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Document Type and Number:
WIPO Patent Application WO/2021/199056
Kind Code:
According to one aspect of the invention a composition is provided which may be used for treating Novel Coronavirus patients. The composition comprises (a) cytokine-expressing, proliferation incompetent, whole Novel Coronavirus cells; (b) an anti-PD-1 antibody that specifically binds to human Programmed Death 1 (PD-I); and (c) a TLR (toll like receptor) agonist; wherein the whole Novel Coronavirus cells are formulated with the TLR agonist.

Application Number:
Publication Date:
October 07, 2021
Filing Date:
July 31, 2020
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International Classes:
A61K39/00; A61K39/215; A61P31/12
Domestic Patent References:
Foreign References:
Other References:
DR. KAMAL KANT KOHLI: "Doxycycline and Ivermectin Combo May Be New Effective Covid 19 Treatment", MEDICAL DIALOGUES, 18 May 2020 (2020-05-18), pages 1 - 3, XP009537174
"COVID-19 Registered Trials - and analysis", THE CENTRE FOR EVIDENCE-BASED MEDICINE, 17 March 2020 (2020-03-17)
CERVANTES-BARRAGAN LUISA, ZÜST ROLAND, MAIER REINHARD, SIERRO SOPHIE, JANDA JOZEF, LEVY FREDERIC, SPEISER DANIEL, ROMERO PEDRO, RO: "Dendritic Cell-Specific Antigen Delivery by Coronavirus Vaccine Vectors Induces Long-Lasting Protective Antiviral and Antitumor Immunity", MBIO, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 1, no. 4, 29 October 2010 (2010-10-29), US , pages 1 - 10, XP055936640, ISSN: 2161-2129, DOI: 10.1128/mBio.00171-10
BUKREYEV A, ET AL: "Granulocyte-macrophage colony-stimulating factor expressed by recombinant respiratory syncytial virus attenuates viral replication and increases the level of pulmonary antigen-presenting cells", JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 75, no. 24, 1 December 2001 (2001-12-01), US , pages 12128 - 12140, XP002335337, ISSN: 0022-538X, DOI: 10.1128/JVI.75.24.12128-12140.2001
ZHOU MING, ZHANG GUOQING, REN GUIPING, GNANADURAI CLEMENT W., LI ZHENGUANG, CHAI QINGQING, YANG YANG, LEYSON CHRISTINA M., WU WENX: "Recombinant Rabies Viruses Expressing GM-CSF or Flagellin Are Effective Vaccines for Both Intramuscular and Oral Immunizations", PLOS ONE, vol. 8, no. 5, 20 May 2013 (2013-05-20), pages e63384-1 - e63384-12, XP055936645, DOI: 10.1371/journal.pone.0063384
HUSSAIN BASHA SYED: "Corona virus drugs – a brief overview of past, present and future", JOURNAL OF PEERSCIENTIST, 1 April 2020 (2020-04-01), pages 1 - 16, XP055936647
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1. A composition comprising: a cytokine-expressing, proliferation incompetent, whole Novel Coronavirus cells; an anti~PD~l antibody that specifically binds to human Programmed Death 1 (PD-1); and a TLR (toll like receptor) agonist; wherein the whole Novel Coronavirus cells are formulated with the TLR agonist.

2. The composition of claim 1 wherein the cytokine is GM-CSF (granulocyte money t cell stimulating factor).

3. The composition of claim 1 wherein the whole Novel Coronavirus cells are autologous to the patient.

4. The composition of claim I wherein the whole Novel Coronavirus cells are formulated with a TLR7/8 agonist.

5. The composition of ciaim 1 wherein the whole Novel Coronavirus cells are formulated with a TLR4 agonist.

6. The composition of ciaim 1 wherein the whole Novel Coronavirus cells are formulated with a TLR4 and a TLR7/8 agonist.

7. The composition of claim 1 wherein the TLR. agonist is selected from the group consisting of GLA, R848, and a combination thereof.

8. The composition of claim 1 wherein the Novel Coronavirus cells are melanoma cells.

9. The composition of claim 1 wherein the TLR agonist and whole infected cells are formulated with an emulsion vehicle.

10. The composition of claim 1 wherem the TLR agonist and whole infected cells are formulated with Lipofectamine™.

11. The composition of claim 1 wherem the TLR agonist and whole infected ceils are formulated with a cationic lipid.

12. A method comprising: administering to a Novel Coronavirus patient immunotherapeutic agents: cytokine-expressing, proliferation incompetent, whole Novel Coronavirus cells; an anti-PD-1 antibody that specifically binds to human Programmed Death 1 (PD- 1); and a TL (toll like receptor) agonist; wherein the whole Novel Coronavirus cells are formulated with the TLR agonist.

13. The method of claim 12 wherein the cytokine is GM-CSF (granulocyte moncyte cell stimulating factor),

14. The method of claim 12 wherein the whole Novel Coronavirus ceils are autologous to the patient.

15. The method of claim 12 wherein the Novel Coronavirus cells are formulated with a TLR7/8 agonist.

16. The method of claim 12 wherein the whole cancer ceils are formulated with a TLR4 agonist.

17. The method of claim 12 wherein the whole cancer ceils are formulated with a TLR4 and a TLR7/8 agonist.

18. The method of claim 12 wherein the TLR agonist is selected from the group consisting of GLA, R848, and a combination thereof.

19. The method of claim 12 wherein the Novel Coronavirus cells are melanoma cells.

20. The method of claim 12 wherein the TLR agonist and whole tumor cells are formulated with an emulsion vehicle.

21. The method of claim 12 wherein the TLR agonist and whole tumor cells are formulated with Lipofectamine™.

22. The method of claim 12 wherein the TLR agonist and whole tumor cells are formulated with a cafiomc lipid.

23. A kit comprising agents: cytokine-expressing, proliferation incompetent, Novel Coronavirus cells; an a ti-PD-1 antibody that specifically binds to human Programmed Death 1 (PD-1); and a TLR (toll like receptor) agonist.

24. The kit of claim 23 further comprising instructions for administering and/or formulating the agents.

25. The kit of claim 23 further comprising an emulsion vehicle.

26. The kit of claim 23 further comprising Lipofectamine™.

27. The kit of claim 23 further comprising a cationic lipid.

28. The antiprotozoal Ivermectin with Doxycycline an antibiotic will act as an effective compound for curing COVID - 19

29. The antiprotozoal Ivermectin with Doxycycline an antibiotic including Vitamin C, B6 and E will act as an effective compound for curing COVID - 19"


Field of the Invention

This invention is related to the vaccine of Novel Coronavirus. In particular, it relates to advanced immunotherapy.

Background of the Invention and Related Prior Art

Due to its extensive history of safety as well as availability in multiple tumor types, lethal iy irradiated tumor cell vaccines engineered to secrete GM-CSF (GVAX) is one vaccine platform that has potential for combinatorial therapy with immune checkpoint blockade antibodies. However, local GM-CSF secreted by GVAX can mobilize myeloid precursors into macrophages and dendritic cells, but this cytokine may not induce their activation. Thus, a major limitation of GVAX is in the activation of antigen presenting cells (APC) necessary for optimal tumor antigen presentation in the afferent arm of the immune system. One simple strategy that phenocopies the robust immunological responses seen in vaccines against infectious agents is to combine multiple TLR agonists with a Novel Coronavirus cell - based vaccine. Clinically, multiple adjuvants have been developed for cancer patients to augment the potency of cancer vaccines, and many of these adjuvants are typically TLR agonists.

One real concern with non-targeted TLR stimulation is the procarcmogenic consequences of chronic TLR stimulation in the tumor cells. Stimulation of TLR4 receptors expressed on tumor cells has shown to promote carcinogenesis. TLR signaling in the hematopoietic compartment, however, has been shown to elicit antitumor responses, which have translated into multiple clinical trials. In order to target the dendritic cells in the tumor microenvironment and test whether TLR4 stimulation in the tumor microenvironment can induce a procarcmogenic effect in vivo, our group injected LPS formulated GVAX intratumoraily and found that intratumoral injection of TLR4 ligand absorbed GVAX improved the local anti-tumor response in vivo in three different murine models.

There is a continuing need in the art to obtain safer and more effective treatments of mutating range of Novel Coronaviruses.

The history of human coronaviruses began in 1965 when Tyrrell and Bynoe 1 found that they could passage a virus named B814. It was found in human embryonic tracheal organ cultures obtained from the respiratory tract of an adult with a common cold. The presence of an infectious agent was demonstrated by inoculating the medium from these cultures intranasally in human volunteers; colds were produced in a significant proportion of subjects, but Tyrrell and Bynoe were unable to grow the agent in tissue culture at that time. At about the same time, Hamre and Procknow were able to grow a virus with unusual properties in tissue culture from samples obtained from medical students with colds. Both B814 and Hamre' s virus, which she called 229E, were ether-sensitive and therefore presumably required a lipid-containing coat for infectivity, but these 2 viruses were not related to any known myxo- or paramyxoviruses. While working in the laboratory of Robert Chanock at the National Institutes of Health, McIntosh et al reported the recovery of multiple strains of ether-sensitive agents from the human respiratory tract by using a technique similar to that of Tyrrell and Bynoe. These viruses were termed “OC” to designate that they were grown in organ cultures.

Within the same time frame, Almeida and Tyrrell 4 performed electron microscopy on fluids from organ cultures infected with B814 and found particles that resembled the infectious bronchitis virus of chickens. The particles were medium sized (80-150 nm), pleomorphic, membrane- coated, and covered with widely spaced club-shaped surface projections. The 229E agent identified by Hamre and Procknow and the previous OC viruses identified by McIntosh et al had a similar morphology (Fig. 1).

Coronavirus OC16. Reprinted with permission from Proc Natl Acad Sci USA. 1967;57;933-940.

In the late 1960s, Tyrrell was leading a group of virologists working with the human strains and a number of animal viruses. These included infectious bronchitis virus, mouse hepatitis virus and transmissible gastroenteritis virus of swine, all of which had been demonstrated to be morphologically the same as seen through electron microscopy. 5 6 This new group of viruses was named coronavirus ( corona denoting the crown-like appearance of the surface projections) and was later officially accepted as a new genus of viruses.

Ongoing research using serologic techniques has resulted in a considerable amount of information regarding the epidemiology of the human respiratory coronaviruses. It was found that in temperate climates, respiratory coronavirus infections occur more often in the winter and spring than in the summer and fall. Data revealed that coronavirus infections contribute as much as 35% of the total respiratory viral activity during epidemics. Overall, he proportion of adult colds produced by coronaviruses was estimated at 15%.

In the 3 decades after discovery, human strains OC43 and 229E were studied exclusively, largely because they were the easiest ones to work with. OC43, adapted to growth in suckling mouse brain and subsequently to tissue culture, was found to be closely related to mouse hepatitis virus. Strain 229E was grown in tissue culture directly from clinical samples. The 2 viruses demonstrated periodicity, with large epidemics occurring at 2- to 3-year intervals. 9 Strain 229E tended to be epidemic throughout the United States, whereas strain OC43 was more predisposed to localized outbreaks. As with many other respiratory viruses, reinfection was common. 10 Infection could occur at any age, but it was most common in children.

Despite the extensive focus placed exclusively on strains 229E and OC43, it was clear that there were other coronavirus strains as well. As shown by Bradburne, 11 coronavirus strain B814 was not serologically identical with either OC43 or 229E. Contributing to the various strain differences in the family of coronaviruses, McIntosh et al found that 3 of the 6 strains previously identified were only distantly related to OC43 or 229E.

Epidemiologic and volunteer inoculation studies found that respiratory coronaviruses were associated with a variety of respiratory illnesses; however, their pathogenicity was considered to be low. 2 8 13 14 The predominant illness associated with infections was an upper respiratory infection with occasional cases of pneumonia in infants and young adults. 15 16 These viruses were also shown to be able to produce asthma exacerbations in children as well as chronic bronchitis in adults and the elderly. 17-19

While research was proceeding to explore the pathogenicity and epidemiology of the human coronaviruses, the number and importance of animal coronaviruses were growing rapidly.

Coronaviruses were described that caused disease in multiple animal species, including rats, mice, chickens, turkeys, calves, dogs, cats, rabbits and pigs. Animal studies included, but were not limited to, research that focused on respiratory disorders. Study focus included disorders such as gastroenteritis, hepatitis and encephalitis in mice; pneumonitis and sialodacryoadenitis in rats; and infectious peritonitis in cats. Interest peaked particularly regarding areas of encephalitis produced by mouse hepatitis virus and peritonitis produced by infectious peritonitis virus in cats.

Pathogenesis of these disease states was various and complex, demonstrating that the genus as a whole was capable of a wide variety of disease mechanisms. Human and animal coronaviruses were segregated into 3 broad groups based on their antigenic and genetic makeup. Group I contained virus 229E and other viruses, group II contained virus OC43 and group III was made up of avian infectious bronchitis virus and a number of related avian viruses. 21



Given the enormous variety of animal coronaviruses, it was not surprising when the cause of a very new, severe acute respiratory syndrome, called SARS, emerged in 2002-2003 as a coronavirus from southern China and spread throughout the world with quantifiable speed. 22

24 This virus grew fairly easily in tissue culture, enabling quick sequencing of the genome. Sequencing differed sufficiently from any of the known human or animal coronaviruses to place this virus into a new group, along with a virus that was subsequently cultured from Himalayan palm civets, from which it presumably had emerged. 25 During the 2002-2003 outbreak, SARS infection was reported in 29 countries in North America, South America, Europe and Asia. Overall 8098 infected individuals were identified, with 774 SARS-related fatalities. 26 It is still unclear how the virus entered the human population and whether the Himalayan palm civets were the natural reservoir for the virus. Sequence analysis of the virus isolated from the Himalayan palm civets revealed that this virus contained a 29- nucleotide sequence not found in most human isolates, in particular those involved in the worldwide spread of the epidemic. In the animal viruses, this nucleotide sequence maintains the integrity of the 10th open reading frame (ORF); whereas in the human strains, the absence of this motif results in 2 overlapping ORFs. The function of the ORFs in the animal and human isolates is unknown, and it is unclear whether the deletion of the 29-nucleotide sequence played a role in the transspecies jump, the capacity of the epidemic strain to spread between humans or the virulence of the virus in humans. Curiously data from seroepidemiologic studies conducted among food market workers in areas where the SARS epidemic likely began indicated that 40% of wild animal traders and 20% of individuals who slaughter animals were seropositive for SARS, although none had a history of SARS -like symptoms. These findings suggest that these individuals were exposed through their occupation to a SARS -like virus that frequently caused asymptomatic infection. Infection control policies may have contributed to the halt of the SARS epidemic. The last series of documented cases to date, in April 2004, were laboratory-acquired.

The SARS epidemic gave the world of coronaviruses an enormous infusion of energy and activity that contributed to the large amount already known about the virology and pathogenesis of coronavirus infections from the expanding area of veterinary virology. 21


Coronaviruses are medium-sized RNA viruses with a very characteristic appearance in electron micrographs of negatively stained preparations (Fig. 1). The nucleic acid is about 30 kb long, positive in sense, single stranded and polyadenylated. The RNA is the largest known viral RNA and codes for a large polyprotein. This polyprotein is cleaved by viral-encoded proteases to form the following: an RNA-dependent RNA polymerase and an ATPase helicase; a surface hemagglutinin-esterase protein present on OC43 and several other group II coronaviruses; the large surface glycoprotein (S protein) that forms the petal-shaped surface projections; a small envelope protein (E protein); a membrane glycoprotein (M protein); and a nucleocapsid protein (N protein) that forms a complex with the RNA. The coding functions of several other ORFs are not clear. The strategy of replication of coronaviruses involves a nested set of messenger RNAs with common polyadenylated 3-ends. Only the unique portion of the 5-end is translated. Mutations are common in nature. In addition, coronaviruses are capable of genetic recombination if 2 viruses infect the same cell at the same time. All coronaviruses develop in the cytoplasm of infected cells (Fig. 2). budding into cytoplasmic vesicles from the endoplasmic reticulum. These vesicles are either extruded or released from the cell within the same time frame, and then the cell is destroyed.

Strain 229E in WI-38 cells. Reprinted with permission from J Virol. 1967 ; 1 : 1019-1027.

All group I coronaviruses, including 229E, use human aminopeptidase N as their cellular receptor. Mouse hepatitis virus, a group II coronavirus, uses a member of the carcinoembryonic antigen family as its receptor. 28 The receptor for OC43 is not known, but it may be 1 of several cell surface molecules, including 9-0-acetylated neuraminic acid and the HFA-I molecule. The


SARS coronavirus uses angiotensin-converting enzyme II as its cellular receptor. ’


Since 2003, 5 new human coronaviruses have been discovered (Table 1). Three of these are group I viruses that are closely related and likely represent the same viral species. In 2004, van der Hoek et al reported the discovery of a new human coronavirus, NL63, isolated from a 7- month-old girl with coryza, conjunctivitis, fever and bronchiolitis. Using a novel genomic amplification technique, these investigators were able to sequence the entire viral genome. Phylogenetic analysis demonstrated that this virus was a group I coronavirus related to 229E and transmissible gastroenteritis virus, a virus of pigs. Screening of 614 respiratory specimens collected between December 2002 and April 2003 turned up 7 additional individuals who tested positive for NL63. All had upper or lower respiratory tract disease or both.

Recent Discoveries of Human Coronaviruses

Shortly after, Fouchier et al reported the identification of a coronavirus, named NL, isolated from an 8-month-old boy with pneumonia and grown from a clinical specimen that was obtained in April 1988. Genomic amplification techniques, based on arbitrarily primed reverse transcriptase-polymerase chain reaction (RT-PCR), were used to identify viral sequences. Full genomic sequence analysis of NF showed that this virus was also a group I coronavirus and closely related to NF63. Four of 139 (2.9%) respiratory specimens collected from November 2000 to January 2002 tested positive for NF. Respiratory tract disease was observed in these 4 children whose ages ranged from 3 months to 10 years. The discovery of both NL63 and NL depended on the propagation of the viruses in cell culture.

With the use of molecular probes that targeted conserved regions of the coronavirus genome, months later, Esper et al found evidence of a human respiratory coronavirus in respiratory specimens obtained from children younger than 5 years of age, which was designated the New Haven coronavirus (HCoV-NH). This approach was based on the theory that the gene for the viral replicase of all coronaviruses has conserved genetic sequences that encode indispensable, essential functions and that these sequences could be targeted for virus identification and discovery. This approach did not require propagation of the virus in cell culture, organ cultures or experimental animals and could be performed directly on respiratory secretions. After the initial identification of novel sequences of HCoV-NH, specific probes were used to screen respiratory specimens collected between January 2002 and February 2003 from children younger than 5 years of age whose respiratory specimen tested negative for respiratory syncytial virus, influenza, parainfluenza and adenoviruses. Of 895 children, 79 (8.8%) tested positive for HCoV- NH by RT-PCR, a majority of whom were sampled in the winter and spring seasons. 34 Sequence and phylogenetic analysis based on the replicase gene showed that HCoV-NH was closely related to both NL63 and NL, although the full genomic sequence of HCoV -NH has not been completed. Cough, rhinorrhea and tachypnea were present in more than one -half of the children infected with HCoV-NH. Eleven children were in the newborn intensive care unit at the time of their sampling and had been hospitalized since birth, suggesting either nosocomial infection or a less likely cause of vertical transmission.

One child, a 6-month-old who tested positive for HCoV-NH, also carried a diagnosis of Kawasaki disease, a vasculitis of early childhood. In a subsequent case-control study, 8 of 11 (72.7%) children with Kawasaki disease tested positive for HCoV-NH while only 1 of 22 (4.5%) age- and time-matched controls tested positive for HCoV-NH (P = 0.0015). 6 By correlating these findings, Graf detected the presence of a peptide corresponding to the spike glycoprotein of NL63, the closely related virus identified in the Netherlands, in tissue from individuals with Kawasaki disease. The summation of these findings suggests that HCoV-NH may play a role in the pathogenesis of Kawasaki disease. Further research is necessary to determine whether HCoV-NH is the cause of Kawasaki disease.


In January 2001, a 71 -year-old man who had recently returned from Shen-zhen, China, a previously SARS-endemic area, presented in Hong Kong with a fever and productive cough. Although his SARS screening was negative, a novel group II coronavirus sequence was amplified by RT-PCR from his respiratory specimen with the use of primers that targeted conserved regions of the viral replicase gene. This novel virus, designated HKU1, was genetically distinct from OC43, the other known human group II coronavirus. This virus could not be propagated in cell culture. Seroepidemiologic studies, based on antibodies reacting with a recombinant HKU1 nucleocapsid, suggested that human infection with HKU1 might be common. However, it is unclear whether the enzyme-linked immunosorbent and Western blot assays used to detect HKU1 antibody were also detecting cross-reactive antibody to OC43 or other human coronaviruses.

Summary of the Invention

According to one aspect of the invention a composition is provided which may be used for treating Novel Coronavirus patients. The composition comprises (a) cytokine-expressing, proliferation incompetent, whole Novel Coronavirus cells; (b) an anti-PD-1 antibody that specifically binds to human Programmed Death 1 (PD-I); and (c) a TLR (toll like receptor) agonist; wherein the whole Novel Coronavirus cells are formulated with the TLR agonist.

According to another aspect of the invention a method is provided. Agents are administered to a Novel Coronavirus patient. The agents are : (a) cytokine-expressing, proliferation incompetent, whole Novel Coronavirus cells; (b) an anti-PD-1 antibody that specifically binds to human Programmed Death 1 (PD-1); and (c) a TLR (toll like receptor) agonist; wherein the whole Novel Coronavirus cells are formulated with the TLR agonist. The whole Novel Coronavirus cells are formulated with the TLR agonist.

According to another aspect of the invention a kit is provided which comprises the agents: (a) cytokine-expressing, proliferation incompetent, whole Novel Coronavirus Cells; (b) an anti-PD-1 antibody that specifically binds to human Programmed Death 1 (PD-1); and (c) a TLR (toll like receptor) agonist; wherein the whole Novel Coronavirus cells are formulated with the TLR agonist. Optionally the whole Novel Coronavirus cells are formulated with the TLR agonist.

The field of coronavirology has advanced significantly in recent years. The SARS epidemic was a dramatic reminder that animal coronaviruses are potential threats to the human population, although the exact mechanism of species-to-species spread of the SARS coronavirus remains obscure. NL63 has been identified in many countries. This virus and the related viruses NL and HCoV-NH are likely the cause of a substantial proportion of respiratory tract disease in infants and children. The impact of HKU1 is not yet known. It seems clear that the coronaviruses infecting humans and causing respiratory disease are heterogeneous and quite widely distributed among groups I and II. It may be that some of the newer coronaviruses represent strains similar to the original B814 and OC strains that could not be further characterized in the 1960s. Additional human coronavirus strains will very likely be discovered, which stresses the need for further investigation into the virology and etiology of these infectious organisms. Detailed Description of the Invention

We have developed in vivo evidence that optimally formulated Novel Coronavirus vaccines combined with PD-1 blockade can be used therapeutically for treating Novel Coronavirus. We have collected evidence demonstrating that the combination regimen can be effective agamst established tumors that are poorly immunogenic. All the components of this combinatorial regimen have been individually tested in patients and found to be clinically safe. The disclosed treatment strategy may work by adaptive immune evasion, although applicants do not intend to be bound by any proposed mechanism of action.

[23] Patients having a variety of Novel Coronavirus may be treated with the combination regimen.

This process are used to cure cancers include colorectal cancer, an aero-digestive squamous cancer, a lung cancer, a brain cancer, a liver cancer, a stomach cancer, a sarcoma, a leukemia, a lymphoma, a multiple myeloma, head-and-neck cancer, an ovarian cancer, cervical cancer, a uterine cancer, a breast cancer, a melanoma, a prostate cancer, a pancreatic carcinoma, and a renal carcinoma. This list is meant to be illustrative rather than limiting. [24] Whole cancer ceils may be allogeneic, syngeneic, or autologous to the treatment recipient. Typically they may be treated to make them proliferation incompetent by a technique which preserves preserve their immunogenicity and their metabolic activity. One typically used technique is irradiation. Such ceils. Typically the same general type of tumor cell is used that the patient bears. For example, a patient suffering from melanoma will typically be administered proliferation incompetent melanoma cells. The cells may express and secrete a cytokine naturally or by transfection with a nucleic acid which directs such expression and secretion. One suitable cytokine is GM-CSF. For example, the tumor cell may express a transgene encoding GM-CSF as described in U.S. Pat. Nos. 5,637,483, 5,904,920, 6,277,368 and 6,350,445, as well as in US Patent Publication No. 20100150946, each of which is expressly incorporated by reference. One example of a GM-CSF- expressing, genetically modified cancer ceil for the treatment of pancreatic cancer is described in U.S. Pat, Nos. 6,033,674 and 5,985,290, both of which are expressly incorporated by reference herein. Other cytokines can be used. Suitable cytokines which may be used include cytokines which stimulate dendritic ceil induction, recruitment, and/or maturation. Such cytokines include, but are not limited to, one or more of GM-CSF, CD40 ligand, IL-12, CCL3, CCL20, and CCL21. Granulocyte- macrophage colony stimulating factor (GM-CSF) polypeptide is a cytokine or fragment having immunomodulatory activity and having at least about 85% amino acid sequence identity to GenBank Accession No. AAA52122.1.

[25] According to one alternative embodiment, cytokines are delivered by inactivated bystander cells which express and secrete one or more cytokines. The bystander cells may provide all of the cytokines which stimulate dendritic cell induction, recruitment, and/or maturation, or may supplement cytokines secreted by the inactivated tumor ceils. Immunomodulatory cytokine expressing bystander cell lines are described in U.S. Pat. Nos. 6,464,973, and 8,012,469, Dessureault et al., Ann. Surg. Oncol. 14: 869-84, 2007, and Eager and Nermmaitis, Mol. Ther. 12: 18-27, 2005, each of which is expressly incorporated by reference.

[26] Antibodies which are suitable for use in the treatment regimen and compositions and kits include any which specifically bind to Programmed Death 1 (PD-1). Exemplary types of antibodies which may be employed include without limitation human, humanized, chimeric, monolclonal, polyclonal, single chain, antibody binding fragments, and diabodies. Typically antibodies are substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof. Antibodies are capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W.E, Paul, ed,, Raven Press, N.Y. (1993); Wilson (1994; J. Immunol, Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys, Methods 25:85-97. An antibody typically specifically binds to an antigen or epitope. Specific binding occurs to the corresponding antigen or epitope even in the presence of a heterogeneous population of proteins and other biologies. Specific binding of an antibody indicates that it binds to its target antigen or epitope with an affinity that is substantially greater than binding to irrelevant antigens The relative difference in affinity is often at least 25% greater, more often at least 50% greater, most often at least 100%. The relative difference can be at least 2x, at least 5x, at least lOx, at least 25x, at least 50x, at least lOOx, at least IGOOx, for example.

Toll like receptors (TLR) are a family of proteins that sense a microbial product and/or initiates an adaptive immune response. TLR activate a dendritic cell (DC). TLRs are conserved membrane spanning molecules containing an ectodomain of leucine -rich repeats, a transmembrane domain and an intracellular TIR. (Toil/IL-IR) domain. TLRs recognize distinct structures in microbes, often referred to as "PAMPs" (pathogen associated molecular patterns). Ligand binding to TLRs invokes a cascade of intra-cellular signaling pathways that induce the production of factors involved in inflammation and immunity.

Exemplary agonists which may be used for these receptors include, without limitation lipoproteins, lipopolypeptides, peptidoglycans, zymosan, lipopolysaccharide, neisseria! porins, flageilin, profiilin, galactoceramide, muramyl dipeptide, giucopyranosyl lipid A (GLA), and resiquimod (R848). Peptidoglycans, lipoproteins, and lipoteichoic acids are cell wall components of Gram-positive. Lipopolysaccharides are expressed by most bacteria. Flageilin is the structural component of bacterial flagella that is secreted by pathogenic and commensal bacterial . A Galactosylcerami.de (a-GalCer) is an activator of natural killer T (NKT) cells. Muramy! dipeptide is a bioactive peptidoglycan motif common to all bacteria. Such agonists mediate innate immune activation via Toll-like Receptors. Specific binding of an agonist for its cognate receptor is often expressed in terms of an affinity. The ligands of the present invention may bind with affinities of between about 10 4 M 1 and about 10 s M Affinity is calculated as ¾ = ko ff /k c ,, (koi- f is the dissociation rate constant, 035 is the association rate constant and K4 is the equilibrium constant). Single or multiple agonists may be used.

In humans, ten TLR have been identified. TLRs that are expressed on the surface of cells include TLR- 1,-2, -4, -5, and -6, while TLR-3, -7/8, and -9 are expressed with the ER compartment. Human dendritic cell subsets can be identified on the basis of distinct TLR expression patterns. By way of example, the myeloid or "conventional" subset of DC (mDC) expresses TLRs 1-8 when stimulated, and a cascade of activation markers (e.g. CD80, CD86, MHC class I and II, CCR7), pro-inflammatory cytokines, and chemokmes are produced. A result of this stimulation and resulting expression is antigen-specific CD4+ and CD8+ T cell primmg. These DCs acquire an enhanced capacity to take up antigens and present them in an appropriate form to T ceils. In contrast, the plasmacytoid subset of DC (pDC) expresses only TLR7 and TLR9 upon activation, with a resulting activation of NK cells as well as T-eells. As dying tumor cells may adversely affect DC function, it has been suggested that activating DC with TLR agonists may be beneficial for priming anti-tumor immunity in an immunotherapy approach to the treatment of cancer. It has also been suggested that successful treatment of breast cancer using radiation and chemotherapy requires TLR4 activation.

TLR agonists known in the art and useful in the present invention include, but are not limited to, the following:

Pam3Cys, a TLR- 1/2 agonist;

CLA, a TLR -2 agonist;

MALP2, a TLR-2 agonist; Pam2Cys, a TL -2 agonist;

LSL-1, a TLR-2 agonist; Hib-OMPC, a TLR-2 agonist; polyribosinic ;polyribocytidic acid (Poly I:C), a TLR-3 agonist; polyadenosine -polyuridylie acid (poly AU), a TLR-3 agonist;

Polyinosinie-Polycytidylic acid stabilized with poiy-L-lysine and carboxymethylcelitilose (Hiltonol©), a TLR-3 agonist; monophosp oryl lipid A (MPL), a TLR-4 agonist;

LPS, a TLR-4 agonist; bacterial flagelim, a TLR-5 agonist; sialyl-Tn (STn), a carbohydrate associated with the MUC1 mucin on a number of h uman cancer cells and a TLR-4 agonist; imiquimod, a TLR-7 agonist; resiquimod, a TLR-7/8 agonist; loxoribine, a TLR-7/8 agonist; and unmethyiated CpG dinucieoti.de (CpG-ODN), a TLR-9 agonist. Lormulation of the Novel Coronavirus cells with the TLR agonist appears to be a contributing factor to enhanced efficacy. Lormulations can be incubated together for periods of times such as ¼, ½, 1, 2, 3, 5, 10, 24 hours, at temperatures such as 4 degrees C. Alternatively, binding in the presence of a lipophilic agent or an emulsifying agent can be employed. Such agents are well known in the art.

V arious dosing schedules may be envisioned, with simultaneous or staggered timing, with single or multiple agents, single cycle or multiple cycles.

Methods of administering treatment agents to cancer patients vary. Exemplary methods include without limitation subcutaneous, intravenous, intramuscular, intraarterial, intradermal, intrathecal, intratumoral, intraperitoneal, sublingual, and epidural administrations. Administration may be to a human, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, or experimental subject. Typically an agent such as an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition is contacted with the subject in an appropriate anatomical location. Administration may be for the purposes of therapy, pharmacokinetic study, diagnostic assay, research, placebo, or experimental method. Agents according to the invention may be, but not need not be, administered as a single composition. Although administration as a single composition is contemplated by the present invention, agents may be delivered to a single subject as separate administrations, which may be at the same or different time, and which may be by the same route or different routes of administration. In some cases, the agents may in fact contact each other within the subject's body, forming a composition in vivo.

The above disclosure generally describes the present invention. All. references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.