are key regulators of a wide range of immune responses and play pivotal roles in autoimmunity and self-tolerance as well as cancer immunology. Evidence suggests that cancer cells use at least the PD-1 / PD-L1 pathway or the PD-1 / PD-L2 pathway to evade antitumor immunity.

[0116] PD-L1 and PD-L2 inhibitors In a preferred embodiment, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or PD-L2. The terms "PD-L1 inhibitor" or "PD-L2 inhibitor" refer to moieties (e.g., compounds, nucleic acids, polypeptides, antibodies) that reduce, inhibit, block, neutralize, or interfere with the activity of, or binding to, PD-L1 or PD-L2 at their receptors, PD-1, or the expression of PD-L1 or PD-L2, including variants, isoforms, species homologs of human PD-L1 or human PD-L2 (e.g., murine homologs), and analogs that share at least one epitope in common with PD-L1 or PD-L2. PD-L1 inhibitors or PD-L2 inhibitors include molecules and macromolecules, such as compounds (small molecule compounds), nucleic acids, polypeptides, antibodies, peptibodies, diabodies, minibodies, single-chain variable fragments (ScFvs), and fragments or variants thereof. Thus, as used herein, a PD-L1 inhibitor or a PD-L2 inhibitor refers to any moiety that antagonizes the activity of PD-L1 or PD-L2, their binding to PD-1, or their expression. The efficacy of a PD-L1 inhibitor or a PD-L2 inhibitor can be measured, for example, by measuring the inhibitor concentration at 50% (half-maximal inhibitor concentration or IC 50) can be measured. PD-L1 inhibitors or PD-L2 inhibitors include the exemplary compounds and compositions described herein. A PD-L1 inhibitory antibody refers to a PD-L1 inhibitor that is a monoclonal or polyclonal antibody, as described herein. Similarly, a PD-L2 inhibitory antibody refers to a PD-L2 inhibitor that is a monoclonal or polyclonal antibody, as described herein.

[0117] More detailed description of various embodiments Pharmaceutical Composition Pharmaceutical compositions containing one or more of the active agents listed above can be administered to a subject by any local or systemic route known in the art, including enteral routes (e.g., oral, feeding tube, enema), topical routes (e.g., devices such as nebulizers for inhalation through the respiratory system, skin patches that act epidermally or transdermally, and suppositories that act rectally or vaginally), and parenteral routes (e.g., subcutaneous, intravenous, intramuscular, intradermal, or intraperitoneal injections; buccal, sublingual, or transmucosal routes; inhalation or intranasal drops or intratracheal routes). The pharmaceutical composition and / or active agent may be micronized by grinding or mashing the solid material, dissolved in a vehicle (e.g., sterile buffered saline or water) for injection or drop (e.g., spray), applied topically, or encapsulated in liposomes or other carriers for targeted delivery. It is appreciated that the preferred route may vary with the age, condition, sex, or health of the subject; the nature of the disease or other pathological conditions, including the number and severity of symptoms; and the active ingredient selected.

[0118] formulation Formulations for administration (i.e., pharmaceutical compositions) may include aqueous solutions, syrups, elixirs, powders, granules, tablets, and capsules, which typically contain conventional excipients such as binders, fillers, lubricants, disintegrants, humectants, suspending agents, emulsifiers, preservatives, buffer salts, flavoring agents, coloring agents, and / or sweeteners. It is appreciated that the preferred formulation may vary with the age, condition, sex, or health of the subject; the nature of the disease or other pathological conditions, including the number and severity of symptoms; and the active ingredient selected.

[0119] Pharmaceuticals In another embodiment, a medicament (e.g., a pharmaceutical composition) containing immune activator(s) (i.e., a checkpoint inhibitor and a tdsRNA) is provided. Other components of the medicament may include excipients and vehicles (e.g., aqueous buffer or water for injection), sterilely packaged in one or more separate containers (e.g., an intranasal applicator or an injection vial). Processes for using and making the medicament are also provided. Further aspects will be apparent from the following description and claims, and any generalizations thereto.

[0120] Effective dose The composition is delivered in an effective amount. The term "effective amount" refers to an amount necessary or sufficient to achieve a desired biological effect. By selecting from among various active compounds and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred administration methods, combined with the teachings provided herein, an effective preventive or therapeutic treatment regimen can be designed that does not cause substantial toxicity but is still effective in treating a particular subject. Based on testing, the toxicity of the inhibitor is also expected to be low. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the specific inhibitor being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective amount of a particular active ingredient without undue experimentation. Generally, it is preferred to use the maximum dose, which is the highest safe dose according to medical judgment.

[0121] For any compound described herein, the therapeutically effective amount can be initially determined from preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined from human data for compounds known to exhibit similar pharmacological activity, such as inhibitors and other related active agents being investigated in humans. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximum efficacy based on the methods described above and other methods is well known in the art and is well within the ability of a person skilled in the art.

[0122] Administration A suitable administration / treatment protocol for treating cancer or tumor in a subject includes, for example, administering to a patient (subject) effective amounts of a tdsRNA and an immune checkpoint inhibitor.

[0123] In some embodiments, the combination therapy of the present invention comprises the administration of tdsRNA and an immune checkpoint inhibitor. Any compound or chemical or pharmaceutical agent in the present disclosure can be administered by any of the disclosed administration methods. The tdsRNA and the immune checkpoint inhibitor can be administered by any suitable method known in the art. For example, the tdsRNA and the immune checkpoint inhibitor can be administered sequentially (at different times) or concurrently (at the same time).

[0124] In some embodiments, the immune checkpoint inhibitor is administered before administration of the tdsRNA. In some embodiments, the immune checkpoint inhibitor is administered simultaneously with administration of the tdsRNA. In some embodiments, the immune checkpoint inhibitor is administered after administration of the tdsRNA.

[0125] In some embodiments, the tdsRNA or immune checkpoint inhibitor is administered continuously. In some embodiments, the tdsRNA or immune checkpoint inhibitor is administered intermittently.

[0126] In some embodiments, the immune checkpoint inhibitor and tdsRNA are co-administered, e.g., the immune checkpoint inhibitor and tdsRNA are administered as two separate formulations. Co-administration can be simultaneous or sequential in any order. In a further embodiment, there is a time during which both (or all) antibodies simultaneously exert their biological activities. The immune checkpoint inhibitor and tdsRNA are co-administered simultaneously or sequentially, e.g., intravenously (iv) via continuous infusion. When both therapeutic agents are co-administered sequentially, the therapeutic agents are administered in two separate administrations separated by a "specified time." The term "specified time" refers to any time between 1 hour and 30 days. For example, one of the agents can be administered within the following time period: The therapeutic agent may be administered within about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. The therapeutic agent may be administered within about 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour. These are the times from the administration of the other therapeutic agent. In some embodiments, the specific time is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In other embodiments, the time period is 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour. In some embodiments, simultaneous administration means at the same time or within a short period of time, typically within less than an hour.

[0127] As used herein, administration period refers to the time during which each member of the composition is administered at least once.The administration period is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days, and in one embodiment, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days, for example, 7 or 14 days.

[0128] In certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) administrations of tdsRNA and multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) administrations of an immune checkpoint inhibitor are given to a subject in need of treatment.

[0129] In certain embodiments, the immune checkpoint inhibitor is administered at a dose of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, or 30 mg / kg. The administration of the immune checkpoint inhibitor can vary from about 0.01 mg / kg to 30 mg / kg, preferably 0.1 mg / kg to 20 mg / kg, and more preferably 1 mg / kg to 10 mg / kg. In certain embodiments, the immune checkpoint inhibitor is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 0.01 mg / kg to 30 mg / kg, e.g., about 0.1 mg / kg to 20 mg / kg, about 1 mg / kg to 10 mg / kg, about 1 mg / kg to 5 mg / kg, or about 1 to 3 mg / kg.

[0130] In certain embodiments, the checkpoint inhibitor is administered once daily, once every two days, once every three days, once every four days, once every five days, once every week, once every two weeks, once every three weeks, or once every four weeks, preferably once every three days. In certain embodiments, the checkpoint inhibitor is administered as a single dose, in two doses, in three doses, in four doses, in five doses, or in six or more doses. The dosing schedule can vary, for example, from once weekly to once every two, three, or four weeks. In one embodiment, the immune checkpoint inhibitor is administered at a dose of about 1 mg / kg to 10 mg / kg every other week.

[0131] In certain embodiments, tdsRNA is administered at a dose of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 1 mg / kg, 2 mg / kg, 2.1 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, or 20 mg / kg. In another embodiment, the dosage of tdsRNA of the present invention administered to prevent and / or treat cancer associated with elevated tdsRNA levels in a patient is about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 7 mg / kg, about 0.1 mg / kg to about 6 mg / kg, about 0.1 mg / kg to about 5 mg / kg. The unit dose is about 0.1 mg / kg, about 0.1 mg / kg to about 4 mg / kg, preferably about 0.1 mg / kg to about 3 mg / kg, about 0.2 mg / kg to 3 mg / kg, about 0.3 mg / kg to about 3 mg / kg, about 0.4 mg / kg to about 3 mg / kg, about 0.6 mg / kg to about 3 mg / kg, about 0.8 mg / kg to about 3 mg / kg, about 0.1 mg / kg to 2 mg / kg, or about 0.1 mg / kg to 1 mg / kg. The total daily dose can vary from 20 mg to 200 mg, preferably from 50 mg to 150 mg, and most preferably from 80 mg to 140 mg. In a preferred embodiment, the tdsRNA of the present invention is administered at a unit dose of about 0.1 mg / kg, about 0.2 mg / kg, about 0.4 mg / kg, about 0.6 mg / kg, about 0.8 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or 5 mg / kg. In one embodiment, the tdsRNA is administered every other week at a dose of about 1 mg / kg to 10 mg / kg.

[0132] In certain embodiments, the tdsRNA is administered once daily, once every two days, once every three days, once every four days, once every five days, once weekly, once every two weeks, or once every four weeks, preferably once every three days. In certain embodiments, the tdsRNA is administered as a single dose, two doses, three doses, four doses, five doses, or six or more doses. The dosing schedule can vary, for example, from once weekly to once every two, three, or four weeks. In one embodiment, the tdsRNA is administered at a dose of about 0.50 mg / kg to 10 mg / kg every other week. In certain embodiments, the dosing frequency can vary from once daily to once monthly.

[0133] An effective amount of the tdsRNA and immune checkpoint inhibitor can be administered to prevent or treat cancer. The appropriate dosage of the tdsRNA and / or immune checkpoint inhibitor can be determined based on the type of disease being treated, the type of tdsRNA and immune checkpoint inhibitor, the severity and course of the disease, the subject's clinical condition, the subject's clinical history and response to treatment, the symptoms involved, the subject's size, sex, and immune status, and the doctor's instructions.

[0134] Preferably, the dosages of the therapeutic agents used in the combination therapies of the present invention are lower than the dosages that have been or are currently used to prevent and / or treat tumors associated with elevated levels of tdsRNA and / or immune checkpoint molecules.

[0135] In some embodiments, methods of treating cancer are performed that have a low likelihood of success, but which are nevertheless deemed to induce an overall beneficial course of treatment given the patient's medical history and life expectancy.

[0136] Thus, in one embodiment, the dose of the tdsRNA and immune checkpoint inhibitor is calculated as mg per kg of body weight, however, in another embodiment, the dose of the tdsRNA and / or immune checkpoint inhibitor is a fixed, constant dose that is fixed regardless of the patient's weight.

[0137] The tdsRNA and the immune checkpoint inhibitor may be administered by the same or different routes of administration. In some embodiments, the tdsRNA is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the immune checkpoint inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally.

[0138] In some embodiments, the immune checkpoint inhibitor is a PD-L1 antagonist or a PD-L2 antagonist (e.g., an anti-PD-L1 antibody). In some embodiments, the anti-PD-L1 antibody or anti-PD-L2 antibody is administered intravenously to a subject at a dose of 120 mg once every three weeks. In some embodiments, the anti-PD-L1 antibody is administered together with tdsRNA (e.g., AMPLIGEN®).

[0139] antibody An "antibody" may be a natural or conventional antibody in which two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (1) and kappa (k). There are five major heavy chain classes (or isotopes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively.

[0140] The light chain contains two domains or regions, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, a variable domain (VH) and three constant domains (collectively referred to as CH, CH1, CH2, and CH3). The variable regions of both the light (VL) and heavy (VH) chains determine antigen binding recognition and specificity. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties, such as antibody chain assembly, secretion, transplacental mobility, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portion of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FR) influence the overall domain structure and therefore the binding site. Complementarity-determining regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site.

[0141] The light and heavy chains of an immunoglobulin each have three CDRs, designated CDR1-L, CDR2-L, and CDR3-L for the light chain and CDR1-H, CDR2-H, and CDR3-H for the heavy chain. Thus, a conventional antibody antigen-binding site contains six CDRs, including a set of CDRs derived from each of the heavy and light chain V regions.

[0142] The term "framework region" (FR) refers to the amino acid sequences inserted between the CDRs, i.e., the portions of the light and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. The light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, and FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively.

[0143] As used herein, a "human framework region" is a framework region that is substantially identical (about 85% or more, particularly 90%, 95%, 97%, 99%, or 100%) to the framework region of a naturally occurring human antibody.

[0144] As used herein, the term "antibody" refers to conventional antibodies and fragments thereof as well as single domain antibodies and fragments thereof, in particular single domain antibodies and the variable heavy chains of chimeric, humanized, bispecific or multispecific antibodies.

[0145] As used herein, antibody or immunoglobulin also includes "single domain antibodies," which are more recently described antibodies whose complementarity-determining regions are portions of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, which are antibodies naturally lacking light chains, single domain antibodies derived from traditional four-chain antibodies, and engineered single domain antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. Single domain antibodies can be naturally occurring single domain antibodies, known as heavy chain antibodies lacking light chains. In particular, Camelidae species, such as camels, dromedaries, llamas, alpacas, and guanacos, produce heavy chain antibodies naturally lacking light chains. Camelid heavy chain antibodies also lack the CH1 domain.

[0146] In the art, the variable heavy chains of these single-domain antibodies, which lack light chains, are known as "VHHs" or "nanobodies." Like conventional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are approximately one-tenth the size of IgG molecules, and as a result, properly folded, functional nanobodies can be produced by in vitro expression while achieving high yields. Furthermore, nanobodies are extremely stable and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 November; 77(1): 13-22).

[0147] The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. The monoclonal antibody may be humanized. In another example, the antibody may be a fragment selected from the group consisting of Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabody, and VHH.

[0148] The term "monoclonal antibody" or "mAb," as used herein, refers to an antibody molecule of a single amino acid composition that is directed against a specific antigen, and should be understood not to require production of the antibody by any particular method. A monoclonal antibody may be produced by a single clone of B cells or a hybridoma, or it may be a recombinant antibody, i.e., produced by protein engineering.

[0149] The term "chimeric antibody" in its broadest sense refers to an engineered antibody containing one or more regions from one antibody and one or more regions from one or more other antibodies. In particular, a chimeric antibody comprises the VH and VL domains of an antibody derived from a non-human animal associated with the CH and CL domains of another antibody, particularly a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, or rabbit. Chimeric antibodies can also represent multispecific antibodies, having specificity for at least two different antigens. In one embodiment, a chimeric antibody has variable domains derived from a mouse and constant domains derived from a human.

[0150] The term "humanized antibody" refers to an antibody that was originally, wholly or partially, of non-human origin but that has been modified to replace certain amino acids, particularly in the framework regions of the heavy and light chains, to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are most often human CH and CL domains. In one embodiment, a humanized antibody has constant domains of human origin.

[0151] An antibody "fragment" comprises a portion of an intact antibody, particularly the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific antibodies and multispecific antibodies formed from antibody fragments. An antibody fragment can also be a single domain antibody, such as a heavy chain antibody or VHH.

[0152] The term "Fab" refers to an antibody fragment having a molecular weight of approximately 50,000 Da and antigen-binding activity, which is obtained by treating IgG with the protease papain, and which comprises approximately the N-terminal half of the H chain and the entire L chain bound together via disulfide bonds.

[0153] The term "F(ab')2" refers to an antibody fragment having a molecular weight of approximately 100,000 Da and antigen-binding activity, which is obtained by treating IgG with the protease pepsin and is slightly larger than Fab, bound via disulfide bonds in the hinge region.

[0154] The term "Fab'" refers to an antibody fragment having a molecular weight of approximately 50,000 Da and antigen-binding activity, which can be obtained by cleaving the disulfide bond in the hinge region of F(ab')2.

[0155] Single-chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers typically expressed by gene fusion, comprising genes encoding VH and VL linked by a peptide-encoding linker. Human scFv fragments of the present invention contain, inter alia, CDRs held in the appropriate conformation by using genetic engineering methods. Bivalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs, such as bivalent sc(Fv)2, with a peptide linker. A "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond.

[0156] "(dsFv)2" denotes two dsFvs coupled by a peptide linker.

[0157] The term "bispecific antibody" or "BsAb" refers to an antibody that combines the antigen-binding sites of two antibodies in a single molecule. Thus, BsAbs are capable of simultaneously binding to two different antigens. Genetic engineering has been used with increasing frequency to design, modify, and generate antibodies or antibody derivatives with desired sets of binding properties and effector functions, as described, for example, in EP 2050764 A1.

[0158] The term "multispecific antibody" denotes an antibody that combines the antigen-binding sites of two or more antibodies in a single molecule.

[0159] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0160] Typically, antibodies are prepared according to conventional methods. Monoclonal antibodies can be produced using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the present invention, mice or other suitable host animals are immunized with the relevant antigen form at appropriate intervals (e.g., twice weekly, weekly, twice monthly, or monthly). The animal may receive a final antigen "booster" within one week before sacrifice. The use of an immunoadjuvant during immunization is often desirable. Suitable immunoadjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or QuilA, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are known in the art. Animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal, or other routes. A given animal may be immunized with multiple forms of antigen via multiple routes.

[0161] In certain embodiments, the present invention provides compositions and methods comprising humanized forms of antibodies. Humanization methods include, but are not limited to, those described in U.S. Patent Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference. The above U.S. Patent Nos. 5,585,089 and 5,693,761, and WO90 / 07861 also propose four possible criteria that can be used in the design of humanized antibodies. The first proposal was to use a framework derived from a specific human immunoglobulin for the acceptor, which is not homologous to the donor immunoglobulin that is usually humanized, or to use a consensus framework derived from many human antibodies. The second proposal was that if an amino acid in the framework of a human immunoglobulin is not a conventional amino acid and the donor amino acid at this position is typical for human sequences, then the donor amino acid, rather than the acceptor amino acid, can be selected. The third proposal was that a donor amino acid, rather than the acceptor amino acid, can be selected at positions in the humanized immunoglobulin chain immediately adjacent to the three CDRs. The fourth proposal was to use donor amino acid residues (resides) at framework positions where the amino acid is predicted to have a side chain atom within 3 Å of the CDR in a three-dimensional model of the antibody and is predicted to be able to interact with the CDR. The above methods are only illustrative of some of the methods that one skilled in the art can use to make humanized antibodies. Those skilled in the art will be familiar with other methods for antibody humanization.

[0162] In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR regions are replaced with amino acids derived from a human immunoglobulin molecule, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not abolish the antibody's ability to bind to a given antigen. Suitable human immunoglobulin molecules include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. A "humanized" antibody retains the same antigen specificity as the original antibody. However, the binding affinity and / or specificity of the antibody may be increased using certain humanization methods, including the "directed evolution" method described by Wu et al., I. Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

[0163] Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, for example, U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, and 6,150,584, the contents of which are incorporated herein by reference, and the references cited therein. These animals have been genetically modified to be functionally deficient in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or part of the human germline immunoglobulin loci, so that immunization of these animals results in the production of fully human antibodies against the antigen of interest. After immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and therefore will not provoke a human anti-mouse antibody (KAMA) response when administered to humans.

[0164] In vitro methods for producing human antibodies also exist. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0165] In one embodiment, the antibody of the present invention is modified to reduce or inhibit the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) functionality (i.e., an antibody with reduced Fc effector function). In particular, the antibody of the present invention does not have an Fc portion or has an Fc portion that does not bind to FcγRI and C1q. In one embodiment, the Fc portion of the antibody does not bind to FcγRI, C1q, or FcγRIII. Antibodies with such functionality are generally known. Naturally occurring such antibodies exist, such as antibodies with an IgG4 Fc region. Antibodies also exist in which the Fc portion has been genetically or chemically altered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) functionality.

[0166] In preferred embodiments, the antibody is an inhibitory antibody, hi some embodiments, the antibody inhibits ligand-receptor binding.

[0167] definition Treat ~ As used herein, the terms "treat," "treating," "treated," or "treatment" refer to therapeutic treatments whose goal is to eliminate or alleviate symptoms. Beneficial or desired clinical results include, but are not limited to, elimination of symptoms, alleviation of symptoms, reduction in the severity of a condition, stabilization (i.e., non-worsening) of the state of a condition, or delay or slowing of the progression of a condition.

[0168] cancer As used herein, "tumor" and "cancer" are used interchangeably unless otherwise specified, and "cancer" refers to the growth, division, or proliferation of abnormal cells in the body, in the form of solid or liquid tumors. Tumors can be benign or malignant. As used herein, the "stromal microenvironment" includes stromal cells that are within the microenvironment of tumor cells and support the growth of tumor cells. Cancers that may be treated by the combinations, pharmaceutical compositions, products, and methods described herein include, but are not limited to, all of the cancers described in this disclosure.

[0169] The present invention can be used to treat neoplastic diseases such as solid or non-solid cancers. As used herein, "treatment" encompasses the prevention, alleviation, control, and / or inhibition of neoplastic diseases. Such diseases include sarcoma, carcinoma, adenocarcinoma, melanoma, myeloma, blastoma, glioma, lymphoma, or leukemia. Exemplary cancers include, for example, carcinoma, sarcoma, adenocarcinoma, melanoma, neural cancer (blastoma, glioma), mesothelioma, and neoplastic disorders of the reticuloendothelial, lymphatic, or hematopoietic system (e.g., myeloma, lymphoma, or leukemia). In certain embodiments, neoplasms, tumors, or cancers include pancreatic cancer; skin cancer; colorectal cancer; ovarian cancer; melanoma; breast cancer; triple-negative breast cancer; head and neck tumor; bladder cancer; renal cell carcinoma; and lung cancer.

[0170] Neoplasms, tumors, and cancers include benign, malignant, metastatic, and non-metastatic types, including any stage (I, II, III, IV, or V) or grade (G1, G2, G3, etc.) of neoplasm, tumor, or cancer, or neoplasm, tumor, cancer, or metastatic cancer that is progressing, worsening, stabilized, or in remission. Cancers that may be treated according to the present invention include, but are not limited to, cells or neoplasms of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal track, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testicles, tongue, or uterus. In addition, cancer specifically includes, but is not limited to, the following histological types of cancer: malignant neoplasms; carcinomas; undifferentiated carcinomas; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; calcifying cell carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; malignant adenocarcinoma, malignant gastrinoma; cholangiocarcinoma, hepatocellular carcinoma; hepatocellular carcinoma in combination with cholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma; and adenomatous polyps. Adenocarcinoma; adenocarcinoma, familial polyposis colon, solid cancer; malignant carcinoid tumor; bronchiolar-alveolar epithelial adenocarcinoma, papillary adenocarcinoma, chromophobe cell carcinoma; acidophil carcinoma; oxyphilic Adenocarcinoma, basophilic carcinoma; clear cell adenocarcinoma, granulocytic carcinoma; follicular adenocarcinoma, papillary / follicular adenocarcinoma, non-encapsulated sclerosing carcinoma; adrenocortical carcinoma; endometroid carcinoma; cutaneous adnexal carcinoma; apocrine adenocarcinoma, sebaceous carcinoma, cerumen Adenocarcinoma, mucoepidermoid carcinoma; cystadenocarcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinic cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia, malignant thymoma; malignant ovarian stromal tumor; malignant theca cell tumor; malignant granulosa cell tumor; malignant androgenitoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant Lipid cell tumor; malignant paraganglioma; extramammary malignant paraganglioma; pheochromocytoma, glomus angiosarcoma, malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma within giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma, malignant fibrous histiocytoma; myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma; mixed tumor; mixed Müllerian tumor;Nephroblastoma, hepatoblastoma, carcinosarcoma, malignant mesenchymoma; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma, embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma, malignant mesonephroma; angiosarcoma, malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma, osteosarcoma, parosteal osteosarcoma, chondrosarcoma, malignant chondroblastoma; mesenchymal Chondrosarcoma, giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastoma, malignant ameloblastoma; ameloblastic fibrosarcoma, malignant pinealoma; chordoma, malignant glioma; ependymoma, astrocytoma, protoplasmic astrocytoma, fibrous astrocytoma, astroblastoma, glioblastoma, oligodendroglioma, oligodendroglioma, undifferentiated neuroectodermal tumor, cerebellar sarcoma; ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactory nerve tumor; malignant meningioma; malignant neurofibrosarcoma, malignant neurilemmoma; malignant granulocytic tumor; malignant lymphoma; Hodgkin's disease; non-Hodgkin's disease; lateral granuloma, small lymphocytic lymphoma, diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides, other types of non-Hodgkin's disease The neoplastic disease may be Hodgkin's lymphoma; malignant histiocytosis, multiple myeloma, mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia, lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. Preferably, the neoplastic disease is a tumor associated with a cancer selected from prostate cancer, liver cancer, kidney cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, brain cancer, hepatocellular carcinoma, lymphoma, leukemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, head and neck cancer, skin cancer, and soft tissue sarcoma, and / or other forms of carcinoma. The tumor may be a metastatic tumor or a malignant tumor.

[0171] More preferably, the neoplastic disease to be treated is pancreatic cancer; skin cancer; colorectal cancer; ovarian cancer; melanoma; breast cancer; triple-negative breast cancer; head and neck tumors; bladder cancer; renal cell carcinoma; and lung cancer.

[0172] Synergy As used herein, the term "synergism" or "synergistic effect," when used in connection with describing the efficacy of a drug combination, means any measured combined effect that exceeds that expected from the sum of the effects of the individual drugs.

[0173] Additive effect As used herein, the term "additive" or "additive effect," when used in connection with describing the efficacy of a drug combination, means any measured combined effect that is similar to that expected from the sum of the effects of the individual drugs.

[0174] subject As used herein, a "subject" is a mammal, preferably a human. In addition to humans, classes of mammals within the scope of the present invention include, for example, farm animals, livestock, laboratory animals, etc. Examples of some farm animals include cows, pigs, horses, goats, etc. Examples of some livestock include dogs, cats, etc. Examples of some laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. In some aspects of this and other embodiments, the subject is a mammal. Preferably, the mammal is selected from the group consisting of humans, primates, farm animals, and livestock. More preferably, the mammal is a human. As used herein, the terms "patient" or "subject" are used interchangeably and refer to a mammal, including, but not limited to, a human or a non-human mammal, such as a bovine, equine, canine, ovine, or feline. Preferably, the patient is a human.

[0175] survival As used herein, "survival" refers to patients who are still alive, including overall survival and progression-free survival. 1-year survival rate and 2-year survival rate refer to the KM estimates of the proportion of subjects who are alive after 12 months or 24 months.

[0176] Survival Extension "Extended survival" means an increase in overall survival and / or progression-free survival in treated patients compared to a control treatment protocol, such as treatment with ipilimumab alone. Survival is monitored for at least about 1 month, 2 months, 4 months, 6 months, 9 months, or at least about 1 year, or at least about 2 years, or at least about 3 years, or at least about 4 years, or at least about 5 years, or at least about 10 years, etc., after initiation of treatment or after initial diagnosis.

[0177] reduce or inhibit "Reducing or inhibiting" refers to the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more. "Reducing or inhibiting" refers to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.

[0178] Improve As used herein, "ameliorate," "ameliorating," and grammatical variations thereof, refer to reducing the severity of symptoms of a disease in a subject.

[0179] Effective or therapeutically effective amount In the present invention, an "effective amount" or "therapeutically effective amount" of a drug, monoclonal antibody or fragment thereof, or compound or composition disclosed herein is the amount of such material sufficient to produce the beneficial or desired results described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will understand that dosage amounts will vary with the route of administration, excretion rate, duration of treatment, the identity of any other drugs administered, the age, size, and species of the mammal, e.g., human patient, and other factors well known in the medical and veterinary arts. Generally, an appropriate dose of any active agent or composition containing the active agent disclosed herein will be the amount of the active agent or composition that is the lowest dose effective to produce the desired effect.

[0180] In some embodiments, a therapeutically effective amount is an amount sufficient to prevent or delay the recurrence of cancer. A therapeutically effective amount can be administered in one or more doses. A therapeutically effective amount of a drug or combination can result in one or more of the following: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibiting, delaying, or slowing, and preferably stopping, to some extent, the infiltration of cancer cells into peripheral organs; (iv) inhibiting (i.e., slowing, and preferably stopping, to some extent) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing or delaying the onset and / or recurrence of tumors; and / or (vii) alleviating, to some extent, one or more symptoms associated with cancer.

[0181] For example, for tumor treatment, a "therapeutically effective dose" may induce tumor regression of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to baseline measurements. Baseline measurements may be from untreated subjects.

[0182] A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such an amount based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0183] to bring into contact with In this embodiment, "contacting" refers to bringing, for example, an immune checkpoint inhibitor and / or one or more additional therapeutic agents into close proximity with the tumor microenvironment. This may be accomplished using traditional drug delivery techniques into a mammal, or in an in vitro setting by applying one or more additional therapeutic agents to the culture medium in which the cancer cells reside.

[0184] chemotherapy drugs For any of the claims, the chemotherapy drug can be any one or more drugs used for chemotherapy. The drug can be in any form, such as, for example, liposomal, sustained-release, or depot form, encapsulated in liposomes. Non-limiting examples of such drugs include at least: ABVD; AC; ACE; abiraterone (Zytiga); Abraxane; Abstral; actinomycin D; Actiq; Adriamycin; afatinib (Giotrif); Afinitor; aflibercept (Zaltrap); Aldara; aldesleukin (IL-2, proleukin, or interleukin-2); alemtuzumab (MabCampath); Alkeran; amsacrine (Amcidin, m-AMSA); Amcidin; anastrozole (Arimidex); Ara C; Aredia; Arimidex; Aromasin; Arsenic trioxide (Trisenox, ATO); Asparaginase (Crisantaspase, Erwinase); Axitinib (Inlyta); Azacitidine (Vidaza); BEACOPP; BEAM; Bendamustine (Levact); Bevacizumab (Avastin); Bexarotene (Targretin); Bicalutamide (Casodex); Bleomycin; Bleomycin, etoposide and platinum (BEP); Bortezomib (Velcade) ;Bosulif;bosutinib (Bosulif);brentuximab (Adcetris);Brufen;buserelin (Suprefact);Busilvex;busulfan (Myleran, Busilvex);CAPE-OX;CAPOX;CAV;CAVE;CCNU;CHOP;CMF;CMV;CVP;cabazitaxel (Jevtana);cabozantinib (Cometriq);Caelyx;Calpol;Campto;capecitabine (Xeloda);Caprelsa;Carbo MV; CarboTaxol; carboplatin; carboplatin and etoposide; carboplatin and paclitaxel; carmustine (BCNU, Gliadel); Casodex; ceritinib (Zykadia); Cerubidin; cetuximab (Erbitux); ChlVPP; chlorambucil (Leukeran); cisplatin; cisplatin and Teysuno;Cisplatin and capecitabine (CX); cisplatin, etoposide, and ifosfamide (PEI); cisplatin, fluorouracil (5-FU), and trastuzumab; cladribine (Leustat, LITAK); Clasteon; clofarabine (Evoltra); Co-codamol (Kapake, Solpadol, Tylex); Cometriq; Cosmegen; crisantaspase; crizotinib (Xalkori); cyclophosphamide; cyclophosphamide, thalidomide, and dexamethasone (CTD); Cyprostat; cyproterone acetate (Cyprostat); cytarabine (Ara C, cytosine arabinoside); cytarabine into spinal fluid; cytosine arabinoside; DHAP; DTIC; dabrafenib (Tafinlar); dacarbazine (DTIC); Dacogen; dactinomycin (actinomycin D, Cosmegen); dasatinib (Sprycel); daunorubicin; De Gramont; Decapeptyl SR; decitabine (Dacogen); degarelix (Firmagon); denosumab (Prolia, Xgeva); Depocyt; dexamethasone; diamorphine; pamidronate disodium; Disprol; docetaxel (Taxotere); docetaxel, cisplatin and fluorouracil (TPF); Doxifos; Doxil; doxorubicin (Adriamycin); doxorubicin and ifosfamide (Doxifos); Drogenil; Durogesic; EC; ECF; EOF; EOX; EP (etoposide and cisplatin); ESHAP; Effentora; Efudix; Eldisine; Eloxatin; Enzalutamide; Epirubicin (Farmorubicin); Epirubicin, cisplatin, and capecitabine (ECX); Epirubicin, carboplatin, and capecitabine (ECarboX); Eposin; Erbitux; Eribulin (Halaven); Erlotinib (Tarceva); Erwinase; Estracyt; Etopophos; Etoposide (Eposin, Etopophos, Vepesid); Everolimus (Afinitor); Evoltra; Exemestane (Aromasin); FAD; FEC; FEC-T chemotherapy;FMD; Folfirinox; FOLFOX; Faslodex; Femara; Fentanyl; Firmagon; Fludara; Fludarabine (Fludara); Fludarabine, cyclophosphamide, and rituximab (FCR); Fluorouracil (5FU); Flutamide; Folinic acid, fluorouracil, and irinotecan (FOLFIRI); Fulvestrant (faslodex); G-CSF; Gefitinib (Iressa); GemCarbo (gemcitabine and carboplatin); GemTaxol; Gemcitabine (Gemzar); Gemcitabine and capecitabine (GemCap); Gemcitabine and cisplatin (GC); Gemcitabine and paclitaxel (GemTaxol); Gemzar; Giotrif; Gliadel; Glivec; Gonapeptyl Depot; Goserelin (Zoladex); Goserelin (Zoladex, Novgos); Granulocyte colony-stimulating factor (G-CSF); Halaven; Herceptin; Hycamtin; Hydrea; Hydroxycarbamide (Hydrea); Hydroxyurea; I-DEX; ICE; IL-2; IPE; Ibandronate; Ibritumomab (Zevalin); Ibrutinib (Imbruvica); Ibuprofen (Brufen, Nurofen); Iclusig; Idarubicin (Zavedos); Idarubicin and dexamethasone; Idelalisib (Zydelig); Ifosfamide (Mitoxana); Imatinib (Glivec); Imiquimod cream (Aldara); Imnovid; Instanyl; Interferon (Intron A); Interleukin; Intron A; ipilimumab (Yervoy); Iressa; irinotecan (Campto); irinotecan and capecitabine (Xeliri); irinotecan de Gramont; irinotecan modified de Gramont; Javlor; Jevtana; Kadcyla; Kapake; Keytruda; lanreotide (Somatuline); Lanvis; lapatinib (Tyverb); lenalidomide (Revlimid); letrozole (Femara); Leukeran; leuprorelin (Prostap, Lutrate); Leustat; Levact;Liposomal doxorubicin; Litak; Lomustine (CCNU); Lynparza; Lysodren; MIC; MMM; MPT; MST Continus; MVAC; MVP; MabCampath; Mabthera; Maxtrex; Medroxyprogesterone acetate (Provera); Megace; Megestrol acetate (Megace); Melphalan (Alkeran); Mepact; Mercaptopurine (Xaluprine); Methotrexate; Methylprednisolone; Mifamurtide (Mepact); Mitomycin C; Mitotane; Mitoxana; Mitoxantrone (Mitozantrone); Morphgesic SR; morphine; Myleran; Myocet; Nab-paclitaxel; Nab-paclitaxel (Abraxane); Navelbine; Nelarabine (Atriance); Nexavar; Nilotinib (Tasigna); Nintedanib (Vargatef); Nipent; Nivolumab (Opdivo); Novgos; Nurofen; Obinutuzumab (Gazyvaro); Octreotide; Ofatumumab (Arzerra); Olaparib (Lynparza); Oncovin; Onkotrone; Opdivo; Oramorph; Oxaliplatin (Eloxatin); Oxaliplatin and capecitabine (Xelox); PAD; PC (paclitaxel and carboplatin, CarboTaxol); PE; PMitCEBO; POMB / ACE; Paclitaxel Cel (Taxol); paclitaxel and carboplatin; pamidronate; Panadol; panitumumab (Vectibix); paracetamol; pazopanib (Votrient); pembrolizumab (Keytruda); pemetrexed (Alimta); pemetrexed and carboplatin; pemetrexed and cisplatin; pentostatin (Nipent); Perjeta; pertuzumab (Perjeta); pixantrone (Pixuvri); Pixuvri; pomalidomide (Imnovid); ponatinib; Potactasol; prednisolone; procarbazine; procarbazine, lomustine, and vincristine (PCV); Proleukin; Prolia; Prostap; Provera; Purinethol; R-CHOP; R-CVP;R-DHAP; R-ESHAP; R-GCVP; RICE; Raloxifene; Raltitrexed (Tomudex); Regorafenib (Stivarga); Revlimid; Rituximab (Mabthera); Sevredol; Clodronate sodium (Bonefos, Clasteon, Loron); Solpadol; Sorafenib (Nexavar); Steroids (Dexamethasone, Prednisolone, Methylprednisolone); Streptozocin (Zanosar); Sunitinib (Sutent); Sutent; TAC; TIP; Tafinlar; Tamoxifen; Tarceva; Targretin; Tasigna; Taxol; Taxotere; Taxotere and cyclophosphamide (TC); Temodal; Temozolomide (Temodal); Temsirolimus; Tepadina; Teysuno; Thalidomide; Thiotepa (Tepadina); Thioguanine (Thioguanine, 6-TG, 6-thioguanine); Tomudex; Topotecan (Hycamtin, Potactasol); Torisel; Trabectedin (Yondelis); Trastuz Mab (Herceptin); trastuzumab emtansine (Kadcyla); treosulfan; tretinoin (Vesanoid, ATRA); triptorelin; Trizenox; Tyrex; Tyverb; VIDE; vandetanib (Caprelsa); Valgatef; VeIP; Vectibix; Velbe; Velcade; vemurafenib (Zelboraf); Vepesid; Vesanoid; Vidaza; vinblastine (Velbe); vincristine; vincristine, actinomycin D (dactinomycin), and cyclophosphamide (VAC) ); Vincristine, actinomycin, and ifosfamide (VAI); Vincristine, doxorubicin, and dexamethasone (VAD); Vindesine (Eldisine); Vinflunine (Javlor); Vinorelbine (Navelbine); Vismodegib (Erivedge); Votrient; XELOX; Xalkori; Xeloda; Xgeva; Xtandi; Yervoy; Yondelis; Z-DEX; Zaltrap; Zanosar; Zavedos; Zelboraf; Zevalin; Zoladex (e.g., breast cancer);Includes Zoladex (e.g., prostate cancer); zoledronic acid (Zometa); Zometa; Zomorph; Zydelig; and Zytiga.

[0185] When numerical ranges are stated herein, it should be understood that all values ​​within the range are also described (e.g., 1-10 also includes every integer value between 1 and 10, as well as all intermediate ranges, such as 2-10, 1-5, and 3-8). The term "about" may refer to statistical uncertainty associated with a measurement, or variation in a numerical quantity, that one of ordinary skill in the art would understand does not affect the operation of the invention or its patentability.

[0186] All modifications and alternatives that come within the meaning of the claims and their legal equivalents are intended to be embraced within their scope. While a claim reciting "comprising" permits the inclusion of other elements within the claim, the invention may also be described by claims that recite the transitional phrases "consisting essentially of" (i.e., permitting the inclusion of other elements within the claim if they do not materially affect the operation of the invention) or "consisting of" (i.e., permitting only the elements recited in the claim, other than impurities or insignificant activity normally associated with the invention), rather than the term "comprising." Any of these three transitional phrases may be used to claim the invention.

[0187] It is understood that no element described herein should be construed as a limitation of the claimed invention unless it is expressly recited in the claims. Accordingly, the claims as issued are the basis for determining the scope of legal protection, not limitations from the specification read into the claims. In the event of a conflict, the prior art is expressly excluded from the present invention to the extent of specific embodiments that anticipate or destroy the claimed invention.

[0188] Moreover, no particular relationship is intended between the limitations of the claims unless such relationship is expressly recited in the claims (e.g., the arrangement of elements in a product claim or the order of steps in a method claim is not a limitation of the claim unless expressly stated to be so). All possible combinations and permutations of the individual elements disclosed herein are considered aspects of the invention. Likewise, generalizations of the descriptions of the invention are also considered part of the invention.

[0189] From the foregoing, it will be apparent to those skilled in the art that the present invention may be embodied in other specific forms without departing from its spirit or essential characteristics.

[0190] While the invention has been described herein in connection with what is considered to be the most practical and preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but on the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0191] Incorporation by Reference All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions, will control. [Example]

[0192] [Example 1] Experimental results Currently, surgery is the only potentially curative option for pancreatic cancer, but because most pancreatic cancers are detected at an advanced stage of the disease, only approximately 15% of patients are eligible at the time of initial diagnosis. Approximately 20% of patients are diagnosed with locally advanced pancreatic cancer, while the remaining 65% present with metastatic disease.

[0193] The current standard of care (SOC) for locally advanced and metastatic pancreatic cancer is FOLFIRJNOX, a four-drug cocktail associated with significant toxicity. FOLFIRJNOX's approval was based on the ACCORD study, a phase 2 / 3 study published in 2011 (Von Hoff et al., 2011). In this study, FOLFIRJNOX was compared with gemcitabine, which was the standard of care at the time.

[0194] Results from the ACCORD study showed that overall survival (OS) was extended from 6.8 months with gemcitabine to 11.1 months with Forfirinox (p<0.001). However, the complete response rate (CR) was only 0.6%. Furthermore, median OS with second-line therapy after progression on Forfirinox was only 4.05 months. The data clearly demonstrate the critical need for new treatment options for this devastating malignancy.

[0195] One of these novel therapeutic options is immunotherapy, which has shown to be a promising treatment strategy, in which the fundamental element is to boost the patient's immune system by reversing tumor-antigen-specific T cell tolerance induced by their tumors.

[0196] One goal in immunotherapy is to reprogram the tumor microenvironment (TME) to convert a "cold" tumor into a "hot" tumor that is responsive to checkpoint blockade. The goal is to unleash a cellular immune response that attacks and destroys cancer cells, and to stimulate T cells within the tumor. reg While reducing T cells in the tumoreff The goal is to prolong survival by expanding (effector T) cells.

[0197] Surprisingly, AMPLIGEN® stimulated the proliferation of CTLs (Tregs) with a concomitant increase in the Teff / Treg ratio within the TME. eff ) can be promoted selectively.

[0198] T eff The ability to expand tumor-infiltrating CD4+ T cells (high), CD8+ T cells (high), and Tregs (high Teff) / Treg ratios within the TME has significant benefits. In pancreatic cancer, tumor-infiltrating CD4+ T cells (high), CD8+ T cells (high), and Tregs (high Teff) / Treg ratios within the TME are important. reg Cells (low) is an independent prognostic factor for prolonged overall survival.

[0199] In pancreatic cancer, T reg Infiltration of TME is a poor prognostic indicator for survival. reg The cohorts were divided into two groups based on whether their cell levels were higher or lower than the median within the TME. reg The group was high T reg showed significantly better survival than the control group (Hiraoka et al., 2006).

[0200] AMPLIGEN® is eff (effector T) cells, T reg Our observation that increasing the ratio of T cells to regulatory T cells, thereby converting a "cold" intrapancreatic TME into a "hot" intrapancreatic TME, is highly relevant to improving the potential for antitumor responses to checkpoint blockade.

[0201] In preclinical models of pancreatic cancer, the combination of AMPLIGEN® with a checkpoint blockade (anti-PD-L1) is found to be synergistic for extending both overall survival and time to tumor progression.

[0202] The inventors propose the use of AMPLIGEN® in combination with checkpoint blockers to improve the ability to treat cancer. Or more specifically, the inventors propose the use of AMPLIGEN® and checkpoint blockers so that they may act synergistically. That is, the inventors expect that the effect of AMPLIGEN® plus the effect of the checkpoint blocker will exceed the effect of AMPLIGEN® plus the effect of the checkpoint blocker.

[0203] The inventors also found that in melanoma, animal models combining AMPLIGEN® with anti-PD-L1 showed a three-fold increase in overall tumor response rate (RECIST (Response Evaluation Criteria In Solid Tumors) criteria). In addition, in transgenic mouse models of pancreatic cancer, the combination of AMPLIGEN® with anti-PD-L1 agents showed a synergistic extension of median survival. Furthermore, the inventors found that in mouse models of colorectal cancer, the combination of AMPLIGEN® plus anti-PD-L1 showed a more than 2.5-fold extension of median survival compared to anti-PD-L1 alone.

[0204] Fundamentals of Barriers to Immunotherapy in Pancreatic Cancer The TME in pancreatic cancer is reg Immunosuppressive cells, including (regulatory T) cells, predominate and are required to drive antitumor responses. eff (effector T) cells in the TME. reg A small number of patients with low cell abundance had a favorable prognosis.

[0205] Importantly, because high levels of tumor-reactive T cells were readily found in bone marrow samples from patients with pancreatic cancer, the lack of effector T cells in the TME of patients with pancreatic cancer is thought to be related to the inability of these effector T cells to migrate from the bone marrow and blood of pancreatic cancer patients to the TME. Thus, these findings suggest that the failure of immunotherapy in pancreatic cancer is not due to the lack of antigenicity of the tumor itself or the lack of effector T cells directed against tumor antigens, but rather to the inability of T cells in the TME. reg This suggests that this is due to an inability to recruit effector T cells into the TME while simultaneously reducing the levels of T cells.

[0206] Lintatolimod (sold under the trade name AMPLIGEN®) is used in TME. eff cell / T reg Increased cell ratio To obtain biopsy specimens of the TME, colorectal cancer was used as a GI model for pancreatic cancer. We used AMPLIGEN® to induce desirable chemokines, such as CXCL10 (a Teff attractant), in the TME while reducing undesirable chemokines, such as CCL22 (CC motif chemokine ligand 22; a Treg attractant). eff / T reg The ratio was improved, which resulted in the TME eff / T reg It was determined whether an increase in the ratio was observed.

[0207] AMPLIGEN® improves the TME in gastrointestinal cancers, including colorectal cancer. A colorectal cancer trial of AMPLIGEN® plus rIFNa-2b and celecoxib demonstrated an increase in the ratio of CXCL10 to CCL22 within the TME in nine patients with metastatic colorectal cancer compared to historical controls. eff Marker / T reg This was accompanied by an increase in the ratio of markers. See the Examples section below.

[0208] Based on these experiments, AMPLIGEN® (lintatolimod) demonstrates the ability to convert "cold" tumors into "hot" tumors that are much more likely to respond to the presence of checkpoint inhibitors (also called checkpoint blockers or immune checkpoint inhibitors, depending on their function).

[0209] The present inventors have demonstrated that tumor-infiltrating CD4+T (high) / CD8+T (high) / %T in the TME in pancreatic cancer. reg We suggest that T (low) is an independent prognostic factor for prolonged overall survival. reg Invasion of TME is a poor prognostic indicator for survival. reg The cohorts were divided into two groups based on whether their cell levels were higher or lower than the median within the TME. reg The group was high T reg showed significantly better survival than the control group.

[0210] AMPLIGEN® is eff Cellular, T reg The potential to increase the ratio of PD-L1 cells to PD-L1 cells, thereby converting a "cold" intrapancreatic TME to a "hot" intrapancreatic TME, is highly relevant to improving the likelihood of an anti-tumor response to checkpoint blockade. The combination of AMPLIGEN® with a checkpoint blockade [anti-PD-L1 (PD-L1)] was synergistic in extending both overall survival and time to tumor progression.

[0211] Summary of data showing that AMPLIGEN® plus checkpoint blockade (checkpoint inhibitor) synergistically extended survival In a transgenic mouse model of pancreatic cancer, the combination of AMPLIGEN® with anti-PD-LI drugs shows a synergistic extension of median survival.

[0212] In a mouse model of colorectal cancer, the combination of AMPLIGEN® plus anti-PD-I demonstrated a greater than 250% increase in median survival compared to anti-PD-I alone.

[0213] Preclinical cancer studies using mouse models of three different solid tumors demonstrate synergistic anti-tumor activity and / or extended median survival when AMPLIGEN® is combined with checkpoint blockers compared to checkpoint blockers alone.

[0214] In melanoma, animal models combining AMPLIGEN® with anti-PD-L1 showed a three-fold increase in overall response rate (RECIST (Response Evaluation Criteria In Solid Tumors) criteria). Additionally, in pancreatic cancer, studies using transgenic mouse models combining AMPLIGEN® with anti-PD-L1 agents showed a synergistic extension of median survival. Furthermore, in mouse models of colorectal cancer, the AMPLIGEN® combination demonstrated a more than 2.5-fold extension of median survival compared to anti-PD-L1 alone.

[0215] AMPLIGEN® induced antitumor synergy with checkpoint immunosuppressive blockade in melanoma models AMPLIGEN® was synergistic with anti-PD-L1, resulting in an enhanced anti-tumor response in the B16 mouse melanoma model. The reduction in tumor size was significant for the AMPLIGEN® 250 μg + anti-PD-L1 cohort compared to the anti-PD-L1 cohort alone (p=0.023).

[0216] The addition of AMPLIGEN® to anti-PD-L1 increased the objective response rate by 300%, from 10% with anti-PD-L1 alone to 30% with the combination. [Example 2]

[0217] Pancreatic cancer According to the Pancreatic Cancer Action Network, pancreatic cancer is the fourth leading cause of cancer death in the United States. It is the only one of the most commonly diagnosed cancers with a 5-year survival rate of only 6 percent. Based on the latest projections, pancreatic cancer is expected to move from the fourth leading cause of cancer death to the second leading cause of cancer death in the United States by 2020. Thus, the projected number of both new pancreatic cancer cases and pancreatic cancer deaths will more than double by 2030 (Matrisian et al., 2012).

[0218] In the European Union, the incidence of pancreatic cancer continues to rise, with mortality rates predicted to increase by approximately 30% to approximately 112,000 new cases per year by 2025. More specifically, breast cancer deaths are predicted to increase from 92,000 and 91,000 in 2010 and 2017, respectively, to 90,000 in 2025. Pancreatic cancer deaths, on the other hand, are predicted to increase from 76,000 and 91,000 in 2010 and 2017, respectively, to 112,000 in 2025, a 30% increase.

[0219] Pancreatic cancer is associated with a 5% 5-year overall survival rate and therefore contributes significantly to cancer-related mortality. Recent studies have predicted that pancreatic cancer will become the second leading cause of cancer-related deaths before 2030. Currently, surgery is the only potentially curative option, but because most pancreatic cancers are detected at an advanced stage of the disease, only approximately 15% of patients are eligible for surgery at the time of initial diagnosis. Approximately 20% of patients are diagnosed with locally advanced pancreatic cancer, while the remaining 30–50% present with metastatic disease. Clearly, there is a critical need for new treatment options for this devastating malignancy.

[0220] The pancreatic gland itself is located in the abdomen between the stomach and the spinal cord. The pancreas is about 6 inches long and pear-shaped, lying on its side. The pancreas is divided into three compartments: the head, or wide part of the pancreas; the body, or central compartment; and the tail, which is the narrow end of the pancreas (https: / / world wide web.cancer.gov / types / pancreatic / patient / pancreatic-treatment-pdq).

[0221] Pancreatic cancer, or pancreatic carcinoma, is a disease in which malignant (cancer) cells form within the tissues of the pancreas. The pancreas is a gland that aids in digestion. The pancreas produces secretions through exocrine pancreatic cells that break down food. The pancreas also produces hormones, such as insulin and glucagon, that help regulate blood sugar through endocrine pancreatic cells. Most pancreatic cancers begin within the exocrine cells. Due to the absence of symptoms in the early stages of pancreatic cancer, the majority of patients are diagnosed after the cancer has spread locally or to other parts of the body.

[0222] Pancreatic cancer is an extremely serious and fatal disease associated with reduced life expectancy.

[0223] Etiological factors associated with the development of intrapancreatic adenocarcinoma in adulthood include tobacco use and environmental exposure to tobacco, particularly both during childhood and in utero from maternal smoking. Tobacco smoke is estimated to contribute to the development of 20-30% of pancreatic cancers.

[0224] Several infectious diseases, including Helicobacter pylori and hepatitis B, also have a positive association with intrapancreatic adenocarcinoma. Occupational factors are also associated with 12-29% of cases and include exposure to a wide range of chemicals / solvents, such as chlorinated hydrocarbons, polycyclic aromatic hydrocarbons, pesticides, and aliphatic solvents.

[0225] For intrapancreatic adenocarcinoma, demographic risk factors include age between 60 and 80 years, African American race, low socioeconomic status, and Ashkenazi Jewish descent. Some medical conditions associated with an increased risk of pancreatic cancer include diabetes, chronic liver cirrhosis, pancreatitis, and a history of cholecystectomy.

[0226] Finally, genetic predisposition also plays a small role in pancreatic cancer risk, with 10-20% of pancreatic cancers having a familial linkage. The etiologic risk factors for the development of pancreatic cancer are numerous and include the following (percentages shown are those listed when available): tobacco smoke (20-30% contribution); infectious diseases; occupation (12-29% contribution); demographics; medical conditions; and genetics (20-20% contribution).

[0227] Detailed characteristics: pathophysiological, histopathological, and clinical features In recent years, evidence has accumulated that tumor-infiltrating lymphocytes (TILs) have a significant impact on several important clinical attributes of cancer. The type, density, and location of T cells within tumors have been shown to provide good prognostic value, which is superior to and independent of the prognostic value of the TNM classification criteria. In pancreatic cancer, CD8+ T lymphocytes represent the major T lymphocyte subset and are associated with favorable clinical outcomes. However, in general, a more detailed analysis of T cells (TILs), apart from the number of CD8 T cells within the tumor environment, is needed. reg T contrasted with eff It is accepted that TME results in good prognostic or predictive markers in pancreatic cancer. eff Cells and T reg Analysis of both cellular contexts reveals important immune signatures in pancreatic tumors.

[0228] Aside from these local immune markers, prognostic and predictive markers have also been found in peripheral blood (PB). The neutrophil-to-lymphocyte ratio (NLR) in peripheral blood has been shown to be a prognostic marker in pancreatic cancer (Kawahara et al., 2016). The use of biomarkers derived from PB has advantages over local tumor tissue because they are less invasive to patients and can be measured longitudinally along the course of treatment. Currently, the enumeration, activation, and presence of regulatory T cells, as well as the co-signaling signatures of TILs and T cells in PB, are being explored in patients with pancreatic tumors. At least in some cases, PB T cells may reflect the co-signaling signature of TILs and could be used as a surrogate marker for the local immune status at diagnosis and during treatment. Tumor cell-free DNA (cfDNA), found in peripheral blood, is being actively explored and is expected to become widely used in the future as a surrogate (liquid biopsy) for direct tumor biopsy, with the advantage of sampling for metastatic disease.

[0229] Pancreatic cancer is difficult to detect and diagnose for the following reasons: (1) there are no noticeable signs or symptoms in the early stages of pancreatic cancer, (2) the signs of pancreatic cancer, if present, are similar to those of many other diseases, such as pancreatitis or ulcers, and (3) the pancreas is shadowed by other organs in the abdomen and is difficult to clearly visualize on imaging tests.

[0230] In order to properly treat pancreatic cancer, it is preferable to assess whether the cancer is resectable. Diagnostic tools used include imaging, peritoneal cytology, and tumor markers. Imaging can be used to detect tumors and determine whether the tumor is resectable.

[0231] Symptoms of pancreatic cancer include, for example, jaundice; light-colored stools or dark-colored urine; pain in the upper or middle abdomen and back; weight loss for unknown reasons; loss of appetite; and fatigue.

[0232] We hypothesize that the dsRNA AMPLIGEN® primarily activates antigen-presenting cells, leading to an increase in the number of monocytes and dendritic cells, which could subsequently lead to an increase in the number of CD8 T cells and a decrease in the number of regulatory T cells or myeloid-derived suppressor cells.

[0233] Conventional treatments for pancreatic cancer are lacking. The current standard of care (SOC) for locally advanced and metastatic pancreatic cancer is Forfirinox, a four-drug cocktail with significant toxicity. The approval of Forfirinox was based on the ACCORD study, a phase 2 / 3 study published in 2011 (Von Hoff et al., 2011). In this study, Forfirinox was compared with gemcitabine, which was the SOC at the time.

[0234] [Table 1]

[0235] Table 1 shows the results of the ACCORD study. Overall survival (OS) was extended from 6.8 months with gemcitabine to 11.1 months with Forfirinox (p<0.001). However, the complete response rate (CR) was only 0.6%. Furthermore, as shown in Table 2, the median OS with second-line treatment after progression on Forfirinox was only 4.05 months.

[0236] [Table 2]

[0237] These methods are unsatisfactory as evidenced by the high mortality rate.

[0238] Unfortunately, the rapidly developing field of immunotherapy using checkpoint blockade has not met with success in patients with pancreatic adenocarcinoma, who show poor response rates to checkpoint blockade using anti-PD1, anti-PD-L1, and anti-CTLA-4 agents.

[0239] The TME in pancreatic cancer is reg Immunosuppressive cells, including T cells, predominate and are required to drive antitumor responses. eff lacking T cells (Liyanage et al., 2002; Hiraoka et al., 2006). reg A small number of patients with low cell abundance had a favorable prognosis (Hiraoka et al., 2006).

[0240] Importantly, high levels of tumor-reactive T cells were readily found in bone marrow samples from patients with pancreatic cancer, suggesting that T cells in the TME of patients with pancreatic cancer are highly reactive. effector The lack of cells causes these T effector This is thought to be related to the inability of cells to migrate from the bone marrow and blood of pancreatic cancer patients into the TME.

[0241] Therefore, these findings suggest that the failure of immunotherapy in pancreatic cancer may be due to the lack of antigenicity of the tumor itself or to T effector Not due to a lack of T cells, but due to the reg while simultaneously reducing the levels of T effector This suggests that this is due to an inability to recruit cells to the TME.

[0242] In pancreatic cancer, the inventors have demonstrated that tumor-infiltrating CD4+ T cells (high), CD8+ T cells (high), and T reg We note that both low and high cell percentage are independent prognostic factors for prolonged overall survival (Ino et al., 2013). Furthermore, in pancreatic cancer, T regInfiltration of TME is a poor prognostic indicator for survival. reg The cohorts were divided into two groups based on whether their cell levels were higher or lower than the median within the TME. reg The group was high T reg showed significantly better survival than the control group (Hiraoka et al., 2006).

[0243] The present inventors have demonstrated that AMPLIGEN® eff Cellular, T reg Experiments were conducted to determine whether increasing the ratio of AMPLIGEN® to PD-L1 cells could convert a "cold" intrapancreatic TME into a "hot" intrapancreatic TME, which is highly relevant to improving the likelihood of an anti-tumor response to checkpoint blockade. As shown below, in a preclinical model of pancreatic cancer, the combination of AMPLIGEN® with a checkpoint blockade (anti-PD-L1) was synergistic in extending both overall survival and time to tumor progression (Figure 1).

[0244] Figure 1 shows that AMPLIGEN® was tested with anti-PD-L1 against pancreatic tumors in mice, and AMPLIGEN® was shown to synergistically extend survival and time to tumor progression (p=0.029 and 0.0418, respectively). Note that all four cohorts (control, AMPLIGEN®, anti-PD-L1, AMPLIGEN® + anti-PD-L1) were studied in the same, parallel experiment. Separate figures (Figures 1A, 1B, 1C, 1D, 1E, and 1F) are used for increased clarity.

[0245] The combination of AMPLIGEN® and a checkpoint inhibitor was found to synergistically extend the time to progression in a mouse model of pancreatic cancer. See Table 3. In this experiment, a subtherapeutic dose of AMPLIGEN® was administered to a mouse model of pancreatic cancer. Because the dose was subtherapeutic, there was no effect on the time to progression, which remained at 33 days, the same as in untreated mice. Similarly, administration of a subtherapeutic dose of a checkpoint inhibitor also had no effect on the time to progression, which remained the same as in the untreated group, at 33 days. However, administration of the same subtherapeutic dose of AMPLIGEN® in combination with the same subtherapeutic dose of a checkpoint inhibitor induced a synergistic extension of the time to progression to 73 days.

[0246] [Table 3]

[0247] A low systemic immune-inflammatory index (SIII) predicts prolonged survival in pancreatic cancer. Systemic immune-inflammatory index (SIII) can be used as a prognostic marker to predict survival in resectable pancreatic cancer. A low SIII (≤900) predicts prolonged survival. SIII = neutrophil-to-lymphocyte ratio (NLR) × platelet count in peripheral blood. The patient cohort with low SIII (N=164) had significantly longer survival compared with the patient cohort with high SIII (n=141) (p<0.001). See Figure 2 (where SIII = neutrophil-to-lymphocyte ratio (NLR) × platelet count in peripheral blood).

[0248] Clinical Treatment Results with AMPLIGEN®: Nine pancreatic cancer patients receiving 400 mg of AMPLIGEN® (IV) twice weekly reduced SIII levels and stabilized metastatic disease for up to 18 weeks. See Figure 3.

[0249] A decrease in SIII is a desirable prognostic sign for prolonged survival.

[0250] Preclinical Models AMPLIGEN® was also tested in mice with pancreatic tumors in combination with anti-PD-L1 and was shown to synergistically extend survival. See Figure 1, panel labeled "Percent Survival." The same was true for time to tumor progression. See Figure 1, panel labeled "Time to Tumor Progression." [Example 3]

[0251] melanoma Similar to the success in pancreatic cancer described above, demonstrating synergy using AMPLIGEN® plus checkpoint blockade, the inventors also saw a positive synergistic anti-tumor response in a melanoma animal model.

[0252] Lintatolimod in combination with an anti-PD-L1 antibody was investigated for its antitumor activity against established subcutaneous B16 melanoma tumors in C57BL / 6 mice. Mice (10 animals per group) were inoculated with 0.4 x 10 B16-F10 tumor cells in the shaved posterior flank. 6 Seven days later (when tumors reached 0.3-0.5 cm in their largest diameter), mice were randomized for tumor size, individually tagged, and assigned to one of six treatment groups: No treatment (negative control) Lintatolimod alone, 100 μg per dose, for four doses Lintatolimod alone, 250 μg per dose, for four doses Anti-PD-L1 mAb alone Lintatolimod plus anti-PD-L1 mAb, 100 μg per dose, for four doses Lintatolimod plus anti-PD-L1 mAb, 250 μg per dose, for four doses Mice were assigned to one of the following treatment groups: 1) Rintatolimod was administered intravenously at 100 or 250 micrograms per dose, repeated four times, five days apart. Anti-PD-L1 mAb (clone 10F.9G2, BioXCell) was administered intraperitoneally at 200 micrograms per dose on days 1 and 3 after each Rintatolimod injection. Tumors were measured three times weekly using a set of calipers to measure two perpendicular diameters and recorded as tumor area. Mice exhibiting ulcerated tumors or tumors greater than 2 cm in diameter (in any direction) were euthanized in accordance with IACUC (Institutional Animal Care and Use Committee) policy.

[0253] Results are presented as tumor size for individual mice throughout the treatment period, mean tumor size within each group, and survival to day 30 (time to euthanasia).

[0254] result: Tumor response at day 30 By day 30, one complete tumor regression was observed in each of the three cohorts receiving anti-PD-L1 mAb. The only cohort that resulted in more than one significant tumor regression was the lintatolimod 250 μg + anti-PD-L1 group. As shown in Table 4, the lintatolimod 250 μg + anti-PD-L1 group had two mice with major partial responses (PRs), which reduced tumor size by 70 and 86% (per RECIST v1.1 criteria), in addition to a complete response (CR).

[0255] Tumor Response Summary: AMPLIGEN® was synergistic with anti-PD-L1, resulting in an enhanced anti-tumor response in the B16 mouse melanoma model.

[0256] The reduction in tumor size was significant for the AMPLIGEN® 250 μg + anti-PD-L1 cohort compared to the anti-PD-L1 cohort alone (p=0.023).

[0257] The addition of AMPLIGEN® to anti-PD-L1 increased the objective response rate three-fold, from 10% with anti-PD-L1 alone to 30% with the combination.

[0258] [Table 4] [Example 4]

[0259] Results from Clinical Trial Demonstrate Positive Effects of AMPLIGEN® on Colorectal Cancer TME Similar to the success in pancreatic cancer described above, the inventors also saw positive results in colorectal cancer. As shown in Figures 4 and 5, a colorectal cancer trial of AMPLIGEN® plus rIFNa-2b and celecoxib demonstrated an increase in the ratio of CXCL10 (C-X-C motif chemokine 10) to CCL22 (C-C motif chemokine ligand 22) within the TME in nine patients with metastatic colorectal cancer compared to historical controls. eff Marker / T reg This was accompanied by an increase in the ratio of markers. Figure 4 depicts a significant improvement (p=0.0015) in the ratio of CXCL10 chemokine (a "benign" C-X-C motif chemokine 10):CCL22 chemokine (a "malignant" C-C motif chemokine ligand 22) in tumor samples compared to similarly collected historical data. See also Figure 5, which depicts the ratio of chemokines and T-cell markers in excised tumors after AMPLIGEN treatment (patients versus historical controls).

[0260] Figure 5 shows that AMPLIGEN® (lintatolimod) has the ability to convert "cold" tumors into "hot" tumors that are much more likely to respond to checkpoint blockade.

[0261] The inventors also found that AMPLIGEN® plus checkpoint blockade extended survival in an animal model for colorectal cancer.

[0262] In a mouse model of colorectal cancer, the combination of AMPLIGEN® plus an anti-mouse PD-1 monoclonal antibody demonstrated a greater than 250% increase in median survival compared to anti-PD-1 alone. See Figure 6. [Example 5]

[0263] bladder cancer Similar to the success in pancreatic cancer and melanoma described above, the inventors also saw positive results in bladder cancer.

[0264] AMPLIGEN® significantly inhibited the growth of human bladder tumor xenografts in nude mice and appeared to act, at least in part, through an immune-enhancing mechanism. [Example 6]

[0265] kidney cancer Similar to the success in pancreatic cancer described above, the inventors have also seen positive results in kidney cancer (also referred to in this disclosure as renal cell carcinoma, renal cell carcinoma, or kidney cancer).

[0266] renal cell carcinoma Regarding the antitumor activity of AMPLIGEN® against human renal cell carcinoma xenografts in nude mice, AMPLIGEN® resulted in statistically significant tumor growth inhibition (p<0.001) and prolonged survival (p<0.002) (Hubbell, 1990).

[0267] Figures 7 and 8 illustrate results for lintatolimod (AMPLIGEN®) administered as monotherapy, confirming its ability to enhance antitumor immune mechanisms and prolong survival. The results indicate that lintatolimod exerts a direct antitumor effect, and that its enhancement of the innate immune response (natural killer cells, also known as NK cells) may play a key role in tumor regression. As shown in Figures 7 and 8, lintatolimod was effective in both inhibiting tumor growth (tumor regression was observed in each mouse) and prolonging survival; 90% of mice treated with lintatolimod were tumor-free, whereas 100% of the control group died following tumor growth. [Example 7]

[0268] Combinatorial immunotherapy of polyI:polyC12U AMPLIGEN® (lintatolimod) and a blocker of programmed death-ligand 1 against established melanoma tumors in a mouse model In this experimental sample, the inventors were able to show that AMPLIGEN® induces anti-tumor synergy when administered with checkpoint blockade. Specifically, the inventors: (1) AMPLIGEN® is synergistic with anti-PD-L1, resulting in an enhanced anti-tumor response in a mouse melanoma model; (2) the anti-tumor effect was significantly greater for the AMPLIGEN® 250 μg + anti-PD-L1 cohort compared with the anti-PD-L1 cohort alone and the AMPLIGEN® 250 μg cohort alone (p=0.023); (3) The addition of AMPLIGEN® to anti-PD-L1 synergistically increased the number of responding tumors that decreased in size as early as day 9 found.

[0269] The study was carried out as follows:

[0270] AMPLIGEN® and anti-PD-L1 antibodies were tested for anti-tumor activity against established subcutaneous B16 melanoma tumors in C57BL / 6 mice. Briefly, mice (10 animals per group) were inoculated with 0.4 x 10 B16-F10 tumor cells in the shaved posterior flank. 6 (i.e., 400,000) cells were inoculated. Seven days later, mice were randomized into six treatment groups: (Group 1) no treatment (negative control); (Group 2) AMPLIGEN® alone at 100 μg per dose for four doses; (Group 3) AMPLIGEN® alone at 250 μg per dose for four doses; (Group 4) anti-PD-L1 mAb alone; (Group 5) AMPLIGEN® plus anti-PD-L1 mAb at 100 μg per dose for four doses; (Group 6) AMPLIGEN® plus anti-PD-L1 mAb at 250 μg per dose for four doses. mAb refers to monoclonal antibody.

[0271] AMPLIGEN® was administered IV at 100 or 250 μg per dose for four doses, five days apart. Anti-PD-L1 mAb was administered IP at 200 μg per dose on days 1 and 3 after each dose of AMPLIGEN®. Tumors were measured three times weekly, measuring two perpendicular diameters using calipers. Mice presenting with ulcerated tumors or tumors greater than 2 cm in diameter were euthanized starting on day 14. This confounded the analysis of tumor size after day 12. Results are presented as tumor size for individual mice throughout the treatment period, up to day 30.

[0272] The data show that by day 9, the AMPLIGEN® 250 μg + anti-PD-L1 cohort resulted in significant tumor regression (70%) compared to the AMPLIGEN® 250 μg alone cohort (0%) and the anti-PD-L1 alone cohort (20%).

[0273] [Table 5]

[0274] Synergy was also seen in tumor size reduction. Briefly, within the AMPLIGEN® 250 μg + anti-PD-L1 cohort, there was a marked increase in the number of tumors that decreased in size.

[0275] [Table 6]

[0276] In conclusion, AMPLIGEN® was synergistic with anti-PD-L1, resulting in an enhanced anti-tumor response in this melanoma model. At both days 9 and 12, the anti-tumor effect was significantly greater for the AMPLIGEN® 250 μg + anti-PD-L1 cohort compared to the anti-PD-L1 cohort alone (p=0.023). Tumor shrinkage was seen at days 9 and 12 with the AMPLIGEN® 250 μg + anti-PD-L1 cohort, progressing to one CR and two PRs by day 30. Thus, the AMPLIGEN® 250 μg + anti-PD-L1 cohort resulted in a 30% overall response rate at day 30, compared to one CR or 10% overall response rate seen with the anti-PD-L1 cohort alone. [Example 8]

[0277] Clinical antitumor responses in patients treated with the combination of Ampligen(tdsRNA) plus checkpoint blockade inhibitors Checkpoint blockade inhibitors, or "checkpoint inhibitors," are molecules that can inhibit or block immune checkpoint proteins, such as PD-1 or PD-L1. Currently, FDA-approved checkpoint inhibitors block CTLA4, PD-1, and PD-L1. The goal of these drugs is to unleash a cellular immune response that attacks and destroys cancer cells. However, currently approved checkpoint inhibitors, such as pembrolizumab and nivolumab, only induce antitumor responses in a minority of patients.

[0278] Thus, one goal of immunotherapy is to reprogram the tumor microenvironment (TME) to convert "cold" (unresponsive) tumors into "hot" tumors that become responsive to checkpoint blockade. Figures 4 and 5 show examples of Ampligen's ability to convert "cold" tumors into "hot" tumors by increasing the ratio of Teff cells:Treg cells within the TME. Figures 1 and 6 and Tables 3, 4, 5, and 6 show examples of Ampligen's ability to synergistically boost the anti-tumor activity of checkpoint inhibitors in animal models.

[0279] Figures 9 and 10 show the ability of treatment with Ampligen plus checkpoint inhibitors to induce clinical responses in patients with triple-negative breast cancer (TNBC) and metastatic recurrent ovarian cancer (MROC), two different types of cancer that do not respond to checkpoint inhibitors as single agents.

[0280] Figures 9A and 9B show CT scan images over time of a woman with a large left-sided breast cancer tumor mass (far right image) before treatment with four cycles of chemokine-modulating therapy using Ampligen plus pembrolizumab. The middle CT scan, taken at the time of treatment, shows that the size of the large tumor mass decreased by 23%. Furthermore, after completion of four cycles of immunotherapy with Ampligen plus pembrolizumab, the entire tumor became necrotic, and dead tumor tissue began to slough off from the chest wall in a dramatic fashion. The far left CT image shows that the size of the tumor mass decreased by more than 97%. In addition, the size of the metastatic breast cancer nodules in the lungs also decreased (Figure 9B), and the pleural effusion also disappeared.

[0281] Pembrolizumab is not approved by the FDA for breast cancer due to its extremely low response rate. In TNBC, the probability of obtaining a clinical response of FDA-approved magnitude using pembrolizumab alone is less than 1%. Ampligen as a single agent has also not shown antitumor activity against breast cancer. Therefore, this is an example of clinical antitumor synergy using Ampligen + checkpoint inhibitor therapy. Furthermore, this was the first patient treated with the combination of Ampligen + checkpoint inhibitor.

[0282] Figures 10A and 10B show a partial anti-tumor response (42% reduction in size) after only two cycles of Ampligen / pembrolizumab / cisplatin in a woman with metastatic recurrent ovarian cancer (MROC). Again, this is the first patient with MROC to be treated with Ampligen plus a checkpoint inhibitor. After four cycles of immunotherapy, this patient achieved a complete remission.

[0283] Pembrolizumab has limited antitumor activity in ovarian cancer and is not approved for this indication. In patients who have relapsed after initial cisplatin chemotherapy, the probability that cisplatin alone would have any significant activity is small. Ampligen was added to this combination in an attempt to induce a synergistic antitumor response, and the induction of a complete response (CR) is evidence of a synergistic anticancer effect.

[0284] While the invention has been described herein in connection with what is considered to be the most practical and preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but on the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. 1. An agent for treating cancer in a subject in need thereof, comprising a combination of a first compound and a second compound as active compounds, administering to a subject a first compound and a second compound, in any order, together or separately; Including, the first compound comprises an effective amount of a checkpoint inhibitor, optionally associated with at least one pharmaceutically acceptable carrier, the checkpoint inhibitor being an anti-PD-1 antibody or an anti-PD-L1 antibody; the second compound is an effective amount of lintatolimod, optionally accompanied by at least one pharmaceutically acceptable carrier; Agent.

2. 2. The agent of claim 1, wherein treating cancer comprises at least one selected from the group consisting of inhibiting tumor growth in a subject; inducing the effect of a checkpoint inhibitor in a subject; enhancing the effect of a checkpoint inhibitor in a subject; prolonging the effect of a checkpoint inhibitor in a subject; and activating a response to a checkpoint inhibitor in a subject.

3. The agent according to claim 1 or 2, wherein the cancer is at least one selected from the group consisting of pancreatic cancer; skin cancer; colorectal cancer; ovarian cancer; melanoma; breast cancer; triple-negative breast cancer; head and neck tumor; bladder cancer; renal cell carcinoma; and lung cancer, and preferably the cancer is selected from pancreatic cancer, colorectal cancer, melanoma, bladder cancer, or renal cell carcinoma.

4. An agent described in any one of claims 1 to 3, wherein lintatolimod forms a complex with a stabilizing polymer.

5. 5. The agent of claim 4, wherein the stabilizing polymer is selected from the group consisting of polylysine; polylysine plus carboxymethylcellulose; polyarginine; polyarginine plus carboxymethylcellulose; and combinations thereof.

6. A method described in any one of claims 1 to 5, wherein the effective amount of lintatolimod is a synergistic therapeutically effective amount.

7. 7. The agent according to any one of claims 1 to 6, wherein the combination of administered lintatolimod and checkpoint inhibitor produces a synergistic effect in treating cancer or inhibiting tumor cell proliferation.

8. The synergistic effect is Prolonging the survival of the subject; Increased target progression time; Inhibition of tumor growth; Induction of tumor cell death; Increased tumor regression; Prevention of tumor recurrence; Prevention of tumor growth; Prevention of tumor spread; Delayed tumor recurrence; Slowing tumor growth; Slowing tumor growth; and Promoting tumor disappearance The agent according to claim 6 or 7, selected from the group consisting of:

9. 9. The agent according to any one of claims 1 to 8, wherein the effective amount of the checkpoint inhibitor is a synergistic therapeutically effective amount.

10. 10. The agent of any one of claims 1 to 9, wherein the administered checkpoint inhibitor provides an additive or synergistic effect in treating cancer or inhibiting tumor growth.

11. The administration is intravenous; Intradermal administration; Subcutaneous administration; 11. The agent according to any one of claims 1 to 10, which is administered intramuscularly; intranasally; intraperitoneally; intracranially; intravesically; orally; or topically.

12. A method according to any one of claims 1 to 11, wherein lintatolimod and a checkpoint inhibitor are administered simultaneously or separately.

13. Lintatolimod and a checkpoint inhibitor are administered separately at different time intervals, 13. The agent of any one of claims 1 to 12, wherein lintatolimod is administered at a frequency selected from the group consisting of once a month, once every three weeks, once every two weeks, once every week, twice a week, three times a week, four times a week, five times a week, six times a week, and daily.

14. Lintatolimod and a checkpoint inhibitor are administered separately, 2 months; 1 month; 3 weeks; 2 weeks; 1 week; 3 days; 1 day; 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, and 30 minutes The agent according to any one of claims 1 to 13, which is administered within a period selected from the group consisting of:

15. The method of claim 14, wherein a second compound comprising lintatolimod is administered intravenously to a subject one to five times weekly at a dosage that averages about 25 to 700 milligrams of lintatolimod per day for up to one month or for a period of more than one month.

15. The agent according to any one of claims 1 to 14.

16. The method of claim 1, wherein a second compound comprising lintatolimod is administered to a subject 1 to 5 times weekly at a dose that provides an average of about 25 to 700 milligrams of tdsRNA per day continuously for at least one month.

16. The agent according to any one of claims 1 to 15.

17. Lintatolimod and a checkpoint inhibitor, together, are used to treat cancer or inhibit the growth of tumor cells, Lintatorimodo alone, Checkpoint inhibitors alone, or Combined lintatolimod alone and checkpoint inhibitor alone 17. The agent according to claim 1, which provides a synergistic effect over the use of

18. The method of any one of claims 1 to 17, wherein the subject is a mammal.

19. The method of any one of claims 1 to 18, wherein the mammal is a human.

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