TUMOR IMMUNOTHERAPY USING SINDBIS VIRAL VECTORS AND AGONIST MONOCLONAL ANTIBODIES

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 3, 2020, is named “27522-0225PCT Sequence Listing_ST25.txt” and is 34 kilobytes in size.

FIELD OF THE INVENTION

The present disclosure describes compositions and methods directed to treating cancer where the compositions include utilizing oncolytic viruses, such as Sindbis virus, and antibodies directed against a co-stimulatory molecule or to an immunesystem agonist molecule, such as OX-40 and 4-1BB (CD137).

BACKGROUND OF THE INVENTION

Immune checkpoint modulation has shown remarkable promise in treating cancer. Although, high response rates with immune checkpoint blockade have been documented in patients with highly immunogenic tumors, often the proportion of patients that respond to treatment is still low. Major challenges to overcome are the lack of T cell infiltration into the tumor microenvironment as well as the immunosuppressive nature of the tumor, which inhibits the intratumoral immune response. Further, tumors tend to quickly escape the immune response by mutating or losing the expression of drug targets or tumor antigens targeted by the immune response. Thus there is a need in the art for compositions and methods that overcome these limitations. The present disclosure addresses these needs.

SUMMARY OF THE INVENTION

The present disclosure provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of (a) a oncolytic viral vector and (b) an antibody directed against a co-stimulatory molecule or a nucleic acid encoding same; or an antibody to an immunesystem agonist molecule or a nucleic acid encoding same.

The oncolytic viral vector can be a Sindbis viral vector. The Sindbis viral vector can be replication defective. The Sindbis viral vector can comprise at least one nucleic acid encoding a therapeutic protein. The Sindbis viral vector can comprise at least one nucleic acid encoding an immunostimulatory or an immunomodulatory protein. The immunostimulatory or immunomodulatory protein can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, or any combination thereof. In a preferred aspect, the immunostimulatory or immunomodulatory protein is IL-12. The Sindbis viral vector can comprise at least one nucleic acid encoding LacZ, Flue or GFP.

The antibody can be an anti-OX40 antibody, an anti-4-1BB antibody, an anti-CD28 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-CD37 antibody, an anti-HVEM antibody, or a combination thereof.

The Sindbis viral vector and the antibody can induce an immune response in a tumor associated antigen (TAA) nonspecific manner. The induced and nonspecific immune response can be a first immune response. The first immune response can be followed by a secondary immuneresponse. The secondary immune response can be the result of one or more TAAs released from the dead tumor cells. The secondary immune response can comprise memory T cells directed against one or more TAAs released from the dead tumor cells.

The present disclosure also provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of (a) a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and (b) an anti-OX40 monoclonal antibody or a nucleic acid encoding same, thereby treating cancer in the subject.

The Sindbis viral vector can be replication defective. The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 and can further comprise the nucleic acid encoding the anti-OX40 monoclonal antibody. The method can comprise administering a Sindbis viral vector comprising the nucleic acid encoding interleukin-12 and administering a Sindbis viral vector comprising the nucleic acid encoding the anti-OX40 monoclonal antibody. The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit (IL-12 α, IL-12, p35 subunit) and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit). The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit of GenBank accession no. M86672 and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit) GenBank accession no. M86671. The nucleic acid encoding interleukin-12 alpha subunit comprises the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid encoding interleukin-12 beta subunit comprises the nucleic acid sequence of SEQ ID NO: 2.

The Sindbis viral vector can comprise a nucleic acid encoding interleukin-12 alpha subunit of amino acid sequence of GenBank accession no. AAA39292.1 and comprise a nucleic acid encoding interleukin-12 beta subunit of amino acid sequence of GenBank accession no. AAA39296.1. The amino acid sequence of the interleukin-12 alpha subunit is of amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the interleukin-12 beta subunit is of amino acid sequence of SEQ ID NO: 4.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 5. The nucleic acid sequence encoding the anti-OX40 variable heavy chain is SEQ ID NO: 6. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 variable light chain comprising the amino acid sequence of SEQ ID NO: 7. The nucleic acid sequence encoding the anti-OX40 variable light chain is SEQ ID NO: 8.

The Sindbis viral vector can comprise a nucleic acid encoding a mouse anti-OX40 heavy chain of amino acid sequence of SEQ ID NO; 5 and a nucleic acid encoding a mouse anti-OX40 light chain of amino acid sequence of SEQ ID NO: 7. The Sindbis viral vector can comprise a nucleic acid encoding a mouse anti-OX40 heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 5 and a nucleic acid encoding a mouse anti-OX40 light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 7.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 9. The nucleic acid sequence encoding the an anti-OX40 antibody heavy chain is SEQ ID NO: 10.

The Sindbis viral vector can comprise a nucleic acid encoding a mouse anti-OX40 antibody light chain comprising the amino acid sequence of SEQ ID No: 11. The nucleic acid sequence encoding the anti-OX40 antibody light chain is SEQ ID NO: 12.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain with an amino acid sequence of SEQ ID NO: 9 and an anti-OX40 antibody light chain with an amino acid sequence of SEQ ID NO: 11. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain of SEQ ID NO: 10 and an anti-OX40 antibody light chain with an amino acid sequence of SEQ ID NO: 12.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain with an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9 and an anti-OX40 antibody light chain with an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10 and an anti-OX40 antibody light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12.

The Sindbis viral vector can comprise the nucleic acid encoding an anti-OX40 antibody heavy chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody variable heavy chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody variable light chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise the nucleic acid encoding an anti-OX40 antibody light chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody variable heavy chain amino acid sequence, and an anti-OX40 antibody variable light chain amino acid sequence, that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain amino acid sequence, and an anti-OX40 antibody light chain amino acid sequence, that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 31. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain is SEQ ID NO: 32. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence of SEQ ID NO: 33. The nucleic acid sequence encoding the human anti-OX40 variable light chain is SEQ ID NO: 34.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 32. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain is SEQ ID NO: 36. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence of SEQ ID NO: 37. The nucleic acid sequence encoding the human anti-OX40 variable light chain is SEQ ID NO: 38.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 36. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 39. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain is SEQ ID NO: 40. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence of SEQ ID NO: 41. The nucleic acid sequence encoding the human anti-OX40 variable light chain is SEQ ID NO: 42.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 40. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 42.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain is SEQ ID NO: 44. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence of SEQ ID NO: 45. The nucleic acid sequence encoding the human anti-OX40 variable light chain is SEQ ID NO: 46.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 44. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence of SEQ ID NO: 47. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain is SEQ ID NO: 48. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence of SEQ ID NO: 49. The nucleic acid sequence encoding the human anti-OX40 variable light chain is SEQ ID NO: 50.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 48. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50.

The Sindbis viral vector and the anti-OX40 monoclonal antibody can be administered sequentially or concurrently. The Sindbis viral vector can be administered systemically. The anti-OX40 monoclonal antibody can be administered systemically. Both the Sindbis viral vector and the anti-OX40 monoclonal antibody, or a nucleic acid encoding same, can be administered systemically. The Sindbis viral vector can be administered parenterally. The anti-OX40 monoclonal antibody can be administered parenterally. Both the Sindbis viral vector and the anti-OX40 monoclonal antibody, or a nucleic acid encoding same, can be administered parenterally. The Sindbis viral vector can be administered intraperitoneally. The anti-OX40 monoclonal antibody can be administered intraperitoneally. Both the Sindbis viral vector and the anti-OX40 monoclonal antibody, or a nucleic acid encoding same, can be administered intraperitoneally.

An antibody of the present disclosure, or a fragment thereof, can be derived from any species, including, but not limited to, a human, a mouse, a rat, a hamster, a dog, a rabbit, a frog, a sheep, a goat, a cow, a horse, a pig, a bird, a donkey, a chicken, a camel, a llama, a dromedary, an alpaca, a shark, a bovine and a turtle. In some aspects, an antibody of the present disclosure, or a fragment thereof, is derived from a human. In some aspects, an antibody of the present disclosure, or a fragment thereof, is derived from a mouse. In some aspects, an antibody of the present disclosure, or a fragment thereof, is derived from a camel, a llama or an alpaca. In some aspects, an antibody of the present disclosure, or a fragment thereof, is derived from a shark. In some aspects, an antibody of the present disclosure, or a fragment thereof, of the present disclosure is a chimeric antibody that is derived from two or more of the aforementioned species. In a non-limiting example, an antibody of the present disclosure, or fragment thereof, can be a chimeric antibody that is derived from a human and a mouse. In some aspects, an antibody of the present disclosure, or a fragment thereof, can be derived from any species other than human and can be further humanized using standard methods known in the art as to reduce the immunogenicity of the antibody.

A “monoclonal antibody” as disclosed herein, can be a full-length antibody or an antigen binding fragment thereof, wherein the “antigen binding fragment” is a fragment of the full length antibody that retains binding to the target antigen of the said monoclonal antibody. For example, a monoclonal antibody against OX-40 or an anti-OX40 monoclonal antibody, as described herein, can be a full length antibody against OX40 antibody or an antigen binding fragment thereof, wherein the fragment retains binding to the OX40 receptor on a cell surface. An “antigen-binding fragment” of an anti-OX-40 antibody, as described herein can include any fragment selected from the group consisting of Fv, Fav, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments.

The cancer can be a solid cancer or a liquid/hematologic cancer. The cancer can comprise metastatic cancer. The cancer can comprise a solid tumor. The cancer can be a carcinoma, a lymphoma, a blastoma, a sarcoma, a leukemia, a brain cancer, a breast cancer, a blood cancer, a bone cancer, a lung cancer, a skin cancer, a liver cancer, an ovarian cancer, a bladder cancer, a renal cancer, a gastric cancer, a thyroid cancer, a pancreatic cancer, an esophageal cancer, a prostate cancer, a cervical cancer or a colorectal cancer. In one preferred aspect, the cancer is colon cancer. In one preferred aspect, the cancer is prostate cancer. In one preferred aspect, the cancer is ovarian cancer.

The present disclosure further provides a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and a nucleic acid encoding an anti-OX40 monoclonal antibody. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and (b) an anti-OX40 monoclonal antibody or a nucleic acid encoding same. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and (b) a Sindbis viral vector comprising a nucleic acid encoding an anti-OX40 monoclonal antibody.

The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit (IL-12 α, IL-12, p35 subunit) and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit). The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit of GenBank accession no. M86672 and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit) GenBank accession no. M86671. The nucleic acid encoding interleukin-12 alpha subunit comprises the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid encoding interleukin-12 beta subunit comprises the nucleic acid sequence of SEQ ID NO: 2.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 36. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 40. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 42.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 44. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 48. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50.

The present disclosure also provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1, thereby treating cancer in the subject. The present disclosure also provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and a nucleic acid encoding NY-ESO-1, thereby treating cancer in the subject.

The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of NCBI Reference accession no. NM_001327.1. The nucleic acid encoding NY-ESO-1 comprises the nucleic acid sequence of SEQ ID NO: 14. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of amino acid sequence of NCBI Reference accession no. NP_001318.1. The amino acid sequence of the NY-ESO-1 comprises the amino acid sequence of SEQ ID NO: 15.

The Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and the Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 can be administered sequentially or concurrently. The Sindbis viral vector comprising a nucleic acid encoding interleukin-12 can be administered systemically. The Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 can be administered systemically. Both the Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and the Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 can be administered systemically. The Sindbis viral vector comprising a nucleic acid encoding interleukin-12 can be administered parenterally. The Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 can be administered parenterally. Both the Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and the Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 can be administered parenterally. The Sindbis viral vector comprising a nucleic acid encoding interleukin-12 can be administered intraperitoneally. The Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 can be administered intraperitoneally. Both the Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and the Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 can be administered intraperitoneally.

The present disclosure further provides a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and a nucleic acid encoding NY-ESO-1. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding interleukin-12 and (b) a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1.

The present disclosure provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of (a) a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 and (b) an anti-OX40 monoclonal antibody or a nucleic acid encoding same, thereby treating cancer in the subject.

The Sindbis viral vector can be replication defective. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 and can further comprise the nucleic acid encoding the anti-OX40 monoclonal antibody. The method can comprise administering a Sindbis viral vector comprising the nucleic acid encoding NY-ESO-1 and administering a Sindbis viral vector comprising the nucleic acid encoding the anti-OX40 monoclonal antibody. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of NCBI Reference accession no. NM_001327.1. The nucleic acid encoding NY-ESO-1 comprises the nucleic acid sequence of SEQ ID NO: 14. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of amino acid sequence of NCBI Reference accession no. NP_001318.1. The amino acid sequence of the NY-ESO-1 comprises the amino acid sequence of SEQ ID NO: 15.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 36. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 40. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 42.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 44. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 48. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50.

The present disclosure further provides a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 and a nucleic acid encoding an anti-OX40 monoclonal antibody. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 and (b) an anti-OX40 monoclonal antibody or a nucleic acid encoding same. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 and (b) a Sindbis viral vector comprising a nucleic acid encoding an anti-OX40 monoclonal antibody. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of NCBI Reference accession no. NM_001327.1. The nucleic acid encoding NY-ESO-1 comprises the nucleic acid sequence of SEQ ID NO: 14. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of amino acid sequence of NCBI Reference accession no. NP_001318.1. The amino acid sequence of the NY-ESO-1 comprises the amino acid sequence of SEQ ID NO: 15.

A “monoclonal antibody” as disclosed herein, can be a full-length antibody or an antigen binding fragment thereof, wherein the “antigen binding fragment” is a fragment of the full length antibody that retains binding to the target antigen of the said monoclonal antibody. For example, a monoclonal antibody against OX-40 or an anti-OX40 monoclonal antibody, as described herein, can be a full length antibody against OX40 antibody or an antigen binding fragment thereof, wherein the fragment retains binding to the OX40 receptor on a cell surface. An “antigen-binding fragment” of an anti-OX40 antibody, as described herein can include any fragment selected from the group consisting of Fv, Fav, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments.

The present disclosure also provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount (a) a Sindbis viral vector and (b) an anti-4-1BB (CD137) monoclonal antibody or a nucleic acid encoding same, thereby treating cancer in the subject. The present disclosure further provides in vitro or ex vivo methods for treating cancer or assessing the treatment of cancer in a subject comprising contacting a biological sample from the subject with (a) a Sindbis viral vector and (b) an anti-4-1BB monoclonal antibody or a nucleic acid encoding same. The Sindbis viral vector does not comprise an endogenous nucleic acid encoding any protein.

The Sindbis viral vector is replication defective. The Sindbis viral vector can comprise a nucleic acid sequence encoding a therapeutic protein, an immunostimulatory protein or an immunomodulatory protein. The Sindbis viral vector can comprise the nucleic acid encoding the therapeutic protein, an immunostimulatory protein or an immunomodulatory protein and further comprise the nucleic acid encoding the anti-4-1BB monoclonal antibody. The Sindbis viral vector can comprise a nucleic acid sequence encoding LacZ (lac operon structural gene lacZ encoding β-galactosidase), Flue (firefly luciferase) or GFP (green fluorescent protein). The Sindbis viral vector can comprise the nucleic acid encoding LacZ, Flue or GFP and further comprise the nucleic acid encoding the anti-4-1BB monoclonal antibody.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1BB antibody heavy chain comprising the heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively. The Sindbis viral vector can comprise the nucleic acid encoding an anti-4-1BB antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 19. The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1BB antibody heavy chain comprising the light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively. The Sindbis viral vector can comprise the nucleic acid encoding an anti-4-1BB antibody light chain comprising the amino acid sequence of SEQ ID NO: 23.

The Sindbis viral vector can comprise the nucleic acid encoding anti-4-1BB antibody heavy chain CDR1, CDR2 and CDR3 amino acid sequences that binds to a target antigen of the amino acid sequence of SEQ ID NO: 24. The Sindbis viral vector can comprise the nucleic acid encoding anti-4-1BB antibody light chain CDR1, CDR2 and CDR3 amino acid sequences that binds to a target antigen of the amino acid sequence of SEQ ID NO: 24. The Sindbis viral vector can comprise the nucleic acid encoding anti-4-1BB antibody heavy chain amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 24. The Sindbis viral vector can comprise the nucleic acid encoding anti-4-1BB antibody light chain amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 24.

The Sindbis viral vector and the anti-4-1BB monoclonal antibody can be administered sequentially or concurrently. The Sindbis viral vector can be administered systemically. The anti-4-1BB monoclonal antibody can be administered systemically. Both the Sindbis viral vector and the anti-4-1BB monoclonal antibody, or a nucleic acid encoding same, can be administered systemically. The Sindbis viral vector can be administered parenterally. The anti-4-1BB monoclonal antibody can be administered parenterally. Both the Sindbis viral vector and the anti-4-1BB monoclonal antibody, or a nucleic acid encoding same, can be administered parenterally. The Sindbis viral vector can be administered intraperitoneally. The anti-4-1BB monoclonal antibody can be administered intraperitoneally. Both the Sindbis viral vector and the anti-4-1BB monoclonal antibody, or a nucleic acid encoding same, can be administered intraperitoneally.

A “monoclonal antibody” as disclosed herein, can be a full-length antibody or an antigen binding fragment thereof, wherein the “antigen binding fragment” is a fragment of the full length antibody that retains binding to the target antigen of the said monoclonal antibody. For example, a monoclonal antibody against 4-1BB or an anti-4-1BB monoclonal antibody, as described herein, can be a full length antibody against 4-1BB antibody or an antigen binding fragment thereof, wherein the fragment retains binding to the 4-1BB receptor on a cell surface. An “antigen-binding fragment” of an anti-4-1BB antibody, as described herein can include any fragment selected from the group consisting of Fv, Fav, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments.

The cancer can be a solid cancer or a liquid/hematologic cancer. The cancer can comprise metastatic cancer. The cancer can comprise a solid tumor. The cancer can be a carcinoma, a lymphoma, a blastoma, a sarcoma, a leukemia, a brain cancer, a breast cancer, a blood cancer, a bone cancer, a lung cancer, a skin cancer, a liver cancer, an ovarian cancer, a bladder cancer, a renal cancer, a gastric cancer, a thyroid cancer, a pancreatic cancer, an esophageal cancer, a prostate cancer, a cervical cancer or a colorectal cancer. In one preferred aspect, the cancer is a lymphoma. In one preferred aspect, the cancer is a B cell lymphoma.

The present disclosure provides a Sindbis viral vector comprising a nucleic acid encoding a therapeutic protein, an immunostimulatory protein or an immunomodulatory protein and a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure provides a Sindbis viral vector comprising a nucleic acid encoding encoding LacZ, Flue or GFP and a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding a therapeutic protein, an immunostimulatory protein or an immunomodulatory protein and (b) a Sindbis viral vector comprising a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding LacZ, Flue or GFP and (b) a Sindbis viral vector comprising a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure further provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding a therapeutic protein, an immunostimulatory protein or an immunomodulatory protein and (b) an anti-4-1BB monoclonal antibody or a nucleic acid encoding same. The present disclosure provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding encoding LacZ, Flue or GFP and (b) an anti-4-1BB monoclonal antibody or a nucleic acid encoding same.

A “monoclonal antibody” as disclosed herein, can be a full-length antibody or an antigen binding fragment thereof, wherein the “antigen binding fragment” is a fragment of the full length antibody that retains binding to the target antigen of the said monoclonal antibody. For example, a monoclonal antibody against 4-1BB or an anti-4-1BB monoclonal antibody, as described herein, can be a full length antibody against 4-1BB antibody or an antigen binding fragment thereof, wherein the fragment retains binding to the 4-1BB receptor on a cell surface. An “antigen-binding fragment” of an anti-4-1BB antibody, as described herein can include any fragment selected from the group consisting of Fv, Fav, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments.

Any of the above aspects can be combined with any other aspect.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-1D. SV.IL12 induces a modest therapeutic efficacy and increases OX40 expression on CD4 T cells. FIG. 1A depicts treatment schema. BALB/c mice were given intraperitoneal (i.p.) injections of SV, IL-12 (50 ng), or SV.IL12 at various times after injection of 7×10⁴CT.26.Fluc on day 0. FIG. 1B depicts survival plots of control and treated mice bearing CT26.Fluc tumors. The x-axis shows days of treatment and y-axis shows percentage survival. Statistical significance between SV.IL12 and all other groups was determined with the Mantel-Cox test. Results are representatives of at least two independent experiments. FIG. 1C-D depict effect on OX40 expression of treatment of CT26 tumor-bearing mice with SV, IL-12 (50 ng), or SV.IL12 on 4 consecutive days (days 1, 2, 3, and 4). On day 7, spleens were excised and a single-cell suspension was stained and analyzed by flow cytometry. As controls, naive and untreated (control) tumor-bearing mice were used. FIG. 1C depicts percentage of OX40 expression by CD4 T cells (left), regulatory T cells (TREG; middle), and CD8 T cells (right). The x-axis shows the various treatment groups and the y-axis shows percentage of OX40+ cells.

FIG. 1D depicts representative flow cytometry plots indicating OX40 staining in different T cell subsets. Bars represent means and each symbol represents an individual mouse. Statistical significance was determined with the Kruskal-Wallis test followed by the Dunns' test or the Mann-Whitney test. Results are representatives of at least two independent experiments.

FIG. 2A-2C. SV infects monocytes/macrophages in mediastinal lymph nodes and quickly activates T cells. FIG. 2A depicts that tumor free mice were treated i.p. with SV expressing the firefly luciferase (Fluc) protein. 4 hours later, bioluminescent images were taken to monitor Fluc expression from SV. To determine the source of the signal, the mediastinal lymph nodes (LN) and adipose tissue were extracted and imaged separately. FIG. 2B depicts percentage of GFP expression by Ly6G-CD11b+F4/80+ cells. FIG. 2C depicts percentage of CD69 expression by CD4 (left graph) and CD8 (right graph) T cells. Tumor free mice were treated i.p. with SV expressing GFP for 4 consecutive days. On day 5, mediastinal and inguinal LN were extracted and a single cell suspension was stained and analyzed by flow cytometry. As control, naïve mice were used. Statistical significance was determined with the Mann-Whitney test. Results are representatives of at least two independent experiments.

FIG. 3A-3D. IL-12 and IFN-γ production derived from SV.IL12 infection. FIG. 3A depicts IL-12 levels in supernatant of infected cells measured by ELISA. 5×10⁵MyC-CaP cells were infected with SV.IL12 at various MOI (10; 1; 0′1) for 2 hours. As control, MyC-CaP cells were infected with SV or left uninfected (mock). SV was washed away and replaced with fresh media. After 24 hours incubation, supernatant was collected and IL-12 was measured by ELISA. FIG. 3B depicts IL-12 levels in plasma was measured by ELISA. FIG. 3C depicts percentage of Tbet expression by CD4 T cells in cell suspensions from mediastinal LN stained and analyzed by flow cytometry. FIG. 3D depicts IFNγ enzyme-linked immunospot analysis of splenocytes from control and treated mice as indicated (n=3-10 mice per group). Tumor bearing mice were treated with SV.IL12 on 4 consecutive days (days 1; 2; 3; 4). As control, naïve, untreated (control), IL-12 (50 ng) and SV treated mice were used. On day 7, plasma, spleen and mediastinal LN were collected from each mice. Statistical significance was determined with the Kruskal-Wallis test followed by the he Dunns' test or Mann-Whitney test. Results are representatives of at least two independent experiments.

FIG. 4A-4B. SV infectivity of MyC-CaP.Fluc tumors. MyC-CaP.Fluc and CT.26.Fluc cells were challenged with serially diluted single round replication SV.GFP (10⁻¹-10⁻⁴) and incubated overnight. 16 hours post infection the percentage of GFP-positive cells was analyzed by flow cytometer for each dilution. FIG. 4A depicts representative flow cytometry plots of GFP positive MyC-CaP.Fluc and CT.26.Fluc cells per dilution and uninfected controls are shown. FIG. 4B depicts plotted infectivity curve of GFP-positive cells.

FIG. 5A-5E. SV.IL12 in combination with anti-OX40 antibody cures established tumors in vivo. FIG. 5A depicts the experimental protocol for the prostate and colon cancer model. FVB/NJ or BALB/c mice were given an i.p. injection of SV.IL12 and/or anti-OX40 at various times after injection of 10⁵MyC-CaP.Fluc or 7×10⁴CT26.Fluc cells on day 0, respectively. FIG. 5B depicts CT26.Fluc tumor growth curves shown as fold changes relative to the luminescence on day 0 of the same mouse. Each line represents an individual mouse. Left graphs: control (n=14) (top) and SV.IL12 (n=20) (bottom). Right graphs: anti-OX40 (n=10) (top) and SV.IL12+anti-OX40 (n=11) (bottom). FIG. 5C depicts the representative bioluminescence images of control and treated CT26.Fluc-bearing mice. FIG. 5D depicts survival plots of control and treated mice bearing peritoneally disseminated CT26.Fluc tumors. FIG. 5E depicts survival plots of control and treated mice bearing peritoneally disseminated MyC-CaP.Fluc tumors. Statistical significances between SV.IL12+anti-OX40 and anti-OX40 or SV.IL12 were determined with the Mantel-Cox test. Results are representatives of at least two independent experiments.

FIG. 6A-6B. Tumor growth of MyC-CaP.Fluc tumor bearing mice during treatment. FVB/NJ mice were given injection of SV.IL12 and/or anti-OX40 intraperitoneally (i.p.) at various times after injection of 10⁵MyC-CaP.Fluc cells on day 0. FIG. 6A depicts tumor growth curves are shown as fold changes relative to the luminescence on day 0 of the same mouse. Each line represents an individual mouse. Left graphs: Control (n=10) (top) and SV.IL12 (n=10) (bottom). Right graphs: aOX40 (n=10) (top) and SV.IL12+aOX40 (n=10) (bottom). FIG. 6B depicts representative bioluminescence images of control and treated CT.26.Fluc bearing mice. Results are representatives of at least two independent experiments.

FIG. 7A-7D. The therapeutic efficacy of SV.IL12 in combination with anti-OX40 is maintained at reduced treatment regimen in CT26.Fluc bearing mice. FIG. 7A depicts the treatment schema. BALB/c mice were given i.p. injection of SV.IL12 (day 1 and 8) and/or anti-OX40 (day 2 and 9) of 7×10⁴CT26.Fluc on day 0. FIG. 7B depicts representative bioluminescence images of control and treated CT26.Fluc bearing mice. FIG. 7C depicts tumor growth curves are shown as fold changes relative to the luminescence on day 0 of the same mouse. Each line represents an individual mouse. Left graphs: Control (n=14) (top) and SV.IL12 (n=15) (bottom). Right graphs: aOX40 (n=17) (top) and SV.IL12+aOX40 (n=15) (bottom). FIG. 7D depicts survival plots of control and treated mice bearing CT26.Fluc tumors. Statistical significance between SV.IL12+aOX40 and SV.IL12 was determined with the Mantel-Cox test. Results are representatives of at least two independent experiments.

FIG. 8A-8B. Therapeutic efficacy of SV.IL12 in combination with anti-OX40 is dependent on CD4 and CD8 T cells. FIG. 8A depicts that mice injected with anti-CD4 (0.4 mg) or anti-CD8 (0.1 mg) depleting antibody. As a control, rat IgG2b (0.4 mg) isotype control was used. The frequency of CD4 and CD8 T cells were assessed by flow cytometry in splenocytes after 24, 48, 72 and 96 hours. FIG. 8B depicts that BALB/c mice were inoculated with 7×10⁴CT.26.Fluc on day −4. Depletion antibody anti-CD4 or anti-CD8 were injected i.p. on day −3, 1, 5, 9, 13 and 17. Mice were left untreated (control) or were treated with SV.IL12 and anti-OX40 on day 4 and 11. Tumor growth curves are shown as fold changes relative to the luminescence on day 0 of the same mouse. Each line represents an individual mouse.

FIG. 9A-9I. Combination therapy markedly changes the transcriptome signature of T cells favoring effector T cells with a Th1 type phenotype. FIGS. 9A-9I depict RNA sequencing of T cells isolated from spleens derived from untreated tumor bearing mice (control) compared with mice treated with SV.IL12 and/or anti-OX40 on day 7. FIG. 9A depicts principal component analysis (PCA) of normalized read counts from the CT26.Fluc tumor model. FIG. 9B depicts PCA of normalized read counts from the MyC-CaP.Fluc tumor model.

FIG. 9C depicts MA plots of differentially expressed genes (DEG; >2-fold) in T cells of control versus anti-OX40 treated mice (top graph), SV.IL12 treated mice (middle graph) or SV.IL12+anti-OX40 treated mice (bottom graph) in the CT.26 model. Significantly (p<0.05) upregulated and downregulated DEG are depicted in red or blue, respectively. FIG. 9D depicts Pathway and network analysis based on DEG in T cells isolated from CT26.Fluc-bearing mice treated with combination therapy. Downregulated (blue) and upregulated (red) pathways are shown, respectively. FIG. 9E depicts Heatmap analysis of selected genes based on normalized read counts linked to T cell differentiation and activation as well as T cell lineage transcription factors. FIGS. 9F-I depict data from tumor bearing mice that were treated with SV.IL12 and/or anti-OX40. As control, naïve and untreated (control) tumor bearing mice were used. On day 7, spleens were excised and a single cell suspension was stained and analyzed by flow cytometry. FIG. 9F depicts percentage of CD44 or Ki-67 expression by T cells from the CT26.Fluc tumor model. FIG. 9G depicts percentage of CD44 or Ki-67 expression by T cells from the MyC-CaP.Fluc tumor model. FIG. 9H depicts the percentage of ICOS and T-bet expression by CD4 T cells.

FIG. 9I depicts representative flow cytometry plots ICOS and T-bet expression by CD4 T cells. Bars represent means and each symbol represent an individual mouse. Statistical significance was determined with the Kruskal-Wallis test followed by the Dunns' test. Results are representatives of at least two independent experiments.

FIG. 10A-10B. Combination therapy induces systemic CD4 and CD8 T cell activation. Tumor bearing mice were left untreated or treated with SV.IL12 and/or anti-OX40. On day 7, spleens were excised and a single cell suspension was stained and analyzed by flow cytometry. As control, naïve and untreated (control) tumor bearing mice were used. FIGS. 10A and 10B depict representative flow cytometry plots of CD44 and Ki-67 expression on CD4 and CD8 T cells in the CT.26.Fluc and MyC-CaP.Fluc tumor model, respectively.

FIG. 11A-11G. SV.IL12 in combination with anti-OX40 promotes metabolic reprogramming of T cells. Tumor bearing mice were left untreated or treated with SV.IL12 and/or anti-OX40. T cells were isolated from spleens on day 7 or otherwise indicated. FIG. 11A depicts selected gene set enrichment analysis (GSEA) of oxidative phosphorylation and glycolysis pathways based on DEG in control versus SV+anti-OX40. FIG. 11B depicts mitochondrial respiration assessed by measuring the median values of oxygen consumption rates (OCR) in T cells of indicated groups using an extracellular flux analyzer. Oligomycin, FCCP, Antimycin A and Rotenone were injected as indicated to identify energetic mitochondrial phenotypes. FIG. 11C depicts Mitotracker Green FM staining of T cells from indicated groups using flow cytometry. FIG. 11D depicts Mitotracker Deep Red FM staining of T cells from indicated groups using flow cytometry. FIG. 11E depicts western blot of c-Myc protein expression in T cells of control or mice treated with anti-OX40, SV.IL12 or SV.IL12+anti-OX40. GAPDH (bottom) is loading control. FIG. 11F depicts baseline extracellular acidification rates (ECAR) in T cells of indicated groups derived from the CT26.Fluc and MyC-CaP.Fluc tumor models. FIG. 11G depicts energy profile (OCR versus ECAR) of T cells from naïve or CT26.Fluc bearing mice treated with SV.IL12+anti-OX40 on day 7, 14 and 40. Error bars indicate SEM. Results are representatives of at least two independent experiments in FIGS. 111B-G.

FIG. 12A-12D. Combination therapy rewires T cells metabolically. FIGS. 12A and 12B depict the metabolic activity of T cells isolated from spleens of mice bearing CT.26.Fluc or MyC-CaP.Fluc tumor respectively, on day 7. Baseline OCR (left) and respiratory capacity (right) was measured in T cells of indicated groups using an extracellular flux analyzer. FIG. 12C depicts representative flow cytometry plots of Mitotracker Green FM and Mitotracker Deep Red staining in CD4 and CD8 T cells isolated from spleens of CT26.Fluc tumor bearing mice. FIG. 12D depicts energy profile (OCR versus ECAR) of T cells from naïve (bottom graph) or CT26.Fluc bearing mice treated with SV.IL12+anti-OX40 (top graph) on day 7, 14 and 30. Data represent the mean of three different experiments. Bars represent means±SEM (A, B, D). Results are representatives of at least two independent experiments.

FIG. 13A-13G. Reprogrammed T cells in SV+anti-OX40 treated mice display enhanced CD4 mediated cytokine production and anti-tumor activity. Tumor bearing mice were left untreated or treated with SV.IL12 and/or anti-OX40. Spleens were excised on day 7 for further analysis. FIG. 13A depicts Heatmap analysis of selected genes based on normalized read counts linked to cytokine expression and enhanced anti-tumor activity. FIG. 13B depicts RNA sequencing performed on isolated T cells from CT26.Fluc tumor-bearing mice. FIG. 13C depicts Interferon-gamma (IFN-γ enzyme-linked immunospot analysis of splenocytes from control and treated mice as indicated (n=5-10 mice per group). Additionally, IFN-γ enzyme-linked immunospot analysis was measured in splenocytes depleted of CD4 or CD8 T cells in the CT26.Fluc (Top) and MyC-CaP.Fluc (Bottom) tumor models. FIG. 13D depicts representative flow cytometry plots of T-bet and granzyme B (GrB) expression by CD4 T cells from indicated groups using flow cytometry. FIG. 13E depicts Percentage of T-bet and granzyme B (GrB) expression by CD4 T cells from indicated groups using flow cytometry of FIG. 13D. FIG. 13F depict cytotoxic activity of T cells from control and treated mice (n=5-10 mice per group) co-cultured at an effector-to-target cell ratio of 10:1 with either CT26.FLUC. FIG. 13G depicts MyC-CaP.Fluc tumor cell lines for 2 days. Additionally, T cells were depleted of CD4 or CD8 T cells and co-cultured as previously described. Cytotoxic activity was assessed based on viability of tumor cells, which was determined by measuring the luciferase activity and is shown relative to naïve T cells. Bars represent means±SEM in FIGS. 13B, 13F and 13G, and each symbol represent an individual mouse in FIG. 13E. Statistical significance was determined with the Kruskal-Wallis test followed by the he Dunns' test. Results are representatives of at least two independent experiments.

FIG. 14A-14F. CD8 T cells show enhanced cytotoxic potential in mice treated with SV.IL12 and anti-OX40. Tumor bearing mice were left untreated or were treated with SV.IL12 with or without anti-OX40. Mice were sacrificed on day 7 to analyze the T cell immune response in spleen. FIGS. 14A-14B depict percentage of granzyme B and T-bet expression, by CD8 T cells from CT26.Fluc tumor bearing mice. FIGS. 14C-14D depict percentage of granzyme B and T-bet expression, respectively by CD8 T cells from MyC-CaP.Fluc tumor bearing mice. FIGS. 14B and 14D depict representative flow cytometry plots. FIGS. 14E and 14F depict percentage of NKG2D (left graph) or T-bet (right graph) expression by CD8 T cells in the mice bearing CT26.Fluc and MyC-CaP.Fluc tumor, respectively. Bars represent means and each symbol represent an individual mouse. Statistical significance was determined with the Kruskal-Wallis test followed by the he Dunns' test. Results are representatives of at least two independent experiments.

FIG. 15A-15F. Mice treated with SV.IL12 in combination with anti-OX40 display enhanced T cell migration and intratumoral T cell activity. CT26.Fluc bearing mice were left untreated or were treated with SV.IL12 and/or anti-OX40. On day 7, spleens were excised and a single cell suspension was stained and analyzed by flow cytometry. FIG. 15A depicts percentage of CXCR3 expression by CD4 (left graph) and CD8 (right graph) T cells. FIG. 15 B depicts representative flow cytometry plots. Tumors were harvested after 2 weeks of treatment from control and treated mice. FIG. 15C depicts intratumoral gene expression of CXCL9 (Top) and CXCL10 (bottom) analyzed by real time PCR. Data are normalized to GAPDH. FIG. 15D depicts intratumoral T cell immune responses from indicated groups that were assessed by flow cytometry. Percentage of CD4 expression by T cells (left graph), Ki-67 expression (middle graph) and granzyme B expression (right graph) by CD4 T cells. FIG. 15E depicts multiplex immunofluorescence staining of tumors isolated from CT26.Fluc tumor bearing mice. FIG. 15F depicts multiplex immunofluorescence staining of tumors isolated from MyC-CaP.Fluc tumor bearing mice. Representative images of T cell infiltration are shown for control as well as anti-OX40, SV.IL12 and SV.IL12+anti-OX40. Proteins of interest were stained and are indicated by color in each image: K-i67 (red), CD3 (green), CD8 (magenta) and DAPI nuclear staining appears in blue. Bars represent means±SEM in FIG. 15C, and each symbol represent an individual mouse in FIGS. 15A and 15D). Statistical significance was determined with the Kruskal-Wallis test followed by the Dunns' test. Results are representatives of at least two independent experiments

FIG. 16A-16D. T cells show enhanced migration into tumors and exert anti-tumor activity in mice treated with SV.IL12+anti-OX40. Tumor bearing mice were left untreated or were treated with SV.IL12 with or without anti-OX40. Mice were sacrificed on day 7 and 14 to analyze the T cell immune response in spleen. FIG. 16A-16C depict percentage of CXCR3 expression by CD4 (left graph) and CD8 (right graph) T cells measured by flow cytometry in the CT26.Fluc tumor model on day 14, in the MyC-CaP.Fluc tumor model on day 7 (B) and in the MyC-CaP.Fluc tumor model on day 14, respectively. FIG. 16D depicts percentage of CD8 expression by T cells (left graph), Ki-67 expression (middle graph) and granzyme B expression (right graph) by CD8 T cells. Tumors were harvested after 2 weeks of treatment from control and treated mice. T cell immune responses from indicated groups were assessed by flow cytometry. Bars represent means and each symbol represent an individual mouse. Statistical significance was determined with the Kruskal-Wallis test followed by the he Dunns' test. Results are representatives of at least two independent experiments.

FIG. 17. Combination therapy stimulates granzyme B expression in MyC-CaP.Fluc tumors. FIG. 17 depicts tumors stained by multiplex immunofluorescence. MyC-CaP.Fluc tumors were harvested after 2 weeks of treatment from control and treated mice. Representative images are shown for untreated (control), anti-OX40, SV.IL12 and SV.IL12+anti-OX40 treated mice. Proteins of interest were stained and are indicated by color in each image: F4/80 (red) and granzyme B (green). DAPI nuclear staining appears in blue.

FIG. 18A-18B. SV.IL12 triggers innate immune response and induces iNOS expression in MyC-CaP.Fluc tumors. MyC-CaP.Fluc tumors in FIG. 18A and CT26.Fluc tumors in FIG. 18B, were harvested after 2 weeks of treatment from control and treated mice. Tumors were stained by multiplex immunofluorescence. Representative images are shown for untreated (control), anti-OX40, SV.IL12 and SV.IL12+anti-OX40 treated mice. Proteins of interest were stained and are indicated by color in each image: iNOS (Cyan), Arginase 1 (green), and CD11b (magenta). DAPI nuclear staining appears in blue.

FIG. 19. Treatment schema of C57/B16 (female) mice re-injected with Alm5-2Fluc-17 tumor. Alm5-2Fluc-17 tumor reinjection was done from 9 C57/B16 mice in 80 mice (16 cages) on day 0. Treatment started on day 9 after cells implantation. Antibodies (250 ug/dose) treatment (blue dots) was done 3 times/week for 3 weeks starting at day 10 after cells re-injected. Sindbis Vector was administered 4 days/week for 4 weeks (red dots) starting day 9 after cells (mornings). IVIS imaging was done on indicated days after tumor implantation.

FIG. 20. Combination of IL-12 and anti-OX40 expressed by Sindbis viral vectors synergistically enhances survival of subjects with established tumors. FIG. 20. depicts Percentage survival rate of C57/B16 (female) mice re-injected with Alm5-2Fluc-17 tumor and treated with SV.IL12 vector; SV.IL-12 vector and anti-OX40 IgG; RepOX40IgG_Rep-IL12 (fragmented SV expressing OX-40 IgG and fragmented SV expressing IL-12, 50% mix of both vectors) or RepOX40IgG_SV-IL12 (fragmented SV expressing OX-40 IgG and full length SV expressing IL-12, 50% mix of both vectors), with n=5 mice in each treatment group. Untreated mice were used as a control. The mice were re-injected with tumor cells and treated according to the scheme in FIG. 19.

FIG. 21A-21C. A20 lymphoma cells were SV infection resistant. FIG. 21A depicts A20 cells and BHK cells were infected with SV carrying GFP overnight. GFP expression was observed under fluorescent microscope. FIG. 21B depicts SV-GFP infectivity to BHK cells was verified by flow cytometry. FIG. 21C depicts SV-GFP infectivity to A20 cells in vivo were measured by flow cytometry. 10⁷A20 cells (express CD45.2) were inoculated to CByJ.SJL(B6)-Ptprca/J (CD45.1 BALB/C) mice. Recipient mice were treated with SV-GFP 4 days later. GFP expression was measured the next day.

FIG. 22A-22C. Sindbis virus (SV) and α4-1BB combination completely cured BALB/C mice A20 lymphoma. FIG. 21A depicts representative bioluminescence images of groups as indicated. Intensity scale, day 0, 7, 21, min: 400, max: 7000; day 14, min: 100, max: 1000; day 28, min: 3000, max: 50000. FIG. 2B depicts tumor growth measured by relative firefly luciferase (fLuc) activity (normalized to day 0 fLuc activity). Untreated, n=16; SV, n=18; α4-1BB Ab, n=13; SV plus α4-1BB Ab, n=13. FIG. 2C depicts survival curve of all groups (the ratio is shown as survived number/total number).

FIG. 23A-23E. SV alone and SV plus α4-1BB mAb stimulated cell cycle progression, cytokine production, and activation. FIG. 23A depicts the numbers of significant differential (SD) expressed genes (upregulated and downregulated) of SV vs. untreated are as indicated. SD expressed genes were selected based on Deseq2 analysis (q<0.05), |Log 2FC|≥1. FIG. 23B depicts the enrichment scores for gene cluster of cell cycle for SV vs. untreated, SV+α4-1BB vs. untreated and SV+α4-1BB vs. SV respectively (“cell cycle” is the gene cluster with the highest enrichment score for these 3 comparisons). FIG. 23C depicts the heat map representing SD expressed cytokine and chemokine genes (left, SV vs. untreated; right, SV+α4-1BB vs. α4-1BB, Log 2FC≥1). Expression values are shown by Z-score. Genes are hierarchically clustered by one minus Pearson correlation. Red arrow, Ccl8, IL4, IL13 and IL21 expression. FIG. 23D depicts the percentage of CD69+ T cells from all groups on day 2 after starting treatment was measured by flow cytometry. FIG. 23E depicts GSEA enrichment plot of KEGG (SV+α4-1BB vs. untreated) TCR receptor signaling pathway. *, p<0.05; **, p<0.01, *** p<0.001.

FIG. 24A-24C. SV infection enhanced cell cycle progression and migration. FIG. 24A depicts DAVID KEGG analysis. FIG. 24B depicts GSEA enrichment plot of KEGG (SV vs. Untreated) cell cycle pathway (SV vs. Untreated). FIG. 24C depicts cell movement pathway was significantly enhanced by IPA (SV vs. Untreated).

FIG. 25A-25B. Significant differential (SD) upregulated genes are clustered by DAVID analysis. FIG. 25A depicts enrichment score of gene clusters for SV+a 4-1BB vs Untreated. FIG. 25B depicts enrichment score of gene clusters for SV+a 4-1BB vs SV.

FIG. 26A-26D. Untreated group had low ratio of T cells and high ratio of regulatory T cells on day 28. FIGS. 26A-26C depict the frequency of CD4, CD8, and Treg respectively measured by flow cytometry. FIG. 26D depicts the Treg/CD8 ratio as indicated.

FIG. 27A-27D. Sindbis virus plus α4-1BB combination induced higher cytotoxicity. FIG. 27A depicts splenocytes mixed with fLuc-A20 lymphoma cells according to the ratio as indicated (splenocytes:lymphoma cells). Cytotoxicity corresponds to the reduction of normalized Luc activity (fLuc activity of A20 lymphoma cells only is normalized to 1). SV+ tumor, α4-1BB+ tumor, SV+α4-1BB+ tumor: tumor inoculated mice. SV, α4-1BB, SV+α4-1BB: mice without tumor inoculation. FIG. 27B depicts splenocytes harvested from all groups after 7 days treatment. The percentage of NKG2D+ cells was measured by flow cytometry (CD8 T cell gated). FIG. 27C depicts the percentage of granzyme B+ and perforin+ cells was measured by flow cytometry (CD8 T cell gated). FIG. 27D depicts cytotoxicity associated genes upregulated in SV+α4-1BB treated group. The heat map depicts the relative expression level of cytotoxicity associated genes. Expression values are shown by Z-score. Genes are hierarchically clustered by one minus Pearson correlation (day 7). Red square, granzyme b and perforin expression. Red arrow, Ifng and Stat4 expression. **, p<0.01; ****,p<0.0001.

FIG. 28A-28F. Sindbis virus plus α4-1BB combination induced Th1 differentiation and IFNγ production. FIG. 28A depicts IFNγ Elispot analysis of splenocytes harvested at day 2, 7, 14 and 28 from all groups as indicated. Upper panel, IFNγ Elispot image on day 7 after treatment. 1, 2, 3: three individual mice. Lower panel, IFNγ spots number from indicated groups over the course of treatment (2×10⁵splenocytes per well). No stimulator was added. FIG. 28B depicts IFNγ production from CD4/CD8 T cell population in splenocytes and purified CD4/CD8 T cells. All groups were cultured in media for 5 h in the presence of brefeldin A. FIG. 28C depicts IFNγ production from purified CD4 T cells at different stimulation conditions. FIG. 28D depicts upregulated Th1 pathway gene set under SV, α4-1BB and SV+α4-1BB stimulation. Expression values are shown by Z-score. Genes are hierarchically clustered by one minus Pearson correlation (day 7). FIG. 28E depicts T-bet expression for all groups as indicated. FIG. 28F depicts EOMES expression for all groups as indicated. CD8 T cell gated. FIGS. 28E and 28F, day 7 after treatment. *, p<0.05; **, p<0.01, ****, p<0.0001.

FIG. 29 depicts IFNγ production from splenocytes of all groups with or without tumor inoculation on day 7 after treatment was measured by Elispot. With tumor: tumor was inoculated on day 0. Without tumor: tumor was not inoculated. No stimulator was added in Elispot assay.

FIG. 30A-30B. IFNγ production measurement. FIG. 30A, IFNγ production (at day 7) by all groups, as indicated, was measured by Elispot. FIG. 30B, IFNγ production of purified T cells (CD8 T cell portion) on day 7 after treatment was measured by flow cytometry.

FIG. 31A-31I. SV and α4-1BB mAb stimulated chemotaxis, adhesion and enhanced T cell infirtration and activation in tumor. FIG. 31A depicts heat map of the expression pattern of SV+α4-1BB upregulated chemokine and chemokine receptor genes (Expression values are shown by Z-score.) Genes are hierarchically clustered by one minus Pearson correlation (day 7). FIG. 31B depicts the percentage of CCR5+ cells was measured by flow cytometry (day 7). FIGS. 31C and 31D depict the percentage of CD11a+ and ICAM-1+ cells, respectively measured by flow cytometry. FIG. 31E depicts the relative expression of CD11a (ltgal) and ICAM-1 was shown by heat map measured by RNA-Seq. Expression values are shown by Z score. FIG. 31F depicts the percentage of OX40+ and ICOS+ T cells were measured by flow cytometry. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. FIG. 31G depicts the frequency of CD3 and CD8 T cells to total harvested cells from tumor measured by flow cytometry. FIG. 31H depicts the CD8/Treg ratio of tumor infiltrated T cells. FIG. 31I depicts the percentage of granzyme B+CD8 T cells as indicated. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

FIG. 32A-32E. The phenotype of tumor infiltrated T cells. FIGS. 32A-32E depict the percentage of Ki67+, Foxp3+, T-bet+, EOMES+, NKG2D+ T cells respectively, measured by flow cytometry.

FIG. 33A-33C. Sindbis virus plus α4-1BB synergistically enhanced T cell glycolysis and oxidative phosphorylation. FIG. 33A depicts GSEA enrichment plot of KEGG (SV+α4-1BB vs. untreated) glycolysis pathway. FIG. 33B depicts the canonical pathways of SV plus α4-1BB Ab stimulation clustered by IPA. Red square, oxidative phosphorylation. FIG. 33C depicts both oxygen consumption rate (oxidative phosphorylation) and Extracellular Acidification Rate (glycolysis) measured by seahorse XFe24. All groups are as indicated (n=4).

FIG. 34A-34B. SV plus low dose α4-1BB mAb cured A20 tumor bearing mice. FIG. 34A depicts Bioluminescence images of mice showing tumor load in A20 tumor bearing mice treated with SV plus low dose α4-1BB mAb, as compared to control (untreated) and SV alone. FIG. 34B depicts tumor growth (Relative Luciferase activity) in each treatment group as indicated. Each line is a single mice.

FIG. 35A-35D. Cured mice are completely protected from A20 lymphoma rechallenge. FIG. 35A depicts Bioluminescence images of groups, previously treated as indicated, were re-challenged with A20 lymphoma cells. FIG. 35B depicts IFNγ production from purified T cells of all groups(To SV+α4-1BB, 4 months after treatment finished), in the absence or presence of A20 tumor cells (5×104 per well), was measured by Elispot assay. FIG. 35C depicts cytotoxicity assay was performed the same as FIG. 27A. Left 2 panels, total splenocytes were used. Right, purified T cells were used. Left upper, A20 Fluc cells and left lower, CT26 Fluc cells were used for co-culture. FIG. 35D depicts significant differential (SD) upregulated gene sets are clustered by DAVID KEGG analysis. *, p<0.05; **, p<0.01; ****, p<0.0001

FIG. 36. Combination of NY-ESO-1 and IL-12 expressed by separate Sindbis viral vectors synergistically enhances survival of subjects with established tumors. FIG. 36 depicts the percentage survival rate of C57/B16 (albino-female) mice re-injected with Alm5-2Fluc-17 tumor cells and treated with SV-IL-12, SV-NY-ESO-1 or a 50% mixture of both the vectors in one injection, SV-IL-12 and SV-NY-ESO-1, as indicated. Untreated mice were used as control. A total of n=5 mice in each group were tested for percentage survival days after tumor transplantation

FIG. 37. Combination of NY-ESO-1 and IL-12 expressed by the same Sindbis viral vectors synergistically enhances survival of subjects with established tumors. FIG. 37 depicts the percentage survival rate of C57/B16 (albino-female) mice re-injected with Alm5-2Fluc-17 tumor cells and treated with SV-IL-12, SV-NY-ESO-1 or a Sindbis viral vector expressing both IL-12 and NY-ESO-1 (SV-NYESO-SGP2-IL12), as indicated. Untreated mice were used as control. A total of n=5 mice in each group were tested for percentage survival days after tumor transplantation.

FIG. 38. pSP6-R_IL12 Sindbis replicon vector expressing IL12 a and b subunits. FIG. 38 depicts plasmid map with SP6, promoter for in vitro transcription; Replicase, SV RNA polymerase; Psg, subgenomic promoter for IL12 expression; linker, joins IL12 a and b subunits; AmpR, ampicillin resistance gene. Numbers show nucleotide positions of genes in the replicon plasmid.

FIG. 39. Sindbis Repicon vector expressing full length antibody to OX40 IgG2a. FIG. 39 depicts plasmid map with T7 promoter for in vitro transcription; Replicase, SV RNA polymerase; Psg subgenomic promoter for expression of anti-OX40 heavy chain IgG2a; 2Psg, second subgenomic promoter for expression of light chain anti-OX40. AmpR, ampicillin resistance gene; ColE1, plasmid origin of replication. Numbers show nucleotide positions of genes in the replicon plasmid.

FIG. 40. Sindbis replicon vector expressing single chain antibody to OX40. FIG. 40 depicts plasmid map with T7, promoter for in vitro transcription; Replicase, SV RNA polymerase; Psg, subgenomic SV promoter; IL12 signal peptide, signal peptide fused to the sequence encoding anti-OX40 single chain antibody; AmpR, ampicillin resistance gene; ColE1, plasmid origin of replication. Numbers show nucleotide positions of genes in the replicon plasmid.

FIG. 41. Sindbis Replicon Vector expressing NY-ESO-1. FIG. 41 depicts plasmid map with T7, promoter for in vitro transcription; Replicase, SV RNA polymerase; Psg, subgenomic promoter for transcription; Hu NY-ESO-1, coding sequence for human NY-ESO-1 tumor associated antigen, Poly A, poly A tail transcribed onto NY-ESO mRNA; AmpR, ampicillin resistant gene; ColE, plasmid origin of replication. Numbers show nucleotide positions of genes in the replicon plasmid.

FIG. 42. pT7StuIR1-FcOX40L_T2A_NY-ESO1. Sindbis replicon vector expressing the OX40 Ligand fused with the Fc receptor sequence and NY-ESO-1. FIG. 42 depicts plasmid map with T7, promoter for in vitro transcription; Replicase, SV RNA polymerase; Psg, subgenomic SV promoter; FcOX40L coding sequence; T2A, termination peptide sequence; NY-ESO-1, coding sequence; AmpR, ampicillin resistance; ColE1, plasmid origin of replication. Numbers show nucleotide positions of genes in the replicon plasmid.

DETAILED DESCRIPTION OF THE INVENTION

Oncolytic virus (OV) therapy has become a novel immunotherapeutic approach to treat cancer. A rationale for oncolytic virus is that they can infect and lyse the tumor cell. They have been made to selectively replicate in tumor cells either through the direction of tumor specific promoters or through direct intratumoral administration. Most OVs encounter a number of barriers to systemic administration. Once lysed by OVs, tumor cells release tumor associated antigens (TAAs) that can stimulate cytotoxic T cells. OV infection also induces an inflammatory response that helps to trigger an immune anti-tumor response. Several OV clinical trials are underway and have shown promising results. However, whether OV therapy can effectively treat tumors that they are unable to infect remains an unresolved limitation.

Sindbis virus (SV) belongs to alphavirus genus and is one type of OV. Although it does not lyse infected tumor cells, it can cause their apoptotic death. It offers several important benefits. SV is known as one of the least virulent alphaviruses with clinical signs and symptoms usually unapparent. It has been estimated that there are 17 times more subclinical than symptomatic SV infections. In general, when symptoms do occur in humans they consist of a self-limiting, mild, febrile disease with vesicular exanthema and arthralgia from which most patients recover within 14 days. The disease is in part self-limiting because SV is an RNA virus that does not integrate in the host genome and hence its presence is transitory. The lack of an integrative step in its replication cycle also avoids insertional mutagenesis risks. In addition, SV vectors of the present disclosure were generated from the laboratory strain AR339, which is not known to cause disease in humans. These vectors were further attenuated by rendering them replication-defective.

SV vectors can target tumors systemically and can reach metastatic tumor cells throughout the body. They can target tumors without infecting normal tissues. However, susceptibility to infection by SV vectors depends on a number of factors including laminin receptor expression and distribution, as well as, defects in IFN signaling in tumors. The present disclosure demonstrates that SV vectors can effectively help cure tumors that they are unable to infect and further demonstrates that the combination antibodies and SV vectors provide a surprising synergistic therapeutic effect against cancer.

The present disclosure provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of (a) a oncolytic viral vector and (b) an antibody directed against a co-stimulatory molecule or a nucleic acid encoding same; or an antibody to an immune system agonist molecule or a nucleic acid encoding same.

The oncolytic viral vector can be a Sindbis viral vector. The Sindbis viral vector can be replication defective. Sindbis viral vectors were produced as described in U.S. Pat. No. 8,093,021 (incorporated herein by reference in its entirety). The Sindbis viral vector can comprise at least one nucleic acid encoding a therapeutic protein. The Sindbis viral vector can comprise at least one nucleic acid encoding an immunostimulatory or an immunomodulatory protein. The immunostimulatory or immunomodulatory protein can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, or any combination thereof. In a preferred aspect, the immunostimulatory or immunomodulatory protein is IL-12. The Sindbis viral vector can comprise at least one nucleic acid encoding LacZ, Flue or GFP.

The antibody can be an anti-OX40 antibody, an anti-4-1BB antibody, an anti-CD28 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-cd37 antibody, an anti-HVEM antibody, or a combination thereof.

The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit (IL-12 α, IL-12, p35 subunit) and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit). The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit of GenBank accession no. M86672 and interleukin-12 beta subunit of GenBank accession no. M86671. The nucleic acid encoding interleukin-12 alpha subunit comprises the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid encoding interleukin-12 beta subunit comprises the nucleic acid sequence of SEQ ID NO: 2.

The nucleic acid encoding interleukin-12 alpha subunit (IL-12 α, IL-12, p35 subunit) comprises the nucleic acid sequence of SEQ ID NO: 1 shown in the following Table.

The nucleic acid encoding interleukin-12 alphasubunit

(IL-12α, IL-12, p35 subunit)

1
gcaagagaca cagtcctggg aaagtctgcc ggctatccag acaattataa aaatgtgtct

61
cccaaggtca gcgttccaac agcctcaccc tcggcatcca gcagctcctc tcagtgccgg

121
tccagcatgt gtcaatcacg ctacctcctc tttttggcca cccttgccct cctaaaccac

181
ctcagtttgg ccagggtcat tccagtctct ggacctgcca ggtgtcttag ccagtcccga

241
aacctgctga agaccacaga tgacatggtg aagacggcca gagaaaaact gaaacattat

301
tcctgcactg ctgaagacat cgatcatgaa gacatcacac gggaccaaac cagcacattg

361
aagacctgtt taccactgga actacacaag aacgagagtt gcctggctac tagagagact

421
tcttccacaa caagagggag ctgcctgccc ccacagaaga cgtctttgat gatgaccctg

481
tgccttggta gcatctatga ggacttgaag atgtaccaga cagagttcca ggccatcaac

541
gcagcacttc agaatcacaa ccatcagcag atcattctag acaagggcat gctggtggcc

601
atcgatgagc tgatgcagtc tctgaatcat aatggcgaga ctctgcgcca gaaacctcct

661
gtgggagaag cagaccctta cagagtgaaa atgaagctct gcatcctgct tcacgccttc

721
agcacccgcg tcgtgaccat caacagggtg atgggctatc tgagctccgc ctgaaagctc

781
aaggccctct gccacagcgc cctcctcaca cagataggaa acaaagaaag attcataaga

841
gtcaggtggt cttggcctgg tgggcttaag ctccttcagg aatctgttct cccatcacat

901
ctcatctccc caaaggtggc acagctacct cagcatggtg ccctccatcg cttctctcat

961
attcactata caagttgttt gtaagttttc atcaaaatat tgttaagggg cgaagacgtc

1021
ctcccctcaa tgtgttagca gaagagcaag aactgataag ctattgtttt tgtgccaaag

1081
tgtttatgaa aacactcagt caccccttat ttaaaaatat ttattgctat attttatact

1141
catgaaagta catgagccta tttatattta tttattttct atttattata atatttctta

1201
tcagatgaat ttgaaacatt ttgaaacata ccttattttg tggttctaat aaagtaatgt

1261
tatca (SEQ ID NO: 1)

The nucleic acid encoding interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit) comprises the nucleic acid sequence of SEQ ID NO: 2 shown in the following Table.

The nucleic acid encoding interleukin-12 beta subunit

(IL-12β, IL-12, p40 subunit)

1
gcacatcaga ccaggcagct cgcagcaaag caagatgtgt cctcagaagc taaccatctc

61
ctggtttgcc atcgttttgc tggtgtctcc actcatggcc atgtgggagc tggagaaaga

121
cgtttatgtt gtagaggtgg actggactcc cgatgcccct ggagaaacag tgaacctcac

181
ctgtgacacg cctgaagaag atgacatcac ctggacctca gaccagagac atggagtcat

241
aggctctgga aagaccctga ccatcactgt caaagagttt ctagatgctg gccagtacac

301
ctgccacaaa ggaggcgaga ctctgagcca ctcacatctg ctgctccaca agaaggaaaa

361
tggaatttgg tccactgaaa ttttaaaaaa tttcaaaaac aagactttcc tgaagtgtga

421
agcaccaaat tactccggac ggttcacgtg ctcatggctg gtgcaaagaa acatggactt

481
gaagttcaac atcaagagca gtagcagttc ccctgactct cgggcagtga catgtggaat

541
ggcgtctctg tctgcagaga aggtcacact ggaccaaagg gactatgaga agtattcagt

601
gtcctgccag gaggatgtca cctgcccaac tgccgaggag accctgccca ttgaactggc

661
gttggaagca cggcagcaga ataaatatga gaactacagc accagcttct tcatcaggga

721
catcatcaaa ccagacccgc ccaagaactt gcagatgaag cctttgaaga actcacaggt

781
ggaggtcagc tgggagtacc ctgactcctg gagcactccc cattcctact tctccctcaa

841
gttctttgtt cgaatccagc gcaagaaaga aaagatgaag gagacagagg aggggtgtaa

901
ccagaaaggt gcgttcctcg tagagaagac atctaccgaa gtccaatgca aaggcgggaa

961
tgtctgcgtg caagctcagg atcgctatta caattcctca tgcagcaagt gggcatgtgt

1021
tccctgcagg gtccgatcct aggatgcaac gttggaaagg aaagaaaagt ggaagacatt

1081
aaggaagaaa aatttaaact caggatggaa gagtccccca aaagctgtct tctgcttggt

1141
tggctttttc cagttttcct aagttcatca tgacaccttt gctgatttct acatgtaaat

1201
gttaaatgcc cgcagagcca gggagctaat gtatgcatag atattctagc attccacttg

1261
gccttatgct gttgaaatat ttaagtaatt tatgtattta ttaatttatt tctgcatttc

1321
acatttgtat accaagatgt attgaatatt tcatgtgctc gtggcctgat ccactgggac

1381
caggccctat tatgcaaatt gtgagcttgt tatcttcttc aacagctctt caatcagggc

1441
tgcgtaggta cattagcttt tgtgacaacc aataagaaca taatattctg acacaagcag

1501
tgttacatat ttgtgaccag taaagacata ggtggtattt ggagacatga agaagctgta

1561
aagttgactc tgaagagttt agcactagtt tcaacaccaa gaaagacttt ttagaagtga

1621
tattgataag aaaccagggc cttctttaga agggtaccta aatttaaaag aattttgaaa

1681
ggctgggtat cggtggtata tgcttttaat tccagcactc aggagaccaa ggcaggcaga

1741
tctctgtgag tttgaggaca gcctggtgta cagagggagt tccagcacag ccagtgccac

1801
acagaaattc tgtctcaaaa acaattaaaa aaaaaaaaaa (SEQ ID NO: 2)

The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit of amino acid sequence of GenBank accession no. AAA39292.1 and interleukin-12 beta subunit of amino acid sequence of GenBank accession no. AAA39296.1. The amino acid sequence of the interleukin-12 alpha subunit is of amino acid sequence of SEQ ID NO: 3. The amino acid sequence of the interleukin-12 beta subunit is of amino acid sequence of SEQ ID NO: 4.

The Sindbis viral vector can comprise the nucleic acid encoding the interleukin-12 alpha subunit of amino acid sequence of GenBank accession no. AAA39292.1 and a nucleic acid encoding the interleukin-12 beta subunit of amino acid sequence of GenBank accession no. AAA39296.1. The amino acid sequence of the interleukin-12 alpha subunit comprises the amino acid sequence of SEQ ID NO: 3 shown in the following Table.

The amino acid sequence of the interleukin-12 alpha subunit

1
MCQSRYLLFL ATLALLNHLS LARVIPVSGP ARCLSQSRNL LKTTDDMVKT AREKLKHYSC

61
TAEDIDHEDI TRDQTSTLKT CLPLELHKNE SCLATRETSS TTRGSCLPPQ KTSLMMTLCL

121
GSIYEDLKMY QTEFQAINAA LQNHNHQQII LDKGMLVAID ELMQSLNHNG ETLRQKPPVG

181
EADPYRVKMK LCILLHAFST RVVTINRVMG YLSSA (SEQ ID NO: 3)

The amino acid sequence of the interleukin-12 beta subunit comprises the amino acid sequence of SEQ ID NO: 4 shown in the following Table.

The amino acid sequence of the interleukin-12 beta subunit

1
MCPQKLTISW FAIVLLVSPL MAMWELEKDV YVVEVDWTPD APGETVNLTC DTPEEDDITW

61
TSDQRHGVIG SGKTLTITVK EFLDAGQYTC HKGGETLSHS HLLLHKKENG IWSTEILKNF

121
KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK FNIKSSSSSP DSRAVTCGMA SLSAEKVTLD

181
QRDYEKYSVS CQEDVTCPTA EETLPIELAL EARQQNKYEN YSTSFFIRDI IKPDPPKNLQ

241
MKPLKNSQVE VSWEYPDSWS TPHSYFSLKF FVRIQRKKEK MKETEEGCNQ KGAFLVEKTS

301
TEVQCKGGNV CVQAQDRYYN SSCSKWACVP CRVRS (SEQ ID NO: 4)

The Sindbis viral vector can comprise a nucleic acid encoding an interleukin-12 alpha subunit that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 3 and a nucleic acid encoding an interleukin-12 alpha subunit that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 4.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 variable heavy chain comprising the amino acid sequence of SEQ ID NO:5 shown in the following Table.

anti-OX40 variable heavy chain amino acid (mouse anti-OX40

variable heavy chain)

1
MAEVQLVESG GGLVQPGGSL RLSCAASGFT FSNYTMNWVR QAPGKGLEWV

51
SAISGSGGST YYADSVKGRF TISRDNSKNT LYLQMNSLRA EDTAVYYCAK

101
DRYSQVHYAL DYWGQGTLVT V (SEQ ID NO: 5)

The nucleic acid sequence encoding the anti-OX40 variable heavy chain comprises the nucleic acid sequence of SEQ ID NO:6 shown in the following Table.

anti-OX40 variable heavy chain nucleic acid

(mouse anti-OX40 variable heavy chain)

5′atggccgaggtgcagctggtggagagcggcggcggcctggtgcagcc

cggcggcagcctgaggctgagctgcgccgccagcggcttcaccttcagc

aactacaccatgaactgggtgaggcaggcccccggcaagggcctggagt

gggtgagcgccatcagcggcagcggcggcagcacctactacgccgacag

cgtgaagggcaggttcaccatcagcagggacaacagcaagaacaccctg

tacctgcagatgaacagcctgagggccgaggacaccgccgtgtactact

gcgccaaggacaggtacagccaggtgcactacgccctggactactgggg

ccagggcaccctggtgaccgtg 3′ (SEQ ID NO: 6)

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 variable light chain comprising the amino acid sequence of SEQ ID NO:7 shown in the following Table.

anti-OX40 variable light chain amino acid

(mouse anti-OX40 variable light chain)

(SEQ ID NO: 7)

1
DIQMTQSPDS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW

YLQKAGQSPQ

51
LLIYLGSNR

The nucleic acid sequence encoding the anti-OX40 variable light chain comprises the nucleic acid sequence of SEQ ID NO:8 shown in the following Table.

anti-OX40 variable light chain nucleic acid

(mouse anti-OX40 variable light chain)

(SEQ ID NO: 8)

5′gacatccagatgacccagtcccccgactccctgcccgtgacccccggc

gagcccgcctccatctcctgccggtcctcccagtccctgctgcactccaa

cggctacaactacctggactggtacctgcagaaggccggccagtcccccc

agctgctgatctacctgggctccaaccgggcctccggcgtgcccgaccgg

ttctccggctccggctccggcaccgacttcaccctgaagatctcccgggt

ggaggccgaggacgtgggcgtgtactactgccagcagtactacaaccacc

ccaccaccttcggccagggcaccaagctggagatcaagcgg-3′

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 variable heavy chain of amino acid sequence of SEQ ID NO: 5 and a nucleic acid encoding an anti-OX40 variable light chain of amino acid sequence of SEQ ID NO: 7. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 5 and a nucleic acid encoding an anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%˜ or 100% identical to amino acid sequence of SEQ ID NO: 7.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain comprising the amino acid sequence of SEQ ID NO:9 shown in the following Table.

anti-OX40 antibody heavy chain amino acid sequence

(mouse anti-OX40 IgG2a antibody heavy chain)

(SEQ ID NO: 9)

MGQSRYLLFLATLALLNHLSLA
MAEVQLVESGGGLVQPGGSLRLSCAASG

FTFSNYTMNWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSK

NTLYLQMNSLRAEDTAVYYCAKDRYSQVHYALDYWGQGTLVTVAAKTTAP

SVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAV

LQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKI

custom-character

PAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVV

DVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM

SGKEEKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT

LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEK

KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

The nucleic acid sequence encoding the anti-OX40 antibody heavy chain comprises the nucleic acid sequence of SEQ ID NO: 10 shown in the following Table.

anti-OX40 antibody heavy chain nucleic acid sequence (mouse anti-OX40 IgG2a

antibody heavy chain)

(SEQ ID NO: 10)

embedded image

ggccgaggtgcagctggtggagagcggcggcggcctggtgcagcccggcggcagcctgaggctgagctgcgccgcc

agcggcttcaccttcagcaactacaccatgaactgggtgaggcaggcccccggcaagggcctggagtgggtgagcg

ccatcagcggcagcggcggcagcacctactacgccgacagcgtgaagggcaggttcaccatcagcagggacaacag

caagaacaccctgtacctgcagatgaacagcctgagggccgaggacaccgccgtgtactactgcgccaaggacagg

tacagccaggtgcactacgccctggactactggggccagggcaccctggtgaccgtggccgccaagaccaccgccc

ccagcgtgtaccccctggcccccgtgtgcggcgacaccaccggcagcagcgtgaccctgggctgcctggtgaaggg

ctacttccccgagcccgtgaccctgacctggaacagcggcagcctgagcagcggcgtgcacaccttccccgccgtg

ctgcagagcgacctgtacaccctgagcagcagcgtgaccgtgaccagcagcacctggcccagccagagcatcacct

embedded image

gtgctgatgatcagcctgagccccatcgtgacctgcgtggtggtggacgtgagcgaggacgaccccgacgtgcaga

tcagctggttcgtgaacaacgtggaggtgcacaccgcccagacccagacccacagggaggactacaacagcaccct

gagggtggtgagcgccctgcccatccagcaccaggactggatgagcggcaaggagttcaagtgcaaggtgaacaac

embedded image

tgcccccccccgaggaggagatgaccaagaagcaggtgaccctgacctgcatggtgaccgacttcatgcccgagga

catctacgtggagtggaccaacaacggcaagaccgagctgaactacaagaacaccgagcccgtgctggacagcgac

ggcagctacttcatgtacagcaagctgagggtggagaagaagaactgggtggagaggaacagctacagctgcagcg

tggtgcacgagggcctgcacaaccaccacaccaccaagagcttcagcaggacccccggcaagtaa-3′

In SEQ ID NOs: 9 and 10, the underlined residues indicate IL-2 signal peptide; the Bold residues indicate variable antigen binding region; the non-underlined residues indicate mouse heavy chain IgG2a constant region, GB Accession BC080671; and the bold and underlined residues indicate the Hinge and disulfide bond region. The double underlined residue in SEQ ID NO: 9 indicates change from C to T to remove ApaI site. The dotted underlined residues in SEQ ID NO: 9 indicate Kozak sequence.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody light chain comprising the amino acid sequence of SEQ ID NO:11 shown in the following Table.

anti-OX40 antibody light chain amino acid sequence

(mouse anti-OX40 IgG2a antibody light chain)

(SEQ ID NO: 11)

MGQSRYLLFLATLALLNHLSLA
DIQMTQSPDSLPVTPGEPASISCRSSQS

LLHSNGYNYLDWYLQKAGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTL

KISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRADAAPTVSIFPPSSE

QLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTY

SMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

The nucleic acid sequence encoding the mouse anti-OX40 antibody light chain comprises the nucleic acid sequence of SEQ ID NO 12 shown in the following Table

anti-OX40 antibody light chain nucleic acid sequence (mouse anti-OX40 IgG2a antibody light

chain)

(SEQ ID NO: 12)

embedded image

acatccagatgacccagtcccccgactccctgcccgtgacccccggcgagcccgcctccatctcctgccggtcct

cccagtccctgctgcactccaacggctacaactacctggactggtacctgcagaaggccggccagtccccccagc

tgctgatctacctgggctccaaccgggcctccggcgtgcccgaccggttctccggctccggctccggcaccgact

tcaccctgaagatctcccgggtggaggccgaggacgtgggcgtgtactactgccagcagtactacaaccacccca

ccaccttcggccagggcaccaagctggagatcaagcgggccgacgccgcccccaccgtgtccatcttccccccct

cctccgagcagctgacctccggcggcgcctccgtggtgtgcttcctgaacaacttctaccccaaggacatcaacg

tgaagtggaagatcgacggctccgagcggcagaacggcgtgctgaactcctggaccgaccaggactccaaggact

ccacctactccatgtcctccaccctgaccctgaccaaggacgagtacgagcggcacaactcctacacctgcgagg

ccacccacaagacctccacctcccccatcgtgaagtccttcaaccggaacgagtgctaa-3′

In SEQ ID NOs: 11 and 12, the underlined residues indicate IL-2 signal peptide; the Bold residues indicate variable antigen binding region; the non-underlined residues indicate light constant region, GB Accession BC091750.1. The dotted underlined residues in SEQ ID NO: 12 indicate Kozak sequence.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO:13 shown in the following Table.

amino acid sequence of the Target antigen

(Target Antigen: OX40/CD134, Receptor for

TNFSF4/OX40L/GP34)

(SEQ ID NO: 13)

1
MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND

RCCHECRPGN

51
GMVSRCSRSQ NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR

SGSERKQLCT

101
ATQDTVCRCR AGTQPLDSYK PGVDCAPCPP GHFSPGDNQA

CKPWTNCTLA

151
GKHTLQPASN SSDAICEDRD PPATQPQETQ GPPARPITVQ

PTEAWPRTSQ

201
GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL

RRDQRLPPDA

251
HKPPGGGSFR TPIQEEQADA HSTLAKI

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable heavy chain comprising the amino acid sequence of SEQ ID NO: 31 shown in the following Table.

Human anti-OX40 antibody, 1-12Z5

(ATCC No. PTA-7216) Heavy chain variable region

amino acid sequence. Leader sequence in bold.

(SEQ ID NO: 31)

1

MTMITPSLVP SSDPLVTAAS VLEFALLIRL TIGQAVVSTQ

STGGGLVQPG RSLRLSCAAS

61
GFTLDDYGMH WVRQAPGKGL EWVSGISWNS DSIGYVDSVK

GRFTISRDNA KNSLYLQMNS

121
LRVEDTALYY CVKDISGWYS FDYWGQGTLV TVSS

The nucleic acid sequence encoding the human anti-OX40 antibody heavy chain variable region comprises the nucleic acid sequence of SEQ ID NO: 32 shown in the following Table.

Human anti-OX40 antibody 1-12Z5

(ATCC No. PTA-7216) Heavy chain variable region

nucleic acid sequence

(SEQ ID NO: 32)

5′-atgaccatgattacgccaagcttggtaccgagctcggatccactagt

aacggccgccagtgtgctggaattcgcccttctaatacgactcactatag

ggcaagcagtggtatcaacgcagagtacggggggaggcttggtacagcct

ggcaggtccctgagactctcctgtgcagcctctggattcacccttgatga

ttatggcatgcactgggtccggcaagctccagggaagggcctggagtggg

tctcaggtattagttggaatagtgatagtataggctatgtggactctgtg

aagggccgattcaccatctccagagacaacgccaagaactccctgtatct

gcaaatgaacagtctgagagttgaggacacggccttgtattactgtgtaa

aagatattagtggctggtacagctttgactactggggccagggaaccctg

gtcaccgtctcctca-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable light chain comprising the amino acid sequence of SEQ ID NO: 33 shown in the following Table.

Human anti-OX40 antibody 1-12Z5

(ATCC No. PTA-7216) Light chain variable region

amino acid sequence

(SEQ ID NO: 33)

1

MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT

LSCRASQSVS SYLAWYQQKP

61
GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP

EDFAVYYCQQ RSNWPITFGQ

121
GTRLEIK

The nucleic acid sequence encoding the human anti-OX40 antibody light chain variable region comprises the nucleic acid sequence of SEQ ID NO: 34 shown in the following Table.

Human anti-OX40 antibody 1-12Z5

(ATCC No. PTA-7216) Light chain variable region

nucleic acid sequence

(SEQ ID NO: 34)

5′atggaagccccagctcagcttctcttcctcctgctactctggctccca

gataccaccggagaaattgtgttgacacagtctccagccaccctgtcttt

gtctccaggggaaagagccaccctctcctgcagggccagtcagagtgtta

gcagctacttagcctggtaccaacagaaacctggccaggctcccaggctc

ctcatctatgatgcatccaacagggccactggcatcccagccaggttcag

tggcagtgggtctgggacagacttcactctcaccatcagcagcctagagc

ctgaagattttgcagtttattactgtcagcagcgtagcaactggccgatc

accttcggccaagggacacgactggagattaaa-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35 shown in the following Table.

Human anti-OX40 antibody 112F32

(ATCC No. PTA-7217) Heavy chain variable region

amino acid sequence

(SEQ ID NO: 35)

1

MEWGPCWVFL VVILEGVQCG VQLVESGGGL VQPGGSLRLS

CAASGFTFSS YSMNWVRQAP

61
GKGLEWVSYI SSSSSTIYYA DSVKGRFTIS RDNAKNSLYL

QMNSLRDEDT AVYYCARGVY

121
HNGWSFFDYW GQGTLLTVSS

The nucleic acid sequence encoding the human anti-OX40 antibody heavy chain variable region comprises the nucleic acid sequence of SEQ ID NO: 36 shown in the following Table.

Human anti-OX40 antibody 112F32

(ATCCNo. PTA-7217) Heavy chain variable region

nucleic acid sequence

(SEQ ID NO: 36)

5′atggagtgggggccgtgctgggttttccttgttgttattttagaaggt

gtccagtgtggggtgcagctggtggagtctgggggaggcttggtacagcc

tggggggtccctgagactctcctgtgcagcctctggattcaccttcagta

gctatagcatgaactgggtccgccaggctccagggaaggggctggagtgg

gtttcatacattagtagtagtagtagtaccatatactatgcagactctgt

gaagggccgattcaccatctccagagacaatgccaagaactcactgtatc

tgcaaatgaacagcctgagagacgaggacacggctgtgtattactgtgcg

agaggagtgtatcacaatggctggtccttctttgactactggggccaggg

aaccctactcaccgtctcctca-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable light chain comprising the amino acid sequence of SEQ ID NO: 37 shown in the following Table.

Human anti-OX40 antibody 112F32

(ATCC No. PTA-7217) Light chain variable region

amino acid sequence.

(SEQ ID NO: 37)

1

MDMRVLAQLL GLLLLCFPGA RCDIQMTQSP SSLSASVGNR

VTITCRASQD ISSWLAWYQQ

61
KPEKAPKSLI YAASSLQSGV PSRFSGSGSG TDFTLTISSL

QPEDFATYYC QQYNSYPLTF

121
GQGTRLEIKR

The nucleic acid sequence encoding the human anti-OX40 antibody light chain variable region comprises the nucleic acid sequence of SEQ ID NO: 38 shown in the following Table.

Human anti-OX40 antibody 12F32 LV

(ATCC No. PTA-7217) Light chain variable region

nucleic acid sequence

(SEQ ID NO: 38)

5′-atggacatgagggtcctcgctcagctcctggggctcctgctgctctg

tttcccaggtgccagatgtgacatccagatgacccagtccccatcctcac

tgtctgcatctgtaggaaacagagtcaccattacttgtcgggcgagtcag

gatattagcagctggttagcctggtatcagcagaaaccagagaaagcccc

taagtccctgatctatgctgcatccagtttgcaaagtggggtcccatcaa

ggttcagcggcagtggatctgggacagatttcactctcaccatcagcagc

ctgcagcctgaagattttgcaacttattactgccaacagtataatagtta

ccccctcaccttcggccaagggacacgactggagattaaacga-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable heavy chain comprising the amino acid sequence of SEQ ID NO: 39 shown in the following Table.

Human anti-OX40 antibody 112Y1 31

(ATCC NO. PTA-7218) Heavy chain variable region

amino acid sequence

(SEQ ID NO: 39)

1

MDTLCSTLLL LTIPSWVLSQ ITLKESGPTL VKPTQTLTLT

CTFSGFSLST SGVGVGWIRQ

61
PPGKALEWLA LIYWDDHSPY SPSLKSRLTI TKDTSKNQVV

LTMTNMDPVD TATYYCARTR

121
GAFDIWGQGT MVTVSS

The nucleic acid sequence encoding the human anti-OX40 antibody heavy chain variable region comprises the nucleic acid sequence of SEQ ID NO: 40 shown in the following Table.

Human anti-OX40 antibody 112Y1 31

(ATCC NO. PTA-7218) Heavy chain variable region

nucleic acid sequence

(SEQ ID NO: 40)

5′-atggacactctttgctccacgctcctgctgctgaccatcccttcatg

ggtcttgtcccagatcaccttgaaggagtctggtcctacgctggtgaaac

ccacacagaccctcacgctgacctgcaccttctctggattctcactcagc

actagtggagtgggtgtgggctggatccgtcagcccccaggaaaggccct

ggagtggcttgcactcatttattgggatgatcatagcccctacagcccat

ctctgaagagcaggctcaccatcaccaaggacacctccaaaaaccaggtg

gtccttacaatgaccaacatggaccctgtggacacagccacatattactg

tgcacgcacccggggggcttttgatatctggggccaagggacaatggtca

ccgtctcttca-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable light chain comprising the amino acid sequence of SEQ ID NO: 41 shown in the following Table.

Human anti-OX40 antibody 112Y1 31 (ATCC NO. PTA-7218) Light chain

variable region amino acid sequence

(SEQ ID NO: 41)

1

MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQGVS SYLAWYQQKP

61
GQAPRLLIYD ASNRATGIPA RFSGSGPGTD FTLTISSLEP EDFAVYYCQQ RSNWHPTFGQ

121
GTKVEIK

The nucleic acid sequence encoding the human anti-OX40 antibody light chain variable region comprises the nucleic acid sequence of SEQ ID NO: 42 shown in the following Table.

Human anti-OX40 antibody 112Y1 31 (ATCC NO.

PTA-7218) Light chain variable region nucleic

acid sequence

(SEQ ID NO: 42)

5′-atggaagccccagcgcagcttctcttcctcctgctactctggctccc

agataccaccggagaaattgtgttgacacagtctccagccaccctgtctt

tgtctccaggggaaagagccaccctctcctgcagggccagtcagggtgtt

agcagctacttagcctggtaccagcagaaacctggccaggctcccaggct

cctcatctatgatgcatccaacagggccactggcatcccagccaggttca

gtggcagtgggcctgggacagacttcactctcaccatcagcagcctagag

cctgaagattttgcagtttattactgtcagcagcgtagcaactggcatcc

gacgttcggccaagggaccaaggtggaaatcaaacgaactgtggctgcac

catc-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43 shown in the following Table.

Human anti-OX40 antibody 112V8 (ATCC NO. PTA-7219) Heavy chain

variable region amino acid sequence

(SEQ ID NO: 43)

1

MDTLCSTLLL LTIPSWVLSQ ITLKESGPTL VKPKQTLTLT CTFSGFSLST SGMGVGWIRQ

61
PPGKALEWLA VIYWDDHQLY SPSLKSRLTI TKDTSKNQVV LTMTNMDPVD TATYYCAHRR

121
GAFQHWGQGT LVTVSSASTK

The nucleic acid sequence encoding the human anti-OX40 antibody heavy chain variable region comprises the nucleic acid sequence of SEQ ID NO: 44 shown in the following Table.

Human anti-OX40 antibody 112V8 (ATCC NO. PTA-7219)

Heavy chain variable region nucleic acid sequence

(SEQ ID NO: 44)

5′-atggacacactttgctccacgctcctgctgctgaccatcccttcatg

ggtcttgtcccagatcaccttgaaggagtctggtcctacgctagtgaagc

ccaaacagaccctcacgctgacctgcaccttctctggattctcactcagc

actagtggaatgggtgtgggctggatccgtcagcccccaggaaaggccct

ggagtggcttgcagtcatttattgggatgatcatcaactctacagtccat

ctctgalgagcaggctcaccatcaccaaggacacctccaaaaaccaggtg

gtccttacaatgaccaacatggaccctgtggacacagccacatattactg

tgcacacagacgaggggccttccagcactggggccagggcaccctggtca

ccgtctcctcagcttccaccaagggc-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable light chain comprising the amino acid sequence of SEQ ID NO: 45 shown in the following Table.

Human anti-OX40 antibody 112V8 (ATCC NO. PTA-7219) Light chain

variable region amino acid sequence

(SEQ ID NO: 45)

1

METPAQLLFL LLLWLPDTTG EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK

61
PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYDSSLTFGG

121
GTKVEIKRT

The nucleic acid sequence encoding the human anti-OX40 antibody light chain variable region comprises the nucleic acid sequence of SEQ ID NO: 46 shown in the following Table.

Human anti-OX40 antibody 112V8 (ATCC NO. PTA-7219)

Light chain variable region nucleic acid sequence

(SEQ ID NO: 46)

5′-atggagtgggggccgtgctgggttttccttgttgttattttagaagg

tgtccagtgtgggatggaaaccccagcgcagcttctcttcctcctgctac

tctggctcccagataccaccggagaaattgtgttgacgcagtctccaggc

accctgtctttgtctccaggggaaagagccaccctctcctgcagggccag

tcagagtgttagcagcagctacttagcctggtaccagcagaaacctggcc

aggctcccaggctcctcatctatggtgcatccagcagggccactggcatc

ccagacaggttcagtggcagtgggtctgggacagacttcactctcaccat

cagcagactggagcctgaagattttgcagtgtattactgtcagcagtatg

atagctcgctcactttcggcggagggaccaaggtggagatcaaacgaac

t-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable heavy chain comprising the amino acid sequence of SEQ ID NO: 47 shown in the following Table.

Human anti-OX40 antibody 112Y55 (ATCC No. PTA-7220) Heavy chain

variable region amino acid sequence

(SEQ ID NO: 47)

1

MDTLCSTLLL LTIPSWVLSQ ITLKESGPTL VKPTQTLTLS CTFSGFSLST SGVGVGWIRQ

61
PPGKALEWLA LIHWDDAERY SPSLKSRLTI TKDTSKNQVV LTMTNMDLVD TATYYCAHTR

121
GAFDIWGQGT MVTVSS

The nucleic acid sequence encoding the human anti-OX40 antibody heavy chain variable region comprises the nucleic acid sequence of SEQ ID NO: 48 shown in the following Table.

Human anti-OX40 antibody 112Y55 (ATCC No.

PTA-7220) Heavy chain variable region nucleic

acid sequence

(SEQ ID NO: 48)

5′-atggacacactttgctccacgctcctgctgctgaccatcccttcatg

ggtcttgtcccagatcaccttgaaggagtctggtcctacgctggtgaaac

ccacacagaccctcacgctgtcctgcaccttctctgggttctcactcagc

actagtggagtgggtgtgggctggatccgtcagcccccaggaaaggccct

ggaatggcttgcactcattcattgggatgatgctgagcgctacagtccat

ctctgaagagcaggctcaccatcaccaaggacacctccaaaaaccaggtg

gtccttacaatgaccaacatggaccttgtggacacagccacatattactg

tgcacacacccggggggcttttgatatctggggccaagggacaatggtca

ccgtctcttca-3′

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 antibody variable light chain comprising the amino acid sequence of SEQ ID NO: 49 shown in the following Table.

Human anti-OX40 antibody 112Y55 (ATCC No. PTA-7220) Light chain

variable region amino acid sequence

(SEQ ID NO: 49)

1

METPAQLLFL LLLWLPDTTG EIVLTQSPGT LSLSPGERAI LSCRASQSVS SSFLAWYQQK

61
PGQAPRLLIY GAFSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYDSSRTFGQ

121
GTKVEIK

The nucleic acid sequence encoding the human anti-OX40 antibody light chain variable region comprises the nucleic acid sequence of SEQ ID NO: 50 shown in the following Table.

Human anti-OX40 antibody 112Y55 (ATCC No.

PTA-7220) Light chain variable region nucleic

acid sequence

(SEQ ID NO: 50)

5′-atggaaaccccagcgcagcttctcttcctcctgctactctggctccc

agataccaccggagaaattgtgttgacgcagtctccaggcaccctgtctt

tgtctccaggggaaagagccatcctctcctgcagggccagtcagagtgtt

agcagcagcttcttagcctggtaccaacagaaacctggccaggctcccag

gctcctcatctatggtgcatttagcagggccactggcatcccagacaggt

tcagtggcagtgggtctgggacagacttcactctcaccatcagcagactg

gagcctgaagattttgcagtgtattactgtcagcagtatgatagctcacg

gacgttcggccaggggaccaaggtggaaatcaaa-3′

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody variable heavy chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 IgG2a antibody heavy chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody variable light chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise the nucleic acid encoding an anti-OX40 IgG2a antibody light chain with an amino acid sequence that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody variable heavy chain amino acid sequence, and a mouse anti-OX40 antibody variable light chain amino acid sequence, that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13. The Sindbis viral vector can comprise a nucleic acid encoding an anti-OX40 antibody heavy chain amino acid sequence, and an anti-OX40 antibody light chain amino acid sequence, that binds to a target antigen of the amino acid sequence of SEQ ID NO: 13.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 36. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 39. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 40. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 42.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 43. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 44. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 45. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 46.

The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable heavy chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47. The nucleic acid sequence encoding the human anti-OX40 variable heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to is SEQ ID NO: 48. The Sindbis viral vector can comprise a nucleic acid encoding a human anti-OX40 variable light chain comprising the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49. The nucleic acid sequence encoding the human anti-OX40 variable light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 50.

Sindbis Viral Vector and Anti-OX40 Monoclonal Antibody

The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit (IL-12 α, IL-12, p35 subunit) and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit). The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit of GenBank accession no. M86672 and interleukin-12 beta subunit of GenBank accession no. M86671. The nucleic acid encoding interleukin-12 alpha subunit comprises the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid encoding interleukin-12 beta subunit comprises the nucleic acid sequence of SEQ ID NO: 2.

Sindbis virus can be administered at least one time, at least two times, at least three times, at least four times or at least five times per week. Sindbis virus can be administered for at least one week, at least two weeks, at least three weeks, or at least four weeks. Sindbis virus can be administered from 10⁶-10⁹TU/mL. Preferably, Sindbis virus can be administered from 10⁶-10⁹TU/mL.

An anti-OX40 monoclonal antibody can be administered at least one time, at least two times, at least three times, at least four times or at least five times per week. An anti-OX40 monoclonal antibody can be administered for at least one week, at least two weeks, at least three weeks, or at least four weeks. An anti-OX40 monoclonal antibody can be administered from 25 μg-500 μg, 25 μg-450 μg, 50 μg-400 μg, from 50 μg-350 μg, from 50 μg-300 μg, from 50 μg-250 μg, from 50 μg-200 μg, from 50 μg-150 μg or from 50 μg-100 μg. An anti-OX40 monoclonal antibody can be administered at 250 μg. An anti-OX40 monoclonal antibody can be administered at 250 μg once a week for one week. An anti-OX40 monoclonal antibody can be administered at 250 μg once a week for two weeks. An anti-OX40 monoclonal antibody can be administered at 250 μg once a week for three weeks. An anti-OX40 monoclonal antibody can be administered at 250 μg three times a week for one week. An anti-OX40 monoclonal antibody can be administered at 250 μg three times a week for two weeks. An anti-OX40 monoclonal antibody can be administered at 250 μg three times a week for three weeks.

The results provided in the instant disclosure demonstrate that administration of the a Sindbis virus expressing IL-12 (SV.IL12) markedly increases the expression of OX40 on CD4 T cells and demonstrate that administration of a combination of SV.IL12 and anti-OX40 monoclonal antibody resulted in complete tumor regression in colon cancer, prostate cancer and ovarian cancer in vivo models and led to a greater than 60% survival rate (in some instances to a greater than 90% survival rate). This combined therapeutic effect was dramatically more effective when compared to either SV.IL12 or anti-OX40 monoclonal antibody treatment alone. These results also confirm that the oncolytic activity of the Sindbis virus is not required to induce a robust and effective anti-tumor response.

The results provided in the instant disclosure demonstrate that the combination of SV.IL12 or anti-OX40 monoclonal antibody treatment markedly changes the transcriptome signature of T cells and favors the differentiation of terminal effector T cells (e.g., effector T cells with a Th1 type phenotype). In particular, pathways upregulated by the combination treatment were dominated by DNA replication, chromosomal organization and cell cycle regulation, but also included various metabolic and immunological processes, such as mitochondrial respiration, nucleotide metabolism and adaptive immune responses. Specifically, only T cells from combined therapy expressed the gene signature of terminally differentiated effector T cells, which are characterized by high expression of the killer lectin-like receptor (KLRG1) and low expression of the interleukin 7 receptor (IL-7R). Furthermore, genes encoding products associated with the differentiation and function of effector cells, such as Batf Id2, Tbet, Gzmb and Ifng, were also highly expressed in T cells following combination therapy. Furthermore, CD4 T cells also expressed a marked anti-tumor effector phenotype (ICOS*Tbet*) which was on average 2 to 3-fold higher during combined therapy compared with SV.IL12 or anti-OX40 treatment.

The tumor microenvironment can be a very challenging milieu for an effector T cell as it is characterized by hypoxia, acidosis and low levels of nutrient sources such as glucose and glutamine. Even if T cell activation and initiation of effector function is allowed, T cells may be unable to generate the bioenergetics intermediates necessary to carry out effector function in the tumor microenvironment. Thus, providing a metabolic support for T cells is crucial for the success of cancer treatments. The results provided in the instant disclosure demonstrate that the combination of SV.IL12 or anti-OX40 monoclonal antibody promotes metabolic reprogramming of T cells. Specifically, the basal rate of oxygen consumption (OCR) was enhanced and spare respiratory capacity was dramatically increased in T cells following combination treatment. The combination also induced elevated protein expression of c-MYC as well as rate of extracellular acidification (ECAR). Collectively, these results show that SV.IL12 induces enhanced oxidative phosphorylation in CD8 T cells and the combination treatment is required to push CD4 T cells towards glycolysis by increasing the protein expression of c-MYC. Thus, the combination of SV.IL12 or anti-OX40 monoclonal antibody metabolically rewires T cells to an energetic state using both metabolic pathways, oxidative phosphorylation and glycolysis.

The results provided in the instant disclosure demonstrate that metabolic reprogrammed T cells display enhanced CD4 mediated cytokine production and anti-tumor activity following treatment with the combination of SV.IL12 and anti-OX40 monoclonal antibody. Specifically, genes encoding pro-inflammatory cytokines ifng and il2 were upregulated in T cells and the secretion of interferon-γ (IFNγ) by splenocytes was increased following combination treatment. Additional, the levels of the cytotoxic proteases, granzyme A and B, were upregulated following combination treatment. Further, granzyme B positive cells were detected in CD8 as well as CD4 T cells, indicating the presence of cytotoxic CD4 T cells following combination treatment. In addition, tumor growth was markedly reduced when co-cultured with splenocytes from mice receiving combined therapy. Surprisingly, tumor growth inhibition was mediated by CD4 T cells. Together, these results clearly show that T cells from combined therapy elicit enhanced anti-tumor and functional activity, such as granzyme B and IFNγ production driven by CD4 T cells.

The results provided in the instant disclosure demonstrate that treatment with the combination of SV.IL12 and anti-OX40 monoclonal antibody results in enhanced T cell migration and intratumoral T cell immunity. Specifically, CXCR3 levels were significantly upregulated on CD4 T cells following combination therapy. In contrast, CXCR3 expression on CD8 T cells only appeared later on in treatment, indicating that CD4 T cells are first recruited to the inflamed site followed by CD8 T cells. Combination therapy also enhanced the production of CXCL9 and CXCL10 in the tumor microenvironment, indicating that CXCR3 positive T cells migrate to the tumor site. These results clearly show that the combination of SV.IL12 and anti-OX40 monoclonal antibody alter the tumor microenvironment by facilitating T cell infiltration via modulation of the CXCR3/CXCL9-11 axis. Not only did combination therapy increase T cell infiltration but CD4 as well as CD8 T cells also demonstrated enhanced functional activity in the tumor, as judged by the Ki-67 and granzyme B expression. These results indicate that the presence of activated T cells in the tumor microenvironment exert anti-tumor activity which inhibits tumor growth. Enhanced iNOS production was also demonstrated in tumors treated with combination therapy. Interestingly, the amount of iNOS inversely correlated with arginase1 production, indicating a repolarization of tumor associated macrophages from the M2-like (pro-tumor) into Ml-like (anti-tumor) phenotype during combination therapy.

Thus, the data provided herein clearly shows that even in absence of direct Sindbis virus infectivity, SV.IL12 in combination with an anti-OX40 monoclonal antibody alter the tumor microenvironment by enhancing T cell infiltration and intratumoral T cell immunity, especially against low immunogenic tumors. The synergistic therapeutic efficacy of the systemic administration of the combination is driven by T cell modulation and reprogramming of its metabolic state, in order to enhance the anti-tumor response in the periphery and in the tumor microenvironment. Furthermore, the use of Sindbis virus allows these metabolically reprogrammed T cells to better infiltrate the tumor microenvironment, which is crucial for an adequate immunotherapy.

Sindbis Viral Vector and Anti-4-1BB Monoclonal Antibody

The present disclosure also provides methods for treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount (a) a Sindbis viral vector and (b) an anti-4-1BB monoclonal antibody or a nucleic acid encoding same, thereby treating cancer in the subject. The present disclosure further provides in vitro or ex vivo methods for treating cancer or assessing the treatment of cancer in a subject comprising contacting a biological sample from the subject with (a) a Sindbis viral vector and (b) an anti-4-1BB monoclonal antibody or a nucleic acid encoding same. Preferably, the Sindbis viral vector does not comprise an endogenous nucleic acid encoding any protein. Sindbis viral vectors were produced as described in U.S. Pat. No. 8,093,021 (incorporated herein by reference in its entirety).

The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1BB antibody heavy chain comprising the heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 amino acid sequences as follows: HCDR1: GFIFSYFDMA (SEQ ID NO: 16), HCDR2: SISPDGSIPYYRDSVK (SEQ ID NO: 17) and HCDR3: RSYGGYSELDY (SEQ ID NO: 18).

The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1BB heavy chain comprising the amino acid sequence of SEQ ID NO:19 shown in the following Table.

anti-4-1BB heavy chain amino acid

(SEQ ID NO: 19)

DVQLVESGGGLVQPGRSLKLSCAASGFIFSYEDMAWVRQAPTKGLEWVAS

ISPDGSIPYYRDSVKGRFTVSRENAKSSLYLQMDSLRSEDTATYYCARRS

YGGYSELDYWGQGVMVTVSS.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1BB antibody light chain comprising the light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 amino acid sequences as follows: LCDR1: QASQDIGNWLA (SEQ ID NO: 20), LCDR2: GSTSLAD (SEQ ID NO: 21) and LCDR3: LQAYGAPW (SEQ ID NO: 22).

The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1BB light chain comprising the amino acid sequence of SEQ ID NO:23 shown in the following Table.

anti-4-1BB light chain amino acid

(SEQ ID NO: 23)

DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYHQKPGKSPQLLIYG

STSLADGVPSRFSGSSSGSQYSLKISRLQVEDIGIYYCLQAYGAPWTEGG

GTKLELK

The Sindbis viral vector can comprise a nucleic acid encoding anti-4-1BB antibody heavy chain CDR1, CDR2 and CDR3 amino acid sequences that binds to a target antigen comprising the amino acid sequence of SEQ ID NO:24 shown in the following Table.

anti-4-1BB target antigen

(SEQ ID NO: 24)

1
MGNNCYNVVV IVLLLVGCEK VGAVQNSCDN CQPGTFCRKY NPVCKSCPPS

51
TFSSIGGQPN CNICRVCAGY FRFKKFCSST HNAECECIEG FHCLGPQCTR

101
CEKDCRPGQE LTKQGCKTCS LGTFNDQNGT GVCRPWTNCS LDGRSVLKTG

151
TTEKDVVCGP PVVSFSPSTT ISVTPEGGPG GHSLQVLTLF LALTSALLLA

201
LIFITLLFSV LKWIRKKFPH IFKQPFKKTT GAAQEEDACS CRCPQEEEGG

251
GGGYEL

The Sindbis viral vector can comprise a nucleic acid encoding a 4-1BB heavy chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 19 and a nucleic acid encoding a 4-1BB light chain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid sequence of SEQ ID NO: 23.

The Sindbis viral vector can comprise the nucleic acid encoding anti-4-1BB antibody light chain CDR1, CDR2 and CDR3 amino acid sequences that binds to a target antigen (4-1BB antigen) of the amino acid sequence of (SEQ ID NO: 24). The Sindbis viral vector can comprise the nucleic acid encoding anti-4-1BB antibody heavy chain amino acid sequence that binds to a target antigen (4-1BB antigen) of the amino acid sequence of (SEQ ID NO: 24). The Sindbis viral vector can comprise the nucleic acid encoding anti-4-1BB antibody light chain amino acid sequence that binds to a target antigen (4-1BB antigen) of the amino acid sequence of (SEQ ID NO: 24).

A “monoclonal antibody” as disclosed herein, can be a full-length antibody or an antigen binding fragment thereof, wherein the “antigen binding fragment” is a fragment of the full length antibody that retains binding to the target antigen of the said monoclonal antibody. For example, a monoclonal antibody against 4-1BB or an anti-4-1BB monoclonal antibody, as described herein, can be a full length antibody against 4-1BB antibody or an antigen binding fragment thereof, wherein the fragment retains binding to the 4-1BB receptor on a cell surface. An “antigen-binding fragment” of an anti-4-1BB antibody, as described herein can include any fragment selected from the group consisting of Fv, Fav, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments.

The cancer can be a solid cancer or a liquid/hematologic cancer. The cancer can comprise metastatic cancer. The cancer can comprise a solid tumor. The cancer can be a carcinoma, a lymphoma, a blastoma, a sarcoma, a leukemia, a brain cancer, a breast cancer, a blood cancer, a bone cancer, a lung cancer, a skin cancer, a liver cancer, an ovarian cancer, a bladder cancer, a renal cancer, a gastric cancer, a thyroid cancer, a pancreatic cancer, an esophageal cancer, a prostate cancer, a cervical cancer or a colorectal cancer. In one preferred aspect, the cancer is a lymphoma. In one preferred aspect, the cancer is a B cell lymphoma.

The present disclosure provides a Sindbis viral vector comprising a nucleic acid encoding a therapeutic protein, an immunostimulatory protein or an immunomodulatory protein and a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure provides a Sindbis viral vector comprising a nucleic acid encoding LacZ, Flue or GFP and a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding a therapeutic protein, an immunostimulatory protein or an immunomodulatory protein and (b) a Sindbis viral vector comprising a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding LacZ, Flue or GFP and (b) a Sindbis viral vector comprising a nucleic acid encoding an anti-4-1BB monoclonal antibody. The present disclosure further provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding a therapeutic protein, an immunostimulatory protein or an immunomodulatory protein and (b) an anti-4-1BB monoclonal antibody or a nucleic acid encoding same. The present disclosure provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding encoding LacZ, Flue or GFP and (b) an anti-4-1BB monoclonal antibody or a nucleic acid encoding same.

The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1 in antibody heavy chain comprising the heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively. The Sindbis viral vector can comprise the nucleic acid encoding an anti-4-1BB antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 19. The Sindbis viral vector can comprise a nucleic acid encoding an anti-4-1BB antibody heavy chain comprising the light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively. The Sindbis viral vector can comprise the nucleic acid encoding an anti-4-1BB antibody light chain comprising the amino acid sequence of SEQ ID NO: 23.

The immunostimulatory or immunomodulatory protein can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6 IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, or any combination thereof. In a preferred aspect, the immunostimulatory or immunomodulatory protein is IL-12. Additional cytokines include IL-I8-IL-36. In addition to CCL17, other chemokines can also be used, including, but not limited to, CCL1-CCL27 and other CC chemokines, CXCLI-CXCL13 and other CXC chemokines, C chemokines, and CX3C chemkines. Cytokine or chemokine receptors and soluble receptors can also be used. Additional immune modulators that can be used include TGF-β and TNFα. In addition, different combinations of the above-mentioned (or alternative) cytokines can be used.

A “monoclonal antibody” as disclosed herein, can be a full-length antibody or an antigen binding fragment thereof, wherein the “antigen binding fragment” is a fragment of the full length antibody that retains binding to the target antigen of the said monoclonal antibody. For example, a monoclonal antibody against 4-1BB or an anti-4-1BB monoclonal antibody, as described herein, can be a full length antibody against 4-1BB antibody or an antigen binding fragment thereof, wherein the fragment retains binding to the 4-1BB receptor on a cell surface. An “antigen-binding fragment” of an anti-4-1BB antibody, as described herein can include any fragment selected from the group consisting of Fv, Fav, F(ab′)2, Fab′, dsFv, scFv, sc(Fv)2, scFv-CH3, scFv-Fc, and diabody fragments.

An anti-4-1BB monoclonal antibody can be administered at least one time, at least two times, at least three times, at least four times or at least five times per week. An anti-4-1BB monoclonal antibody can be administered for at least one week, at least two weeks, at least three weeks, or at least four weeks. An anti-4-1BB monoclonal antibody can be administered from 25 μg-500 μg, 25 μg-450 μg, 50 μg-400 μg, from 50 μg-350 μg, from 50 μg-300 μg, from 50 μg-250 μg, from 50 μg-200 μg, from 50 μg-150 μg or from 50 μg-100 μg. An anti-4-1BB monoclonal antibody can be administered at 50 μg. An anti-4-1BB monoclonal antibody can be administered at 50 μg once a week for three weeks. An anti-4-1BB monoclonal antibody can be administered at 250 μg. An anti-4-1BB monoclonal antibody can be administered at 250 μg three times week for two weeks. An anti-4-1BB monoclonal antibody can be administered at 350 μg. An anti-4-1BB monoclonal antibody can be administered at 350 μg three times week for two weeks.

The results provided in the instant disclosure demonstrate that administration of the combination of Sindbis virus and anti-4-1BB monoclonal antibody resulted in complete tumor regression in an lymphoma in vivo model and that this therapeutic effect was dramatically more effective when compared to either Sindbis virus or anti-4-1BB monoclonal antibody treatment alone. Tumor elimination involves a synergistic effect of the combination that significantly boosts T cell cytotoxicity, IFNγ production, T cell proliferation, migration, and glycolysis. As described in more detail below, the data identified the molecular pathways, including upregulated cytokines, chemokines and metabolic pathways in T cells that are triggered by the combined therapy and help to achieve a highly effective anti-tumor response.

The results provided in the instant disclosure demonstrate that administration of the combination of Sindbis virus and anti-4-1BB monoclonal antibody resulted in increased T cell cycle progression, cytokine production and activation. T cell proliferation is critical for an effective anti-tumor response.

The results provided in the instant disclosure demonstrate that administration of the combination of Sindbis virus and anti-4-1BB monoclonal antibody resulted in increased cytotoxicity (e.g., increased cytotoxic T cell function). Specifically, genes such as Gzmb (granzyme B), Prfl (perforin) and Klrkl (NKG2D) are significantly upregulated in T cells (particularly CD8 T cells) following administration of Sindbis virus and anti-4-1BB monoclonal antibody.

The results provided in the instant disclosure demonstrate that administration of the combination of Sindbis virus and anti-4-1BB monoclonal antibody resulted in increased IFNγ production from T cells and Th1 differentiation. The combination of Sindbis virus and anti-4-1BB monoclonal antibody upregulated the expression of STAT4, Ccr5, Cxcr3, Havcr2(Tim3), IL12rbl and Klrcl in T cells, which are required for the development of Th1 cells from naïve CD4+ T cells and IFNγ production. This increase was independent of the presence or absence of TAA. The combination of Sindbis virus and anti-4-1BB monoclonal antibody increased IFNγ production from both CD4 and CD8 T cells (with a larger portion CD4 T cells producing IFNγ) and demonstrated that antigen presenting cells (APCs) are essenTh1tial for helping T cells product IFNγ. The combination of Sindbis virus and anti-4-1BB monoclonal antibody also increased T-bet in T cells. T-bet is a key transcription factor which is essential for type I immune response (IFNγ production, T cell cytotoxicity) and memory T cell differentiation. Thus, this indicates that the combination of Sindbis virus and anti-4-1BB monoclonal antibody boosts the type I immune response, which is critical for controlling tumor growth. The combination of Sindbis virus and anti-4-1BB monoclonal antibody also increased Eomesodermin (EOMES) in T cells. EOMES, another important transcription factor, is upregulated in activated T cells and is essential for memory CD8 T cell development.

The results provided in the instant disclosure demonstrate that administration of the combination of Sindbis virus and anti-4-1BB monoclonal antibody resulted in increased chemotaxis, adhesion and enhanced T cell infiltration and activation in tumors. Specifically, the combination significantly upregulates CD11a and ICAM-1(CD54) in both CD4 and CD8 T cells, which are two adhesion molecules expressed on activated T cells and are essential for the formation of immune synapses between T cells and APCs and are also required for T cell/T cell homotypic aggregation and activation. The combination of Sindbis virus and anti-4-1BB monoclonal antibody also significantly upregulated OX40 and ICOS in T cells. OX40 engagement promotes effector T cell function and survival and ICOS is another key CD4 T cell costimulatory molecule. Tumor infiltrating lymphocytes play a critical anti-tumor role and are an important marker for prognosis. The percentage of CD3 and CD8 T cells increased about two-fold following combination treatment. Thus, these results demonstrate that combination treatment enhanced T cell infiltration, division, activation, cytotoxicity and downregulated the inhibitory Treg population.

The results provided in the instant disclosure demonstrate that administration of the combination of Sindbis virus and anti-4-1BB monoclonal antibody resulted in enhanced T cell glycolysis and oxidative phosphorylation. T cell activation requires a quick consumption of energy through both enhanced glycolysis and oxidative phosphorylation. Metabolic switch is a major feature of T cell activation and memory T cell development. Upregulation of glycolysis genes quickly produce ATP and supports T cell migration and cytotoxicity in hypoxic or acidific microenvironments (such as in and around a tumor). The instant results demonstrate that combination treatment significantly increased both oxygen consumption rate (OCR, represents oxidative phosphorylation) and extracellular acidification rate (ECAR, represents glycolysis). This indicates that both glycolysis and oxidative phosphorylation are activated in combination treated T cells.

The results provided in the instant disclosure demonstrate that mice cured by the administration of the combination of Sindbis virus and anti-4-1BB monoclonal antibody are completely protected from cancer rechallenge demonstrating that these mice acquired long lasting antitumor immunity.

The quick inhibition of tumor growth is critical for cancer therapy because tumor cells undergo exponentially rapid division. However, the induction of adaptive immunity and establishment of tumor specific immunity takes a long time. An ideal therapy requires an early, quick reduction of tumor burden, and a later induction of anti-tumor specificity that prevents relapse. The data presented herein demonstrates that the combination of Sindbis virus and anti-4-1BB monoclonal antibody treatment induced massive T cell activation due to viral induced immune response. This massive activation helps to control the tumor in a TAA nonspecific manner.

It was shown herein that both NKG2D (KLRKI) and granzyme B are highly expressed under combination treatment. This massive nonspecific activation is critical for controlling tumor growth at an early time point (day 7). This step is also important for inducing anti-tumor specificity that is mediated by TAAs released from dead tumor cells due to nonspecific killing. After tumor regression, T cells from treated animals were able maintain the ability to produce IFNγ and acquired immunological memory to rapidly reject tumor rechallenges. IFNγ production from purified T cells of cured mice was significantly enhanced after encountering tumor cells. This demonstrates that anti-tumor specificity is fully established in cured mice.

The data also shows that Sindbis viral infection of tumor cells, inclusion of dendtric cells and lymphodepletion are not necessary for successful cancer treatment. The omission of these additional features decreases costs, any risks related to toxicity and infection.

Thus, the data provided herein demonstrates that the combination of Sindbis virus and anti-4-1BB monoclonal antibody completely eradicated a B-cell lymphoma in a preclinical mouse model, a result that could not be achieved with either treatment alone. Tumor elimination involves a synergistic effect of the combination that significantly boosts T cell cytotoxicity, IFN-γ production, migration, tumor infiltration and oxidative phosphorylation. In addition, all mice that survived after treatment developed long lasting antitumor immunity.

Sindbis Viral Vector and Sindbis Viral Vector NY-ESO-1

The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit (IL-12 α, IL-12, p35 subunit) and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit). The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit of GenBank accession no. M86672 and interleukin-12 beta subunit of GenBank accession no. M86671. The nucleic acid encoding interleukin-12 alpha subunit comprises the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid encoding interleukin-12 beta subunit comprises the nucleic acid sequence of SEQ ID NO: 2.

Nucleic acid sequence encoding NY-ESO-1 (NM_001327.1)

(SEQ ID NO: 14)

1
agcagggggc gctgtgtgta ccgagaatac gagaatacct cgtgggccct gaccttctct

61
ctgagagccg ggcagaggct ccggagccat gcaggccgaa ggccggggca cagggggttc

121
gacgggcgat gctgatggcc caggaggccc tggcattcct gatggcccag ggggcaatgc

181
tggcggccca ggagaggcgg gtgccacggg cggcagaggt ccccggggcg caggggcagc

241
aagggcctcg gggccgggag gaggcgcccc gcggggtccg catggcggcg cggcttcagg

301
gctgaatgga tgctgcagat gcggggccag ggggccggag agccgcctgc ttgagttcta

361
cctcgccatg cctttcgcga cacccatgga agcagagctg gcccgcagga gcctggccca

421
ggatgcccca ccgcttcccg tgccaggggt gcttctgaag gagttcactg tgtccggcaa

481
catactgact atccgactga ctgctgcaga ccaccgccaa ctgcagctct ccatcagctc

541
ctgtctccag cagctttccc tgttgatgtg gatcacgcag tgctttctgc ccgtgttttt

601
ggctcagcct ccctcagggc agaggcgcta agcccagcct ggcgcccctt cctaggtcat

661
gcctcctccc ctagggaatg gtcccagcac gagtggccag ttcattgtgg gggcctgatt

721
gtttgtcgct ggaggaggac ggcttacatg tttgtttctg tagaaaataa aactgagcta

781
cgaaaaaaaa aaaaaaaaaa aaaaaa

The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of amino acid sequence of NCBI Reference accession no. NP_001318.1. The amino acid sequence of the NY-ESO-1 comprises the amino acid sequence of SEQ ID NO:15 shown in the following Table.

Amino acid sequence of NY-ESO-1 (NP_001318.1)

(SEQ ID NO: 15)

1
mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgaga arasgpggga

61
prgphggaas glngccrcga rgpesrllef ylampfatpm eaelarrsla qdapplpvpg

121
vllkeftvsg niltirltaa dhrqlqlsis sclqqlsllm witqcflpvf laqppsgqrr

A replication defective Sindbis viral vector as described herein can be any replication defective Sindbis viral vector including a replication defective viral vector described, for example, in U.S. Pat. Nos. 7,303,898, 7,306,792, and 8,093,021. Replication defective vectors are preferred for use in the present invention in order to prevent infection of healthy tissues.

The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit (IL-12 α, IL-12, p35 subunit) and interleukin-12 beta subunit (IL-12 β, IL-12, p40 subunit). The Sindbis viral vector can comprise the nucleic acid encoding interleukin-12 alpha subunit of GenBank accession no. M86672 and interleukin-12 beta subunit of GenBank accession no. M86671. The nucleic acid encoding interleukin-12 alpha subunit comprises the nucleic acid sequence of SEQ ID NO: 1. The nucleic acid encoding interleukin-12 beta subunit comprises the nucleic acid sequence of SEQ ID NO: 2.

The present disclosure further provides a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 and a nucleic acid encoding an anti-OX40 monoclonal antibody. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 and (b) an anti-OX40 monoclonal antibody or a nucleic acid encoding same. The present disclosure also provides a composition comprising (a) a Sindbis viral vector comprising a nucleic acid encoding NY-ESO-1 and (b) a Sindbis viral vector comprising a nucleic acid encoding an anti-OX40 monoclonal antibody. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of NCBI Reference accession no. NM_001327.1. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of NCBI Reference accession no. NM_001327.1. The nucleic acid encoding NY-ESO-1 comprises the nucleic acid sequence of SEQ ID NO: 14. The Sindbis viral vector can comprise the nucleic acid encoding NY-ESO-1 of amino acid sequence of NCBI Reference accession no. NP_001318.1. The amino acid sequence of the NY-ESO-1 comprises the amino acid sequence of SEQ ID NO: 15.

The results provided in the instant disclosure demonstrate that administration of a combination of IL-12 and NY-ESO-1, expressed by separate Sindbis virus vector synergistically enhances the survival rate of a subject bearing an established tumor. The results described herein show that mice transplanted with Alm5-2Fluc-17 ovarian cancer cells by reinjection to establish tumor as depicted in FIG. 19, when treated post-tumor establishment with a SV vector expressing NY-ESO-1 (SVNYESO) showed no enhancement of survival, with a percentage survival rate similar to untreated tumor bearing mice, thereby showing that some tumors are resistant to treatment with SV expressing a TAA, like NY-ESO-1. The results described herein show that treatment of the tumor bearing mice, with a SV expressing NYESO (SV-NYESO_SV-IL12) showed improvement in survival rate. The results show that surprisingly treatment with a 50% mix in one injection of a SV expressing IL-12 (SV-IL-12) and a SV expressing NYESO (SV-NYESO_SV-IL12), demonstrated synergistically enhanced survival as compared to mice treated with the SV-IL-12 or SV-NYESO. The results described herein clearly show the possibility of using a combination of SV vectors expressing IL-12 and NY-ESO-1, for treatment of cancers that may be resistant to treatment with a SV expressing a tumor associated antigen.

The results provided in the instant disclosure demonstrate that administration of a combination of IL-12 and NY-ESO-1, expressed by the same Sindbis virus vector synergistically enhances the survival rate of a subject bearing an established tumor. The results show that mice bearing established tumors of Alm5-2Fluc-17 ovarian cancer cells, when treated with a Sindbis viral vector that expresses both IL-12 and NYESO (SV-NYESO_SGP2_IL12), demonstrated synergistically enhanced survival as compared to mice treated with the SV-IL-12 or SV-NYESO. The results described herein clearly show the possibility of using a single SV vectors expressing both IL-12 and NY-ESO-1, for treatment of cancers that may be resistant to treatment with a SV expressing a tumor associated antigen.

Treating cancer means treating at least one symptom of cancer. Treating at least one symptom of cancer can include any of the following, or any combination thereof: inhibiting tumor growth, reducing tumor size, reducing tumor number, reducing tumor burden, preventing cancer recurrence, preventing metastasis of a primary tumor.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia and germ cell tumors. More particular examples of such cancers include adrenocortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma, endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney chromophobe, kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, acute myeloid leukemia, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, pheochromocytoma, paraganglioma, prostate adenocarcinoma, rectum adenocarcinoma, sarcoma, skin cutaneous melanoma, stomach adenocarcinoma, testicular germ cell tumors, thyroid carcinoma, thymoma, uterine carcinosarcoma, uveal melanoma. Other examples include breast cancer, lung cancer, lymphoma, melanoma, liver cancer, colorectal cancer, ovarian cancer, bladder cancer, renal cancer or gastric cancer. Further examples of cancer include neuroendocrine cancer, non-small cell lung cancer (NSCLC), small cell lung cancer, thyroid cancer, endometrial cancer, biliary cancer, esophageal cancer, anal cancer, salivary, cancer, vulvar cancer, cervical cancer, Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Adrenal gland tumors, Anal cancer, Bile duct cancer, Bladder cancer, Bone cancer, Bowel cancer, Brain tumors, Breast cancer, Cancer of unknown primary (CUP), Cancer spread to bone, Cancer spread to brain, Cancer spread to liver, Cancer spread to lung, Carcinoid, Cervical cancer, Children's cancers, Chronic lymphocytic leukemia (CLL), Chrome myeloid leukemia (CML), Colorectal cancer, Ear cancer, Endometrial cancer, Eye cancer, Follicular dendritic cell sarcoma, Gallbladder cancer, Gastric cancer, Gastro esophageal junction cancers, Germ cell tumors, Gestational trophoblastic disease (GIT)), Hairy cell leukemia, Head and neck cancer, Hodgkin lymphoma, Kaposi's sarcoma, Kidney cancer, Laryngeal cancer, Leukemia, Gastric linitis plastica, Liver cancer, Lung cancer, Lymphoma, Malignant schwannoma, Mediastinal germ cell tumors, Melanoma skin cancer, Men's cancer, Merkel cell skin cancer, Mesothelioma, Molar pregnancy, Mouth and oropharyngeal cancer, Myeloma, Nasal and paranasal sinus cancer, Nasopharyngeal cancer, Neuroblastoma, Neuroendocrine tumors, Non-Hodgkin lymphoma (NHL), Esophageal cancer, Ovarian cancer, Pancreatic cancer, Penile cancer, Persistent trophoblastic disease and choriocarcinoma, Pheochromocytoma, Prostate cancer, Pseudomyxoma peritonei, Rectal cancer. Retinoblastoma, Salivary gland cancer, Secondary' cancer, Signet cell cancer, Skin cancer, Small bowel cancer, Soft tissue sarcoma, Stomach cancer, T cell childhood non Hodgkin lymphoma (NHL), Testicular cancer, Thymus gland cancer, Thyroid cancer, Tongue cancer, Tonsil cancer, Tumors of the adrenal gland, Uterine cancer. Vaginal cancer, Vulval cancer, Wilms' tumor, Womb cancer and Gynaecological cancer. Examples of cancer also include, but are not limited to, Hematologic malignancies, Lymphoma, Cutaneous T cell lymphoma, Peripheral T cell lymphoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, Multiple myeloma, Chrome lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, Myelodysplastic syndromes, Myelofibrosis, Biliary tract cancer, Hepatocellular cancer, Colorectal cancer, Breast cancer, Lung cancer, Non-small cell lung cancer, Ovarian cancer, Thyroid Carcinoma, Renal Cell Carcinoma, Pancreatic cancer, Bladder cancer, skin cancer, malignant melanoma, merkel cell carcinoma, Uveal Melanoma or Glioblastoma multiforme.

The nucleotide sequences encoding the TAAs to be expressed by a Sindbis viral vector as described herein are well known in the art and can be easily obtained from the literature. For example, the sequence of NY-ESO-1, a testicular antigen aberrantly expressed in human cancers was published in 1997 (http://www.pnas.org/content/94/5/1914.full, Yao-Tseng Chen, Matthew J. Scanlant, Ugur Sahin, Ozlem Tiireci, Ali O. Guret, Solam Tsangt, Barbara Williamsont, Elisabeth Stockertt, Michael Pfreundschuh, and Lloyd J. Old, PNAS 1997.), whereas the Carcinoembryonic antigen sequence was published in 1987 (http://mcb.asm.org/content/7/9/3221.short Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen. N Beauchemin, S. Benchimol, D Cournoyer, A Fuks and C P Stanners, Molecular and Cellular Biology.

Although in mice a single i.p. injection of the SV/TAA as described herein, is sufficient to elicit a detectable CD8+ mediated immune response directed against the tumor, other regimens may be necessary for achieving a maximal response. For example, between 1 and about 8 i.p. injections over a time period of between 1 week and many weeks, with the possibility of injecting one or more booster injections 1 or more years later, may be preferably administered for a maximum effect.

EXAMPLES
Example 1: Sindbis with Anti-OX40 Enable Immune Responses to Cold Tumors

This study, investigates the therapeutic efficacy of a replication-deficient oncolytic viral vector called Sindbis Virus. Because Sindbis Virus (SV) is a blood-borne pathogen, vectors from this virus can be administered in the bloodstream via the intravenous (i.v.) and intraperitoneal (i.p.) routes, which greatly facilitates their delivery [Tseng, J C, et al., Nature Biotech., 2004]. Furthermore, SV was genetically modified to be replication-defective by splitting its genome and deleting the packaging signal to block viral assembly after viral replication [Bredenbeek P J, et al., J. Virol. 1993]. This study shows that SV expressing the pro-inflammatory cytokine IL-12 (SV.IL12) activates T cells as well as enhances the expression of OX40 on CD4 T effector cells and, therefore, potentiates efficacy of the agonistic anti-OX40 antibody therapy. The data indicates that combination of SV.IL12 and anti-OX40 activates tumor immunity against low immunogenic tumors through the metabolic rewiring of T cells into highly activated effector cells. Furthermore, SV.IL12 in combination with anti-OX40 induces a marked immune cell infiltration into the tumor microenvironment. Considering that tumors tend to quickly escape the immune response by mutating or losing the expression of drug targets or tumor antigens targeted by the immune response, the treatment approach disclosed herein reduces the risk of developing tumor resistances and offers an attractive and safe strategy to change the immunogenic phenotype of various cancers without prior knowledge of tumor antigens.

The studies presented herein describe several, non-limiting examples of anti-OX-40 antibody, Sindbis viral vector (SV), Sindbis viral vector expressing IL-12 (SV.IL-12), Sindbis viral vector expressing an anti-OX-40 antibody and Sindbis viral vector expressing both IL-12 and an anti-OX-40 antibody. These examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not and are not intended to limit the claimed invention.

Materials and Methods

Cell Lines

Baby hamster kidney (BHK), BALB/c colon carcinoma [CT26.WT (ATCC® CRL-2638™)] and FVB prostate carcinoma [MyC-CaP (ATCC® CRL-3255™)] cell lines were obtained from the American Type Culture Collection (ATCC). Firefly luciferase (Fluc)-expressing CT26 and MyC-CaP cells (CT26.Fluc and MyC-CaP.Fluc) were generated by stable transfection of pGL4.20_Fluc plasmid.

BHK cells were maintained in minimum essential a-modified media (a-MEM) (Corning CellGro) with 5% fetal bovine serum (FCS, Gibco) and 100 mg/ml penicillin-streptomycin (Corning CellGro). CT26.Fluc and MyC-CaP.Fluc cells were maintained in Dulbecco's modified Eagles medium containing 4.5 g/l Glucose (DMEM, Corning CellGro) supplemented with 10% FCS, 100 mg/ml penicillin-streptomycin, 7.5 μg/ml Puromycin or 400 g/ml Gentamycin, respectively. All cell lines were cultured at 37° C. and 5% C02.

SV Production

SV-LacZ production and titering were done the same as previously described [Scherwitzl I, Mal Ther Oncolytics. 2018]. SV.IL12 and SY.Lacz vectors were produced as previously described [Subramanian A et al., Proc Natl Acad Sci US A. 2005; Leonard W J et al., F1000Res. 2016; Rowell J F et al., J. Immunol. 1999; Metcalf T U et al., J. Virol. 2013]. All SV viral vectors used in these studies are replication-defective. Vectors were produced as previously described. SV.IL12 plasmid used in this study has been published in 2002 [Tseng J C et al., J Natl Cancer Inst. 2002]. To construct a Sindbis viral vector containing genes for interleukin 12 (IL-12), the Sindbis viral vector SinRep/2PSG was first constructed, which contains a secondary subgenomic promoter that is responsive to the Sindbis replicase. Two DNA oligonucleotide primers (sequence 5′ CGCGTAAAGCATCTCTACGGTGGTCCTAATAGTGCATG-3′; SEQ ID NO: 29) and its complementary strand 5′CACTATTAGGACCACCGTCGAGATGCTTTA-3′; SEQ ID NO: 30) containing the subgenomic promoter sequence were annealed and ligated into the MluI and SphI sites of the SinRep plasmid. The murine IL-12 α subunit gene (mp35; ATCC 87596) and the IL-12 β subunit gene (mp40; ATCC 87595) were subcloned into the MluI and the StuI sites of SinRep/2PSG, respectively, to produce the Sin-Rep/IL12 plasmid.

SV empty is the same plasmid without an additional gene of interest (e.g.IL12). SV.Luc was generated as described [Tseng, J C et al., Nature Biotech., 2004]. SV.GFP was generated as published in 2012 [Suzme R et al., Cancer Gene Ther., 2012]. Briefly, plasmids carrying the replicon (e.g. SinRep-IL12 or SinRep-IL-12) or DHBB helper RNAs were linearized with XhoI. In vitro transcription was performed using the mMessage mMachine RNA transcription kit (Ambion). Helper and replicon RNAs were then electroporated into BHK cells and incubated at 37° C. in αMEM supplemented with 10% FCS. After 12 hours, the media was replaced with OPTI-MEM (GIBCO-BRL) supplemented with CaCl₂) (100 mg/l) and cells were incubated at 37° C. After 24 hours, the supernatant was collected, centrifuged to remove cellular debris, and frozen at −80° C. Vectors were titrated as previously described [Tseng J C et al., J. Natl. Can. Inst., 2002].

In Vivo Experiments and Tumor Models

All experiments were performed in accordance with the Institutional Animal Care and Use Committee of New York University Health. Six to 12-week old female BALB/c mice were purchased from Taconic (Germantown, N.Y.) and age matched male FVB/NJ mice were purchased from Jackson Laboratory.

Tumor Inoculation and Animal Studies

Treatment started on day 4 after i.p. inoculation of 7×10⁴CT26.Fluc cells or 105 cells of MyC-CaP.Fluc in 500 μl OPTI-MEM. For treatments, mice were randomized and SV (10⁷TU/ml), in a total volume of 500 μl, was injected i.p. into the left side of the animal once for CT26.Fluc and 4 days a week (days 1, 2, 3, 4) for a total of 4 weeks for MyC-CaP.Fluc inoculated mice. The immune checkpoint inhibitor anti-OX40 (clone OX-86, BioXCell) was injected i.p. into the left side of the animal at a dose of 250 μg per injection (1×/week for the CT26.Fluc and 3×/week for MyC-CaP.Fluc tumor bearing mice). Therapeutic efficacy of the treatment was monitored in two ways: tumor luminescence and survival. Noninvasive bioluminescent imaging was performed using the IVIS Spectrum imaging system (Caliper Life Science) at the indicated time points and tumor growth was quantified using the Living Image 3.0 software (Caliper Life Science) as previously described 86. Relative tumor growth for each mouse was calculated dividing total body counts of a given day by total body counts of the first IVIS image. Survival was monitored and recorded daily.

Flow Cytometry

For flow cytometry analysis, spleens and tumors were harvested from mice and processed as previously described [Scherwitzl I, et al., Mol. Ther. Oncol, 2018]. The extracted tumors were chopped into small pieces and incubated with a digestive mix containing RPMI with collagenase IV (50 μg/ml) and DNAse I (20 U/ml) for 1 hour at 37° C. Tumor samples had additional hyaluronidase V (50 μg/ml) in the digestive mix.

Spleens and digested tumors were mashed through a 70-μm strainer before red blood cells were lysed using ammonium-chloride-potassium (ACK) lysis (Gibco). Cells were washed with PBS containing 1% FCS and surface receptors were stained using various antibodies. Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD44, ICOS, OX40, CD69, Foxp3, Granzyme B and Tbet, were purchased from Biolegend. Fluorochrome-conjugated antibodies against mouse CD8a were purchased from BD Biosciences. Mitotracker Deep Red FM, Mitotracker Green and Fluorchrome-conjugated antibodies against CXCR3 and Ki67 were purchased from Thermofisher. Stained cells were fixed with PBS containing 4% Formaldehyde. For intracellular staining, the forkhead box P3 (FOXP3) staining buffer set was used (eBioscience). Flow cytometry analysis was performed on a LSR II machine (BD Bioscience) and data were analyzed using FlowJo (Tree Star).

T Cell Isolation

Total T cells were freshly isolated with the EasySep™ mouse T Cell Isolation Kit. Freshly isolated lymphocytes were depleted of either CD4 or CD8 specific T cells using EasySep™ mouse CD4 and CD8 Positive Selection Kits II. Isolation of T cells and depletions were performed according to the manufacturer's protocols (Stemcell Technologies).

Enzyme-Linked Immunospot (ELISPOT)

Enzyme-linked immunospot was performed as previously described [Scherwitzl I, et al., Mol. Ther. Oncol, 2018]. Splenocytes and T cells were prepared as described for flow cytometry. Mouse IFNγ ELISPOT was performed according to the manufacturer's protocol (BD Bioscience). Lymphocytes (4×105 cells) and isolated (8×104) T cells were plated per well overnight in RPMI supplemented with 10% FCS. No additional stimulus was used in the ELISPOT. As positive control, cells were stimulated with 5 ng/ml PMA+1 g/ml Ionomycin.

Ex Vivo Cytotoxic Assay

T cells were isolated on day 7 and day 14 during treatment. 8×105/ml T cells were co-cultured with CT26.Fluc cells (2×104/ml) or MyC-CaP.Fluc cells (2×104/ml) in a 24 well plate for 2 days in 1 ml RPMI supplemented with 10% FCS. Cells were washed with PBS and lysed with 100 l of M-PER Mammalian Protein Extraction Reagent (Promega) per well. Cytotoxicity was assessed based on the viability of CT26 cells, which was determined by measuring the luciferase activity in each well. Luciferase activity was measured by adding 100 l of Steady-Glo reagent (Promega) to each cell lysate and measuring the luminescence using a GLOMAX portable luminometer (Promega).

CD8+ and CD4+ T-Cell Depletion In Vivo

CD8+ T cells were depleted using anti-CD8 antibody (clone 2.43) (Bio X cell, Lebanon, N.H.). 0.1 mg antibody in 0.2 ml PBS was injected into each mouse, starting 1 day before the first SV treatment, and then every 4 days for 2 weeks. CD4+ T cells were depleted using anti-CD4 antibody (clone GK 1.5) (Bio X cell, Lebanon, N.H.). 0.4 mg were injected into each mouse, starting day 1 before the first treatment. Control mice were injected with PBS and isotype controls.

Quantitative Real-Time PCR

RNA was extracted from tumor samples using RNeasy Kit (Qiagen), followed by cDNA synthesis with the iScript II Kit (Bio-Rad). qRT-PCR was performed using iQ™ SYBR Green Supermix (Biorad) and an StepOne™ Real-Time PCR Detection System (Applied Biosystems). PCR conditions were as follows: 95° C. for 10 min, followed by 40 cycles (94° C. for 30 s, 58° C. for 30 s) of amplification. For quantitation, CT values were normalized to GAPDH and expression was analysed using the 2-ΔΔCT method. Primers for CXCL9, CXCL10 and GAPDH were used. CXCL9 (Forward: GAAGTCCGCTGTTCTTTTCC; SEQ ID NO: 25 Reverse: TTGACTTCCGTTCTTCAGTG; SEQ ID NO: 26), CXCL10 (Forward: GCTGCAACTGCATCCATATC; SEQ ID NO: 27; Reverse: AGGAGCCCTTTTAGACCTTT; SEQ ID NO: 28).

Transcriptome Analysis of T Cells

Total RNA was extracted from freshly isolated T cells on day 7 of treatment from spleens using RNeasy Kit (Qiagen). For each group, 3 BALB/C mice or 3 FVB/J mice were used for biological repeats. RNA-seq was done by NYUMC Genome Center. RNA quality and quantity was analyzed. RNAseq libraries were prepared and loaded on the automated HiSeq 4000 Sequencing System (Illumina) and run as single 50 nucleotide reads.

Alignment and Differential Expression Analysis

Sequences were aligned to the mm10 mouse genome using Bowtie software, Version 1.0.0⁸⁷[Langmead R et al., Genome Biol. 2009] with two mismatches allowed. Uniquely mapped reads were further processed by removing PCR duplicates with Picard (“Picard Tools.” Broad Institute, GitHub repository. http://broadinstitute.github.io/picard/) MarkDuplicates and transcripts were counted using HTSeq⁸⁸and differential gene expression was performed between all groups using DESeq [Anders S et al., Genome Biol. 2010]. Differences in gene expression were considered significant if padj<0.05.

GSEA and Enrichment Map Analysis

The network-based method enrichment map 90 was used for gene-set enrichment visualization and interpretation of data. As a follow up analysis of Gene-Set Enrichment Analysis2 (GSEA) [Mootha V K et al., Nat. Genet., 2003] it reduces redundancy and helps in the interpretation of large gene sets and helps to quickly identify major enriched functional themes in the gene expression data. To perform this analysis, we first assigned a unique row identifier for each transcript and obtained differentially expressed genes through DESeq [Anders S et al., Genome Biol. 2010]. These genes were then ranked and GSEA was performed in Gene Pattern 92 server using GSEA pre-ranked module. We then obtained the gene identifiers corresponding to the gene names using the Bioconductor package ‘org.Mm.eg.db’ and the resulting positively and negatively regulated gene identifiers were used to generate enrichment maps in Cytoscape [Shannon P et al., Genome Res. 2003]. Expression heatmap is drawn by Morpheus (https://software.broadinstitute.org/morpheus/). Highest and lowest expression for each gene (row min. and row max.) were displayed as red or blue color, respectively.

Measurement of Oxygen Consumption and Extracellular Acidification Rates of T Cells

T cell metabolic output was measured by Seahorse technology as previously described [Scharping N E et al., Cancer Immunol. Res., 2017]. Purified T cells were plated at 6×10⁵cells/well in a Seahorse XF24 cell culture microplate. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an Agilent Seahorse XFe24 metabolic analyzer following the procedure recommended by the manufacturer (Agilent). For the mitochondrial stress test, 1) oligomycin (1 μM), 2) FCCP (1.5 μM) and 3) rotenone (100 nM) and antimycin A (1 μM) were injected sequentially through ports A, B and C.

Immunoblot Analysis

Cells were lysed in M-PER© Mammalian Protein Extraction Reagent according to the manufacturer's protocol. Lysates were separated by SDS-PAGE on 4-12% Bio-Rad gels, transferred to polyvinylidene difluoride (PVDF) membranes, blocked in 5% Milk in TBS buffer with 0.1% Tween-20 (TBST). Primary antibodies to c-Myc (Santa Cruz Biotechnology) and GAPDH (Thermofisher) were added at room temperature or overnight at 4° C. Secondary fluorescent antibodies (IRDye®, LI-COR) were added in 5% Milk in TBST for 1 h at room temperature. Odyssey® Classic Infrared Imaging System was used for visualization.

Histochemistry and Multiplex immunofluorescence (MIF)

Tumors of mice were collected, fixed in 4% PFA for 2 days and embedded in paraffin, sectioned and H&E stained. For Multiplex immunofluorescence staining and imaging, five micron paraffin sections were stained with Akoya Biosciences® Opal™ multiplex automation kit on a Leica BondRX® autostainer, according to the manufacturers' instructions. Prior to incubation with the first primary antibody, sections underwent heat retrieval with Bond Epitope Retrieval Buffer 2 (Leica ER2, AR9640) and blocking. Primary antibodies in Panel 1 were against CD3 (1:200, Biorad, MCA1477T), CD8 (1:2000, Cell Signaling, 98941S), Ki67 (1:200, Abcam, AB16667). Primary antibodies in Panel 2 recognized iNos (1:1000, Genetex, GTX130246), Arg1 (1:750, Genetex, GTX109242), Granzyme B (1:1000, Abcam, AB4059), CD11b (1:10,000, Abcam, AB133357), F480 (1:250, Cell Signaling, 70076S). Each primary antibody was followed by a cocktail of horse radish peroxidase-conjugated secondary antibodies against mouse and rabbit IgG (RTU, Akoya/Perkin Elmer, Cat # ARH1001) and then tyramide mediated signal amplification (TSA) with covalent linkage of the individual Opal fluorophor (each 1:250, Opal 520 (FP1496001KT), 540 (FP1487001KT), 570 (FP1494001KT), 620 (FP1488001KT), 650 (FP1495001KT) or 690 (FP1497001KT), Akoya/Perkin Elmer Cat #'s) to the tissue antigen. Antibodies were subsequently stripped using either ER1 (Leica, AR9961) or ER2 (Leica, AR9640) heat retrieval buffer and the next round of immunostaining initiated. After completion of the sequential incubations and stripping, slides were counterstained with spectral DAPI (Akoya/PerkinElmer, FP1490). Monoplex controls were used to confirm appropriate staining for antibodies integrated into the multiplex panels. Multispectral imaging was performed on a Vectra3 imaging system (Akoya/PerkinElmer) at 20×. The fluorophore emission signatures were captured by a multispectral camera and then unmixed with InForm software (Akoya/PerkinElmer). Autofluorescence, obtained from an unstained slide, was removed from the composites and pseudo-colored images exported as tif files.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 7.0 as described in Figure legends. All data are shown as mean±s.e.m. Figures were prepared using GraphPad Prism 7, Adobe Photoshop and ImageJ Software. Treated groups were compared, using a one-way analysis using Prism7 (GraphPad Software), to naïve mice. Differences with a P value of <0.05 were considered significant: *P<0.05; **P<0.005; ***P<0.0001.

SV Expressing IL-12 Enhances the Expression of OX40 on CD4 T Cells

The study described herein investigated the therapeutic effect of SV.IL12 in immune-competent tumor bearing mice (colon cancer; CT26). To exploit SV.IL12 for cancer therapy, tumor cells were i.p. implanted and after tumor establishment (4 days after tumor cell injection [day 0]), SV, SV.IL12 or IL-12 were i.p. injected on 4 consecutive days (day 1, 2, 3 and 4) for a total of 4 weeks (FIGS. 1A and B). While untreated (control), SV and IL-12 treated mice succumb to cancer after 3 weeks, treatment with SV.IL12 slightly prolonged survival time with an overall long-term survival rate of 7.1%. These data suggest that SV expressing IL-12 is needed to induce the observed therapeutic efficacy. Shortly after i.p. injection, SV infects macrophages in mediastinal lymph nodes where T cells get subsequently activated (FIG. 2). Even though SV.IL12 infected cells secrete significant amounts of IL-12 as observed in in vitro experiments (FIG. 3A), i.p. injection of SV.IL12 did not significantly change levels of plasma IL-12 in mice (FIG. 3B). Thus, suggesting that IL-12 produced by SV acts locally and stimulates transduced macrophages (FIG. 2) that present tumor antigens to corresponding T cells and activates them further. That shapes the subsequent anti-tumor immune response, such as promoting the differentiation into Th1 cells as well as increasing IFNγ production (FIGS. 3C and 3D)^{27, 28, 29, 30}. After one week of treatment, we analyzed the T cell response for their expression of inhibitory and activation markers. OX40 was markedly upregulated on CD4 T cells during SV.IL12 treatment, which was mostly among the effector CD4 T cells and less on the regulatory T cells (FIGS. 1C and 1D). Interestingly, SV treatment also induced OX40 upregulation on CD4 T cells but to a lesser extent (FIGS. 1C and 1D). On the basis of the results above and previous studies, reporting a beneficial effect of anti-OX40 in cancer treatment [Aspeslagh S et al., Eur. J Cancer, 2016], it was hypothesized that the agonistic anti-OX40 antibody could augment the therapeutic efficacy of SV.IL12 and help induce anti-tumor immune responses without to require knowledge of the tumor antigens.

Intraperitoneal Delivery of SV.IL12 and Anti-OX40 Antibody Cures Established Cancers

To investigate whether the oncolytic activity of SV.IL12 in combination with anti-OX40 is required for successful anti-cancer therapy, SV non-susceptible (colon cancer; CT26) and susceptible (prostate cancer; MyC-CaP) tumor cell lines were used in this study (FIG. 4) [Granot T et al., Mol. Ther., 2014; Huang P Y et al., Mol. Ther., 2012] Immuno-competent female BALB/c and male FVB/NJ mice were implanted with either CT26 or MyC-CaP tumor cell lines, which expressed the firefly luciferase (Fluc) protein, respectively. This allowed monitoring tumor growth in vivo using noninvasive bioluminescent imaging. Once tumors became established (day 0), mice were treated with SV.IL12 in combination with anti-OX40. SV.IL12 was i.p. injected on 4 consecutive days (day 1, 2, 3 and 4) for a total of 4 weeks (FIG. 5A). Anti-OX40 was injected 3 times a week (day 0, 2 and 4) for a total of 2 weeks. In both tumor models, all untreated animals experienced progressive tumor growth and succumbed to cancer on week 3 (FIGS. 5 and 6). Mice bearing CT26.Fluc or MyC-CaP.Fluc tumors showed some delay in tumor growth when treated with i.p. injected SV.IL12 or anti-OX40 alone but with only a moderate effect on long-term survival (FIGS. 5 and 6). However, the combination of SV.IL12 with anti-OX40 resulted in complete regression of tumors in both tumor models (FIGS. 5 and 6). Tumors occasionally did recur in mice treated with combination therapy after treatment was completed, resulting in a long-term survival rate of 91.6% and 50% in the CT26 and MyC-CaP tumor model, respectively. In conclusion, combination of SV.IL12 with anti-OX40 elicits a strong therapeutic efficacy against two distinct solid tumors. Furthermore, these findings confirm that the oncolytic activity of SV is not required to induce a robust and effective anti-tumor response. Due to the fact that anti-OX40 monotherapy already resulted in a 20-50% survival rate, whether the addition of SV.IL12 would allow reduction of treatment frequencies while still maintaining the strong therapeutic efficacy of combination therapy, was investigated. This is especially important for lowering risks of adverse events as well as being more convenient for patients in clinics. Interestingly, therapeutic efficacy in the CT26.Fluc tumor model was maintained with only one injection per week of SV.IL12 and anti-OX40 (FIG. 7). This is in contrast with MyC-CaP.Fluc tumor bearing mice for which the full treatment regimen was required (data not shown). Thus, in the following experiments mice bearing CT26 tumors were treated with one injection a week whereas MyC-CaP tumor bearing mice received the full treatment regimen.

Combination Therapy Markedly Changes the Transcriptome Signature of T Cells

The requirement of T cells during SV.IL12 with anti-OX40 treatment was assessed. The presence of both CD4 and CD8 T cells was required for eliciting the observed therapeutic efficacy as mice treated with the corresponding depleting antibodies were unable to control tumor growth (FIG. 8). To better understand the impact of the combination therapy on T cells, RNA sequencing was performed on isolated T cells from spleens derived from naïve, control as well as anti-OX40 and SV with or without anti-OX40 treated mice on day 7. Principal component analysis (PCA) of normalized reads showed a distinct segregation between combined therapy and all other groups in both tumor models (FIGS. 9A and 9B). These data suggest that combined therapy induces a distinct T cell response in the periphery independently of tumor model and mouse strain suggesting an indirect and immunity driven role of SV vectors with a negligible direct effect of the vector in tumors (FIG. 9C). Indeed, gene expression profiles of control versus anti-OX40, SV or SV in combination with anti-OX40 were clearly distinct, with the most differentially expressed genes (DEG) in the latter one (503, 49 and 2100 DEG, respectively) (FIG. 9C). Of 2100 DEG in control versus combination therapy, more genes were downregulated than upregulated (1637 vs. 463)>two-fold. Similarly, in control versus anti-OX40 as well as control versus SV more DEG were downregulated than upregulated (393 versus 110 and 30 versus 19, respectively).

Unbiased pathway enrichment and network analyses of DEG from control versus combination therapy was performed to determine biological processes in T cells that are influenced by this treatment (FIG. 9D). Although both upregulated and downregulated DEG were included in the analysis, the vast majority of pathways were upregulated in T cells treated with combination therapy with the exception of four clusters (TGFbeta signaling, ribosomal biogenesis, translation and chromatin modification). The upregulated pathways were dominated by DNA replication, chromosomal organization and cell cycle regulation, but also included various metabolic and immunological processes, such as mitochondrial respiration, nucleotide metabolism, and adaptive immune responses.

Combination Therapy Enhances Systemic T Cell Responses, Favoring Th1 Like ICOS CD4 T Cells

As T cells from combination therapy express a marked change in their transcriptome signature compared with all other groups, markers for T cell differentiation and activation (e.g., PD-1, ICOS, OX40, TIM3, KLRG1, IL7R) as well as T cell lineage transcription factors (e.g., EOMES, TBET, GATA3, BCL6, RORC, FOXP3) were analyzed (FIG. 9E). Only T cells from combined therapy expressed the gene signature of terminally differentiated effector T cells, which are characterized by high expression of the killer lectin-like receptor (KLRG1) and low expression of the interleukin 7 receptor (IL-7R) [Joshi N S et al., Immunity, 2007]. Furthermore, genes encoding products associated with the differentiation and function of effector cells, such as Batf, Id2, Tbet, Gzmb and Ifng, were also highly expressed in T cells isolated from mice treated with combined therapy compared with all other groups. The enhancement of effector T cells in combined therapy was confirmed by flow cytometry in both tumor models, as judged by the increased expression of the activation and proliferation markers CD44 and Ki-67, respectively (FIGS. 9F, 9G and 10). Interestingly, CD4 T cells also expressed a marked anti-tumor effector phenotype (ICOS⁺Tbet⁺) which was on average 2 to 3-fold higher during combined therapy compared with SV.IL12 or anti-OX40 treatment (FIGS. 9H and 9I). Previous studies reported a correlation between expansion of ICOS⁺Tbet⁺ CD4 T cells and clinical benefit in cancer patients who received anti-CTLA4 therapy [Wei S C et al., Cell, 2017; Ng Tang G et al., Cancer. Immunol. Res. 2003; Carthon B C et al., Clin. Cancer Res., 2010]. In summary, SV.IL12 in combination with anti-OX40 induces a marked systemic T cell response and favors the differentiation of terminal effector T cells. Furthermore, combined therapy induces a sustained increase in the frequency of ICOS⁺Tbet⁺ CD4 T cells which has also been reported to be elevated during successful anti-CTLA4 cancer therapy.

CD4 and CD8 T Cells are Metabolically Reprogrammed in Mice Treated with SV.IL12 and Anti-OX40

The tumor microenvironment can be a very challenging milieu for an effector T cell as it is characterized by hypoxia, acidosis and low levels of nutrient sources such as glucose and glutamine [Delgoffe, G M et al, Cancer Immunol. Res., 2016; Scharping, N. E, Vaccines, 2016; Chang, C H et al., Cell, 2015]. Even if T cell activation and initiation of effector function is allowed, T cells may be unable to generate the bioenergetic intermediates necessary to carry out effector function in the tumor microenvironment. Thus, providing a metabolic support for T cells is crucial for the success of cancer treatments as previously reported [Scharping, N. E. et al., Immunity, 2016; Ho, P C et al., Cell, 2015; Siska P J., et al, Trends immunol., 2015; Zhao et al., Nat. Immunol., 2016]. To test if SV.IL12 in combination with anti-OX40 influences the metabolic state of T cells, Gene Set Enrichment Analysis (GSEA) of the RNA sequencing data was performed between T cells from combined therapy and control. GSEA analysis showed significantly higher expression of genes involved in oxidative phosphorylation and glycolysis pathways during combination therapy (FIG. 11). To confirm GSEA analysis, peripheral T cells from both tumor models were metabolically profiled using Seahorse analysis on day 7 (FIGS. 111B, 12A and 12B). Oxidative phosphorylation and glycolytic profiles in T cells from naïve, control and mice treated with SV.IL12 and/or anti-OX40 were determined by measuring the rate of oxygen consumption (OCR) and the rate of extracellular acidification (ECAR), respectively. Basal OCR was enhanced in T cells from combined therapy and SV.IL12 treatment, but only the former harbored a dramatic increase in spare respiratory capacity in the CT26 model (FIGS. 11B and 12A). This was in contrast to T cells from combined therapy in the MyC-CaP.Fluc tumor model which expressed 3.75-fold higher basal OCR with no spare respiratory capacity (FIG. 12B). The reason for this discrepancy between the two models might be the differences in the number of treatments as MyC-CaP.Fluc bearing mice receive 3 and 4 times more injections of anti-OX40 and SV.IL12, respectively.

Analysis of mitochondrial mass (FIGS. 11C and 12C) and activity (FIG. 11D and FIG. 12C), using flow cytometry with the mitochondrial stain Mitotracker Green and DeepRed respectively, revealed that SV.IL12 with or without anti-OX40 induced higher mitochondrial mass and activity in CD8 T cells but not in CD4 T cells. These data suggest that the observed increase in basal OCR was mainly driven by CD8 T cells. Interestingly, a slight decrease of active mitochondria occurred in CD4 T cells from mice treated with combined therapy, which might explain the increase in spare respiratory capacity in this group. To test if the reduction of active mitochondria in CD4 T cells is associated with a switch towards glycolysis, the master regulator for glycolysis c-MYC and basal ECAR were measured in T cells from all groups. Indeed, the addition of anti-OX40 to SV.IL12 induced elevated protein expression of c-MYC as well as basal ECAR (FIGS. 11E and 11F). T cells from naïve and control as well as SV.IL12 or anti-OX40 treated mice showed no signs of elevations. Collectively, these findings reveal that SV.IL12 induces enhanced oxidative phosphorylation in CD8 T cells, whereas the addition of anti-OX40 to SV.IL12 is needed to push CD4 T cells towards glycolysis by increasing the protein expression of c-MYC.

To determine the kinetics of peripheral T cell metabolism over the course of treatment with SV.IL12 and anti-OX40, OCR and ECAR were measured on day 7, 14 and 40 in CT26.Fluc bearing mice (FIG. 11G). As shown above, T cells on day 7 shifted towards a glycolytic state which is associated with the initial effector phase. Two weeks into the treatment, T cells switched to a highly energetic state utilizing both metabolic pathways, oxidative phosphorylation and glycolysis, as reported for highly activated T cells [Buck M D et al., J. Exp. Med., 2015] Once tumors were fully rejected and mice were tumor-free for a month, T cells returned to a more quiescent state, such as naïve cells. Interestingly, T cells from MyC-CaP.Fluc bearing mice switched to a highly energetic state early on during treatment (day 7) and remained in this metabolic phenotype 2 weeks after treatment has stopped (FIG. 12D). The reason for this discrepancy might be the differences in the number of treatments applied in both tumor models as MyC-CaP.Fluc bearing mice receive 3 and 4 times more anti-OX40 and SV.IL12, respectively. T cells from control as well as anti-OX40 or SV.IL12 treated mice in both tumor models remained in a quiescent state over the course of treatment (FIG. 12E). In summary, SV.IL12 in combination with anti-OX40 metabolically rewires T cells to an energetic state using both metabolic pathways, oxidative phosphorylation and glycolysis. This phenotype does not occur in SV.IL12 or anti-OX40 treated mice, which succumb to cancer. Thus, the changed metabolic state of T cells correlate with an efficient anti-tumor response and better survival rate.

Metabolic Reprogrammed T Cells in SV.IL12 with Anti-OX40 Treated Mice Display Enhanced CD4 Mediated Cytokine Production and Anti-Tumor Activity

To test if metabolic reprogrammed T cells in combined therapy possess enhanced effector functions, cytokine production and cytotoxicity were analyzed in T cells isolated from spleens on day 7. Genes encoding pro-inflammatory cytokines ifng and il2 were upregulated in T cells from mice treated with SV in combination with anti-OX40 (FIG. 13A). ELISPOT analysis of interferon-γ (IFNγ) by splenocytes confirmed RNA sequencing data, showing the strongest IFNγ secretion in mice treated with combined therapy in both tumor models (FIGS. 13B and 13C). Splenocytes from SV.IL12 treated mice also produced IFNγ but to a lesser extent. Interestingly, the main producer of IFNγ were CD4 T cells as depletion of CD4 T cells but not CD8 T cells abolished IFNγ secretion in splenocytes from mice treated with combined therapy.

In addition, RNA levels of the cytotoxic proteases, granzyme A and B, were upregulated in mice treated with combination therapy compared with all other groups (FIG. 13A). Protein expression of granzyme B correlated with RNA levels as measured by flow cytometry in both tumor models (FIGS. 13D, 13E, and 14A-14D). Further, granzyme B positive cells were detected in CD8 as well as CD4 T cells, suggesting the presence of cytotoxic CD4 T cells in mice treated with combined therapy [Brown D M et al., Cell Immunol, 2010; Mucida D. et al., Nat Immunol, 2013; Reis B S et al., Nat Immunol 2013] Upregulation of granzyme B was associated with downregulation of the transcription factor Foxo1 which is known to control granzyme transcription through repression of the transcription factor T-bet (FIG. 13A) [Rao R R et al., Immunity, 2012]. Last, the enhanced cytotoxic potential of T cells from combined therapy was also supported by elevated expression of the NKG2D receptor which has been shown to trigger TCR-independent cytotoxicity in activated T cells (FIGS. 13A, 14E and 14F) [Verneris M R et al., Blood 2004].

Having observed upregulation of granzymes and cytotoxic receptors in combination therapy, the function of T cells was investigated using an ex vivo tumor growth assay. Splenocytes obtained from all groups were co-cultured at an effector-to-target cell ratio of 10:1 with either CT26.FLUC (FIG. 13F) or MyC-CaP.Fluc (FIG. 13G) tumor cell lines. The anti-tumor activity of splenocytes was determined by measuring the luciferase activity of cell lines, which correlates with tumor growth. Tumor growth was markedly reduced when co-cultured with splenocytes from mice receiving combined therapy compared with splenocytes from naïve, control and mice treated with anti-OX40 in both tumor models. The anti-tumor activity of splenocytes from mice treated with SV.IL12 alone was weaker than that from combined therapy. Surprisingly, tumor growth inhibition was mediated by CD4 T cells as depletion of CD4 T cells but not CD8 T cells abolished the inhibitory effect on tumor cells. Together, these results clearly show that T cells from combined therapy elicit enhanced anti-tumor and functional activity, such as granzyme B and IFNγ production driven by CD4 T cells.

Mice Treated with SV.IL12 in Combination with Anti-OX40 Display Enhanced T Cell Migration and Intratumoral T Cell Immunity

Only a minority of the total of treated patients respond to current immunotherapy and the presence of TILs has been shown to be one of the main factors that influence the responsiveness towards various therapies in multiple cancers [Galon, J et al., Science 2006; Hwang W T et al., Gynecol Oncol 2012]. Due to the fact that SV elicited anti-tumor responses do not necessarily require direct infection of the tumor or intratumoral injection, whether SV.IL12 therapy in combination with anti-OX40 could nevertheless alter the local tumor microenvironment and favor intratumoral immunity, was investigated. To assess whether SV.IL12 in combination with anti-OX40 induces T cell infiltration into the tumor, the chemokine receptor CXCR3 on peripheral T cells was analyzed after one week of treatment. In the CT26.Fluc model CXCR3 levels were significantly upregulated on CD4 T cells during combination therapy compared with all other groups and CXCR3 levels remained elevated over the course of treatment (FIGS. 15A, 15B and 16A). In contrast, CXCR3 expression on CD8 T cells only appeared later on in treatment, suggesting that CD4 T cells are first recruited to the inflamed site followed by CD8 T cells (FIG. 16A). MyC-CaP.Fluc tumor bearing mice showed elevated levels of CXCR3 on CD4 and CD8 T cells after one week of combination treatment (FIGS. 16B and 16C). Furthermore, SV.IL12 treatment also increased CXCR3 expression on T cells but to lesser extent. The reason for this discrepancy between the two models might be the differences in the number of treatments as MyC-CaP.Fluc bearing mice receive 3 and 4 times more injections of anti-OX40 and SV.IL12, respectively. Cells expressing CXCR3 follow the gradient of their ligands CXCL9, CXCL10 and CXCL11 [Groom, J. R. & Luster, A. D. Exp. Cell Res. 2011]. Indeed, combination therapy also enhanced the production of CXCL9 and CXCL10 in the tumor microenvironment, as judged by real-time PCR, suggesting that CXCR3 positive T cells migrate to the tumor site (FIG. 15C). Treatment of SV.IL12 or anti-OX40 alone did not alter the expression of these ligands. In line with these observations, an overall increase in T cells was observed in CT26.Fluc and MyC-CaP.Fluc peritoneally disseminated tumors from mice treated with combined therapy compared with control and anti-OX40 treated mice (FIGS. 15E and 15F). SV.IL12 treated mice also showed enhanced T cell infiltration but to a lesser extent. Furthermore, dissecting CD4 and CD8 T cells by flow cytometry revealed that combination therapy increases the proportion of CD4 T cells in CT26.Fluc tumors, which is consistent with the elevated CXCR3 expression on peripheral CD4 T cells (FIG. 15D). These results clearly show that SV.IL12 in combination with anti-OX40 alter the tumor microenvironment by facilitating T cell infiltration via modulation of the CXCR3/CXCL9-11 axis. Not only did combination therapy increase T cell infiltration in both tumor models but CD4 as well as CD8 T cells also demonstrated enhanced functional activity in the tumor, as judged by the Ki-67 and granzyme B expression (FIGS. 15D, 16D and 17). In line with these results, a decrease in proliferation, as judged by the expression of Ki-67 in tumor cells, was observed in CT26.Fluc and MyC-CaP.Fluc tumor cells when treated with combined therapy compared with all other treatments (FIGS. 15E and 15F). These results suggest that the presence of activated T cells in the tumor microenvironment exert anti-tumor activity which inhibits tumor growth. Besides from T cell activation, we also observed enhanced iNOS production in MyC-CaP.Fluc tumors treated with combination therapy compared with control or anti-OX40 treatment (FIG. 18). SV.IL12 treatment alone also induced iNOS production but to a lesser extent. Interestingly, the amount of iNOS inversely correlated with ariginase1 production, suggesting a repolarization of tumor associated macrophages from the M2-like (pro-tumor) into M1-like (anti-tumor) phenotype during combination therapy. These trends were only observed in MyC-CaP.Fluc and not in CT26.Fluc tumors, which might be a consequence of SV directly infecting MyC-CaP cells.

The study described herein provides a practical strategy for cancer immunotherapy using an OV and anti-OX40. This strategy takes advantage of the preexisting T cell immune repertoire in vivo, removing the need to know about present tumor antigens. The study described herein shows that the combination of replication-deficient SV.IL12 and anti-OX40 amplifies these antitumor T cells and induces their action throughout the body against two distinct solid tumors, reversing effectively local tumor-mediated immune suppression. This effect was specific for combination therapy and was not observed during SV.IL12 or anti-OX40 treatment alone.

The high metabolic activity of cancer cells together with the poor vasculature blood supply in the tumor microenvironment can induce nutrient deprivation [Delgoffe, G M et al., Cancer Immunol Res. 2016; Scharping, N E & Delgoffe, GM, Vaccines, 2016; Chang, C H et al., Cell, 2015]. These conditions can impair TCR signaling, glycolytic and mitochondrial metabolism, as well as decrease mitochondrial mass, all hallmarks of T effector cells, resulting in impaired anti-tumor effector functions of tumor-specific T cells. 39-42 Scharping, N. E. et al., Immunity 2016; Ho, P. C. et al., Cell 2015; Siska, P J & Rathmell, J C, Trends Immunol., 2015; Zhao, E. et al., Nat Immunol, 2016]. The data in two distinct models of cancer immunotherapy disclosed in the study described herein, shows that SV.IL12 in combination with OX40 signaling provides the necessary metabolic support to T cells to generate an efficient antitumor response. This metabolic support is characterized most prominently by elevated mitochondrial function and mass in CD8 T cells as well as a switch to aerobic glycolysis in CD4 T cells. T cells from mice treated with SV.IL12 in combination with anti-OX40 demonstrated enhanced protein expression of c-Myc compared with all other groups. Thus, the study described herein clearly shows that T cells are metabolically reprogrammed in the periphery during combination therapy.

The study described herein strongly shows that the therapeutic efficacy of SV.IL12 with anti-OX40 is driven by T cell modulation and reprogramming of its metabolic state, in order to enhance the anti-tumor response in the periphery and in the tumor microenvironment. Furthermore, the use of SV allows these metabolically reprogrammed T cells to better infiltrate the tumor microenvironment, which is crucial for an adequate immunotherapy. Anti-OX40 antibody is currently being studied in phase 1 and 2 clinical trials. SV will be tested as a single agent in its first clinical trial in the third quarter of 2020. The results from our current preclinical studies provide a strong rationale for combining SV.IL12 with agonistic anti-OX40 antibodies in a therapeutic format in patients with solid tumors. In summary, the studies described herein clearly show that even in absence of direct SV tumor targeting, SV.IL12 in combination with anti-OX40, or SV vector encoding IL-12 and anti-OX40, can alter the tumor microenvironment in distinct solid tumors through an indirect and immunity driven mechanism that enhances T cell infiltration and intratumoral T cell immunity.

Example 2: Combination of IL-12 and Anti-OX40 Expressed by Sindbis Viral Vectors Synergistically Enhances Survival of Subjects with Established Tumors

The study described herein investigates the effect of administering IL-12 and anti-OX40 antibody, both expressed by Sindbis viral vectors, on established tumors. This strategy is particularly advantageous for treatment of cancers like ovarian cancer, wherein the combination of SV.IL-12 and anti-OX40 antibody is not found to be as effective, as observed in colon and prostate cancers. The administration of SV/IL-12 and an anti-OX40 antibody enhanced clearance of established tumor of colon and prostate cancer cell lines, CT26 and MyC-Cap respectively. C57/B16 albino (female) mice re-injected with Alm5-2Fluc-17 ovarian cancer cells to establish a tumor (FIG. 19), and treated with: a) a SV vector expressing IL-12; b) a combination of SV vectors expressing IL-12 and an anti-OX-40 antibody (aOX40_REP-IL12), c) a 50% mixture of either a fragmented SV expressing OX-40 IgG plus a fragmented SV expressing IL-12 (RepOX4OIgG_Rep-IL12) or d) a 50% mix of a fragmented SV expressing OX-40 IgG plus a full length SV expressing IL-12 (RepOX4OIgG_SV-IL12). The percentage survival rate of the treatment groups RepOX4OIgG_SV-IL12 and aOX40_Rep-IL12 were comparable, and higher than the SV-IL-12 and RepOX4OIgG_Rep-IL12 treatment group. However, the results showed that, the RepOX4OIgG_SV-IL12 treatment group showed the highest enhancement of survival rate (FIG. 20).

The study described herein, provides plasmid constructs for expressing IL-12, and anti-OX40 in a SV vector. The study described herein, provides plasmid constructs encoding IL-12 a and b subunits (FIG. 38), anti-OX40 IgG2a heavy and light chains (FIG. 39) and a single chain antibody to OX40 (FIG. 40).

SV.IL12 plasmid used in this study has been published in 2002 [Tseng J C et al., J Natl Cancer Inst. 2002]. To construct a Sindbis viral vector containing genes for interleukin 12 (IL-12), the Sindbis viral vector SinRep/2PSG was first constructed, which contains a secondary subgenomic promoter that is responsive to the Sindbis replicase. Two DNA oligonucleotide primers (sequence 5′ CGCGTAAAGCATCTCTACGGTGGTCCTAATAGTGCATG-3′; SEQ ID NO: 29) and its complementary strand 5′CACTATTAGGACCACCGTCGAGATGCTTTA-3′; SEQ ID NO: 30) containing the subgenomic promoter sequence were annealed and ligated into the MluI and SphI sites of the SinRep plasmid. The murine IL-12 α subunit gene (mp35; ATCC 87596) and the IL-12 β subunit gene (mp40; ATCC 87595) were subcloned into the MluI and the StuI sites of SinRep/2PSG, respectively, to produce the Sin-Rep/IL12 plasmid.

The H and L chains of the OX40 Ab are expressed from a single SV using two subgenomic promoters. The synthesized sequences were designed to encode an IL-12 secretory signal peptide upstream of both H and L polypeptide sequences preceded by a ribosome binding site and the start codon. The variable Ab binding sequences that functionally bind to activate the OX40 Receptor contain complementarity determining regions that are not unique. The variable chain is linked to the respective L (GenBank accession BAR42292) and H chain (GenBank accession CAC20702) constant region sequences of mouse IgG2a; the murine IgG2a isotype is comparable to the BioXcell OX40 Ab used in parallel in vivo experiments.

In summary results described herein clearly show the possibility of using a combination of SV vectors expressing IL-12 and anti-OX40 antibody or a SV vector expressing both expressing IL-12 and anti-OX40 antibody, for treatment of cancers that may be resistant to treatment with anti-OX40 antibody administered directly.

Example 3. Molecular and Metabolic Pathways Mediating Curative Treatment of a Non-Hodgkin B Cell Lymphoma by Sindbis Viral Vectors and Anti-4-1BB Monoclonal Antibody

The studies described herein use an antibody directed at 4-1BB (CD137, TNFRSF9), a T cell costimulatory molecule. 4-1BB agonist stimulation greatly enhances NK and cytotoxic T cell activity. There are preclinical studies showing that α4-1BB effectively treats lymphoma and that depletion of Treg cells enhances the therapeutic effect of α4-1BB [Houot R et al., Blood, 2009]. The A20 tumor cells uses in the study described herein were derived from a spontaneously arising reticulum cell sarcoma (a non-Hodgkin lymphoma) in a BALB/c mouse.

Previously, SV carrying NYESO-1 was used, which encodes the cancer testis TAA, NYESO-1, to cure CT26 tumors expressing NYESO-1 [Scherwitzl I et al., Mol. Ther. Oncolytics, 2018]. The studies described herein show that systemically disseminated A20 lymphoma can be completely cured by SV plus α4-1BB mAb combination therapy without the need to produce a SV that encodes a TAA known to be present in the A20 lymphoma cells. Further, neither intratumoral injection of the SV vectors nor infection of the tumors is required as the A20 B lymphoma cells used in the current model are resistant to SV infection.

One difference in the current study, compared with those previously published, is the use of SV vector combination therapy that involves an agonistic mAb for a costimulatory receptor versus targeting checkpoint blockade molecules such as CTLA4 and PD-1. The studies described herein show that agonistic mAbs in combination with SV vectors trigger a cascade of events that results in curative results. The findings disclosed herein reveal the potential of SV combination therapy to cure tumors for which TAAs are completely unknown.

Materials and Methods

Firefly Luciferase (Fluc)-Expressing A20 Cells Generation

A20 cells were transfected with pGL4-neo_Fluc plasmid (Promega) by electroporation via Nucleofector™ kit V (Lonza). Fluc-A20 cell clones were selected and maintained in RPMI1640 (Cellgro)+10% FBS (Gibco)+250 μg/ml G418 (Gibco). One A20 clone stably expressed fLuc and was used for tumor inoculation and consecutive experiments.

SV Production

SV-LacZ production and titering were done the same as previously described [Scherwitzl I et al., Mol. Ther. Oncolytics, 2018].

SV-GFP Infection

A20 cells and control BHK cells were infected by SV carrying GFP for 1 h. The GFP expression was observed the next day by fluorescence microscopy.

A20 Tumor Inoculation and In Vivo Imaging System (IVIS) Imaging

3×10⁶fLuc-A20 cells were inoculated to BALB/C mice by i.p injection. Tumor growth was monitored as previously described [Scherwitzl I et al., Mol. Ther. Oncolytics, 2018].

SV and α4-1BB Ab treatment

Treatment was started after successful tumor inoculation (4 days after tumor cell injection, confirmed by IVIS imaging). Tumor growth was measured every week by noninvasive bioluminescent imaging. SVLacZ was injected 4 times per week, for totally 3 weeks. The virus (10⁷-10⁸TU/mL) in a total volume of 500 μL was i.p. injected. For 2 groups (4-1BB and SV plus 4-1BB), 350 μg/mouse 41BB Ab was injected 3 times/week for 2 weeks. InVivo MAb anti-mouse 4-1BB was ordered from BioXCell (Clone: LOB12.3, Cat. No. BE0169). In low dose treatment protocol, SVLacZ was injected i.p. 3 times per week, for totally 3 weeks. 41BB Ab (50 g/mouse) was injected once a week for 3 weeks.

Elispot

Mouse IFNγ ELISPOT was performed according to the manufacturer's protocol (BD Biosciences). 2×10⁵splenocytes or 1×10⁵T cells were plated per well O/N in RPMI supplemented with 10% FBS. For a positive control, splenocytes were stimulated with 5 ng/ml PMA+1 μg/ml Ionomycin.

Flow Cytometry

Fluorochrome-conjugated antibodies against mouse CD3, CD4, CD8, CD25, CD44, CD62L, ICOS, CD11 a, ICAM-1 were purchased from Biolegend (San Diego, Calif.). Fluorochrome-conjugated antibodies against mouse Foxp3, EOMES and CCR5 were purchased from Thermofisher. BUV395 conjugated antibody against mouse CD8a was purchased from BD Biosciences. For surface staining, cells were washed and stained with anti-mouse direct conjugated antibodies. Cells were analyzed using the LSRII flow cytometer (BD Biosciences) and data were analyzed using Flowjo software (Treestar, Ashland, Oreg.). For intracellular cytokines staining, stimulated cells were fixed with cytofix/cytoperm solution (BD Biosciences), permeablized with perm/wash buffer (BD Biosciences) and stained with anti-mouse IFNγ antibodies. For nuclear antigen, cells were fixed and permeabilized by Foxp3 fixation/permeabilization buffer (eBioscience) and stained with anti-Foxp3, T-bet, Ki67 and EOMES antibody.

RNA Isolation and Transcriptome Analysis

Total RNA was harvested by RNAeasy isolation kit (Qiagen, Valencia, Calif.). For each group, 3 BALB/C mice were used as biological repeats. RNA-seq was performed by NYUMC Genome Technology Center (GTC). To identify significant differences in expression between any pair of groups, differential expression analysis was performed using Deseq2 and an adjusted p value cutoff of 0.05 was applied [19, Love M I et al, Genome Biol. 2014] (q<0.05). To increase stringency, only genes with a Log 2 fold change ≥1 (upregulated) or ≤−1 (downregulated) were selected for further analysis. Gene cluster analysis was performed by DAVID analysis using the selected differentially expressed genes [Huang da W et al., Nucleic Acids Res 2009, Huang da W et al., Nat Protoc. 2009]. RNA-seq results (normalized counts) were used as input to perform with Gene Set Enrichment Analysis (GSEA) [Subramanian A, et al., Proc Natl Acad Sci USA. 2005]. Molecular Signatures Database (MSigDB)v4.0 were used as screening database. For each gene, the gene expression value is normalized by the relative log 2 fold change compared to the median value of this gene. Expression heatmap is drawn by Morpheus (https://software. broadinstitute.org/morpheus/). Cannonical pathway and disease and biological functional analysis were generated by ingenuity pathway analysis (IPA; Ingenuity Systems, Redwood City, Calif.) using the statistical differential expressed genes list. To increase the sample representativeness, for IPA, we choose nominal p<0.05 as cutoff value.

Tumor Infiltrating Lymphocyte (TIL) Harvest

To investigate the phenotype of TIL, all treatments were started 11 days after tumor inoculation, After 7 days treatment, tumor mass was harvested and the phenotype of TIL were analyzed as previously described [18, Scherwitzl I, Mol Ther Oncolytics. 2018].

T Cell Seahorse Assay

T cells were isolated from spleen by using pan T cell isolation kit (Stemcells). T cells were plated at 6×10⁵cells/well in 24 well plate. Oxygen consumption rate (OCR) and excellular acidification rate (ECAR) were measured by Agilent Seahorse XFe24.

Statistical Analysis

For the two group comparison, statistical difference was determined by unpaired two tail Student t-test. The multiple sample comparison was analyzed by one way ANOVA. P<0.05 was determined to be significant for all experiments. All values were calculated with Excel (Microsoft) and Prism software (GraphPad).

SV and α4-1BB mAb Combination Completely Cured A20 Lymphoma

To explore if SV has therapeutic effect on tumors not targeted or infected by SV vectors, the A20 B cell lymphoma was used, which is highly resistant to SV infection (FIG. 21). To monitor tumor growth in vivo, a firefly luciferase (f-Luc) expression vector was transfected into the A20 lymphoma cell line by electroporation. A stable f-Luc expressing A20 clone was isolated through G418 selection. 3×10⁶/mouse f-Luc A20 tumor cells were inoculated by intraperitoneal (i.p.) injection. Tumor growth was monitored by IVIS imaging once per week. Tumors were successfully established after 4 days inoculation (FIG. 22A). After tumors were established, SV and α4-1BB mAb treatment started (designated as day 0). A therapeutic protocol similar to that previously described [Scherwitzl I et al., Mol Ther Oncolytics. 2018], was used. SV plus α4-1BB mAb combination achieved the best therapeutic effect (FIG. 22B). All mice in that group showed complete tumor regression in 2 weeks. Although, both SV or α4-1BB treatments alone achieved obvious therapeutic effects compared with untreated mice, they were not as effective as the combination and a fraction of mice in these two groups eventually succumbed to tumor (FIG. 22C).

SV Alone and SV Plus α4-1BB mAb Stimulated Cell Cycle Progression, Cytokine Production, and Activation

In the study described herein, SV significantly inhibited tumor growth by day 7 (FIG. 22A). T cells play a critical role in SV induced anti-tumor immunity. T cell response reaches a peaked on day 7 after infection [Scherwitzl I et al., Mol Ther Oncolytics. 2018]. To explore how SV induced T cell responses that help to eradicate A20 lymphoma, RNA-Seq was performed using purified splenic T cells from all groups on day 7. Compared with untreated samples, 271 genes upregulated (q<0.05 and Log 2 Fold Change≥1) and 28 genes downregulated (q<0.05 and Log 2 Fold Changes≤−1) were identified in the SV infected group through Deseq2 analysis (FIG. 23A, Table 1).

TABLE 1

The SD expressed genes list for SV vs. untreated group by RNA-Seq (q

< 0.05, Log2FC ≥ 1 and Log2FC ≤ −1).

gene
baseMean
log2FC
log2FCunshrunk
pvalue
padj

Gm2762
38.5
3.15
4.04
0
0

Il21
229
2.57
3.66
1.81E−13
2.63E−11

Ascl2
24.4
2.55
3.83
2.83E−12
3.8E−10

Akr1c18
25.3
2.4
4.32
6.37E−11
6.98E−09

Angptl2
741.2
2.35
2.59
0
0

E2f8
535.1
2.33
2.61
0
0

Vat1l
14.9
2.33
4.01
1.65E−10
1.68E−08

Aurkb
806.6
2.3
2.46
0
0

Gzmb
4113.9
2.26
2.51
0
0

Cdc25c
143.9
2.08
2.51
3E−15
5.19E−13

Prr11
288
2.04
2.36
0
3.3E−14

Ighg2c
873
2.04
4.64
6.37E−08
4.29E−06

Tpx2
1113.7
2.03
2.19
0
0

Bub1
362.3
2.02
2.22
0
0

Bub1b
1344.3
2.01
2.14
0
0

Nusap1
813.1
2
2.12
0
0

Kntc1
402.4
2
2.22
0
0

Kif18b
606.6
1.99
2.14
0
0

Gzmk
1785.2
1.99
2.28
0
4E−15

Nuf2
279.6
1.97
2.25
0
1.8E−14

Mcm10
477.4
1.96
2.11
0
0

Mki67
5899.1
1.96
2.12
0
0

Ccnb2
1255.4
1.95
2.1
0
0

Rad54l
350.2
1.95
2.16
0
0

Hmmr
311.7
1.94
2.19
0
1E−15

Chn1
24
1.94
2.52
2.4E−09
2.01E−07

Ccna2
1680.2
1.93
2.04
0
0

Pclaf
522.9
1.93
2.17
0
5E−15

Cenpf
495.6
1.92
2.07
0
0

Kif2c
341.3
1.92
2.1
0
0

Cdkn3
129.2
1.92
2.16
0
0

Vdr
250.7
1.92
2.2
1.02E−13
1.57E−11

Fignl1
145.7
1.91
2.15
0
0

Ttk
211.5
1.9
2.14
0
5E−15

Diaph3
356.3
1.89
2.03
0
0

Oip5
50.6
1.88
2.49
1.92E−10
1.94E−08

Kif4
650.2
1.87
1.98
0
0

Rrm2
1718.2
1.87
2.01
0
0

Sccpdh
123.3
1.86
2.05
0
3.6E−14

Clspn
637.1
1.83
1.98
0
0

Padi4
34.4
1.83
2.41
6.18E−08
4.18E−06

Shcbp1
274.8
1.82
1.98
0
0

Rad51
401.4
1.82
1.99
0
0

Tox2
889.5
1.82
2.01
0
1.5E−14

Spc24
506
1.81
1.92
0
0

Sgo1
162.8
1.81
2.01
0
5E−15

Depdc1a
29.6
1.81
2.69
1.72E−08
1.26E−06

Lrr1
59
1.8
2.37
9.15E−10
8.14E−08

Cdca8
1251.5
1.79
1.91
0
0

Kif20a
898.5
1.78
1.9
0
0

Espl1
809.6
1.78
1.9
0
0

Cdk1
1228.8
1.78
1.92
0
0

Sapcd2
275.5
1.78
1.98
0
5E−14

Pbk
107.5
1.78
2.08
1.08E−13
1.63E−11

Gfra4
95
1.78
2.1
9.39E−11
9.94E−09

Fcer1a
69.7
1.77
2.87
1.43E−06
6.68E−05

Lag3
2916.7
1.76
1.84
0
0

Rad51ap1
205.6
1.76
1.92
0
0

Ube2c
1391
1.75
1.95
5E−15
9.54E−13

Knl1
203.3
1.75
1.98
2.7E−14
4.39E−12

Kif15
595.3
1.74
1.87
0
0

Cep55
503.9
1.74
1.88
0
0

Ticrr
558.4
1.74
1.88
0
0

Neil3
228.1
1.72
1.89
0
7E−15

Spc25
211.3
1.72
1.94
1.08E−13
1.63E−11

Lpar3
17.1
1.72
2.4
4.07E−07
2.23E−05

Ncapg
383.1
1.71
1.87
0
4E−15

Iqgap3
284.2
1.71
1.86
0
4E−15

Nek2
554.5
1.71
1.87
0
2.9E−14

1500009L16Rik
143.2
1.69
2.2
2.79E−08
1.97E−06

Ckap2l
455.4
1.68
1.86
3E−15
6.37E−13

Ncaph
822.4
1.67
1.76
0
0

Uhrf1
1603.6
1.67
1.77
0
0

Pimreg
230
1.67
1.88
1.32E−13
1.96E−11

Top2a
4726.3
1.66
1.73
0
0

Spag5
1183.2
1.66
1.77
0
0

Kif14
299.1
1.66
1.84
3E−15
6.22E−13

Il10
1005.5
1.66
1.83
3.1E−14
4.98E−12

Pif1
204
1.66
1.94
1.16E−11
1.41E−09

Parpbp
117.3
1.66
1.94
1.52E−11
1.79E−09

Ly6a
2208.2
1.65
2.56
2.41E−06
0.000106

Melk
329.8
1.64
1.75
0
0

Ccnb1
160.8
1.63
1.88
1.91E−11
2.2E−09

Cit
784.3
1.62
1.74
0
4E−15

Esco2
149.5
1.61
1.83
4.58E−12
5.99E−10

CT030166.6
24.2
1.61
2.49
2.45E−06
0.000107

Trip13
233.7
1.6
1.74
0
5E−14

Cenpe
685.5
1.59
1.71
0
1E−15

Fads2
70.5
1.57
1.98
1.07E−07
6.9E−06

Cdc45
628.7
1.56
1.62
0
0

Cdca5
791.4
1.56
1.63
0
0

Cdca3
1038.6
1.56
1.64
0
0

Coro2b
349.5
1.56
1.68
1.3E−14
2.31E−12

Cenph
201.9
1.55
1.73
2.02E−12
2.73E−10

Troap
236.4
1.54
1.73
6.69E−12
8.53E−10

Birc5
1540.7
1.54
1.73
3.18E−11
3.56E−09

Ifi27l2a
8730.4
1.54
1.88
1.34E−07
8.38E−06

Mcpt1
510.2
1.54
3.77
4.63E−05
0.001264

Plk1
794.8
1.53
1.65
1E−15
1.43E−13

Ankle1
238.9
1.53
1.72
1.64E−11
1.92E−09

Cd109
30.7
1.53
2.47
3.8E−05
0.001099

Cdca2
444.5
1.52
1.64
0
2.9E−14

Siglec1
622.7
1.52
1.69
1.49E−11
1.77E−09

Ska1
210.9
1.52
1.73
2.11E−10
2.12E−08

Igkv14-111
309.6
1.51
2.66
7.06E−05
0.001789

Alox5
104.2
1.5
2.08
6.41E−06
0.000241

Stmn1
257.1
1.49
1.61
1.1E−14
2E−12

Ect2
255.1
1.49
1.71
1.15E−09
9.97E−08

Asf1b
1276.4
1.48
1.55
0
0

Ncapg2
526.2
1.48
1.58
0
1.5E−14

Cenpm
289.6
1.48
1.64
8.64E−12
1.07E−09

Cenpp
81.2
1.48
1.69
2.19E−10
2.19E−08

F13a1
227.4
1.48
2.13
1.59E−05
0.000526

Oas1a
498.3
1.47
1.73
2.33E−08
1.66E−06

Oas2
1573.5
1.47
2.24
3.32E−05
0.000987

Cenpi
136.8
1.46
1.61
1.49E−12
2.03E−10

Tex15
58.4
1.46
1.88
2.46E−07
1.43E−05

Lif
439.6
1.45
1.5
0
0

Tk1
1004.9
1.45
1.54
0
1E−15

Kif22
1022.2
1.45
1.56
1.09E−13
1.63E−11

BC030867
129.6
1.45
1.65
7E−10
6.44E−08

Tnp2
17.6
1.45
2.43
6.42E−05
0.001655

Tph1
43
1.45
2.97
0.000121
0.0028

lghv1-39
28.6
1.45
2.67
0.000133
0.003011

Tyms
809.6
1.44
1.51
0
0

Kif11
1109.9
1.44
1.51
0
0

Ighv2-6
141.1
1.44
1.89
9.88E−06
0.000349

Gng3
34.8
1.44
2.26
1.32E−05
0.000449

Spdl1
204.5
1.43
1.54
1E−14
1.71E−12

Cdh23
89.8
1.43
1.55
8.05E−12
1.01E−09

Aspm
319.4
1.43
1.59
9.94E−11
1.04E−08

Dsp
79.2
1.43
1.78
2.44E−06
0.000107

Ighv1-19
10.2
1.43
2.88
0.000154
0.003401

Depdc1b
350.8
1.42
1.54
3.09E−13
4.45E−11

Ctsg
151.2
1.42
2.54
0.000128
0.002935

Mxd3
280.1
1.41
1.5
2E−15
3.2E−13

Chaf1a
1027.2
1.4
1.49
2E−15
4.68E−13

Aunip
24.6
1.4
2.68
8.31E−05
0.00205

Mt3
18.3
1.4
2.93
0.000162
0.003547

Tcf19
1158.6
1.39
1.45
0
0

Gpm6b
315
1.39
1.48
1.5E−13
2.21E−11

Cd5l
2824.6
1.39
1.52
7.15E−11
7.78E−09

Msr1
48.9
1.39
1.89
1.13E−05
0.000392

Ighg1
2604.8
1.39
3.8
0.00023
0.004805

Ncapd2
3741.3
1.38
1.42
0
0

Oas3
1474.3
1.38
1.71
2.37E−06
0.000105

Necab3
27.4
1.38
1.88
1.94E−05
0.000622

Tpsab1
262.4
1.37
1.66
1.78E−06
8.17E−05

Gm4951
44.3
1.37
1.8
3.61E−06
0.000148

Exo1
187.1
1.36
1.54
1.17E−08
8.79E−07

Adam33
60.3
1.36
1.62
1.37E−06
6.46E−05

Klrb1a
36.2
1.36
2
7.82E−05
0.001956

Ndc80
345.5
1.35
1.44
2.6E−14
4.36E−12

Slc16a2
105.6
1.35
1.56
1.32E−07
8.29E−06

Klhl23
20.6
1.35
2.46
0.000156
0.003429

Ckap2
394.5
1.32
1.39
0
2.3E−14

Cdc6
570.6
1.32
1.4
2.3E−14
3.85E−12

E2f7
275.3
1.32
1.48
3.98E−09
3.21E−07

Mpo
1003.3
1.32
2.2
0.000333
0.006459

Mastl
108.5
1.31
1.51
7.78E−08
5.19E−06

Cenpn
307
1.3
1.38
6.9E−14
1.09E−11

Rad51b
65.9
1.3
1.52
2.32E−07
1.37E−05

Pgam2
13
1.3
2.75
0.000571
0.009954

Smtn
271.7
1.29
1.4
2.59E−10
2.57E−08

Car5b
136
1.29
1.45
6.02E−10
5.61E−08

Wfdc17
99.9
1.29
1.47
2.18E−07
1.3E−05

Xkr5
39.4
1.29
1.62
5.06E−06
0.000197

Ifit1bl1
137.8
1.29
1.67
2.19E−05
0.000693

Dhfr
503.3
1.28
1.35
2.1E−14
3.58E−12

Ccne1
503.1
1.28
1.37
8.53E−12
1.06E−09

Il4
152.5
1.28
1.52
3.69E−06
0.000151

Ms4a3
31.1
1.28
2.52
0.000653
0.011034

Fancd2
392.9
1.27
1.33
0
3.4E−14

Syce2
175.1
1.27
1.39
1.01E−09
8.79E−08

Slc43a3
380.4
1.26
1.36
1.77E−11
2.05E−09

Mcpt2
69.1
1.26
3.47
0.000769
0.012455

Dna2
343.7
1.25
1.36
3.64E−10
3.52E−08

Stil
422.9
1.25
1.39
3.95E−08
2.72E−06

Ms4a4a
77.2
1.24
1.41
1.03E−07
6.69E−06

Ifit3
421.9
1.24
2
0.000649
0.010983

Ighv1-9
232.8
1.24
2.33
0.001087
0.016261

Brca1
443.2
1.23
1.32
6.31E−12
8.12E−10

Cks1b
772.5
1.22
1.32
1.23E−09
1.05E−07

Scin
117.4
1.22
1.59
0.000102
0.002435

Fanca
375.8
1.21
1.29
3.34E−12
4.44E−10

Sostdc1
55.7
1.2
1.42
2.05E−05
0.000654

Tmprss4
19.7
1.2
1.67
0.000585
0.010122

Pcdhgc4
19.5
1.2
1.98
0.000598
0.010298

C3
1368.2
1.19
1.36
9.68E−07
4.74E−05

Fbn2
27
1.19
1.43
6.33E−05
0.001639

Igf2bp2
71.9
1.19
1.64
0.000461
0.008431

Pask
202.9
1.18
1.28
1.46E−09
1.24E−07

Atp6v1g3
12.9
1.18
2.18
0.001663
0.022323

Tmem121
19.5
1.17
1.86
0.001247
0.017975

E2f1
876.4
1.16
1.19
0
0

Zan
929.3
1.16
1.27
3.35E−08
2.33E−06

Plac8
2583
1.16
1.28
1.19E−07
7.61E−06

Rad54b
114.1
1.16
1.29
1.79E−07
1.09E−05

Lig1
3384.1
1.15
1.2
7E−15
1.29E−12

Gins2
413.5
1.15
1.22
1.04E−11
1.28E−09

Gpsm2
436.7
1.15
1.23
2.7E−11
3.06E−09

Arhgap11a
847.4
1.15
1.22
7.3E−11
7.83E−09

Gbp11
355.1
1.15
1.23
6.55E−10
6.06E−08

Gm15987
44.4
1.15
1.51
0.000198
0.004191

Efcab11
31.5
1.15
1.62
0.000442
0.008194

Sncb
15
1.15
2.01
0.001303
0.018592

Il13
60.8
1.15
1.81
0.001306
0.01862

Smpdl3b
185.8
1.14
1.28
1.47E−07
9.11E−06

Ispd
31.4
1.14
1.42
0.000161
0.003543

Igf2bp3
26.6
1.14
1.91
0.001053
0.015932

Platr11
10.2
1.14
2.09
0.00249
0.029873

Cpa3
467.2
1.14
2.12
0.002534
0.03018

Tacc3
1922.3
1.13
1.16
0
1E−15

Rmi2
184.3
1.13
1.21
5.58E−10
5.23E−08

Nsl1
195.3
1.13
1.21
1.45E−09
1.23E−07

AC151730.1
71.6
1.13
1.55
0.000357
0.006861

Myh3
15.8
1.13
1.64
0.001352
0.019086

Ighg2b
522.3
1.13
2.65
0.002343
0.028708

Klrg1
746.6
1.12
1.21
3.27E−10
3.18E−08

Ociad2
184.7
1.12
1.27
1.87E−08
1.35E−06

Mis18bp1
180.6
1.12
1.24
1.95E−07
1.17E−05

Ccl1
31
1.12
1.77
0.001407
0.019614

Prtn3
290.9
1.12
2.13
0.002845
0.032835

Cdc20
1268.9
1.1
1.14
7.2E−14
1.12E−11

Tfec
71.7
1.1
1.32
6.43E−05
0.001657

Isg15
787.9
1.1
1.38
0.000249
0.005119

Klra7
38.4
1.1
1.58
0.00111
0.016512

Hist1h2bj
17.7
1.1
1.75
0.001687
0.022479

Kbtbd6
12
1.1
2.04
0.002527
0.030129

Stx11
1153.5
1.09
1.14
3.55E−12
4.68E−10

Lgals1
11014.9
1.09
1.15
9.3E−10
8.22E−08

Ccl8
29.5
1.09
4.6
0.003929
0.041586

Nt5dc2
188.1
1.08
1.22
4.38E−06
0.000174

Dnph1
136.4
1.08
1.29
6.66E−05
0.001706

Bcat1
363.8
1.07
1.14
8.94E−10
8.07E−08

Kifc1
310.5
1.07
1.17
8.33E−08
5.52E−06

Mybl2
481.2
1.07
1.17
5.93E−07
3.09E−05

Mx1
719.3
1.07
1.66
0.002705
0.031699

Mcm5
5776.8
1.06
1.1
0
9.6E−14

Cdkn2c
421.3
1.06
1.11
1.25E−11
1.49E−09

Ccl2
77.5
1.06
1.47
0.001637
0.022077

Ifit1
298.3
1.06
1.61
0.002709
0.031699

Ighv5-2
17.8
1.06
2.05
0.004935
0.049159

Ccr5
1338.8
1.05
1.1
1.43E−12
1.97E−10

Serpinb6b
1199
1.05
1.12
3.96E−09
3.2E−07

Smc2
1176.2
1.05
1.12
4.67E−09
3.71E−07

Isg20
861.4
1.05
1.15
1.91E−06
8.67E−05

Klrc3
41.9
1.05
1.37
0.000456
0.008347

Rtp4
1189.3
1.05
1.43
0.001656
0.022272

Nrg1
17.4
1.05
1.91
0.00423
0.043995

Gins1
191.5
1.04
1.17
3.75E−06
0.000152

Knstrn
855.1
1.03
1.06
5.4E−14
8.61E−12

Phf19
475.4
1.03
1.08
7.44E−11
7.92E−09

Art2a-ps
407.6
1.03
1.17
4.28E−05
0.001207

Rfc4
533.3
1.02
1.04
0
0

Tnfsf11
468.5
1.02
1.11
4.92E−07
2.61E−05

Fn1
453.7
1.02
1.33
0.001188
0.01738

Gna14
24.8
1.02
1.42
0.002372
0.028943

Bard1
316.8
1.01
1.07
2.26E−09
1.9E−07

Pole
1124.1
1.01
1.07
2.85E−08
2.01E−06

Pdcd1
880.8
1.01
1.08
6.75E−08
4.53E−06

Prc1
830.9
1.01
1.1
1.43E−06
6.68E−05

Art2b
542.6
1.01
1.19
0.000104
0.002467

Mcm8
150.3
1
1.09
8.42E−07
4.25E−05

Xaf1
1257.6
1
1.1
1.6E−06
7.42E−05

Tnfrsf8
167.1
1
1.1
4.36E−06
0.000173

Apitd1
305.6
1
1.14
2.51E−05
0.000779

Gstt3
58.6
1
1.15
5.96E−05
0.001564

Hist1h1b
56.5
1
1.32
0.001234
0.01782

Cfap77
26
1
1.31
0.001439
0.019968

Myo1d
68
1
1.42
0.002852
0.032892

4930438A08Rik
145.8
−1.01
−1.17
0.000312
0.006134

Gm7860
120.6
−1.02
−1.09
1.76E−07
1.08E−05

Gm38405
61.2
−1.03
−1.22
0.000285
0.005674

Gm5608
44.9
−1.03
−1.27
0.000717
0.011861

Fjx1
45.8
−1.03
−1.38
0.002866
0.033002

Gm37510
31.1
−1.03
−1.48
0.004083
0.042776

9230114K14Rik
64.2
−1.04
−1.19
6.16E−05
0.001606

Cd164l2
60
−1.04
−1.24
0.000281
0.005618

Podn
23.2
−1.04
−1.32
0.001135
0.016833

Mfrp
55.6
−1.04
−1.33
0.0016
0.021746

Timm8a2
27.2
−1.05
−1.28
0.000484
0.00875

Gm11210
24.9
−1.06
−1.38
0.000895
0.014062

Trim72
99
−1.08
−1.33
0.000468
0.008514

Rbm44
28.3
−1.09
−1.5
0.001468
0.020306

Pygm
1170.9
−1.12
−1.19
1.17E−10
1.2E−08

Prr15
32.2
−1.12
−1.31
5.32E−05
0.001422

Prdm14
18.6
−1.13
−1.53
0.001494
0.020587

Tm4sf1
20.6
−1.14
−1.56
0.001205
0.017549

Rpl31-ps6
17.6
−1.15
−1.6
0.000885
0.013952

Six4
17.7
−1.17
−1.57
0.000705
0.011726

Tex45
736.4
−1.19
−1.29
6.92E−09
5.36E−07

Apol8
1161.9
−1.2
−1.25
3E−15
4.88E−13

AC158622.5
86.8
−1.23
−1.44
9.3E−06
0.00033

Fgf17
23
−1.27
−1.72
0.000249
0.005119

AC166361.2
101
−1.3
−1.42
2.71E−09
2.24E−07

Gsdmc4
19.2
−1.33
−1.63
1.68E−05
0.00055

Ap3s1-ps2
15.1
−1.34
−1.96
0.000176
0.003785

Pmel
62.3
−1.73
−4.35
1.15E−06
5.52E−05

NIH DAVID cluster analysis was performed using the upregulated gene list. Enriched clusters were ranked based on enrichment score. Cell cycle gene cluster achieved the highest enrichment score (FIGS. 23B and 24A). This result was confirmed by KEGG gene set enrichment analysis (GSEA) (FIG. 24B). Cell cycle gene set ranks as the highest (enrichment score=0.64, FDR q value=0.1, nominal p value=0). These results indicate that SV infection enhances T cell cycle progression. SV induced upregulation of a series of cytokine and chemokine/chemokine receptors (FIG. 23C, left). To identify cytokines/chemokines that are upregulated by the administration of SV vectors, we compared SV plus α4-1BB mAb versus α4-1BB mAb (FIG. 23C, right). CCL8, IL-4, IL-13 and IL-21 were among those RNAs whose expression was upregulated by SV treatment. IL-21 anti-tumor effect is dependent on the activation of T, B and NK cells [Leonard W J et al., F1000Res. 2016]. IL-4, IL-10, IL-21 upregulation is consistent with previous reports [Rowell J F et al., J Immunol. 1999, Metcalf T U et al., J Virol. 2013]. In addition, Ingenuity Pathway Analysis (IPA) indicates that SV treatment enhances T cell movement by altering the expression of a number of molecules involved migration (Table 2, FIG. 24C), including a number of chemokines and chemokine receptors.

TABLE 2

The upregulated cell movement pathway for SV vs. untreated

group by IPA. SV induced SD upregulated gene sets are

clustered by DAVID analysis (SV vs. Untreated). Gene

clusters are ranked by enrichment score.

Prediction

(based on

Genes in
measurement
Expr Log

ID
dataset
direction)
Ratio
Findings

Il21
IL21
Increased
2.57
Increases

(2)

Lag3
LAG3
Decreased
1.76
Decreases

(3)

Il10
IL10
Decreased
1.66
Decreases

(2)

Ccl1
CCL1
Increased
1.12
Increases

(1)

Ccr5
CCR5
Increased
1.05
Increases

(3)

Pdcd1
PDCD1
Decreased
1.01
Decreases

(3)

Tnfrsf8
TNFRSF8
Increased
1
Increases

(1)

Cxcr5
CXCR5
Increased
0.98
Increases

(1)

Ccr2
CCR2
Increased
0.9
Increases

(9)

Cxcr3
CXCR3
Increased
0.77
Increases

(1)

Sh2d1a
SH2D1A
Increased
0.68
Increases

(1)

Batf
BATF
Increased
0.67
Increases

(7)

Pycard
PYCARD
Increased
0.64
Increases

(3)

Ccr4
CCR4
Increased
0.62
Increases

(4)

Ccl5
CCL5
Increased
0.62
Increases

(2)

Itgb1
ITGB1
Increased
0.51
Increases

(1)

S1pr2
S1PR2
Increased
0.5
Increases

(1)

Lcp1
LCP1
Affected
0.5
Affects

(1)

Tnfsf14
TNFSF14
Increased
0.5
Increases

(1)

Hspd1
HSPD1
Decreased
0.41
Decreases

(1)

Cbfb
CBFB
Decreased
0.4
Decreases

(1)

Jak3
JAK3
Increased
0.36
Increases

(1)

Rap1a
RAP1A
Decreased
0.31
Decreases

(3)

Was
WAS
Increased
0.29
Increases

(2)

Etv6
ETV6
Increased
0.26
Increases

(3)

Rac2
RAC2
Increased
0.25
Increases

(36)

Apbb1ip
APBB1IP
Increased
−0.21
Decreases

(2)

Pecam1
PECAM1
Decreased
−0.34
Increases

(10)

Ldlr
LDLR
Decreased
−0.41
Increases

(1)

Bach2
BACH2
Increased
−0.46
Decreases

(1)

S1pr1
S1PR1
Decreased
−0.58
Increases

(6)

Gpr132
GPR132
Increased
−0.6
Decreases

(1)

To understand why SV plus α4-1BB mAb achieves the best therapeutic effect, Deseq2 analysis was run for SV plus α4-1BB mAb vs. untreated samples. 1046 upregulated genes (q<0.05 and Log 2 Fold Change≥1) and 877 downregulated genes (q<0.05 and Log 2 Fold Change≤−1) in the SV plus α4-1BB mAb group were identified (FIG. 23A, Table 3). T cells from animals treated with SV+α4-1BB mAb vs. treated were also compared with SV only and 316 upregulated genes (p<0.05 and Log 2 Fold Change≥1) and 439 downregulated genes (p<0.05 and Log 2 Fold Change≤−1) in the SV+α4-1BB mAb treated group were found (FIG. 23A, Table 4).

TABLE 3

The SD expressed genes list for SV + α4-1BB vs. untreated group by RNA-

Seq (q < 0.05, Log2FC ≥ 1 and Log2FC ≤ −1).

gene
baseMean
log2FC
log2FCunshrunk
pvalue
padj

Gzmk
1785.2
4.75
5.32
0
0

Gzmb
4113.9
4.37
4.81
0
0

Ccl8
29.5
3.64
8.24
0
0

Ociad2
184.7
3.53
3.76
0
0

Aurkb
806.6
3.51
3.73
0
0

Ret
794.7
3.51
4.41
0
0

E2f8
535.1
3.5
3.88
0
0

Tpx2
1113.7
3.34
3.58
0
0

Kif2c
341.3
3.33
3.58
0
0

Kif18b
606.6
3.29
3.51
0
0

Hmmr
311.7
3.26
3.61
0
0

Cdc25c
143.9
3.25
3.81
0
0

Pbk
107.5
3.24
3.63
0
0

Cenpf
495.6
3.23
3.46
0
0

Prr11
288
3.23
3.68
0
0

Ccna2
1680.2
3.18
3.34
0
0

Ccnb2
1255.4
3.16
3.38
0
0

Bub1
362.3
3.16
3.44
0
0

Pif1
204
3.16
3.57
0
0

Klrg1
746.6
3.15
3.34
0
0

Nusap1
813.1
3.14
3.31
0
0

Bub1b
1344.3
3.13
3.32
0
0

Rad54l
350.2
3.13
3.43
0
0

Cdk1
1228.8
3.11
3.32
0
0

Mki67
5899.1
3.11
3.36
0
0

Rad51
401.4
3.08
3.34
0
0

Ttk
211.5
3.08
3.4
0
0

Nuf2
279.6
3.08
3.46
0
0

Fignl1
145.7
3.07
3.37
0
0

Shcbp1
274.8
3.05
3.26
0
0

Cdkn3
129.2
3.05
3.35
0
0

Rrm2
1718.2
3.04
3.26
0
0

Ube2c
1391
3.03
3.35
0
0

Pclaf
522.9
3.03
3.36
0
0

Lrr1
59
3.03
3.74
0
0

Cdca8
1251.5
3.02
3.21
0
0

Cep55
503.9
3.02
3.24
0
0

Mem10
477.4
3.01
3.22
0
0

Sapcd2
275.5
2.99
3.28
0
0

Pimreg
230
2.99
3.3
0
0

Kif4
650.2
2.97
3.13
0
0

Kntc1
402.4
2.96
3.25
0
0

Ccr5
1338.8
2.95
3.07
0
0

Kif14
299.1
2.94
3.2
0
0

Sgo1
162.8
2.94
3.21
0
0

Spc24
506
2.93
3.08
0
0

Espl1
809.6
2.93
3.12
0
0

Clspn
637.1
2.93
3.14
0
0

Wdr95
226
2.88
3.09
0
0

Nek2
554.5
2.88
3.13
0
0

Ccnb1
160.8
2.88
3.25
0
0

Ncapg
383.1
2.86
3.09
0
0

Top2a
4726.3
2.85
2.96
0
0

Ncaph
822.4
2.85
3
0
0

Kif20a
898.5
2.85
3.03
0
0

Diaph3
356.3
2.85
3.04
0
0

Akr1c18
25.3
2.85
4.88
6E−15
1.84E−13

Ckap2l
455.4
2.83
3.1
0
0

Oip5
50.6
2.83
3.55
0
0

Ermn
17.7
2.82
5.41
3.2E−14
8.82E−13

Spag5
1183.2
2.81
2.97
0
0

Birc5
1540.7
2.81
3.13
0
0

Dpysl5
94.9
2.78
3.57
0
0

Cdca5
791.4
2.77
2.88
0
0

Iqgap3
284.2
2.77
2.98
0
0

Kif15
595.3
2.76
2.95
0
0

Plk1
794.8
2.75
2.94
0
0

Troap
236.4
2.75
3.04
0
0

Cenpe
685.5
2.73
2.91
0
0

Esco2
149.5
2.73
3.04
0
0

Gng3
34.8
2.72
3.74
0
1E−15

Cit
784.3
2.71
2.9
0
0

Kif22
1022.2
2.71
2.91
0
0

Car5b
136
2.71
2.94
0
0

Ncald
297.2
2.71
2.95
0
0

Cdca2
444.5
2.7
2.87
0
0

Knl1
203.3
2.7
3.01
0
0

Hist1h1b
56.5
2.7
3.28
0
0

Aunip
24.6
2.69
4.29
1.5E−14
4.26E−13

1500009L16Rik
143.2
2.68
3.37
0
0

Osr2
54.7
2.68
4.06
8E−15
2.43E−13

Depdc1b
350.8
2.67
2.86
0
0

Ticrr
558.4
2.67
2.87
0
0

Melk
329.8
2.66
2.82
0
0

Tpsab1
262.4
2.66
3.16
0
0

Smpdl3b
185.8
2.65
2.9
0
0

Spc25
211.3
2.65
2.95
0
0

Cdc45
628.7
2.64
2.73
0
0

Ccr2
1142.1
2.64
2.83
0
0

Il21
229
2.64
3.76
3.3E−14
9.01E−13

BC030867
129.6
2.63
2.93
0
0

Uhrfl
1603.6
2.61
2.76
0
0

Mxd3
280.1
2.61
2.76
0
0

Tyms
809.6
2.6
2.7
0
0

Neil3
228.1
2.6
2.82
0
0

Trip13
233.7
2.59
2.78
0
0

Rad51ap1
205.6
2.58
2.78
0
0

Asf1b
1276.4
2.57
2.69
0
0

Kif11
1109.9
2.55
2.67
0
0

Ska1
210.9
2.55
2.86
0
0

Stmn1
257.1
2.53
2.71
0
0

Ccl5
22142.8
2.53
2.75
0
0

Ly6a
2208.2
2.53
3.76
5.03E−13
1.2E−11

Igf2bp3
26.6
2.52
3.61
1.47E−13
3.72E−12

Aspm
319.4
2.51
2.77
0
0

Parpbp
117.3
2.51
2.86
0
0

Ect2
255.1
2.49
2.82
0
0

Cdca3
1038.6
2.48
2.6
0
0

E2f7
275.3
2.48
2.74
0
0

Depdc1a
29.6
2.48
3.46
2E−15
7.6E−14

Gm33460
48.9
2.47
2.96
0
0

Tex15
58.4
2.47
3
0
0

Tk1
1004.9
2.46
2.59
0
0

Lag3
2916.7
2.45
2.56
0
0

Spdl1
204.5
2.45
2.61
0
0

Gm15056
28.9
2.45
4.48
4.74E−11
8.5E−10

Ckap2
394.5
2.44
2.55
0
0

Ankle1
238.9
2.44
2.71
0
0

Mt3
18.3
2.42
4.28
3.9E−11
7.15E−10

Cenph
201.9
2.41
2.66
0
0

Arsb
1795.9
2.4
2.55
0
0

Cenpi
136.8
2.4
2.6
0
0

Gm6637
196.3
2.4
2.69
0
0

Ndc80
345.5
2.39
2.53
0
0

Chaf1a
1027.2
2.39
2.53
0
0

Cenpm
289.6
2.39
2.61
0
0

CT030166.6
24.2
2.37
3.39
1.66E−12
3.74E−11

Ms4a3
31.1
2.37
4.07
2E−10
3.29E−09

Il13
60.8
2.36
3.42
3.84E−11
7.06E−10

Ctsg
151.2
2.36
3.9
2.01E−10
3.3E−09

Col6a5
17.7
2.36
6.13
4.48E−10
6.83E−09

Ncapd2
3741.3
2.33
2.39
0
0

Tcf19
1158.6
2.32
2.41
0
0

Gbp11
355.1
2.31
2.46
0
0

Fhl2
217.6
2.3
2.44
0
0

Ncapg2
526.2
2.29
2.43
0
0

Cks1b
772.5
2.28
2.46
0
0

Gzma
3261.2
2.28
3.76
8.07E−10
1.18E−08

Nkg7
8456.1
2.27
2.34
0
0

Pask
202.9
2.27
2.43
0
0

Il10
1005.5
2.27
2.5
0
0

Prc1
830.9
2.26
2.46
0
0

Wipf3
37.6
2.26
3.33
3.67E−12
7.88E−11

Mpo
1003.3
2.26
3.54
7.46E−10
1.09E−08

Osbpl3
1358.5
2.25
2.33
0
0

Mastl
108.5
2.25
2.54
0
0

Serpinb6b
1199
2.24
2.38
0
0

Adgrg1
449.6
2.24
2.4
0
0

Slc16a2
105.6
2.24
2.53
0
0

Cel1
31
2.24
3.2
7.42E−11
1.29E−09

Cdc6
570.6
2.22
2.34
0
0

Bspry
174.2
2.22
2.64
1E−15
3.6E−14

Ccne1
503.1
2.21
2.36
0
0

Cenpp
81.2
2.21
2.47
0
0

Lxn
79
2.21
2.45
0
0

Lgals1
11014.9
2.2
2.34
0
0

Adap1
1257.2
2.18
2.3
0
0

F13a1
227.4
2.17
3.04
1.97E−10
3.24E−09

Cdc20
1268.9
2.16
2.24
0
0

Tigit
2942.9
2.16
2.38
0
0

Cdkn2a
59.4
2.15
2.56
3.4E−14
9.34E−13

Exo1
187.1
2.14
2.4
0
0

Clip4
39.8
2.14
2.75
7.69E−12
1.58E−10

F2rl2
42.8
2.13
2.58
1.57E−13
3.95E−12

Hist1h2bj
17.7
2.13
2.99
4.05E−10
6.22E−09

Gldc
15.3
2.13
3.09
1.37E−09
1.92E−08

Dhfr
503.3
2.12
2.24
0
0

Lig1
3384.1
2.11
2.19
0
0

Cenpn
307
2.11
2.23
0
0

Tox2
889.5
2.11
2.32
0
0

Cdkn2c
421.3
2.1
2.2
0
0

Kifc1
310.5
2.1
2.26
0
0

Slc22a3
85.3
2.1
2.57
1.47E−12
3.33E−11

Msr1
48.9
2.1
2.73
1.56E−11
3.04E−10

Trp73
22
2.1
3.54
2.07E−08
2.37E−07

Prtn3
290.9
2.1
3.62
2.37E−08
2.68E−07

Csf2
72.9
2.09
2.47
8E−14
2.1E−12

Fancd2
392.9
2.08
2.16
0
0

Gm2788
29.7
2.08
2.77
5.16E−11
9.2E−10

Nfe2
91.3
2.08
2.85
9.6E−10
1.38E−08

Rnase2a
5.3
2.08
6.48
1.58E−08
1.84E−07

Mis18bp1
180.6
2.07
2.26
0
0

Samd14
203.2
2.07
2.39
3E−15
8.5E−14

Oas1a
498.3
2.07
2.42
4E−15
1.1E−13

Hist1h3c
11.8
2.07
3.72
2.61E−08
2.93E−07

Tacc3
1922.3
2.06
2.12
0
0

Wdr31
15.1
2.06
3.44
1.23E−08
1.46E−07

Knstrn
855.1
2.05
2.12
0
0

Anxa2
5777.8
2.05
2.21
0
0

Tpbg
19.3
2.04
3.63
2.53E−08
2.85E−07

Cst7
1893.9
2.02
2.09
0
0

Slc43a3
380.4
2.02
2.16
0
0

Muc13
227
2.02
2.24
0
0

Rad51b
65.9
2.02
2.3
0
5E−15

Xkr5
39.4
2.02
2.42
2.9E−13
7.07E−12

Chil3
42.4
2.02
3.21
4.25E−08
4.61E−07

Bard1
316.8
2.01
2.11
0
0

Acot7
2318.6
2.01
2.12
0
0

Arhgap11a
847.4
2.01
2.13
0
0

Rad54b
114.1
2.01
2.21
0
0

Pcdhgc4
19.5
2.01
2.97
5.51E−09
6.96E−08

Mcm5
5776.8
2
2.06
0
0

Rmi2
184.3
2
2.12
0
0

Dna2
343.7
2
2.16
0
0

S100a6
3227.2
2
2.17
0
0

Mybl2
481.2
2
2.19
0
0

Brca1
443.2
1.99
2.11
0
0

Esm1
680.1
1.99
2.16
0
0

E2fl
876.4
1.98
2.04
0
0

F10
33.6
1.98
3.1
4.08E−08
4.44E−07

Lif
439.6
1.97
2.05
0
0

Pglyrp1
1046.5
1.97
2.15
0
0

Fam19a3
168.7
1.97
2.22
1E−15
1.9E−14

Spp1
51
1.97
2.41
2.47E−11
4.68E−10

Faxc
12.4
1.97
3.44
1.3E−07
1.3E−06

Stil
422.9
1.96
2.17
0
0

Dapk2
315.5
1.95
2.04
0
0

Serpinb9
1224.7
1.95
2.06
0
0

Fanca
375.8
1.95
2.06
0
0

Bcat1
363.8
1.95
2.06
0
0

Neurl1b
49.9
1.95
2.46
2.74E−10
4.38E−09

Gins1
191.5
1.94
2.14
0
0

Tg
98.4
1.94
2.15
0
1E−15

Dnph1
136.4
1.94
2.27
5.77E−13
1.36E−11

Tmem40
47.5
1.94
2.52
2.2E−09
2.98E−08

Hist1h3g
9.3
1.94
3.19
1.43E−07
1.41E−06

Ighg1
2604.8
1.94
4.76
2.48E−07
2.33E−06

Psrc1
122.1
1.93
2.07
0
0

Serpina3f
452
1.93
2.12
0
0

Robo3
27.3
1.93
2.44
7.7E−10
1.13E−08

Gcg
62.2
1.93
5.91
2.19E−07
2.08E−06

Smc2
1176.2
1.92
2.03
0
0

Sgo2a
143.4
1.92
2.11
0
0

Nt5dc2
188.1
1.92
2.15
0
5E−15

Klrc2
53.2
1.92
2.27
5.7E−13
1.35E−11

Prss57
9.8
1.92
3.88
2.9E−07
2.7E−06

Gpsm2
436.7
1.91
2.02
0
0

Nsl1
195.3
1.9
2.03
0
0

C3
1368.2
1.9
2.15
6E−15
1.81E−13

Angpt1
63.2
1.9
2.28
1.89E−11
3.65E−10

Popdc2
54.7
1.89
2.36
3.65E−10
5.66E−09

Kbtbd6
12
1.89
3.03
1.35E−07
1.34E−06

Stk32c
341
1.87
1.99
0
0

Syce2
175.1
1.87
2.03
0
0

Gp9
24.8
1.87
2.53
3.19E−08
3.56E−07

Poc1a
388.3
1.86
1.95
0
0

2610318N02Rik
193.2
1.86
2.02
0
0

Ifng
1413.7
1.86
2.11
5.7E−14
1.51E−12

Hcn2
36.3
1.86
2.42
6.4E−09
7.97E−08

Klhl23
20.6
1.86
3.13
1.4E−07
1.38E−06

Nrg1
17.4
1.86
3
3.31E−07
3.06E−06

Insrr
64.7
1.85
2.05
3E−15
1.03E−13

Cldnd2
53.5
1.85
2.12
3.45E−13
8.31E−12

Ifit1bl1
137.8
1.85
2.34
1.18E−09
1.67E−08

Scn11a
5.9
1.85
5.36
7.48E−07
6.42E−06

Pdzph1
10.7
1.85
4.11
9.95E−07
8.34E−06

Gins2
413.5
1.84
1.94
0
0

Gm12250
177.3
1.84
2.07
6E−15
1.73E−13

Dach1
18.3
1.84
2.8
3.05E−07
2.83E−06

Rasgef1a
270.7
1.83
1.94
0
0

Pcyt1b
106.8
1.83
2.01
0
0

Oas3
1474.3
1.83
2.25
3.83E−10
5.9E−09

Cd70
12.5
1.83
2.73
3.29E−07
3.05E−06

Tuba8
42.2
1.83
2.76
4.67E−07
4.21E−06

Tph1
43
1.83
3.53
1.35E−06
1.11E−05

Zbtb32
667.1
1.82
1.9
0
0

Tfr2
139.5
1.82
2.41
2.31E−08
2.62E−07

Mmrn1
13.3
1.82
2.99
7.75E−07
6.64E−06

Gm24289
6.9
1.82
5.21
1.22E−06
1.01E−05

Dlgap5
705.3
1.81
1.9
0
0

Ska3
252.1
1.81
1.91
0
0

Fam81a
55.4
1.81
2.16
1.92E−10
3.16E−09

Plppr3
42.4
1.81
2.74
7.18E−07
6.2E−06

Mpl
45.7
1.81
2.69
7.96E−07
6.8E−06

Hist1h2ag
6.4
1.81
3.59
1.52E−06
1.23E−05

Fbxo41
15.3
1.8
2.59
1.51E−07
1.48E−06

Mcm3
5809.4
1.79
1.84
0
0

Ska2
254.2
1.79
1.91
0
0

Art2b
542.6
1.79
2.08
6.27E−12
1.3E−10

Necab3
27.4
1.79
2.36
1.87E−08
2.16E−07

Lilr4b
1553.9
1.78
1.91
0
0

AA467197
30.3
1.78
2.21
9E−09
1.09E−07

Maats1
7.8
1.78
3.55
2.36E−06
1.84E−05

Col6a2
16.9
1.78
3.82
2.57E−06
1.99E−05

Cdca7
631.9
1.77
1.89
0
0

Serpina3g
2916.5
1.77
1.92
0
0

Dyrk3
249.1
1.77
1.95
2E−15
5.8E−14

Apitd1
305.6
1.77
1.98
9.1E−14
2.37E−12

AC153938.2
40.5
1.77
2.19
2.58E−09
3.47E−08

Eomes
4109.7
1.75
1.8
0
0

Pola1
686.1
1.75
1.83
0
0

Pole
1124.1
1.75
1.86
0
0

Eme1
219.4
1.75
1.88
0
0

Map6
151
1.75
1.88
0
0

Gm4951
44.3
1.75
2.24
1.88E−09
2.57E−08

Tff3
16.7
1.75
2.36
1.47E−07
1.45E−06

Nmral1
561.7
1.74
1.86
0
0

Plac8
2583
1.74
1.92
2E−15
6.3E−14

Klrc1
236.6
1.74
1.95
5E−14
1.34E−12

S100a4
1170.3
1.74
1.96
7.04E−13
1.66E−11

Mns1
129.3
1.74
2.05
2.42E−10
3.91E−09

Miat
31.2
1.74
2.07
5.13E−10
7.74E−09

Efcab11
31.5
1.74
2.33
6.62E−08
6.98E−07

Erfe
15.4
1.74
2.77
9.89E−07
8.3E−06

Kcnk5
399.5
1.73
1.81
0
0

Stx11
1153.5
1.73
1.81
0
0

Smtn
271.7
1.73
1.87
0
0

Anln
217.2
1.73
1.89
1E−15
3.1E−14

Gm17745
84
1.73
1.92
1.9E−14
5.44E−13

Krt18
26.4
1.73
2.17
1.75E−08
2.03E−07

2900011O08Rik
14.8
1.73
2.39
9.4E−07
7.92E−06

Tpi1
2445.6
1.72
1.78
0
0

Chsy1
2251.6
1.72
1.8
0
0

Gtse1
554
1.72
1.81
0
0

Pdcd1
880.8
1.72
1.84
0
0

Vdr
250.7
1.72
1.97
2.66E−11
5E−10

Ltb4r1
44.5
1.72
2.06
1.19E−09
1.68E−08

2010110K18Rik
30.1
1.72
2.11
9.25E−09
1.12E−07

Alox5
104.2
1.72
2.35
2.33E−07
2.2E−06

Pde10a
17
1.72
2.48
7.24E−07
6.24E−06

Dscc1
150.3
1.71
1.83
0
0

Mcm6
5226.1
1.7
1.74
0
0

Tfdp1
1161.4
1.7
1.76
0
0

E2f2
2088.1
1.7
1.78
0
0

1700011L03Rik
6.7
1.7
5.77
5.28E−06
3.81E−05

Col6a1
15.6
1.7
4.62
6.57E−06
4.64E−05

Chst11
1091.1
1.69
1.75
0
0

Il12rb1
1086.8
1.69
1.76
0
0

Gm4841
29.9
1.69
2.35
3.46E−07
3.19E−06

Treml1
31.4
1.69
2.36
1.75E−06
1.4E−05

Fcer1a
69.7
1.69
2.77
3.83E−06
2.85E−05

Rfc4
533.3
1.68
1.72
0
0

Rgs16
7742.7
1.68
1.75
0
0

Ezh2
1820.5
1.68
1.76
0
0

Lockd
222.2
1.68
1.85
3.1E−14
8.5E−13

Fam57b
23.3
1.68
5.08
4.18E−07
3.8E−06

Dixdc1
18.6
1.68
2.66
2.93E−06
2.25E−05

Trpc6
7.4
1.68
4.05
5.76E−06
4.12E−05

Adra1b
14.9
1.68
2.83
6.8E−06
4.78E−05

Ttc39c
656.7
1.67
1.72
0
0

Wee1
206.8
1.67
1.75
0
0

Neb
354.6
1.67
1.9
2.03E−11
3.9E−10

Prdm1
912.1
1.66
1.74
0
0

Gimap7
2437.5
1.66
1.76
0
0

Mcm8
150.3
1.66
1.79
0
4E−15

Tnfrsf8
167.1
1.66
1.82
1E−14
3.02E−13

Lgalsl
189.5
1.66
1.82
2.5E−14
7.06E−13

Ptgr1
201.9
1.66
1.84
1.25E−13
3.19E−12

Fads2
70.5
1.66
2.07
1.89E−08
2.18E−07

Usp18
491.6
1.66
2.34
1.26E−06
1.03E−05

Serpinb9b
63.4
1.66
2.45
1.55E−06
1.26E−05

Vwa2
4.2
1.66
5.89
3.9E−06
2.9E−05

Saa3
14.8
1.66
4.96
6.94E−06
4.87E−05

Mad2l1
857.6
1.65
1.69
0
0

Hells
609.3
1.65
1.71
0
0

Cisd1
468.5
1.65
1.73
0
0

Slc35d3
110.5
1.65
1.77
0
5E−15

Sncb
15
1.65
2.65
2.82E−06
2.17E−05

Ppbp
30.6
1.65
2.38
3.36E−06
2.54E−05

Tff1
8.6
1.65
3.22
1.24E−05
8.24E−05

Nsd2
2242.8
1.64
1.68
0
0

Phf19
475.4
1.64
1.71
0
0

Ccdc34
308.1
1.64
1.73
0
0

1700001O22Rik
140.3
1.64
1.78
4E−15
1.36E−13

Gm20667
5.2
1.64
4.98
1.13E−05
7.61E−05

Ppp1r3g
7.3
1.64
3.91
1.3E−05
8.58E−05

4930519L02Rik
9.9
1.64
4.33
1.41E−05
9.23E−05

Racgap1
3392.2
1.63
1.67
0
0

Lmnb1
6788.3
1.63
1.69
0
0

Kif23
1414.3
1.63
1.69
0
0

Ryk
207.2
1.63
1.76
1E−15
2.6E−14

Heatr9
204.6
1.63
1.85
1.58E−11
3.07E−10

Ifi27l2a
8730.4
1.63
1.99
2.36E−08
2.67E−07

Tnip3
36.1
1.63
2.1
2.07E−07
1.97E−06

Vat1l
14.9
1.62
3.1
9.48E−06
6.47E−05

Tpsb2
486.9
1.62
2.93
2.12E−05
0.000133

AW112010
5981.3
1.61
1.69
0
0

9630013D21Rik
122.8
1.61
1.8
4.6E−12
9.71E−11

Mtfp1
42.1
1.61
1.98
4.08E−08
4.44E−07

Col1a2
95
1.61
2.11
6.19E−07
5.41E−06

Ascl2
24.4
1.61
2.62
1.12E−05
7.53E−05

Ccdc18
45.6
1.6
1.91
1.15E−08
1.37E−07

Myct1
34.7
1.6
2.16
1.88E−06
1.49E−05

Hist1h2bm
8.5
1.6
3.51
2.45E−05
0.000151

Elane
132
1.6
3.14
2.69E−05
0.000165

Serping1
49.6
1.59
1.9
6.03E−09
7.54E−08

Rab44
136.4
1.59
2.08
8.37E−07
7.11E−06

Dcn
49
1.59
2.48
3.24E−06
2.45E−05

6530402F18Rik
553.7
1.58
1.63
0
0

Klrk1
793.9
1.58
1.68
0
0

Nup37
207.1
1.58
1.74
3.05E−13
7.43E−12

Bst1
74.2
1.58
1.75
1.64E−12
3.71E−11

Il1rl1
328.6
1.58
1.74
1.73E−12
3.88E−11

Klrc3
41.9
1.58
1.99
7.66E−08
7.98E−07

Adgrg7
5.3
1.58
5.46
9.43E−06
6.44E−05

Gp1bb
21.6
1.58
2.44
2.04E−05
0.000128

Sh2d1a
1356.7
1.57
1.64
0
0

Areg
192.8
1.57
1.78
1.04E−10
1.78E−09

Mlkl
114.8
1.57
1.78
1.33E−10
2.24E−09

D630039A03Rik
66.5
1.57
1.86
1.8E−08
2.09E−07

Sdsl
60
1.57
2.27
7.96E−06
5.53E−05

Oas2
1573.5
1.57
2.38
8.81E−06
6.05E−05

Tjp2
411.7
1.56
1.67
1E−15
3E−14

Fut7
69.8
1.56
1.8
1.61E−09
2.23E−08

Ces2g
190
1.56
2.29
8.88E−06
6.1E−05

Tmem59l
15.2
1.56
2.3
1.24E−05
8.24E−05

Kcnj5
5.3
1.56
5.41
1.25E−05
8.31E−05

Maob
9.3
1.56
3.57
3.89E−05
0.000229

Dut
2331.7
1.55
1.6
0
0

4933404O12Rik
326.5
1.55
1.66
0
1.4E−14

Alox8
57.9
1.55
1.79
1.05E−09
1.5E−08

Runx2os1
41.2
1.55
2.01
1.56E−06
1.26E−05

Etv4
18.3
1.55
2.25
1.04E−05
7E−05

Ccnf
676.9
1.54
1.62
0
0

Spns2
183.2
1.54
1.86
1.1E−07
1.11E−06

H1fx
65.9
1.54
1.97
1.34E−06
1.1E−05

Chtf18
707.9
1.53
1.58
0
0

Gbp2b
1682.8
1.53
1.59
0
0

Cmtm7
2372.8
1.53
1.59
0
0

Gstt3
58.6
1.53
1.73
3.64E−10
5.65E−09

Chek2
113.8
1.53
1.77
3.66E−09
4.79E−08

Rab39b
35.1
1.53
1.86
7.94E−08
8.25E−07

Vash1
14.6
1.53
2.36
2.63E−05
0.000162

Incenp
3803.5
1.52
1.56
0
0

Zwilch
309.4
1.52
1.59
0
0

Brip1
231.5
1.52
1.6
0
0

Gm14005
181.4
1.52
1.61
0
2E−15

Foxm1
1814.7
1.52
1.62
0
5E−15

1700020L24Rik
90.2
1.52
1.72
8.22E−10
1.2E−08

Il4
152.5
1.52
1.8
3.53E−08
3.9E−07

Mx1
719.3
1.52
2.3
2.01E−05
0.000127

Gna15
580.1
1.51
1.6
0
1E−15

Dctpp1
771.7
1.51
1.64
2.43E−13
5.96E−12

Angptl2
741.2
1.51
1.67
1.1E−11
2.2E−10

Mgarp
30.5
1.51
1.87
4.92E−07
4.41E−06

Gm19585
364.2
1.5
1.56
0
0

Zfp367
650.8
1.5
1.58
0
0

S100a10
7676.1
1.5
1.58
0
0

C330027C09Rik
421.9
1.5
1.63
1.44E−13
3.66E−12

Cenpk
43.5
1.5
1.73
9.19E−09
1.11E−07

Ly6e
13759
1.5
1.83
2.5E−07
2.35E−06

Ccl4
2257.9
1.5
1.9
1.59E−06
1.28E−05

Casp7
625.6
1.49
1.53
0
0

Zfand4
84.9
1.49
1.61
1.84E−13
4.58E−12

Tstd3
269.1
1.49
1.65
1.75E−11
3.39E−10

Lmtk3
107.1
1.49
1.75
4.88E−08
5.24E−07

AC153498.1
62.1
1.49
1.77
7.06E−08
7.4E−07

Grtp1
36.2
1.49
1.96
3.97E−06
2.94E−05

Gm2762
38.5
1.49
2.09
6.91E−06
4.85E−05

D130058E05Rik
5.1
1.49
5.39
2.27E−05
0.000142

Npy
11.8
1.49
2.45
6.48E−05
0.00036

Serpina3h
8.8
1.49
2.77
7.52E−05
0.000411

Ighg2c
873
1.49
3.73
7.66E−05
0.000418

Nkain1
8
1.49
3.23
7.74E−05
0.000421

Podnl1
2382.6
1.48
1.53
0
0

Gzmm
496.8
1.48
1.56
0
0

Haspin
214.2
1.48
1.6
1.76E−13
4.4E−12

Csfl
1178.9
1.48
1.64
5.94E−11
1.05E−09

Lamc2
28.8
1.48
1.89
2.97E−06
2.28E−05

Gm11454
22.4
1.48
1.93
3.25E−06
2.46E−05

Tal1
90.9
1.48
2.14
2.23E−05
0.000139

Ybx3
2394.9
1.47
1.56
0
0

Rln3
181.3
1.47
1.61
1.29E−11
2.56E−10

Rai14
77.8
1.47
1.68
9.24E−09
1.11E−07

Gmpr
24.8
1.47
2.3
5.94E−05
0.000334

Drp2
31.1
1.47
2.94
9.79E−05
0.000518

Cma1
339
1.47
2.71
0.00011
0.000572

Gbp5
1110.5
1.46
1.49
0
0

Alad
1397.6
1.46
1.63
2.48E−10
4E−09

Tnfsf4
81.6
1.46
1.73
2.63E−07
2.47E−06

Rtkn2
30.4
1.46
1.85
1.64E−06
1.32E−05

Klra7
38.4
1.46
2.03
1.43E−05
9.35E−05

Pf4
162.2
1.46
2.25
9.08E−05
0.000485

Gbp2
3001.3
1.45
1.5
0
0

Ercc6l
189.4
1.45
1.55
2.2E−14
6.19E−13

Iigp1
872.5
1.45
1.55
2.8E−14
7.75E−13

Gemin6
89.9
1.45
1.55
8.5E−14
2.2E−12

Paqr4
188.3
1.45
1.58
4.17E−12
8.88E−11

Cd160
483.3
1.45
1.63
1.06E−09
1.51E−08

Ttc16
66.2
1.45
1.72
1.54E−07
1.51E−06

Ulk4
24.5
1.45
1.83
2.23E−06
1.75E−05

Ccnb1ip1
40.8
1.45
1.87
6.02E−06
4.29E−05

Tubb1
17.5
1.45
2.25
5.51E−05
0.000312

Vax2
12.7
1.45
2.38
0.000106
0.000558

Slc16a11
9.1
1.45
2.57
0.000108
0.000563

Col3a1
82.1
1.45
2.78
0.000124
0.000638

Sdf2l1
994.9
1.44
1.51
0
0

Psmc3ip
149.8
1.44
1.53
1E−15
3.8E−14

Tmem107
70.8
1.44
1.63
4.82E−09
6.16E−08

Tmem121
19.5
1.44
2.22
6.17E−05
0.000344

Platr11
10.2
1.44
2.5
0.00012
0.000617

9230102O04Rik
5.8
1.44
3.53
0.000127
0.000649

Ms4a4b
9942.9
1.43
1.46
0
0

Gmnn
974.1
1.43
1.47
0
0

Bak1
2591.4
1.43
1.48
0
0

Casp3
2922.8
1.43
1.48
0
0

Sass6
197.1
1.43
1.51
0
4E−15

2310031A07Rik
64.6
1.43
1.81
4.21E−06
3.1E−05

Gda
44.3
1.43
2.06
3.9E−05
0.00023

Ckm
8
1.43
3.17
0.000143
0.000725

Mfsd13a
222.3
1.42
1.53
1.9E−13
4.74E−12

Gna14
24.8
1.42
1.92
1.63E−05
0.000106

Retnla
7.6
1.42
5.6
8.17E−05
0.000441

4930579G24Rik
292.8
1.41
1.44
0
0

Alcam
430
1.41
1.5
0
1.7E−14

Ak6
275.7
1.41
1.56
3.31E−10
5.2E−09

Ica1
85.1
1.41
1.64
1.66E−07
1.62E−06

Smo
59
1.41
1.66
3.83E−07
3.5E−06

A930002I21Rik
49.6
1.41
1.91
2.49E−05
0.000154

Col1a1
89.2
1.41
1.94
2.5E−05
0.000154

Cd34
96.8
1.41
2.01
5.84E−05
0.000329

Lhx2
15.9
1.41
2.11
6.05E−05
0.000339

Ptger3
20.8
1.41
2.29
0.000156
0.000779

Xdh
1588.9
1.4
1.45
0
0

Ttn
292.1
1.4
1.52
3.22E−11
5.97E−10

Asns
221.7
1.4
1.57
2.68E−09
3.59E−08

Cmc2
422.7
1.4
1.6
2.68E−08
3.01E−07

Cks2
105.1
1.4
1.64
3.29E−07
3.05E−06

Gzmc
56.6
1.4
1.74
4.29E−06
3.16E−05

Hist1h2bb
17.7
1.4
2
7.72E−05
0.00042

Epx
8
1.4
6.63
8.75E−05
0.00047

Gata2
341.6
1.4
2.14
0.000102
0.000536

Hp
143.1
1.4
2.47
0.000179
0.000879

Gm11843
7.5
1.4
4.37
0.000181
0.000888

Batf
1910.4
1.39
1.44
0
0

Hip1
1867.7
1.39
1.46
0
0

Havcr2
486.2
1.39
1.5
1.88E−12
4.18E−11

Acod1
232.1
1.39
1.69
1.35E−06
1.11E−05

Grb10
61
1.39
1.77
5.98E−06
4.26E−05

Reg2
5.6
1.39
5.03
7.66E−06
5.33E−05

N4bp1
3266.2
1.38
1.4
0
0

Prim1
1043.4
1.38
1.41
0
0

Mtfr2
115.7
1.38
1.47
2.56E−13
6.28E−12

Hmgb2
1572.1
1.38
1.49
1.93E−12
4.28E−11

Isg20
861.4
1.38
1.52
2.64E−10
4.24E−09

Cmpk2
311.4
1.38
1.66
1E−06
8.41E−06

Isg15
787.9
1.38
1.72
4.32E−06
3.17E−05

Il1rn
44.9
1.38
1.79
1.05E−05
7.06E−05

Fkbp1b
22.4
1.38
1.9
3.16E−05
0.00019

Nwd1
24.3
1.38
2.01
7.94E−05
0.000431

Tnp2
17.6
1.38
2.34
0.000128
0.000654

Ifit3
421.9
1.38
2.21
0.000147
0.00074

Hist1h2ai
9.8
1.38
2.17
0.000184
0.000901

Cdc20b
14.4
1.38
2.55
0.00019
0.000927

a
12.6
1.38
2.36
0.000204
0.000988

Tubb5
27245.9
1.37
1.4
0
0

Myo1f
6082
1.37
1.41
0
0

Cdc25b
5230.3
1.37
1.42
0
0

E2f3
421.5
1.37
1.42
0
0

Impa1
900.5
1.37
1.42
0
0

Kif24
76.5
1.37
1.56
2.19E−08
2.49E−07

Art2a-ps
407.6
1.37
1.56
4.59E−08
4.94E−07

Gp5
24.7
1.37
1.84
5.7E−05
0.000321

Dio2
18.9
1.37
2.13
0.000105
0.000551

Cdk6
2038
1.36
1.38
0
0

Ppil1
932
1.36
1.4
0
0

Shmt1
546.5
1.36
1.41
0
0

Plxdc1
231.9
1.36
1.46
6.89E−12
1.42E−10

Hopx
270.1
1.36
1.48
2.27E−11
4.33E−10

Serp2
160.9
1.36
1.5
5.14E−10
7.76E−09

Dusp14
437.4
1.36
1.5
1.35E−09
1.89E−08

Lgals7
154
1.36
1.59
1.04E−06
8.72E−06

Nqo1
35.8
1.36
1.7
5.6E−06
4.01E−05

Aldh1a1
40.1
1.36
1.98
0.000105
0.000552

Ifit1
298.3
1.36
2.02
0.000125
0.000642

Psat1
1715.9
1.35
1.37
0
0

Gins3
181.3
1.35
1.46
6.05E−11
1.07E−09

Slc16a3
161.5
1.35
1.49
3.5E−10
5.45E−09

Gbp10
432.2
1.35
1.51
4.13E−09
5.34E−08

Ube2l6
515.6
1.35
1.54
4.68E−08
5.03E−07

Slfn3
67.7
1.35
1.6
6.85E−07
5.93E−06

Nccrp1
65
1.35
1.64
4.48E−06
3.27E−05

Rgs8
18.7
1.35
1.85
9.21E−05
0.000492

Trim58
10.1
1.35
2.9
0.000362
0.001642

Cpa3
467.2
1.35
2.45
0.000376
0.001695

Fkbp5
2388.9
1.34
1.37
0
0

Tmpo
4623.5
1.34
1.39
0
0

Cxcr3
2032
1.34
1.39
0
0

Mrps25
464.1
1.34
1.41
0
7E−15

Zdhhc2
533.9
1.34
1.42
3E−15
8.8E−14

Slc25a13
161.1
1.34
1.42
1.21E−13
3.1E−12

Nudcd1
238.9
1.34
1.43
1.69E−13
4.24E−12

Phf11b
840.6
1.34
1.45
4.58E−12
9.71E−11

Galr3
5.7
1.34
4.53
0.000359
0.001631

Kctd17
526.7
1.33
1.39
0
0

Klre1
117.7
1.33
1.66
8.94E−06
6.13E−05

Gm14130
18.9
1.33
1.81
4.17E−05
0.000244

Myo1d
68
1.33
1.84
7.41E−05
0.000405

Ica1l
24.1
1.33
1.82
0.000114
0.00059

Coa6
201.6
1.32
1.4
5.2E−14
1.39E−12

Mettl7a1
240.2
1.32
1.42
4.43E−12
9.41E−11

Sytl3
850.3
1.32
1.44
5.64E−10
8.46E−09

Lrrc75a
59.6
1.32
1.6
5.23E−06
3.78E−05

Hspa1b
148.2
1.32
1.66
1.9E−05
0.000121

Serpina3i
24.8
1.32
2.37
0.000411
0.00183

Mmp8
16.8
1.32
2.57
0.000494
0.002149

Mcm2
4604.3
1.31
1.34
0
0

Wdr62
861.5
1.31
1.35
0
0

Cdk5r1
454.4
1.31
1.35
0
0

Cenpu
141.6
1.31
1.39
1.83E−13
4.57E−12

Myl6b
55.6
1.31
1.55
2.08E−06
1.64E−05

Nmur1
2.7
1.31
4.59
9.87E−05
0.000522

Prim2
843.4
1.3
1.34
0
0

Rrm1
4168.2
1.3
1.34
0
0

Dnajc15
778.1
1.3
1.35
0
0

Brip1os
583.1
1.3
1.37
1E−15
3.8E−14

Perp
113.8
1.3
1.41
4.33E−10
6.63E−09

Msantd3
9.2
1.3
2.05
0.000408
0.00182

Col6a6
6.3
1.3
4.16
0.000432
0.001913

Gnaz
9.7
1.3
2.35
0.000531
0.002295

Syt11
1025
1.29
1.31
0
0

Rab27a
1564.8
1.29
1.32
0
0

Stip1
4349.6
1.29
1.32
0
0

Cep19
280.3
1.29
1.34
0
0

Selenoh
1560.4
1.29
1.34
0
1E−15

Clec1b
45.4
1.29
1.59
1.54E−05
0.0001

Il5
3.6
1.29
5.79
0.000125
0.000643

Klrb1a
36.2
1.29
1.9
0.000175
0.000861

Serpina3n
16.7
1.29
2.04
0.000428
0.001901

Ms4a2
35.1
1.29
2.29
0.000563
0.002414

Tmem171
12.4
1.29
2.26
0.000567
0.002428

Snai3
164.9
1.28
1.39
2.53E−10
4.06E−09

CT030173.1
47.8
1.28
1.54
5.74E−06
4.11E−05

Iqcg
28.8
1.28
1.6
1.76E−05
0.000113

Mst1r
38.6
1.28
1.61
2.34E−05
0.000146

Serpine2
31.1
1.28
1.66
0.000104
0.000547

Pawr
19.1
1.28
1.82
0.00015
0.000752

D630045J12Rik
11.4
1.28
2.12
0.000411
0.001831

Oasl2
98.6
1.28
3
0.000737
0.003051

Rab19
1074.2
1.27
1.31
0
0

Rap2a
566
1.27
1.31
0
0

Tm6sf1
1262.4
1.27
1.32
0
0

Caskin2
253.4
1.27
1.33
6E−15
1.65E−13

Gm7901
153.5
1.27
1.38
7.96E−10
1.16E−08

Ly6c1
2390.4
1.27
1.41
2.71E−08
3.04E−07

Pxmp2
56.9
1.27
1.44
4.38E−07
3.98E−06

Il3
4
1.27
5.13
0.000123
0.000633

C1ql1
3.8
1.27
5.38
0.000393
0.001762

Car1
559.7
1.27
1.97
0.000423
0.001879

Sla
9431
1.26
1.29
0
0

Runx2
2056.2
1.26
1.3
0
0

Impa2
575
1.26
1.3
0
0

Atp2b4
1723.8
1.26
1.3
0
0

Cpsf2
1192.5
1.26
1.32
2.6E−14
7.37E−13

Bcl2l1
1866.8
1.26
1.33
3.2E−14
8.76E−13

2810006K23Rik
128.8
1.26
1.41
3.26E−08
3.63E−07

Sostdc1
55.7
1.26
1.49
6.65E−06
4.69E−05

Gm15987
44.4
1.26
1.64
4.28E−05
0.00025

Mest
21.1
1.26
1.73
0.000157
0.000783

Fkbp10
29.4
1.26
1.74
0.000252
0.001191

Hnf4a
4.7
1.26
3.99
0.000517
0.002238

Gp1ba
18.9
1.26
2.02
0.000659
0.002769

AC153369.2
8.4
1.26
2.51
0.000813
0.003321

Prss34
356.8
1.26
2.96
0.000841
0.003422

Tarm1
9.2
1.26
3.21
0.000847
0.003442

Nudt5
931.4
1.25
1.28
0
0

Aurka
633.5
1.25
1.29
0
0

C1qtnf6
340.9
1.25
1.3
0
0

Nrm
1120.2
1.25
1.3
0
0

Kifc5b
272.7
1.25
1.31
1E−15
3.3E−14

H2afx
3402.7
1.25
1.31
2E−15
5.8E−14

Gyg
764.1
1.25
1.31
8E−15
2.44E−13

Lsm2
779.8
1.25
1.34
8.52E−12
1.74E−10

Xaf1
1257.6
1.25
1.36
2.65E−09
3.56E−08

Kndc1
53.5
1.25
1.48
2.11E−06
1.66E−05

Xkr8
51.5
1.25
1.48
3.91E−06
2.91E−05

Gm37004
26
1.25
1.56
3.67E−05
0.000218

Scin
117.4
1.25
1.62
6.9E−05
0.000381

Sgms2
21.4
1.25
1.8
0.00038
0.00171

Vcan
11.1
1.25
2.23
0.000894
0.003604

Lrp3
7.7
1.25
2.52
0.000956
0.003818

Nde1
1235.2
1.24
1.26
0
0

Hn1l
449.3
1.24
1.3
6E−15
1.74E−13

Lamc1
923.9
1.24
1.31
1.71E−13
4.3E−12

Alg6
94.2
1.24
1.39
1.32E−07
1.31E−06

AC126459.2
179
1.24
1.4
3.61E−07
3.32E−06

Dsp
79.2
1.24
1.55
4.45E−05
0.000258

Gm14148
21.4
1.24
1.54
5.86E−05
0.00033

AC151730.1
71.6
1.24
1.69
8.59E−05
0.000463

Gm13461
16.5
1.24
1.82
0.000465
0.002039

Fhl1
19.9
1.24
1.74
0.000492
0.002143

Smpx
6.2
1.24
4.22
0.000628
0.002658

Tom1l1
20.3
1.24
1.95
0.00073
0.003028

Hist1h2ab
3.6
1.24
3.05
0.000791
0.003245

Il33
4.5
1.24
3.09
0.000871
0.003531

Epha3
8.5
1.24
2.62
0.000892
0.003598

Hist1h2ae
10.9
1.24
2.06
0.000918
0.003688

Gm13031
8
1.24
2.51
0.000941
0.003764

Gm14569
11.7
1.24
3.38
0.000999
0.003964

Slamf7
804.9
1.23
1.27
0
0

Cxcr6
818
1.23
1.27
0
0

Cpox
574.5
1.23
1.28
0
8E−15

Adam19
5999.2
1.23
1.29
2E−15
5.2E−14

Ms4a4c
692.6
1.23
1.3
4.5E−14
1.22E−12

Penk
1018
1.23
1.3
2.51E−12
5.5E−11

Klrb1f
190
1.23
1.32
3.65E−10
5.66E−09

Gm6166
141.1
1.23
1.32
1.18E−09
1.67E−08

Fam184a
33.5
1.23
1.5
1.8E−05
0.000116

Meis1
68.8
1.23
1.55
8.54E−05
0.00046

Erg
37.2
1.23
1.55
0.000101
0.000531

Enpp6
21.8
1.23
1.82
0.00032
0.001474

Gm6982
3.3
1.23
4.32
0.000669
0.002804

Gm7456
10.4
1.23
2.12
0.000809
0.003311

Spon1
28.9
1.23
2.16
0.000894
0.003604

Unc80
11
1.23
2.08
0.001129
0.004421

Ran
2657.7
1.22
1.24
0
0

Zranb3
259.4
1.22
1.27
0
0

Cbx5
1657.3
1.22
1.27
0
1E−15

Ms4a6d
734.6
1.22
1.28
3E−15
1.07E−13

Fbxo5
492.7
1.22
1.28
1.3E−14
3.61E−13

Hsph1
3580.6
1.22
1.28
4.8E−14
1.29E−12

Pttg1
1600.8
1.22
1.28
1.21E−13
3.11E−12

Cysltr2
169.1
1.22
1.37
3.3E−07
3.06E−06

Phf11a
221.4
1.22
1.4
9.86E−07
8.28E−06

Rgs18
52.3
1.22
1.54
9.22E−05
0.000492

Wdr54
19.6
1.22
1.61
0.000186
0.000909

Clstn3
34.6
1.22
1.6
0.000219
0.001054

Aqp1
400.8
1.22
1.93
0.00081
0.003312

Il20ra
9.2
1.22
1.97
0.001035
0.00409

Hist1h2bk
8.4
1.22
2.66
0.001176
0.004581

Chaf1b
1135
1.21
1.23
0
0

Rnaseh2b
648.9
1.21
1.24
0
0

Tnfrsf9
1547.1
1.21
1.29
1.28E−10
2.17E−09

Smoc2
90.6
1.21
1.34
6.72E−08
7.07E−07

Fancf
96.5
1.21
1.34
9.78E−08
1E−06

Tex13c2
4.1
1.21
3.28
0.000884
0.003573

Mcpt1
510.2
1.21
3.19
0.001329
0.005105

Chit1
6.9
1.21
2.51
0.00139
0.005311

Psma1
2415.5
1.2
1.22
0
0

Zcchc18
377.7
1.2
1.23
0
0

Ppa1
1390.3
1.2
1.25
3E−15
9.8E−14

Alms1
195.5
1.2
1.33
1.17E−07
1.18E−06

Ms4a4a
77.2
1.2
1.36
2.06E−07
1.97E−06

Farp1
144.7
1.2
1.34
4.58E−07
4.13E−06

Lrrc49
50.8
1.2
1.38
1.78E−06
1.42E−05

Tfec
71.7
1.2
1.43
1.18E−05
7.88E−05

Gm15232
43.8
1.2
1.46
1.89E−05
0.00012

Fgd1
24
1.2
1.52
0.000114
0.00059

C78197
19.1
1.2
1.74
0.000569
0.002437

Slc18a2
46.4
1.2
1.9
0.000856
0.003474

Clgn
5.1
1.2
3.51
0.001392
0.005316

Calm3
5962.7
1.19
1.21
0
0

Dtl
1077.7
1.19
1.26
4.67E−11
8.43E−10

Ptpn13
1107.8
1.19
1.3
6.78E−09
8.4E−08

Mrpl34
516.5
1.19
1.33
2.57E−07
2.41E−06

Nphp4
99.7
1.19
1.33
3.18E−07
2.95E−06

Trim46
87.2
1.19
1.34
6.83E−07
5.91E−06

Fn1
453.7
1.19
1.54
0.000169
0.000836

I830127L07Rik
6.6
1.19
5.81
0.00103
0.004071

Reps2
11
1.19
2.16
0.001383
0.005292

Mcm7
3981.2
1.18
1.21
0
0

Trim37
677.9
1.18
1.21
0
0

BC055324
200.3
1.18
1.22
0
7E−15

Gm4737
198.1
1.18
1.25
3.91E−11
7.17E−10

Pycard
1522.9
1.18
1.25
8.74E−11
1.51E−09

Fam185a
192.8
1.18
1.28
2.12E−09
2.88E−08

Bag2
63
1.18
1.31
4.71E−07
4.24E−06

Ifitm1
214.1
1.18
1.44
6.52E−05
0.000362

Alox12
39.7
1.18
1.46
0.00013
0.000665

Cebpe
5.7
1.18
3.45
0.001328
0.005104

Ppp2r2c
6.5
1.18
4.48
0.00155
0.005841

Mcpt2
69.1
1.18
3.32
0.001613
0.006049

Fam46c
5669.2
1.17
1.2
0
0

Plscr1
472.1
1.17
1.22
7.7E−14
2.01E−12

Grb7
380.9
1.17
1.3
7.86E−07
6.72E−06

Phlda3
37.7
1.17
1.38
2.49E−05
0.000154

Rtkn
29.5
1.17
1.44
0.000126
0.000646

Cym
46.2
1.17
1.51
0.000165
0.000821

Cfap77
26
1.17
1.51
0.000167
0.000826

Gm10286
2.8
1.17
4.98
0.000868
0.00352

AC158990.2
7.6
1.17
2.88
0.001943
0.007104

Entpd1
1241.1
1.16
1.2
0
0

Dbi
1291.8
1.16
1.21
2.3E−14
6.44E−13

Hmga1-rs1
312.3
1.16
1.21
1.05E−12
2.43E−11

Tmem237
165.9
1.16
1.22
1.29E−12
2.96E−11

Raph1
355.4
1.16
1.22
5.71E−12
1.19E−10

Echdc1
172.2
1.16
1.23
3.63E−11
6.71E−10

Tuba1b
1989.9
1.16
1.26
1.8E−08
2.09E−07

Ifi214
383.7
1.16
1.31
9.86E−07
8.28E−06

Pvrig
126.3
1.16
1.31
1.56E−06
1.26E−05

Steap3
134.6
1.16
1.4
5.47E−05
0.00031

Fam89a
34.7
1.16
1.45
5.54E−05
0.000313

Gm8719
29.4
1.16
1.47
0.000149
0.000748

Epas1
1633
1.16
1.5
0.000353
0.001608

Adamts3
19.6
1.16
1.69
0.000885
0.003576

Opn3
13.4
1.16
1.74
0.000965
0.003851

Syngr4
9.5
1.16
2.04
0.001733
0.006439

Tmem98
10.9
1.16
1.94
0.002001
0.007282

Rfc5
1166.1
1.15
1.17
0
0

Pdk3
769.1
1.15
1.17
0
0

Samd3
769
1.15
1.2
5E−15
1.56E−13

Psmb9
7187.6
1.15
1.22
2.5E−11
4.73E−10

Slc9a5
265.3
1.15
1.22
7.94E−11
1.38E−09

Fbxl8
274.2
1.15
1.23
6.27E−10
9.33E−09

Myh10
131.2
1.15
1.37
3.82E−05
0.000226

Ighv1-81
14
1.15
3.56
0.00155
0.005841

Palm3
10.2
1.15
1.86
0.001827
0.006731

Ifit3b
107.9
1.15
2.04
0.002247
0.008038

Pla2g16
2185.4
1.14
1.15
0
0

Ptprcap
9906.9
1.14
1.18
0
1E−15

Zbp1
5544.8
1.14
1.22
1.03E−09
1.47E−08

Eif2ak2
397.5
1.14
1.23
5.43E−09
6.87E−08

Ifi209
837.9
1.14
1.24
2.48E−08
2.79E−07

Prkar2b
166.3
1.14
1.28
1.79E−06
1.43E−05

Gm5620
33.2
1.14
1.38
0.000109
0.00057

Tspo2
25.6
1.14
2.05
0.002092
0.007567

Gm12641
4.5
1.14
2.63
0.002216
0.007947

Gm5391
5.3
1.14
2.44
0.002442
0.008648

Chga
5.7
1.14
2.51
0.002652
0.00928

Bhlhe40
29564.9
1.13
1.15
0
0

Scd2
2011
1.13
1.16
0
0

Arl6ip1
6248
1.13
1.17
0
1E−15

Pycrl
578
1.13
1.17
1E−15
1.8E−14

Erg28
683.6
1.13
1.18
3E−15
8.3E−14

Pgk1
407
1.13
1.18
3.15E−13
7.63E−12

Rom1
500.9
1.13
1.2
4.67E−11
8.43E−10

Eef1akmt1
350.7
1.13
1.22
1.39E−08
1.64E−07

Cenpv
293.7
1.13
1.23
6.57E−08
6.93E−07

Xk
84.8
1.13
1.25
2.68E−07
2.51E−06

Clec7a
192.7
1.13
1.26
6.18E−07
5.4E−06

Nudt1
390.3
1.13
1.26
6.42E−07
5.59E−06

Ltbp1
63.3
1.13
1.33
3.43E−05
0.000205

9430037O13Rik
48.3
1.13
1.37
0.000118
0.000609

Dgkg
28.8
1.13
1.49
0.000398
0.001781

Bex4
6.5
1.13
2.52
0.002771
0.009627

Prf1
1704.5
1.12
1.14
0
0

Dpagt1
515.2
1.12
1.18
7.25E−12
1.49E−10

Enkd1
215
1.12
1.2
5.04E−09
6.42E−08

Klra3
69.3
1.12
1.33
3.42E−05
0.000204

G0s2
77.4
1.12
1.38
0.000194
0.000946

4930520O04Rik
19.9
1.12
1.53
0.00072
0.002993

Gm24507
10.1
1.12
1.9
0.002546
0.008969

Irx3
8.6
1.12
1.79
0.002652
0.00928

Zfp575
7.4
1.12
2.11
0.002982
0.010267

4930427A07Rik
1017.7
1.11
1.13
0
0

Fen1
1518.2
1.11
1.13
0
0

Gng2
1586.5
1.11
1.15
0
0

Polq
483.2
1.11
1.17
4.68E−11
8.45E−10

Aplf
178.2
1.11
1.19
2.84E−09
3.78E−08

Optn
199.7
1.11
1.22
3.7E−07
3.4E−06

Dhx58
1168.4
1.11
1.24
8.05E−07
6.86E−06

Nudt7
57.3
1.11
1.34
6.92E−05
0.000382

Prr18
25.2
1.11
1.42
0.000467
0.002048

Fam109b
49.4
1.11
1.49
0.000642
0.002708

Thbs1
150.8
1.11
1.48
0.00069
0.002882

2210011C24Rik
23
1.11
1.46
0.000846
0.003439

Rtp4
1189.3
1.11
1.5
0.000884
0.003572

Myom2
3.3
1.11
2.84
0.002323
0.00828

Ncr1
264.6
1.11
1.76
0.002392
0.008499

Adgrl4
27.6
1.11
1.74
0.002603
0.009132

Gm15941
4.3
1.11
2.54
0.003237
0.011024

Hoxa9
9
1.11
2.09
0.003272
0.011122

Myrf
11.2
1.11
2.56
0.00334
0.011303

Endod1
867.3
1.1
1.12
0
0

Ruvbl2
1746.2
1.1
1.14
0
2E−15

Mrps6
469.4
1.1
1.16
1.21E−11
2.4E−10

Naa38
642.5
1.1
1.18
1.39E−08
1.64E−07

Gcat
256.4
1.1
1.23
2.8E−06
2.16E−05

Tmem163
137
1.1
1.32
0.000126
0.000646

Ccdc80
29.4
1.1
1.48
0.000747
0.003085

Epdr1
29.9
1.1
1.56
0.001471
0.005584

Naaladl1
17.4
1.1
1.55
0.001554
0.005857

Gm5541
13.5
1.1
1.97
0.003169
0.010832

Cfb
9.7
1.1
2.3
0.003841
0.012715

Tipin
1045.9
1.09
1.13
1E−15
4.3E−14

Hikeshi
408
1.09
1.15
6.66E−12
1.38E−10

Epsti1
2970.1
1.09
1.14
2.87E−11
5.39E−10

Mfsd2b
366.8
1.09
1.19
1.24E−07
1.24E−06

Zan
929.3
1.09
1.19
2.42E−07
2.29E−06

1190007I07Rik
77.8
1.09
1.22
2.98E−06
2.28E−05

Kcnip3
48.9
1.09
1.29
3.51E−05
0.000209

Ryr1
67.2
1.09
1.33
0.000206
0.000995

Rsph1
29
1.09
1.35
0.000451
0.001985

Slfn4
78.7
1.09
1.51
0.000918
0.003688

Gm42517
3
1.09
4.44
0.00121
0.004704

Gm31597
30.9
1.09
1.54
0.001558
0.005869

Col8a2
11.9
1.09
2.08
0.003591
0.012023

Gm5787
4.5
1.09
2.84
0.003715
0.012368

Glp1r
11.2
1.09
2.1
0.004071
0.013338

Taf6
1182.3
1.08
1.1
0
0

Plp2
432.8
1.08
1.12
1E−15
2.3E−14

Atcay
258.4
1.08
1.12
3E−15
1.06E−13

Casp4
449.3
1.08
1.13
8.76E−12
1.79E−10

Sytl2
1359.2
1.08
1.14
6.48E−11
1.14E−09

Txn1
4856.9
1.08
1.14
1.86E−09
2.55E−08

Banf1
2759.2
1.08
1.17
1.7E−07
1.65E−06

Tubd1
123.6
1.08
1.19
5.64E−07
4.98E−06

Apol10b
57.6
1.08
1.37
0.000331
0.001516

9130604C24Rik
12.8
1.08
1.68
0.002537
0.008944

AC133083.4
8.9
1.08
2.13
0.003676
0.012261

Mcpt8
355.9
1.08
2.25
0.004527
0.01461

Bub3
2635.7
1.07
1.09
0
0

Sept11
2112.3
1.07
1.11
5.1E−14
1.36E−12

5830432E09Rik
221.9
1.07
1.12
1.77E−12
3.97E−11

Tbc1d31
347.8
1.07
1.11
2.85E−12
6.2E−11

Sema7a
543.5
1.07
1.12
7.39E−12
1.52E−10

PgP
482.7
1.07
1.12
9.9E−11
1.69E−09

Mrpl51
433.6
1.07
1.13
1.05E−09
1.5E−08

AC123720.1
301.8
1.07
1.14
5.56E−09
7.01E−08

Slc29a4
63.5
1.07
1.17
9.44E−07
7.95E−06

Dctd
147.9
1.07
1.19
2.49E−06
1.93E−05

Csrp2
91.9
1.07
1.19
3.25E−06
2.46E−05

Ptrh1
154.5
1.07
1.2
6.71E−06
4.72E−05

Unc5b
14.9
1.07
1.45
0.001958
0.00715

A430018G15Rik
17
1.07
1.55
0.001969
0.007187

Upk1b
3.1
1.07
3.96
0.003029
0.010405

Gm11223
13
1.07
1.72
0.003491
0.011736

Scrn1
9.5
1.07
1.96
0.004421
0.014317

G6b
5.8
1.07
2.42
0.004604
0.014825

Rangap1
5185.4
1.06
1.07
0
0

Cd8b1
8616.8
1.06
1.08
0
0

Mms22l
883
1.06
1.08
0
0

Cchcr1
724.1
1.06
1.09
0
0

Rnpep
2406
1.06
1.1
0
1E−15

Cntln
217.1
1.06
1.09
0
7E−15

Clic1
10203.8
1.06
1.09
1E−15
2.1E−14

Pkmyt1
1872.8
1.06
1.1
6E−15
1.73E−13

Phgdh
591.6
1.06
1.1
6.8E−14
1.79E−12

Lcp1
29534.7
1.06
1.1
9.5E−14
2.46E−12

Trbv3
327
1.06
1.1
1.27E−13
3.23E−12

Gm13394
2531.6
1.06
1.11
1.77E−11
3.42E−10

Gm5855
163.2
1.06
1.12
3.97E−10
6.11E−09

Rad51c
174.3
1.06
1.13
6.37E−09
7.94E−08

Fgl2
387.2
1.06
1.15
4.93E−07
4.42E−06

Fam69b
58.7
1.06
1.17
1.34E−06
1.1E−05

Ddah2
90
1.06
1.21
3.01E−05
0.000182

Mid2
43.1
1.06
1.37
0.000679
0.002842

C430042M11Rik
20.4
1.06
1.44
0.001415
0.00539

1700006J14Rik
34.9
1.06
1.52
0.002226
0.007977

Kifc5c-ps
3.2
1.06
5.01
0.002273
0.008113

Gm4316
14.1
1.06
1.62
0.002956
0.010189

Ighv1-77
5.6
1.06
2.91
0.004291
0.013953

Syngr3
14.2
1.06
1.89
0.004613
0.014847

Garem1
10.4
1.06
2.16
0.004725
0.015161

Gm5732
8.2
1.06
2.1
0.004985
0.01591

9230110C19Rik
6.9
1.06
2.22
0.005156
0.016393

Cdk4
5123.3
1.05
1.07
0
0

Psmb8
13142.6
1.05
1.09
5.3E−14
1.42E−12

Cdc25a
933.6
1.05
1.09
5.4E−14
1.43E−12

Pmf1
1789.9
1.05
1.09
1.87E−12
4.17E−11

Galm
344.1
1.05
1.1
1.09E−11
2.19E−10

Tpgs1
515.9
1.05
1.11
2.78E−09
3.71E−08

2610524H06Rik
134.2
1.05
1.12
3.58E−08
3.95E−07

St14
207.6
1.05
1.16
2.31E−06
1.8E−05

Ifi206
467.7
1.05
1.18
6.08E−06
4.32E−05

F2rl3
91.5
1.05
1.18
1.08E−05
7.26E−05

Ctnnbip1
164
1.05
1.18
1.81E−05
0.000116

Ccl12
4.4
1.05
3.63
0.003485
0.011718

Slc17a6
4.8
1.05
3.66
0.003716
0.012368

Gm867
11.8
1.05
1.74
0.00416
0.013584

Srd5a1
9.7
1.05
1.76
0.004432
0.014342

Bad
457.8
1.04
1.08
1.55E−11
3.03E−10

Pafah2
209.2
1.04
1.11
2.07E−08
2.37E−07

Dzip3
213.7
1.04
1.12
4.28E−08
4.63E−07

Casp1
695.8
1.04
1.13
1.06E−07
1.08E−06

Nrgn
587.4
1.04
1.2
5.55E−05
0.000314

Gm28942
25.8
1.04
1.35
0.000795
0.003259

Gm13361
26.6
1.04
1.39
0.00157
0.005905

AC151730.3
14.5
1.04
1.64
0.003469
0.011674

Cyp4f39
4
1.04
3.04
0.003903
0.012858

Ranbp17
12.9
1.04
1.93
0.005338
0.01689

Gm6419
7.9
1.04
2.1
0.006041
0.0188

Unc119b
2622.3
1.03
1.05
0
0

Ak3
1405.9
1.03
1.05
0
0

Brca2
378
1.03
1.06
0
0

Prelid2
433.7
1.03
1.06
0
1E−15

Hmbs
853.4
1.03
1.07
2.67E−12
5.82E−11

Cobll1
564
1.03
1.07
1.31E−11
2.6E−10

Ttf2
773.8
1.03
1.08
1.77E−10
2.94E−09

Ap1s2
357.9
1.03
1.09
6.21E−10
9.25E−09

Arhgap33
461.4
1.03
1.1
1.1E−07
1.11E−06

Cxcr5
1400.5
1.03
1.11
1.62E−07
1.58E−06

Zfp324
80.8
1.03
1.16
1.23E−05
8.15E−05

1700008J07Rik
46
1.03
1.2
8.13E−05
0.00044

Pla2g1b
3.1
1.03
5.68
0.000671
0.002812

Rasd2
26.4
1.03
1.31
0.001055
0.004161

Gm19412
24.7
1.03
1.45
0.002222
0.007968

Gm21850
5.8
1.03
5.15
0.003849
0.012735

Ninj2
22.5
1.03
1.57
0.003881
0.012815

Cacna1b
12.9
1.03
1.59
0.003981
0.013088

Hspd1
2211.8
1.02
1.03
0
0

Gins4
994.2
1.02
1.03
0
0

Hirip3
1284.6
1.02
1.04
0
1E−15

Dtx3l
2072.2
1.02
1.05
1E−15
3.7E−14

Atad5
649
1.02
1.05
4E−15
1.19E−13

Slc22a15
789
1.02
1.05
2.1E−13
5.2E−12

Nefh
428.6
1.02
1.08
2.24E−09
3.03E−08

Gemin8
131.1
1.02
1.14
1.99E−05
0.000126

Coa3
751.4
1.02
1.17
3.92E−05
0.000231

AL591582.1
67.5
1.02
1.2
0.000137
0.000694

Rabl2
66.4
1.02
1.21
0.000142
0.000719

BC147527
314.7
1.02
1.21
0.000232
0.001111

F5
26.3
1.02
1.32
0.001751
0.006497

Cysltr1
12.2
1.02
1.53
0.003789
0.012571

Ankrd29
17.2
1.02
1.59
0.003942
0.01298

Serpinb1b
5.6
1.02
3.25
0.00527
0.016699

Cxcl11
12.9
1.02
1.71
0.005596
0.017603

A730089K16Rik
8.9
1.02
3.51
0.006351
0.019658

Gm12669
5.9
1.02
2.72
0.006491
0.020036

Draxin
7.5
1.02
2.51
0.006903
0.021116

Lrrn4
10.2
1.02
2.39
0.00739
0.022374

Fbxw8
1446.7
1.01
1.03
0
0

Mis18a
835
1.01
1.03
0
0

Adprh
1898
1.01
1.03
0
1E−15

Gmds
345.1
1.01
1.05
2.03E−12
4.49E−11

Hat1
1129
1.01
1.06
7.79E−10
1.14E−08

Psmb10
4675.3
1.01
1.07
1.49E−09
2.07E−08

F730043M19Rik
151.6
1.01
1.1
5.92E−07
5.2E−06

Map1a
43.3
1.01
1.14
5.25E−05
0.000299

Gata1
221
1.01
1.18
0.000138
0.0007

4930524J08Rik
30.3
1.01
1.2
0.00043
0.001909

Ly6c2
4431.1
1.01
1.23
0.000513
0.002224

Gm30211
96.4
1.01
1.34
0.001432
0.005448

Slc30a2
29
1.01
1.33
0.002911
0.010056

Lpar3
17.1
1.01
1.53
0.003294
0.011174

Gm35037
11.6
1.01
1.65
0.005347
0.016913

Pcdh7
8.7
1.01
1.64
0.006542
0.020151

Ntf5
8.2
1.01
1.67
0.006819
0.020886

Olfr414
5.8
1.01
3.46
0.007252
0.022

AC153562.2
4.6
1.01
2.14
0.007428
0.022477

Cd248
7.6
1.01
2.32
0.007647
0.023068

Ccdc120
9.6
1.01
1.83
0.007745
0.023309

Ahsa1
2898.5
1
1.02
0
0

Cdca4
1330.7
1
1.03
0
0

Ebp
1270.8
1
1.03
8E−15
2.22E−13

Thop1
736
1
1.03
6.1E−14
1.6E−12

Jdp2
335.6
1
1.03
2.76E−13
6.73E−12

Rpa3
304.5
1
1.04
3E−10
4.76E−09

Ece2
225.8
1
1.06
1.04E−08
1.25E−07

Cttn
156.2
1
1.07
2.39E−08
2.7E−07

Ydjc
187.1
1
1.07
1.09E−07
1.1E−06

5033430I15Rik
174.1
1
1.07
4.67E−07
4.21E−06

Orc1
394.8
1
1.09
8.9E−07
7.52E−06

Cx3cr1
141.8
1
1.09
3.2E−06
2.43E−05

Ltbp3
64
1
1.12
2.83E−05
0.000173

Zfp239
60.8
1
1.15
5.9E−05
0.000332

Mansc1
76.8
1
1.17
0.000128
0.000654

Bahcc1
65.2
1
1.17
0.000156
0.00078

Rpl39-ps
163.8
1
1.2
0.000445
0.001964

Ccl2
77.5
1
1.39
0.002955
0.010188

Col5a1
37.5
1
1.48
0.004515
0.014573

Wisp1
171.4
1
1.46
0.004818
0.015407

Hist1h2bl
3.2
1
2.02
0.00672
0.020634

Gm11658
7.3
1
1.65
0.007621
0.023001

Gm44101
5.1
1
2.58
0.007779
0.02339

Atp6v1g3
12.9
1
1.89
0.007957
0.023852

Bbs2
593.4
−1
−1.02
0
0

Cdc42bpb
487.3
−1
−1.06
4.36E−09
5.61E−08

Dapk1
459.6
−1
−1.06
1.22E−08
1.45E−07

Lonrf1
133.9
−1
−1.06
2.72E−08
3.04E−07

Vps37b
99356.7
−1
−1.07
1.11E−07
1.12E−06

C130050O18Rik
95.9
−1
−1.14
1.81E−05
0.000116

Thrb
56.4
−1
−1.13
3.27E−05
0.000196

Gm45743
60.6
−1
−1.14
4.4E−05
0.000256

Gm42743
41.9
−1
−1.13
4.43E−05
0.000257

Nectin1
128.6
−1
−1.15
7.02E−05
0.000387

Proscos
49.9
−1
−1.15
0.000156
0.00078

Snx24
100.2
−1
−1.2
0.000273
0.00128

Gm43919
34.9
−1
−1.19
0.000325
0.001493

Fbxl12os
26.5
−1
−1.25
0.001077
0.004236

Robo4
55.1
−1
−1.28
0.001574
0.00592

Fam149a
48.8
−1
−1.32
0.001849
0.006801

Gm17021
42.4
−1
−1.35
0.00255
0.008981

Gm15918
28.4
−1
−1.36
0.003178
0.010856

Dcst2
12.5
−1
−1.43
0.004038
0.013242

Traj23
11.2
−1
−1.45
0.004281
0.013929

Cyp1a1
14.7
−1
−1.53
0.005398
0.017057

Siglecg
751.8
−1
−1.64
0.005784
0.018124

Gm5538
4.9
−1
−3.78
0.006528
0.020119

2310026I22Rik
9.2
−1
−1.66
0.006733
0.020671

Zfp536
9.7
−1
−1.65
0.006926
0.02116

Slc8a2
9.9
−1
−1.82
0.007393
0.022379

C130074G19Rik
7.9
−1
−1.78
0.008117
0.024268

Gm37078
5.3
−1
−2.53
0.008127
0.024294

Enthd1
11.5
−1
−1.83
0.008208
0.024502

Oprl1
7.9
−1
−1.99
0.008234
0.024563

Gm16069
513.8
−1.01
−1.03
0
1.4E−14

Slc12a7
12768.1
−1.01
−1.05
2.7E−14
7.4E−13

Gm1043
603.2
−1.01
−1.06
2.48E−10
4E−09

Gm16638
160.1
−1.01
−1.07
7.79E−10
1.14E−08

Skil
15256
−1.01
−1.08
3.99E−08
4.35E−07

Gm6085
80.3
−1.01
−1.16
0.000113
0.000586

Arhgap20
94.6
−1.01
−1.18
0.000163
0.00081

AC158605.3
38.2
−1.01
−1.27
0.000516
0.002237

She
55.5
−1.01
−1.25
0.000684
0.002858

Abca9
130.3
−1.01
−1.29
0.000993
0.003946

Chrna9
16
−1.01
−1.35
0.002576
0.009052

Cplx1
15.4
−1.01
−1.49
0.004189
0.013661

Gm2694
5.2
−1.01
−3.48
0.006165
0.019142

Fxyd1
9.9
−1.01
−1.64
0.006183
0.01919

Gm31479
6.5
−1.01
−2.85
0.007441
0.022507

Clec2h
5.3
−1.01
−2.58
0.007558
0.022832

Hba-a2
368.3
−1.01
−2.17
0.007639
0.023049

G730013B05Rik
7.5
−1.01
−1.88
0.00769
0.02318

A430072C10Rik
4.4
−1.01
−2.03
0.007723
0.023258

Slc6a19os
5.5
−1.01
−1.86
0.008023
0.024027

Sema3b
11.3
−1.01
−1.97
0.008087
0.024202

Ldhal6b
9.7
−1.01
−1.95
0.008111
0.024255

Dip2c
434.7
−1.02
−1.08
1.85E−09
2.54E−08

Trio
1619.6
−1.02
−1.08
2.11E−08
2.41E−07

Il13ra1
83.3
−1.02
−1.13
3.14E−06
2.39E−05

Gm45059
77.9
−1.02
−1.12
5.67E−06
4.06E−05

Lrrc25
483.1
−1.02
−1.15
9.03E−06
6.18E−05

Ptpro
88.1
−1.02
−1.16
1.63E−05
0.000106

Susd4
89.1
−1.02
−1.16
2.64E−05
0.000162

AC152827.1
55.1
−1.02
−1.19
0.000137
0.000695

Epb41l1
134.6
−1.02
−1.21
0.000176
0.000867

Cd177
45.2
−1.02
−1.33
0.001186
0.004619

AC104880.1
22.4
−1.02
−1.36
0.00212
0.007654

Gm16060
18
−1.02
−1.37
0.002489
0.008799

Pmepa1os
12.1
−1.02
−1.46
0.003899
0.012852

Hoga1
13.2
−1.02
−1.61
0.004419
0.014313

Gm37109
3.1
−1.02
−2.45
0.005212
0.016548

Plxna2
19.9
−1.02
−1.61
0.005262
0.016685

A430027C01Rik
13.4
−1.02
−1.85
0.006086
0.018924

Gm21984
9.4
−1.02
−1.85
0.006525
0.020112

Gm44860
11.3
−1.02
−2.21
0.006672
0.020505

Gramd2
8.5
−1.02
−2.62
0.006793
0.020823

Mcf2
7.3
−1.02
−3.16
0.006935
0.02118

Ablim3
13.3
−1.02
−2.32
0.006988
0.021312

4922502D21Rik
7.8
−1.02
−1.93
0.007227
0.021936

Cyp4f13
1429.7
−1.03
−1.04
0
0

Gm37494
567.7
−1.03
−1.07
1.06E−11
2.12E−10

Maff
11634.4
−1.03
−1.08
1.68E−11
3.25E−10

Tdrp
4170.6
−1.03
−1.08
1.84E−10
3.04E−09

Dennd2c
139.5
−1.03
−1.1
2.13E−08
2.43E−07

Pla2g7
1374.2
−1.03
−1.11
9.79E−08
1E−06

Apoe
8629.4
−1.03
−1.11
1.62E−07
1.58E−06

AC140264.2
106
−1.03
−1.13
1.08E−06
8.95E−06

Alpk1
263.7
−1.03
−1.12
1.08E−06
8.96E−06

Jmy
4073.6
−1.03
−1.13
2.17E−06
1.7E−05

Fdx1l
80.3
−1.03
−1.13
2.58E−06
1.99E−05

Pnck
68.8
−1.03
−1.15
9.22E−06
6.3E−05

Ttc12
74.5
−1.03
−1.25
0.000243
0.001157

Gm12966
32.5
−1.03
−1.23
0.000274
0.001283

AC117769.3
40.9
−1.03
−1.27
0.000618
0.002621

Foxq1
65.3
−1.03
−1.28
0.0007
0.002921

Gm12000
21.3
−1.03
−1.42
0.002794
0.009693

Mir7046
10.4
−1.03
−1.64
0.005264
0.016687

Gm31532
8.9
−1.03
−1.73
0.006325
0.019582

L3mbtl1
7.3
−1.03
−2.07
0.006502
0.020058

Bnip3l-ps
6.6
−1.03
−1.96
0.006735
0.020672

Sfmbt2
429.2
−1.04
−1.09
2.21E−11
4.22E−10

Cd86
1049.2
−1.04
−1.09
3.35E−10
5.26E−09

Cdc14b
1062
−1.04
−1.1
2.04E−09
2.79E−08

Gm42595
333.4
−1.04
−1.1
2.24E−09
3.02E−08

Tlr13
195.6
−1.04
−1.13
5.29E−08
5.62E−07

Egf
186.8
−1.04
−1.12
1.95E−07
1.87E−06

Fgfr1
240.7
−1.04
−1.13
6.63E−07
5.75E−06

Cap2
83.3
−1.04
−1.14
8.33E−07
7.09E−06

C1qb
6167.6
−1.04
−1.15
4.45E−06
3.26E−05

Fam167a
155
−1.04
−1.16
5.75E−06
4.11E−05

Cspg5
62.6
−1.04
−1.17
2.27E−05
0.000142

Tnnt2
65.1
−1.04
−1.2
2.53E−05
0.000156

Cxcl16
314.6
−1.04
−1.2
5.52E−05
0.000313

A430057M04Rik
48.4
−1.04
−1.27
0.000387
0.001739

Oscp1
44.4
−1.04
−1.29
0.000555
0.002385

H2-Ea-ps
7790.9
−1.04
−1.31
0.000565
0.002422

Il12b
54.2
−1.04
−1.45
0.001388
0.005304

Pde8b
30.7
−1.04
−1.64
0.003238
0.011026

Ptch2
15.2
−1.04
−1.56
0.003758
0.01249

Tmtc1
26.5
−1.04
−1.66
0.004404
0.01428

1810011O10Rik
24.2
−1.04
−1.65
0.004405
0.014281

Gm12167
10.2
−1.04
−1.7
0.005323
0.016848

Gm12089
3.8
−1.04
−2.79
0.005526
0.017413

Gm28035
6
−1.04
−2.57
0.006042
0.0188

C230037L18Rik
243.6
−1.05
−1.1
7.77E−13
1.82E−11

Dyx1c1
106
−1.05
−1.13
3.09E−07
2.87E−06

Hebp1
934.9
−1.05
−1.18
5.55E−06
3.99E−05

Tmem51
428.5
−1.05
−1.17
6E−06
4.27E−05

Naip5
181.7
−1.05
−1.19
1.01E−05
6.87E−05

Cd300c2
673.6
−1.05
−1.2
2.32E−05
0.000144

Card10
54.5
−1.05
−1.19
2.47E−05
0.000153

Coro6
40.8
−1.05
−1.22
8.79E−05
0.000471

Bhlhe41
99.6
−1.05
−1.24
9.66E−05
0.000512

Fhit
63.9
−1.05
−1.24
0.000136
0.000692

Gm18194
29.5
−1.05
−1.28
0.000246
0.001168

Lin7b
27.7
−1.05
−1.28
0.000301
0.001393

Gm6012
40.5
−1.05
−1.29
0.000404
0.001806

Sox5
27.1
−1.05
−1.31
0.000448
0.001973

P2ry2
61.4
−1.05
−1.36
0.000804
0.003293

Gm44214
21.7
−1.05
−1.35
0.000952
0.003805

Blnk
622
−1.05
−1.41
0.001157
0.004518

Acot4
15.6
−1.05
−1.67
0.003129
0.010702

AC160562.1
14.3
−1.05
−1.6
0.003251
0.011059

Myo15
15.2
−1.05
−1.66
0.003332
0.011284

Extl1
17.9
−1.05
−1.78
0.005054
0.0161

Gm15929
8.8
−1.05
−1.92
0.005178
0.016449

Gm43777
8.3
−1.05
−1.79
0.005291
0.016755

Gm2814
9.6
−1.05
−2.68
0.005673
0.017801

Rassf3
6479.1
−1.06
−1.09
0
8E−15

2810021J22Rik
1264.6
−1.06
−1.12
9.26E−11
1.59E−09

Ovgp1
326.4
−1.06
−1.12
1.35E−10
2.27E−09

Mapk12
152.1
−1.06
−1.15
5.43E−08
5.76E−07

AC125071.1
106
−1.06
−1.17
6.39E−07
5.57E−06

Bbs1
97.2
−1.06
−1.17
1.57E−06
1.27E−05

Gm10167
58.6
−1.06
−1.22
2.61E−05
0.00016

Rassf8
83.1
−1.06
−1.23
4.05E−05
0.000238

Mapk15
40.7
−1.06
−1.27
0.000162
0.000807

Ghrl
55
−1.06
−1.33
0.000426
0.001892

Slc4a11
40.7
−1.06
−1.33
0.000485
0.002114

Cybrd1
29.8
−1.06
−1.35
0.000586
0.002499

Gm8251
24.9
−1.06
−1.4
0.000924
0.003706

Tceal1
18.7
−1.06
−1.52
0.002435
0.00863

Pifo
13.3
−1.06
−1.59
0.003076
0.010546

Bicdl2
10.3
−1.06
−1.77
0.003577
0.011987

CT009757.4
3.8
−1.06
−3.2
0.004176
0.013625

Tmem30b
16
−1.06
−1.86
0.004968
0.015862

Gm826
7.6
−1.06
−2.41
0.0051
0.01623

Tmem236
8.3
−1.06
−1.94
0.005165
0.016415

Gm13201
8.2
−1.06
−2.49
0.00522
0.01657

Zbtb20
1429.4
−1.07
−1.1
0
1E−15

Dstyk
697.7
−1.07
−1.1
0
8E−15

1830077J02Rik
399
−1.07
−1.15
9.22E−09
1.11E−07

Stac3
119.4
−1.07
−1.15
1.12E−07
1.13E−06

Xkrx
235.4
−1.07
−1.18
5.59E−07
4.94E−06

Aph1c
92.6
−1.07
−1.18
1.71E−06
1.37E−05

Ly86
762.3
−1.07
−1.21
8.49E−06
5.85E−05

Stab2
1255.1
−1.07
−1.21
8.58E−06
5.91E−05

4921507P07Rik
57.2
−1.07
−1.21
8.97E−06
6.15E−05

Lst1
1097.1
−1.07
−1.21
1.14E−05
7.63E−05

Erbb2
135
−1.07
−1.26
4.73E−05
0.000273

Syde2
248.2
−1.07
−1.24
4.75E−05
0.000274

Hspg2
41.3
−1.07
−1.3
0.00025
0.001187

Cfap74
36.6
−1.07
−1.35
0.000347
0.001581

Rusc2
49.2
−1.07
−1.34
0.000354
0.001609

Cpe
36.2
−1.07
−1.35
0.000616
0.002616

1700061G19Rik
22.8
−1.07
−1.36
0.000645
0.002719

Rab30
116
−1.07
−1.57
0.001884
0.006917

Gm15537
21
−1.07
−1.58
0.002688
0.009387

Dlk1
20.9
−1.07
−1.54
0.002932
0.010115

Akp-ps1
12.6
−1.07
−1.63
0.003213
0.010955

Gm23346
11.3
−1.07
−2.42
0.003589
0.012019

Col2a1
13.8
−1.07
−1.69
0.003605
0.012063

Muc3
12.6
−1.07
−1.82
0.004403
0.014278

Rgsl1
4.5
−1.07
−2.66
0.004427
0.014331

Gm42655
7.7
−1.07
−2.37
0.004556
0.014693

Hbb-bs
1829.7
−1.07
−2.23
0.004758
0.015252

Crip3
8.6
−1.07
−2.83
0.004816
0.015403

4732440D04Rik
113
−1.08
−1.14
2.24E−10
3.65E−09

Mmp15
73.3
−1.08
−1.16
2.28E−08
2.6E−07

Rgl1
1317.4
−1.08
−1.18
1.34E−07
1.33E−06

Gm15706
118.9
−1.08
−1.18
1.66E−07
1.62E−06

Cdk14
85
−1.08
−1.28
5.71E−05
0.000322

Gm18310
30.9
−1.08
−1.37
0.000572
0.002447

4933433G15Rik
17.6
−1.08
−1.52
0.001341
0.005145

Blk
523.7
−1.08
−1.89
0.0037
0.012329

Hrh1
9.5
−1.08
−1.97
0.003762
0.012499

Dusp10
33817.9
−1.09
−1.12
0
1E−15

Gm42829
278.5
−1.09
−1.12
0
1E−15

Klf7
2672.7
−1.09
−1.12
0
1E−15

Gpr157
197.5
−1.09
−1.12
7E−15
2.04E−13

Sez6l2
335
−1.09
−1.13
1.4E−14
3.99E−13

1700109H08Rik
571.4
−1.09
−1.15
4.74E−11
8.5E−10

Arsg
171.4
−1.09
−1.17
4.69E−09
6E−08

Lypd6b
490.3
−1.09
−1.17
1.28E−08
1.51E−07

Gm14168
317.9
−1.09
−1.17
3.99E−08
4.35E−07

Rnd2
50.2
−1.09
−1.21
3.16E−06
2.4E−05

1700003F12Rik
31.2
−1.09
−1.35
0.000266
0.001254

Cd300c
47.9
−1.09
−1.42
0.000366
0.001658

1700028N14Rik
48.1
−1.09
−1.37
0.000451
0.001986

Gm11210
24.9
−1.09
−1.41
0.000578
0.002472

Stac2
76.6
−1.09
−1.44
0.000652
0.002746

Gm13199
23
−1.09
−1.44
0.000847
0.003442

Wfdc3
16.1
−1.09
−1.49
0.00138
0.005282

D630033O11Rik
38.5
−1.09
−1.71
0.002251
0.00805

Pcdhga12
13.8
−1.09
−1.85
0.003166
0.010824

Mcmdc2
8.5
−1.09
−2.47
0.004266
0.013892

AtG
5537.4
−1.1
−1.17
2.92E−10
4.66E−09

Cage1
267.2
−1.1
−1.17
4.95E−09
6.32E−08

Plce1
62.8
−1.1
−1.22
1.85E−06
1.47E−05

Wdfy3
451.6
−1.1
−1.27
1.85E−05
0.000118

Gm13054
37.4
−1.1
−1.34
0.000134
0.000681

Snord72
35.8
−1.1
−1.34
0.000207
0.000998

Tdrd9
21.8
−1.1
−1.44
0.00065
0.00274

Fam229a
25.2
−1.1
−1.44
0.000757
0.003124

Gm38192
22.6
−1.1
−1.57
0.001304
0.005024

A930038B10Rik
14.3
−1.1
−1.62
0.002057
0.007455

AL607131.1
38.2
−1.1
−1.7
0.002202
0.007906

Prox1
12.4
−1.1
−1.72
0.002422
0.008591

Alas2
140.4
−1.1
−1.81
0.002845
0.009851

Gm15696
19.1
−1.1
−1.81
0.00296
0.010201

Arhgap42
15.2
−1.1
−1.96
0.002998
0.010315

Fam13a
8.3
−1.1
−2.15
0.003567
0.011962

Rgcc
877.6
−1.11
−1.14
0
0

Thbs3
181.1
−1.11
−1.16
2.09E−12
4.62E−11

Tnfrsf12a
1193.9
−1.11
−1.18
2.33E−10
3.77E−09

Wnt5b
363.9
−1.11
−1.17
2.97E−10
4.72E−09

D7Bwg0826e
309.2
−1.11
−1.18
6.4E−10
9.5E−09

Cdo1
154.9
−1.11
−1.18
1.84E−09
2.52E−08

Ing4
286.6
−1.11
−1.2
1.16E−08
1.38E−07

Dock4
301.1
−1.11
−1.21
3.54E−08
3.91E−07

Sh2d4b
213.3
−1.11
−1.21
1.23E−07
1.23E−06

Nr4a3
16228.2
−1.11
−1.22
6.69E−07
5.8E−06

Fam213b
477.2
−1.11
−1.23
6.75E−07
5.85E−06

Gk5
113.2
−1.11
−1.23
8.68E−07
7.36E−06

Hey1
1015.7
−1.11
−1.23
1.04E−06
8.68E−06

Bach2it1
141.1
−1.11
−1.23
1.16E−06
9.58E−06

Sorcs2
70.7
−1.11
−1.26
5.81E−06
4.15E−05

Bmp2
169.3
−1.11
−1.27
7.01E−06
4.92E−05

Snta1
208.6
−1.11
−1.27
1.06E−05
7.16E−05

Gm13710
186.2
−1.11
−1.29
1.71E−05
0.00011

Cascl
50.4
−1.11
−1.3
2.08E−05
0.000131

Nr3c2
42.8
−1.11
−1.35
7.47E−05
0.000408

Myo5c
12.5
−1.11
−1.65
0.001671
0.006233

Gm45140
14.1
−1.11
−1.7
0.001727
0.006423

Scx
11.1
−1.11
−1.65
0.002003
0.007288

AC156952.1
12.6
−1.11
−1.89
0.002764
0.009612

D6Ertd474e
4.7
−1.11
−3.38
0.003004
0.010332

Gm9873
11.3
−1.11
−1.97
0.003018
0.010375

Smim6
7.7
−1.11
−2.44
0.003338
0.011301

Gm20513
6.4
−1.11
−2.77
0.003468
0.011673

Hap1
446.1
−1.12
−1.18
2.38E−10
3.85E−09

Epb4113
649.5
−1.12
−1.22
1.19E−07
1.19E−06

Gm9403
86.5
−1.12
−1.24
2.13E−07
2.03E−06

Ccpg1
685.5
−1.12
−1.24
7.55E−07
6.47E−06

Clec4a1
389.4
−1.12
−1.28
6.12E−06
4.35E−05

CAAA01194877.1
40.7
−1.12
−1.37
0.000103
0.000544

Padi1
28.6
−1.12
−1.39
0.000201
0.000973

Cd36
136.3
−1.12
−1.45
0.000339
0.001549

Bcar1
37.6
−1.12
−1.43
0.00035
0.001594

Snx22
65.3
−1.12
−1.61
0.001167
0.004552

Trim7
268.2
−1.12
−1.78
0.001673
0.006239

Actl7b
11.5
−1.12
−1.66
0.001764
0.006538

Cacna1f
12.7
−1.12
−1.62
0.001813
0.006692

Gm10425
5.3
−1.12
−3.54
0.002191
0.007874

Prph
18.6
−1.12
−1.79
0.002429
0.00861

Mfap2
4.7
−1.12
−2.58
0.00275
0.009577

Gm16083
11.2
−1.12
−2.24
0.002964
0.010212

Peg13
3375.2
−1.13
−1.17
0
0

Basp1
514.9
−1.13
−1.21
5.81E−10
8.69E−09

Gm42659
103.8
−1.13
−1.21
4.02E−09
5.22E−08

Tagln
142.5
−1.13
−1.21
1.06E−08
1.27E−07

Syk
3325.1
−1.13
−1.23
5.23E−08
5.56E−07

Gm28731
120.2
−1.13
−1.23
6.72E−08
7.06E−07

Trim36
327.4
−1.13
−1.23
7.28E−08
7.61E−07

Smagp
295.1
−1.13
−1.24
1.66E−07
1.62E−06

Wdfy4
2179.7
−1.13
−1.39
9.1E−05
0.000486

Tenm3
7.8
−1.13
−3.31
0.000878
0.003554

Srms
29.4
−1.13
−1.59
0.000938
0.003755

Ccr10
27.6
−1.13
−1.67
0.001242
0.00481

Scn3a
17.9
−1.13
−1.76
0.001259
0.00487

Six1
18.8
−1.13
−1.61
0.00127
0.004905

Mab21l3
31.1
−1.13
−1.89
0.001644
0.006154

Tcf21
11.3
−1.13
−1.75
0.002034
0.007381

Gm20506
6.3
−1.13
−3.45
0.00239
0.008493

Hbb-bt
232
−1.13
−2.38
0.002847
0.009853

Chka
3724.8
−1.14
−1.18
0
7E−15

Ttc28
2577.9
−1.14
−1.22
6.08E−10
9.07E−09

C330013E15Rik
164.4
−1.14
−1.25
2.81E−07
2.62E−06

E230029C05Rik
167.6
−1.14
−1.28
7.77E−07
6.65E−06

Gm22596
38.9
−1.14
−1.32
1.85E−05
0.000118

Gm37856
32.5
−1.14
−1.38
5.67E−05
0.00032

Acot1
60.2
−1.14
−1.39
6.79E−05
0.000375

Zfp811
50.1
−1.14
−1.41
0.000133
0.000678

Ctnnd2
131.9
−1.14
−1.44
0.000139
0.000703

5430402O13Rik
22.1
−1.14
−1.55
0.000666
0.002793

Btnl4
51.4
−1.14
−1.72
0.000896
0.00361

Tex26
22.2
−1.14
−1.65
0.000907
0.003649

Cecr2
73.9
−1.14
−1.95
0.001666
0.00622

Cd209c
9.8
−1.14
−2.49
0.002577
0.009055

Msi1
220.6
−1.15
−1.2
8.41E−13
1.97E−11

P2ry13
230.8
−1.15
−1.26
1.71E−08
1.99E−07

Sgip1
455.7
−1.15
−1.28
3.33E−07
3.08E−06

C1qc
6828
−1.15
−1.29
6.45E−07
5.61E−06

1700047K16Rik
77
−1.15
−1.31
6.01E−06
4.28E−05

Fam83e
25.7
−1.15
−1.41
8.3E−05
0.000448

Mxra7
39.3
−1.15
−1.43
0.00011
0.000574

Cspg4
3.3
−1.15
−5.62
0.000117
0.000603

Car3
34.9
−1.15
−1.84
0.001092
0.00429

Ephx3
23.3
−1.15
−1.75
0.001523
0.005749

Des
17.8
−1.15
−1.87
0.00184
0.006771

Neurl1a
7.4
−1.15
−2.41
0.002443
0.008651

Stox2
6.7
−1.15
−2.3
0.00249
0.008799

Lepr
5.9
−1.15
−2.45
0.002517
0.008882

Gm45212
5.8
−1.15
−2.56
0.002531
0.008927

Creg1
2831
−1.16
−1.22
4.72E−12
9.94E−11

Txnrd3
195.6
−1.16
−1.25
7.32E−10
1.08E−08

Cd163
1248.1
−1.16
−1.31
6.08E−07
5.33E−06

Rab34
100.4
−1.16
−1.31
1.51E−06
1.23E−05

Fgf17
23
−1.16
−1.55
0.000756
0.00312

Prlr
10.5
−1.16
−2.27
0.001924
0.007048

Il1bos
10.4
−1.16
−2.22
0.002031
0.007373

Gm38299
5.4
−1.16
−3.44
0.002059
0.007461

Gm35584
9
−1.16
−2.58
0.002101
0.007598

Rasl10b
6.3
−1.16
−2.53
0.002111
0.007625

Il22ra2
7.5
−1.16
−2.84
0.002262
0.008085

Tgfbr3
2708.7
−1.17
−1.2
0
0

Bach2
5671.9
−1.17
−1.21
0
6E−15

Gm43352
344.3
−1.17
−1.24
3.79E−11
6.97E−10

Tnfrsf21
641.6
−1.17
−1.25
8.28E−11
1.43E−09

Mpeg1
6302.5
−1.17
−1.25
7.72E−10
1.13E−08

Tbxas1
561.4
−1.17
−1.27
7.87E−09
9.61E−08

Thsd1
89.3
−1.17
−1.3
2.62E−07
2.46E−06

Il1b
670.8
−1.17
−1.31
7.38E−07
6.36E−06

Tbx2
144.3
−1.17
−1.34
3.67E−06
2.74E−05

March1
226
−1.17
−1.41
1.83E−05
0.000117

Serpine1
218.8
−1.17
−1.42
5.48E−05
0.00031

Gm37509
25.6
−1.17
−1.44
6.32E−05
0.000352

Ptgis
82.5
−1.17
−1.47
9.03E−05
0.000483

Crnde
21.9
−1.17
−1.47
0.000116
0.000599

Gm15448
29.6
−1.17
−1.71
0.000537
0.002315

Mir5107
156.7
−1.17
−1.83
0.001041
0.004111

1700001J03Rik
13.9
−1.17
−2.01
0.001786
0.006611

Gm17999
9.8
−1.17
−2.54
0.0018
0.006652

Gm37621
8.8
−1.17
−2.16
0.001873
0.006882

Myzap
9.4
−1.17
−2.33
0.001875
0.006886

Gm15156
8.3
−1.17
−2.23
0.001925
0.00705

4930455G09Rik
13.9
−1.17
−2.58
0.002094
0.007572

Mtss1
2502.5
−1.18
−1.19
0
0

1700056E22Rik
810.3
−1.18
−1.24
3E−14
8.31E−13

Plekha6
574.9
−1.18
−1.24
3.19E−13
7.72E−12

Gnal
314.2
−1.18
−1.26
4.93E−10
7.47E−09

Wnk4
72.2
−1.18
−1.29
1.22E−08
1.45E−07

Clec12a
552.3
−1.18
−1.35
2.93E−06
2.25E−05

9330102E08Rik
20.4
−1.18
−1.59
0.000312
0.001441

Adgrl3
33.2
−1.18
−1.74
0.00049
0.002135

AC152065.1
24.8
−1.18
−1.71
0.000632
0.002673

Gm26685
15.5
−1.18
−1.8
0.000915
0.003677

Gm5466
17.5
−1.18
−1.99
0.001304
0.005023

Reln
18.1
−1.18
−2.28
0.001614
0.006051

Gucy2c
7.2
−1.18
−2.88
0.001714
0.006382

Gm45572
4.9
−1.18
−3.42
0.001752
0.0065

Gm2058
5.4
−1.18
−2.64
0.001753
0.006503

Fcnaos
7.6
−1.18
−2.37
0.001762
0.006532

Gm13868
10
−1.18
−2.61
0.001817
0.006701

Ldlr
1329.5
−1.19
−1.23
0
1E−15

S1pr1
21169
−1.19
−1.24
3.9E−14
1.05E−12

Ksr2
333.8
−1.19
−1.27
7.47E−11
1.3E−09

Lrrc75b
342.7
−1.19
−1.3
5.18E−09
6.59E−08

Dnah17
303
−1.19
−1.3
4.01E−08
4.37E−07

Tmem141
255.2
−1.19
−1.37
1.75E−06
1.4E−05

Gpat3
224.7
−1.19
−1.35
1.96E−06
1.55E−05

Adhfe1
58
−1.19
−1.4
5.68E−06
4.06E−05

Poln
40.7
−1.19
−1.45
5.15E−05
0.000294

Klra17
46.4
−1.19
−1.57
0.000193
0.000938

Syce1l
12.2
−1.19
−1.87
0.000875
0.003544

Gm15523
17.3
−1.19
−1.93
0.001035
0.00409

0610040J01Rik
15.3
−1.19
−2.08
0.001054
0.004156

Gm11525
16.1
−1.19
−2.03
0.001213
0.004715

6330403L08Rik
20.6
−1.19
−2.07
0.001227
0.00476

Gm15848
16.2
−1.19
−3.07
0.001697
0.006322

Gm26799
792.8
−1.2
−1.26
7.8E−14
2.04E−12

Nr1d2
2875.9
−1.2
−1.26
3.15E−13
7.65E−12

Tcf4
708.9
−1.2
−1.3
6.96E−09
8.59E−08

Cd300a
1109.4
−1.2
−1.33
5.7E−08
6.03E−07

Ccnd1
258
−1.2
−1.35
1.29E−07
1.29E−06

Gm12474
77.6
−1.2
−1.34
2.3E−07
2.18E−06

Kcnj9
35.1
−1.2
−1.78
0.000455
0.002001

Baiap2l1
10.8
−1.2
−2.01
0.001135
0.00444

Slc6a1
17.6
−1.2
−2.23
0.001191
0.004634

Gpr152
13.5
−1.2
−2.12
0.001386
0.0053

Gm44735
8.1
−1.2
−2.18
0.001412
0.005383

Gm15675
565.2
−1.21
−1.28
2.06E−13
5.1E−12

Plxna1
900.2
−1.21
−1.3
3.45E−10
5.38E−09

Slc7a7
700.4
−1.21
−1.31
1.07E−09
1.52E−08

P4ha2
71.8
−1.21
−1.35
9.41E−08
9.65E−07

Dbndd2
61.2
−1.21
−1.35
1.45E−07
1.43E−06

Angptl7
118.5
−1.21
−1.38
5.17E−07
4.61E−06

Sirpb1a
105.6
−1.21
−1.39
5.82E−07
5.13E−06

Kcnk13
70
−1.21
−1.41
2.14E−06
1.68E−05

Gm2238
53.2
−1.21
−1.43
9.18E−06
6.28E−05

AC139941.2
39.3
−1.21
−1.54
8.74E−05
0.000469

Plekhg1
29
−1.21
−1.66
0.000292
0.001357

AC154548.2
22
−1.21
−1.72
0.000515
0.002231

Gm36937
12.9
−1.21
−2.31
0.001265
0.004889

Gm36159
8.5
−1.21
−2.36
0.001348
0.005169

Rgs7bp
8.5
−1.21
−2.61
0.001437
0.005464

Amigo2
1152.3
−1.22
−1.27
1E−15
3.2E−14

Pde5a
641
−1.22
−1.29
4E−14
1.08E−12

Myo9a
1226.4
−1.22
−1.29
1.92E−13
4.77E−12

Ptpdc1
185.8
−1.22
−1.3
3.71E−12
7.94E−11

Rab7b
324.8
−1.22
−1.32
6.47E−10
9.59E−09

Ptgs1
1227.5
−1.22
−1.31
6.72E−10
9.94E−09

Siglech
840.4
−1.22
−1.36
9.21E−08
9.46E−07

Gm13340
40.7
−1.22
−1.42
3.06E−06
2.34E−05

Slpi
1443.3
−1.22
−1.51
3.7E−05
0.00022

Crabp2
41.6
−1.22
−1.55
0.000115
0.000594

Cpne9
20.6
−1.22
−1.59
0.000175
0.000861

Wtip
28.7
−1.22
−1.67
0.00019
0.000927

Nox1
20
−1.22
−1.77
0.000288
0.00134

Kcnj2
25
−1.22
−2.13
0.000891
0.003595

Gm38160
15.5
−1.22
−2.33
0.001201
0.004671

Tulp1
4.4
−1.22
−2.59
0.001242
0.00481

Tbx4
5.5
−1.22
−2.3
0.001274
0.004917

Rapgef4
2629.2
−1.23
−1.28
0
1E−15

Zfyve28
1185
−1.23
−1.32
2.46E−11
4.68E−10

Tns3
884
−1.23
−1.34
1.44E−09
2.01E−08

Alpk3
97.8
−1.23
−1.35
3.57E−09
4.69E−08

Ucp3
43.4
−1.23
−1.42
1.96E−06
1.55E−05

Slc26a8
47
−1.23
−1.43
3.29E−06
2.48E−05

Slc16a7
76.5
−1.23
−1.46
6.11E−06
4.34E−05

Ssc5d
41.7
−1.23
−1.5
2.14E−05
0.000134

AC159649.1
24.5
−1.23
−1.54
6.02E−05
0.000337

Clec9a
155.9
−1.23
−1.61
8.74E−05
0.00047

Has3
31.1
−1.23
−1.68
0.000212
0.001023

Muc2
15.7
−1.23
−1.78
0.000371
0.001676

Lsamp
11.3
−1.23
−1.86
0.000524
0.002268

Gm44185
16.5
−1.23
−1.85
0.000526
0.002273

Gm12263
11.8
−1.23
−2.06
0.000838
0.003411

Adam11
386.6
−1.24
−1.3
0
1.4E−14

9230114K14Rik
64.2
−1.24
−1.43
1.77E−06
1.42E−05

AC158605.2
23.8
−1.24
−1.72
0.000182
0.000892

Ccser1
25.6
−1.24
−1.92
0.000376
0.001694

Kncn
8.8
−1.24
−1.98
0.000829
0.00338

5930403N24Rik
16
−1.24
−2.23
0.000843
0.003427

Sbsn
107.9
−1.25
−1.36
1.8E−09
2.47E−08

Adgrl2
148.4
−1.25
−1.38
1.1E−08
1.31E−07

Slc16a9
181.6
−1.25
−1.43
4.53E−07
4.09E−06

Gm17116
29.3
−1.25
−1.58
3.4E−05
0.000203

Cxcl2
1196
−1.25
−1.58
4.79E−05
0.000276

Tpm2
26.7
−1.25
−1.65
9.83E−05
0.00052

AC158622.3
22.2
−1.25
−1.68
0.00016
0.0008

Gm37755
11.4
−1.25
−2.1
0.000797
0.003266

Tmem132b
9.3
−1.25
−3.04
0.001003
0.003981

Gtf2ird1
693.3
−1.26
−1.37
1.66E−10
2.76E−09

Rhbdf1
115.9
−1.26
−1.4
2.97E−08
3.32E−07

Slc23a3
71.8
−1.26
−1.41
1.12E−07
1.13E−06

Spon2
49.3
−1.26
−1.42
1.54E−07
1.51E−06

Gm11342
46.6
−1.26
−1.46
9.11E−07
7.69E−06

Gm15728
46.1
−1.26
−1.49
4.42E−06
3.24E−05

Skor1
38.4
−1.26
−1.48
5.95E−06
4.25E−05

Olfr1033
26.2
−1.26
−1.71
0.000174
0.000858

Gm15327
25.1
−1.26
−1.81
0.000356
0.001619

Gm32999
14.5
−1.26
−2.13
0.000663
0.002785

4833411C07Rik
12.7
−1.26
−2.47
0.000791
0.003247

Gm12290
7.9
−1.26
−2.93
0.000865
0.003511

Slc28a1
7.4
−1.26
−2.34
0.000891
0.003595

Cd55
2669.5
−1.27
−1.32
0
0

Adra2a
136.7
−1.27
−1.34
4.7E−14
1.26E−12

Dnhd1
272.9
−1.27
−1.35
6.78E−13
1.6E−11

Pirb
1339.8
−1.27
−1.41
1.54E−08
1.8E−07

Cd300lf
581.8
−1.27
−1.41
1.64E−08
1.92E−07

Zfhx3
113.3
−1.27
−1.44
1.52E−07
1.49E−06

Amn
69.8
−1.27
−1.5
2.81E−06
2.16E−05

Gm44401
34.9
−1.27
−1.51
3.5E−06
2.62E−05

Cecr6
48.6
−1.27
−1.52
4.28E−06
3.15E−05

Sirpb1b
32.1
−1.27
−1.69
2.66E−05
0.000163

Zfp618
38
−1.27
−1.64
4.75E−05
0.000274

Apoc1
69.6
−1.27
−1.69
7.23E−05
0.000397

Ltbp2
21
−1.27
−1.83
0.000197
0.000955

Gm17455
30.9
−1.27
−1.88
0.000295
0.001371

St18
3.6
−1.27
−4.89
0.000421
0.001874

Gpbar1
9.8
−1.27
−2.27
0.000836
0.003403

Ptk2
617.3
−1.28
−1.32
0
0

Sbf2
450.1
−1.28
−1.33
0
0

AC135019.1
300.9
−1.28
−1.34
0
1E−15

Ctsf
383.8
−1.28
−1.37
4.71E−12
9.94E−11

Npm2
80.8
−1.28
−1.37
9.51E−11
1.63E−09

Rin1
193.4
−1.28
−1.38
5.16E−10
7.79E−09

Adgre1
1926.6
−1.28
−1.4
8.52E−10
1.24E−08

Hpgd
485.4
−1.28
−1.4
2.16E−09
2.93E−08

Gm18407
52.8
−1.28
−1.47
3.97E−07
3.62E−06

AC164573.1
54.6
−1.28
−1.47
7.45E−07
6.4E−06

Arg2
120.5
−1.28
−1.49
7.92E−07
6.76E−06

Flt3
441.7
−1.28
−1.5
8.85E−07
7.48E−06

Gm26615
44.6
−1.28
−1.65
2.71E−05
0.000166

A530064D06Rik
30
−1.28
−1.72
3.42E−05
0.000204

Gm33280
43.4
−1.28
−1.76
9.08E−05
0.000485

Gm15930
22
−1.28
−1.93
0.000219
0.001054

Hoxaas3
15.8
−1.28
−1.9
0.000251
0.001189

Gm11980
17.1
−1.28
−1.92
0.000307
0.001421

Gm43857
14.2
−1.28
−2.08
0.000361
0.001637

Gm37510
31.1
−1.28
−1.87
0.000376
0.001697

Gm12972
20.1
−1.28
−2.06
0.000381
0.001713

Cyp26b1
13.2
−1.28
−1.96
0.000381
0.001714

P4ha3
10.9
−1.28
−1.95
0.000471
0.002065

Prex2
8.7
−1.28
−2.59
0.000692
0.00289

Usp2
832.7
−1.29
−1.39
4.34E−11
7.86E−10

Tlr2
527.6
−1.29
−1.46
7.25E−08
7.59E−07

Matn2
73.1
−1.29
−1.57
2.24E−06
1.75E−05

AC159196.3
73.6
−1.29
−1.53
2.47E−06
1.92E−05

Fam135a
30.1
−1.29
−1.79
7.1E−05
0.000391

Gm4247
22.2
−1.29
−1.72
7.22E−05
0.000397

Fam83c
5.2
−1.29
−3.81
0.000414
0.001843

Otud1
1927.6
−1.3
−1.33
0
0

Fam209
161.5
−1.3
−1.41
5.91E−10
8.83E−09

Spred3
56.8
−1.3
−1.44
8.54E−09
1.04E−07

Lman1l
148.2
−1.3
−1.46
2.39E−08
2.7E−07

C1qa
7016.9
−1.3
−1.46
2.44E−08
2.75E−07

Msantd1
67.1
−1.3
−1.47
1.68E−07
1.64E−06

Nlgn3
46.3
−1.3
−1.5
3.68E−07
3.38E−06

Samd4
57.2
−1.3
−1.64
1.07E−05
7.19E−05

Cyp2d40
3.9
−1.3
−3.38
0.000438
0.001938

Usp28
3778
−1.31
−1.34
0
0

Ntn4
238.3
−1.31
−1.42
1.89E−10
3.13E−09

Gm19325
31.3
−1.31
−1.59
6.63E−06
4.67E−05

Ccdc148
86.4
−1.31
−1.62
6.67E−06
4.7E−05

Matn1
29.8
−1.31
−1.6
7.41E−06
5.18E−05

Art5
20.4
−1.31
−1.74
4.51E−05
0.000261

Slc6a7
16
−1.31
−1.9
0.000175
0.000862

Paqr6
11.7
−1.31
−2.35
0.000444
0.001958

6430548M08Rik
386.7
−1.32
−1.41
4.35E−13
1.04E−11

Jup
1184.5
−1.32
−1.41
4.36E−13
1.04E−11

Lima1
405.9
−1.32
−1.43
4.11E−11
7.48E−10

Tgm2
2908.8
−1.32
−1.44
1.38E−10
2.32E−09

Cfp
5197
−1.32
−1.44
4.73E−10
7.18E−09

Igf1
580.6
−1.32
−1.5
3.7E−08
4.07E−07

Gm44053
47.1
−1.32
−1.6
1.61E−06
1.3E−05

Gpr4
64.4
−1.32
−1.62
4.44E−06
3.25E−05

Mc1r
8.3
−1.32
−2.63
0.000507
0.002201

Ampd1
414.5
−1.33
−1.39
0
0

C6
1221.3
−1.33
−1.46
3.07E−10
4.85E−09

Gfra2
1060.6
−1.33
−1.46
5.81E−10
8.69E−09

Clec4n
902.8
−1.33
−1.49
4.25E−09
5.48E−08

Krt83
243.6
−1.33
−1.49
2.68E−08
3.01E−07

Clec4b1
78.8
−1.33
−1.72
1.43E−05
9.36E−05

Clec4a4
10.7
−1.33
−2.9
0.000393
0.001763

Nr2e3
6.8
−1.33
−4.34
0.000443
0.001955

Irs2
11401.3
−1.34
−1.4
0
1E−15

Fbxl22
362.3
−1.34
−1.41
6E−15
1.88E−13

Csf1r
9890.8
−1.34
−1.45
2.94E−12
6.38E−11

Zfp608
304
−1.34
−1.47
7.59E−10
1.11E−08

Gm42636
106.9
−1.34
−1.48
1.27E−09
1.79E−08

Fgd4
96.4
−1.34
−1.66
3.01E−06
2.3E−05

Gm9530
13.7
−1.34
−2.18
0.00018
0.000884

Gli1
13
−1.34
−2.06
0.00019
0.000928

Epha4
19.9
−1.34
−2.3
0.000222
0.001066

Tmem132c
5.6
−1.34
−3.55
0.000311
0.001435

Vipr1
6553.5
−1.35
−1.4
0
0

Adamdec1
659.5
−1.35
−1.47
7.08E−11
1.24E−09

Fmnl2
329.1
−1.35
−1.5
1.5E−09
2.08E−08

Guca2a
39.9
−1.35
−1.58
4.21E−07
3.82E−06

Tgm4
40.9
−1.35
−1.6
7.48E−07
6.42E−06

Gm12259
65.1
−1.35
−1.67
4.31E−06
3.17E−05

Trf
681.7
−1.35
−1.77
1.94E−05
0.000123

Podn
23.2
−1.35
−1.75
2.38E−05
0.000148

Aldh6a1
522.8
−1.36
−1.47
2.47E−11
4.68E−10

Adh1
149.8
−1.36
−1.49
4.27E−10
6.55E−09

Gm20544
121
−1.36
−1.51
9.16E−10
1.32E−08

Lpcat2
297.8
−1.36
−1.51
1.63E−09
2.25E−08

Rufy4
34.6
−1.36
−1.67
2.98E−06
2.28E−05

Syt1
3.7
−1.36
−5.79
0.000188
0.000918

Ankrd63
174.2
−1.37
−1.48
8.29E−12
1.7E−10

Slco2b1
818.9
−1.37
−1.49
9.09E−12
1.84E−10

Gm44907
211
−1.37
−1.48
2.4E−11
4.58E−10

Gm26888
163
−1.37
−1.5
8.25E−11
1.43E−09

Bcl2a1a
59.4
−1.37
−1.77
7.74E−06
5.39E−05

AC153955.5
18.5
−1.37
−2.1
9.26E−05
0.000494

Lrrc3
23.5
−1.37
−2.17
0.000121
0.000625

Armcx4
774.4
−1.37
−2.13
0.000162
0.000806

Gm12689
10.9
−1.37
−2.37
0.000236
0.001124

Slc40a1
4625.9
−1.38
−1.49
3.67E−12
7.88E−11

Tmem8b
139.4
−1.38
−1.5
8.99E−12
1.83E−10

Gm14027
93.7
−1.38
−1.5
3.48E−11
6.44E−10

Gm15503
167.6
−1.38
−1.51
8.32E−11
1.44E−09

Cd302
387
−1.38
−1.53
1.75E−10
2.9E−09

Mafb
1846.3
−1.38
−1.52
2.27E−10
3.69E−09

Gm15931
220.2
−1.38
−1.57
6.29E−09
7.84E−08

Apol9b
51.1
−1.38
−1.56
1.78E−08
2.06E−07

Pid1
185.8
−1.38
−1.68
8.53E−07
7.24E−06

Hes1
309.1
−1.38
−1.68
1.9E−06
1.51E−05

Jag1
135.9
−1.38
−1.7
4.46E−06
3.26E−05

A530099J19Rik
38
−1.38
−2.21
9.87E−05
0.000522

4930471C04Rik
10.8
−1.38
−2.24
0.000147
0.000741

Slc13a2
13.1
−1.38
−2.43
0.000153
0.000766

Gm15880
16.2
−1.38
−2.53
0.000207
0.000999

H2-Ob
1767
−1.39
−1.47
2E−15
6.2E−14

St8sia6
1584.6
−1.39
−1.47
4E−15
1.36E−13

Kcnip2
299.8
−1.39
−1.49
9.54E−13
2.22E−11

Agap1
495.9
−1.39
−1.54
3.02E−10
4.8E−09

Cyp27a1
464.9
−1.39
−1.57
5.08E−09
6.47E−08

Glis3
183.7
−1.39
−1.6
3.44E−08
3.81E−07

Ttc36
55
−1.39
−1.62
1.95E−07
1.88E−06

S1pr3
58.4
−1.39
−1.66
2.67E−07
2.5E−06

Shisa4
42.4
−1.39
−1.69
1.93E−06
1.53E−05

Notch4
137.5
−1.39
−1.8
8.56E−06
5.9E−05

Unc5a
20.3
−1.39
−2.82
0.000189
0.000922

Gm43364
7
−1.39
−2.54
0.000253
0.001196

Myo10
1090.4
−1.4
−1.49
8E−15
2.44E−13

Ifi207
440.3
−1.4
−1.55
2.58E−10
4.15E−09

Ppfibp2
358.3
−1.4
−1.57
9.23E−10
1.33E−08

Kcnh4
54
−1.4
−1.8
8.38E−06
5.79E−05

Ppp1r14a
48.4
−1.4
−1.89
1.95E−05
0.000124

AC160403.2
22.2
−1.4
−1.93
2.84E−05
0.000173

Gm37900
17.4
−1.4
−1.95
3.9E−05
0.00023

Tal2
7.8
−1.4
−3.32
0.000204
0.000985

Gper1
10.3
−1.4
−2.81
0.000226
0.001083

Gm45053
497.4
−1.41
−1.46
0
0

Erp27
298.4
−1.41
−1.48
0
0

Rims3
1029.1
−1.41
−1.5
2E−15
5.8E−14

Epha2
215.5
−1.41
−1.55
4.1E−11
7.48E−10

Dll4
78
−1.41
−1.6
4.69E−09
6E−08

Cacna1e
284.9
−1.41
−1.69
2.23E−07
2.12E−06

AC159502.1
24.7
−1.41
−1.84
1.34E−05
8.79E−05

Gm14221
27.8
−1.41
−2.18
5.98E−05
0.000335

Gm24371
8.6
−1.41
−2.69
0.000193
0.000938

Haglr
8.2
−1.41
−2.62
0.000212
0.001022

Tnfrsf10b
1091.3
−1.42
−1.48
0
0

Lhfpl3
194
−1.42
−1.5
0
1E−15

Ppp1r32
136.8
−1.42
−1.54
1E−12
2.32E−11

Cystm1
143.7
−1.42
−1.62
6.84E−09
8.46E−08

Bank1
715.4
−1.42
−1.65
3.75E−08
4.12E−07

Trim47
215.2
−1.42
−1.64
3.98E−08
4.35E−07

Gm14548
229.3
−1.42
−1.68
5.07E−08
5.42E−07

A930036K24Rik
102.7
−1.42
−1.69
4.41E−07
4E−06

Bmf
141.4
−1.42
−1.87
8.04E−06
5.59E−05

Tnnt3
267.5
−1.43
−1.49
0
0

Abcg3
1117.1
−1.43
−1.52
0
3E−15

Lilra6
252.1
−1.43
−1.59
5.27E−11
9.37E−10

Kirrel3
96.4
−1.43
−1.63
2.29E−09
3.09E−08

Nlrp3
1148.6
−1.43
−1.64
6.8E−09
8.42E−08

Tspan15
134
−1.43
−1.63
6.99E−09
8.63E−08

Pdzd2
59.3
−1.43
−1.68
4.54E−08
4.89E−07

Adap2
260.1
−1.43
−1.7
7.96E−08
8.26E−07

E230013L22Rik
56
−1.43
−1.81
1.9E−06
1.5E−05

Kcnj10
1374.3
−1.44
−1.55
1.4E−14
4.01E−13

Plbd1
1881.3
−1.44
−1.58
7.79E−12
1.6E−10

Nphp3
103.3
−1.44
−1.58
3.52E−11
6.5E−10

Trim72
99
−1.44
−1.79
3.19E−06
2.42E−05

9330179D12Rik
27.2
−1.44
−1.85
4E−06
2.96E−05

Gm44889
45.5
−1.44
−1.82
4.43E−06
3.25E−05

Mcc
21.2
−1.44
−2.36
4.81E−05
0.000277

Gm14137
13.8
−1.44
−3.59
0.000153
0.000766

Ifngr2
7439.3
−1.45
−1.52
0
0

Ripor3
228.4
−1.45
−1.53
0
0

Atp2a1
251.9
−1.45
−1.54
0
1.7E−14

Lrrc23
180.5
−1.45
−1.54
1E−15
2.6E−14

Map3k9
162.8
−1.45
−1.56
9E−15
2.57E−13

Ccr3
902.9
−1.45
−1.57
1.22E−12
2.78E−11

Gm13919
14.4
−1.45
−2.29
6.69E−05
0.00037

Gm43263
4.5
−1.45
−4.23
0.000109
0.00057

Gm16378
7.4
−1.45
−3.04
0.00012
0.000616

Hpgds
565.3
−1.46
−1.58
3.5E−14
9.57E−13

Grk3
1230.7
−1.46
−1.58
6.3E−14
1.66E−12

Itgb5
955.2
−1.46
−1.58
2.04E−13
5.07E−12

Slc45a3
1350.4
−1.46
−1.61
1.37E−11
2.69E−10

St6galnac2
280.3
−1.46
−1.66
1.57E−09
2.17E−08

B230398E01Rik
42.2
−1.46
−1.79
5.23E−07
4.65E−06

Tm4sf1
20.6
−1.46
−2.07
3.08E−05
0.000186

Elfn2
10.1
−1.46
−2.85
0.000117
0.000607

4930426D05Rik
77.3
−1.46
−3.31
0.000118
0.000608

Neurl3
9998.4
−1.47
−1.51
0
0

Gas6
210.8
−1.47
−1.69
7.28E−09
8.96E−08

Arhgap32
163.9
−1.47
−1.72
1.72E−08
2E−07

Slc8a1
117.6
−1.47
−1.87
8.73E−07
7.39E−06

Riiad1
6.9
−1.47
−3.36
0.000112
0.000581

Gm5150
271.8
−1.48
−1.61
1.69E−13
4.24E−12

Ctsk
47.3
−1.48
−1.67
1.41E−09
1.96E−08

Vwa3b
95.8
−1.48
−1.69
2.72E−09
3.64E−08

Gm42778
66.3
−1.48
−1.69
2.76E−09
3.68E−08

Apol9a
29.5
−1.48
−1.72
3.39E−08
3.76E−07

Capn9
80.5
−1.48
−1.77
3.53E−08
3.9E−07

Cd164l2
60
−1.48
−1.78
2.67E−07
2.5E−06

Jhy
47.7
−1.48
−2.71
6.28E−05
0.00035

Gm26583
155.4
−1.49
−1.61
1E−13
2.59E−12

Axl
7411.5
−1.5
−1.62
4.7E−14
1.27E−12

Cd209b
9.6
−1.5
−3.41
7.28E−05
0.0004

Mpzl1
469.8
−1.51
−1.65
1.12E−12
2.58E−11

Stra6l
77.8
−1.51
−1.78
3.26E−09
4.3E−08

Slc1a3
115.6
−1.51
−1.87
2.05E−07
1.96E−06

Plaur
2412.8
−1.53
−1.62
0
0

Tmem26
319.6
−1.53
−1.69
2.21E−12
4.88E−11

Islr2
51.9
−1.53
−1.85
1.27E−07
1.27E−06

Serinc4
39.2
−1.53
−1.89
2.14E−07
2.04E−06

Gm28496
23.9
−1.53
−1.91
7E−07
6.05E−06

Adam22
142.7
−1.53
−2.01
7.91E−07
6.76E−06

Gm39059
17
−1.53
−2.24
8.57E−06
5.9E−05

Rbm44
28.3
−1.53
−2.17
8.64E−06
5.94E−05

Gm45767
59.3
−1.54
−1.71
2.82E−11
5.29E−10

Rgl3
91.8
−1.54
−1.8
1.32E−09
1.86E−08

Rasal2
84.5
−1.54
−1.79
2.62E−09
3.51E−08

Gpc4
55.1
−1.54
−1.88
6.39E−08
6.74E−07

Mybpc3
45.4
−1.54
−1.95
3.3E−07
3.06E−06

Timm8a2
27.2
−1.54
−1.94
3.96E−07
3.61E−06

Cryaa
25.4
−1.54
−2.02
1.86E−06
1.48E−05

Gm32914
19
−1.54
−2.22
9.67E−06
6.58E−05

Cd207
65.2
−1.54
−3.46
4.96E−05
0.000285

Kcnj16
221.3
−1.55
−1.75
2.52E−11
4.76E−10

Sult6b2
52.2
−1.55
−1.74
3.35E−10
5.26E−09

9430038I01Rik
518.8
−1.55
−1.75
3.56E−10
5.54E−09

Dmpk
201.4
−1.55
−1.77
3.83E−10
5.9E−09

Ace
93.4
−1.55
−2.34
4.87E−06
3.54E−05

Maml3
448.5
−1.56
−1.64
0
0

Vcam1
9147.6
−1.56
−1.73
1.93E−12
4.28E−11

AC160405.2
68.7
−1.56
−1.74
2.56E−11
4.83E−10

Marcks
880
−1.56
−1.77
9.63E−11
1.65E−09

Gm16486
59.7
−1.56
−1.75
1.02E−10
1.74E−09

Myo18b
126.8
−1.56
−1.88
6.78E−09
8.4E−08

Adgrg6
43.3
−1.56
−2.1
9.01E−07
7.61E−06

Kif26a
36.7
−1.56
−2.4
9E−06
6.17E−05

Carns1
4046.3
−1.57
−1.6
0
0

Hcar2
808.2
−1.57
−1.79
2.26E−10
3.67E−09

Pyroxd2
242.8
−1.58
−1.75
1.52E−12
3.44E−11

Lhfp
173.9
−1.58
−1.82
4.67E−10
7.1E−09

Sned1
230.5
−1.58
−1.89
5.36E−09
6.79E−08

Adam30
27.5
−1.59
−2.32
5.57E−06
4E−05

Rasgef1b
1868
−1.61
−1.68
0
0

Shd
168
−1.61
−1.78
9.04E−13
2.11E−11

Ear2
200.6
−1.61
−1.83
1.1E−11
2.2E−10

Dgki
117.3
−1.61
−1.86
5.63E−11
9.99E−10

Mrc1
3441.3
−1.62
−1.79
4.62E−13
1.1E−11

Plpp3
349.2
−1.62
−1.82
3.16E−12
6.83E−11

Armcx1
41.6
−1.62
−2.1
4.7E−07
4.23E−06

AC152979.5
38.7
−1.62
−2.27
1.36E−06
1.11E−05

Cfap61
26
−1.62
−2.46
3.27E−06
2.48E−05

Gm44577
14.8
−1.62
−2.61
5.02E−06
3.64E−05

Gm17040
9.2
−1.62
−2.56
9.58E−06
6.53E−05

Paqr9
427.9
−1.63
−1.84
1.58E−12
3.57E−11

Gm20692
32.4
−1.63
−2.15
3.32E−07
3.07E−06

Treml4
1601.2
−1.64
−1.75
0
0

Pcolce2
67.5
−1.64
−2.02
1.22E−08
1.45E−07

Slc11a1
1813.6
−1.65
−1.79
0
1.3E−14

1700029H14Rik
34.9
−1.65
−2
1.69E−08
1.97E−07

Prrt3
12.9
−1.65
−2.43
3.28E−06
2.48E−05

Hepacam2
34.3
−1.65
−3
7.92E−06
5.51E−05

Ucp1
7.3
−1.65
−4.17
1.21E−05
8.04E−05

Lrp4
160.7
−1.66
−1.93
6.9E−11
1.2E−09

Etv1
13.7
−1.66
−2.72
2.68E−06
2.07E−05

Prig
9.9
−1.66
−2.49
5.13E−06
3.72E−05

Zbtb10
1450.8
−1.67
−1.75
0
0

Nr1d1
927.5
−1.67
−1.77
0
0

Prkcg
96.9
−1.67
−1.9
5.06E−12
1.06E−10

Gpr137b
186
−1.67
−1.92
8.97E−12
1.82E−10

Tnnc2
16.7
−1.67
−2.35
6.15E−07
5.38E−06

Fstl4
14.3
−1.68
−3.16
3.97E−06
2.95E−05

Hfe
584
−1.69
−1.89
1.61E−13
4.06E−12

Prkar1b
244.4
−1.7
−1.87
2E−15
6.5E−14

Scnn1a
79.5
−1.7
−2.03
3.02E−10
4.79E−09

Fndc7
27.5
−1.7
−2.39
2.09E−07
1.99E−06

Mfrp
55.6
−1.7
−2.23
2.88E−07
2.68E−06

Cd209d
96.9
−1.7
−2.46
7.68E−07
6.58E−06

Cd14
1110.8
−1.71
−1.92
9.3E−14
2.42E−12

Slc15a2
279.3
−1.71
−1.97
5.11E−12
1.07E−10

Gm43063
40.1
−1.71
−2.17
8.64E−09
1.05E−07

Gm26575
11.1
−1.71
−3.24
5.02E−06
3.64E−05

Gm8463
8.7
−1.71
−4.85
6.5E−06
4.6E−05

Gm20219
486.3
−1.72
−1.84
0
0

Aatk
130.6
−1.72
−1.94
9.9E−14
2.57E−12

Postn
510.9
−1.72
−1.93
1.24E−13
3.17E−12

Gm43786
17.6
−1.72
−2.36
5.5E−07
4.88E−06

Amz1
508.4
−1.73
−1.88
0
1E−15

Nuak1
190.3
−1.73
−1.88
0
1E−15

Pmel
62.3
−1.73
−4.31
1.27E−06
1.04E−05

Itga9
882.5
−1.74
−1.91
0
1.1E−14

Prr15
32.2
−1.74
−2.1
8.59E−10
1.25E−08

AC125351.1
47.4
−1.74
−2.21
3.88E−09
5.05E−08

Mmp19
385.3
−1.74
−2.31
5.08E−08
5.43E−07

Prdm14
18.6
−1.74
−2.51
1.15E−06
9.57E−06

Gm4258
54.5
−1.75
−1.99
2.33E−12
5.13E−11

Nav1
825.1
−1.76
−1.96
4E−15
1.36E−13

Nptxr
82
−1.76
−2.03
2.98E−12
6.45E−11

AC158622.5
86.8
−1.76
−2.1
2.83E−10
4.52E−09

Gsdmc4
19.2
−1.76
−2.22
1.78E−08
2.07E−07

AC153955.2
13.5
−1.76
−3.93
1.96E−06
1.55E−05

Ppp1r9a
128.9
−1.77
−2.16
2.22E−10
3.61E−09

Gm28050
11.2
−1.77
−2.76
8.49E−07
7.21E−06

Pilra
854.5
−1.78
−1.94
0
0

Trpm2
1260.6
−1.78
−1.96
0
6E−15

Lilra5
286.1
−1.78
−2.02
7.9E−14
2.05E−12

Ap3s1-ps2
15.1
−1.78
−2.77
7.4E−07
6.37E−06

Slc22a23
202.4
−1.79
−1.98
0
2E−15

4930438A08Rik
145.8
−1.79
−2.11
2.09E−10
3.43E−09

A930030B08Rik
34.4
−1.79
−2.41
3.65E−08
4.02E−07

Ptgds
5.4
−1.79
−5.45
1.85E−07
1.79E−06

Vstm4
221
−1.8
−2.08
3.94E−13
9.46E−12

Gm9889
639.6
−1.81
−1.87
0
0

Pira2
53.5
−1.81
−2.39
3.64E−09
4.77E−08

Six4
17.7
−1.81
−2.59
2.33E−07
2.2E−06

Gm7860
120.6
−1.82
−1.97
0
0

B230303O12Rik
93.4
−1.82
−1.99
0
0

Cadm1
1067.9
−1.82
−1.99
0
0

Mertk
1388.9
−1.82
−2.24
2.24E−10
3.64E−09

Mmp12
54
−1.82
−2.56
4.76E−08
5.11E−07

Npc1l1
18.3
−1.82
−2.72
2E−07
1.92E−06

Sema6a
113.4
−1.83
−2.2
1.34E−11
2.65E−10

Trpv4
50.5
−1.83
−2.26
1.61E−10
2.68E−09

Snx31
36.9
−1.85
−2.5
4.1E−09
5.31E−08

Plch2
20.7
−1.85
−2.61
3.52E−08
3.9E−07

Jag2
544.2
−1.86
−1.97
0
0

Fst
29.8
−1.86
−2.48
1.16E−08
1.38E−07

Apol8
1161.9
−1.87
−1.95
0
0

Siglece
886.5
−1.88
−2.03
0
0

Tbc1d9
512.6
−1.88
−2.05
0
0

Tcf712
320.1
−1.88
−2.17
7.7E−14
2.03E−12

Pygm
1170.9
−1.89
−2
0
0

Gm5608
44.9
−1.89
−2.41
1.16E−09
1.65E−08

Gm38405
61.2
−1.9
−2.33
5.15E−11
9.19E−10

Gm13830
33
−1.9
−2.36
3.07E−10
4.85E−09

Sash1
878.3
−1.91
−2.13
0
0

Dnah2
262.4
−1.91
−2.26
3.83E−13
9.2E−12

Gm6377
147.2
−1.91
−2.41
6.81E−11
1.19E−09

Cbln1
33.6
−1.92
−2.75
2.21E−09
3E−08

Zfp819
24.1
−1.93
−2.62
5.98E−09
7.49E−08

AC160028.2
11.6
−1.94
−3.91
2.08E−07
1.99E−06

Tenm4
307.5
−1.95
−2.18
0
1E−15

Dlc1
173.9
−1.95
−2.23
1E−15
4.2E−14

Cd209a
127.2
−1.95
−2.5
5.42E−11
9.63E−10

Pilrb1
346.4
−1.96
−2.22
0
3E−15

Aspa
38.6
−1.97
−2.81
9.1E−10
1.31E−08

Abcc3
2372
−1.98
−2.14
0
0

Tex45
736.4
−1.98
−2.15
0
0

Rnf150
123.1
−1.98
−2.22
0
0

Cd300e
272.8
−1.98
−2.43
1.21E−12
2.77E−11

2900052N01Rik
111.1
−2.01
−2.36
7E−15
1.96E−13

Lrp1
4560.8
−2.04
−2.28
0
0

Gpd1
933.9
−2.05
−2.29
0
0

Fam71a
57.8
−2.05
−2.6
1.94E−11
3.73E−10

Mybpc2
29.1
−2.06
−3.63
9.03E−09
1.09E−07

Itgad
5960.5
−2.07
−2.28
0
0

Cxcl1
159.6
−2.08
−2.59
1.52E−12
3.44E−11

Prss29
62.2
−2.09
−2.5
4E−15
1.24E−13

Pilrb2
349.2
−2.1
−2.38
0
0

Gm10605
75.3
−2.1
−2.44
1E−15
4.2E−14

Tecta
37.6
−2.1
−2.63
7.07E−12
1.46E−10

Adam23
301.5
−2.13
−2.37
0
0

Cbr2
76.5
−2.15
−3.07
5.98E−11
1.06E−09

Spic
2050.5
−2.16
−2.37
0
0

Sema6d
939.7
−2.17
−2.48
0
0

Hmox1
16492.8
−2.19
−2.65
4E−15
1.36E−13

Tgfb2
26.2
−2.19
−2.92
4.6E−12
9.71E−11

Fcna
5828.3
−2.21
−2.4
0
0

Col14a1
699.7
−2.37
−2.68
0
0

Wisp2
41.1
−2.37
−3.69
2.46E−12
5.41E−11

Ptprm
389.7
−2.42
−2.7
0
0

Hs3st2
297.9
−2.45
−2.9
0
0

Adgre4
851.8
−2.47
−2.72
0
0

AC166361.2
101
−2.48
−2.78
0
0

Akr1b7
148.5
−2.51
−2.99
0
0

Gdf15
168.7
−2.59
−3.42
0
5E−15

Nfasc
204.4
−2.61
−3.25
0
0

Il1a
348.3
−2.63
−3.24
0
0

Fjx1
45.8
−2.75
−4.7
7E−15
2.03E−13

TABLE 4

The SD expressed genes list for SV + α4-1BB vs. SV group

by RNA-Seq (q < 0.05, Log2FC ≥ 1 and Log2FC ≤ −1).

gene
baseMean
log2FC
log2FCunshrunk
pvalue
padj

Ret
794.7
3.23
4.01
0
0

Gzmk
1785.2
2.77
3.04
0
0

Ermn
17.7
2.73
4.67
1.64E−13
2.4E−11

Ccl8
29.5
2.55
3.64
1.07E−11
9.75E−10

Wdr95
226
2.44
2.58
0
0

Ociad2
184.7
2.4
2.5
0
0

Col6a5
17.7
2.39
5.83
3.12E−10
1.88E−08

Trp73
22
2.15
3.6
9.67E−09
4.02E−07

Ncald
297.2
2.11
2.28
0
0

Gzmb
4113.9
2.11
2.3
0
0

Wipf3
37.6
2.1
2.84
2.82E−11
2.25E−09

2900011O08Rik
14.8
2.04
2.95
8.03E−09
3.4E−07

Klrg1
746.6
2.03
2.13
0
0

Clip4
39.8
1.98
2.46
1.07E−10
7.2E−09

Cd70
12.5
1.98
3.01
2.87E−08
1.05E−06

Adgrg1
449.6
1.93
2.06
0
0

Ccl5
22142.8
1.91
2.08
0
0

Ccr5
1338.8
1.89
1.96
0
0

Faxc
12.4
1.88
3.08
3.97E−07
1.08E−05

Dpysl5
94.9
1.84
2.16
2.13E−11
1.76E−09

Gm6637
196.3
1.83
2.02
3E−15
7.75E−13

Mpl
45.7
1.8
2.67
8.7E−07
2.14E−05

Cdkn2a
59.4
1.79
2.09
6.97E−11
5.07E−09

1700011L03Rik
6.7
1.77
6.04
2.28E−06
4.92E−05

Treml1
31.4
1.75
2.44
6.78E−07
1.73E−05

Ccr2
1142.1
1.74
1.86
0
0

Saa3
14.8
1.71
5.22
3.99E−06
7.95E−05

Hist1h1b
56.5
1.7
1.96
2.2E−09
1.07E−07

Osr2
54.7
1.69
2.19
3.53E−07
9.87E−06

Col6a1
15.6
1.69
4.32
7.89E−06
0.000144

Tff1
8.6
1.69
3.25
8.36E−06
0.000151

Fam57b
23.3
1.68
5.02
4.51E−07
1.21E−05

Col3a1
82.1
1.68
3.39
9.31E−06
0.000166

Cldnd2
53.5
1.67
1.88
1.05E−11
9.69E−10

Col6a2
16.9
1.67
3.3
1.11E−05
0.000192

Insrr
64.7
1.62
1.77
7.83E−13
9.25E−11

Dixdc1
18.6
1.62
2.45
5.33E−06
0.000102

Gm33460
48.9
1.61
1.8
6.88E−11
5.04E−09

Csf2
72.9
1.61
1.86
3.27E−09
1.52E−07

Gcg
62.2
1.61
3.73
1.52E−05
0.000252

Nkg7
8456.1
1.57
1.62
0
0

Tmem40
47.5
1.57
1.98
8.68E−07
2.14E−05

Stk32c
341
1.55
1.64
0
1E−15

Serpine2
31.1
1.55
2.06
2.38E−06
5.09E−05

Wdr31
15.1
1.55
2.2
1.3E−05
0.00022

S100a4
1170.3
1.54
1.73
2.18E−10
1.37E−08

AA467197
30.3
1.54
1.86
3.68E−07
1.02E−05

Dcn
49
1.54
2.29
5.15E−06
9.93E−05

Gp1bb
21.6
1.54
2.35
3.11E−05
0.000458

Osbpl3
1358.5
1.52
1.57
0
0

Smpdl3b
185.8
1.52
1.62
6E−15
1.27E−12

Gldc
15.3
1.51
1.94
9.21E−06
0.000165

Arsb
1795.9
1.5
1.59
0
2E−15

Bspry
174.2
1.5
1.74
3.88E−08
1.38E−06

Gm15056
28.9
1.5
2.19
4.69E−05
0.000651

Esm1
680.1
1.49
1.62
2.19E−13
3.05E−11

Pif1
204
1.49
1.64
3.87E−11
2.98E−09

2310031A07Rik
64.6
1.49
1.87
1.65E−06
3.7E−05

Pglyrp1
1046.5
1.48
1.61
9.35E−13
1.08E−10

Slc35d3
110.5
1.46
1.56
3.8E−14
6.51E−12

Pbk
107.5
1.46
1.55
8.9E−14
1.4E−11

Gm2788
29.7
1.46
1.78
1.1E−06
2.61E−05

Slc16a11
9.1
1.46
2.52
9.04E−05
0.001144

Miat
31.2
1.45
1.67
5.48E−08
1.88E−06

Samd14
203.2
1.44
1.65
2.06E−08
8.04E−07

Tnfsf4
81.6
1.44
1.71
2.52E−07
7.28E−06

Gp5
24.7
1.44
1.94
1.94E−05
0.000307

Kcnj5
5.3
1.44
3.82
6.73E−05
0.000888

Vax2
12.7
1.43
2.3
0.00012
0.001443

Car5b
136
1.42
1.49
0
7.2E−14

Reg2
5.6
1.42
5.34
5.5E−06
0.000105

Tpbg
19.3
1.42
2.07
7.65E−05
0.00099

Pf4
162.2
1.42
2.19
0.000132
0.001566

Trpc6
7.4
1.42
2.85
0.000145
0.001696

Ppbp
30.6
1.41
1.97
6.29E−05
0.000839

Adap1
1257.2
1.4
1.48
1E−15
2.09E−13

Kif2c
341.3
1.4
1.48
4E−15
8.96E−13

Tff3
16.7
1.4
1.76
1.49E−05
0.000248

Muc13
227
1.39
1.53
8.75E−10
4.68E−08

Serpina3h
8.8
1.39
2.39
0.000211
0.002313

Rgs16
7742.7
1.38
1.44
0
0

Fhl2
217.6
1.38
1.45
0
2.7E−14

Serping1
49.6
1.38
1.61
2.11E−07
6.25E−06

Slc22a3
85.3
1.38
1.65
1.66E−06
3.71E−05

Nfe2
91.3
1.38
1.81
4.07E−05
0.000577

Igf2bp3
26.6
1.37
1.7
2.05E−05
0.000323

Ppp1r3g
7.3
1.37
2.61
0.000309
0.003167

Map6
151
1.36
1.45
4.41E−13
5.67E−11

Fkbp10
29.4
1.36
1.89
6.66E−05
0.00088

Tuba8
42.2
1.36
1.95
0.000162
0.00185

Mmrn1
13.3
1.36
1.98
0.000187
0.002084

Hist1h2ag
6.4
1.36
2.16
0.000327
0.003318

Rasgef1a
270.7
1.34
1.42
4E−15
8.29E−13

Fam81a
55.4
1.34
1.55
1.08E−06
2.55E−05

Col1a2
95
1.34
1.73
2.79E−05
0.000418

Fhl1
19.9
1.34
1.9
0.000148
0.001721

Fam19a3
168.7
1.33
1.48
2.06E−08
8.04E−07

Cdk1
1228.8
1.32
1.41
5.17E−13
6.5E−11

1700001O22Rik
140.3
1.32
1.42
7.29E−11
5.24E−09

Pimreg
230
1.32
1.42
1.77E−10
1.14E−08

F2rl2
42.8
1.32
1.52
1.19E−06
2.78E−05

Dach1
18.3
1.32
1.82
0.000178
0.002004

Cenpf
495.6
1.31
1.39
6.5E−14
1.05E−11

Tpx2
1113.7
1.31
1.39
1.56E−12
1.7E−10

Hmmr
311.7
1.31
1.42
2.97E−10
1.8E−08

Tubb1
17.5
1.31
1.94
0.000239
0.002557

Upk3b
15.3
1.31
2.92
0.000536
0.005053

Kif18b
606.6
1.3
1.37
1.37E−13
2.06E−11

Adgrg7
5.3
1.3
3.04
0.00033
0.003346

Cep55
503.9
1.29
1.36
1.3E−12
1.42E−10

Tpsab1
262.4
1.29
1.5
4E−06
7.97E−05

Unc5b
14.9
1.29
1.8
0.000177
0.001988

Sytl2
1359.2
1.28
1.34
1.2E−14
2.26E−12

Kif14
299.1
1.28
1.36
6.35E−11
4.68E−09

Tg
98.4
1.28
1.39
2.03E−09
9.93E−08

Ube2c
1391
1.28
1.4
6.71E−09
2.91E−07

Ifng
1413.7
1.28
1.45
2.03E−07
6.03E−06

Angpt1
63.2
1.28
1.48
3.02E−06
6.25E−05

Aunip
24.6
1.28
1.61
9.97E−05
0.00124

Birc5
1540.7
1.27
1.41
2.84E−08
1.05E−06

AC153938.2
40.5
1.27
1.51
9.04E−06
0.000162

Gng3
34.8
1.27
1.49
1.41E−05
0.000237

Robo3
27.3
1.27
1.51
2.28E−05
0.000357

Myct1
34.7
1.27
1.65
0.000137
0.001615

Kif22
1022.2
1.26
1.35
3.03E−11
2.39E−09

Rad51
401.4
1.26
1.34
5.95E−11
4.4E−09

Havcr2
486.2
1.26
1.36
1.36E−10
9.01E−09

S100a6
3227.2
1.26
1.37
1.24E−09
6.42E−08

Neurl1b
49.9
1.26
1.52
2.54E−05
0.000389

Irx3
8.6
1.26
2.06
0.000738
0.006563

Scrn1
9.5
1.26
2.43
0.000777
0.006836

Depdc1b
350.8
1.25
1.32
7.29E−12
6.94E−10

Prc1
830.9
1.25
1.36
9.49E−10
5E−08

Lxn
79
1.25
1.35
3.32E−09
1.53E−07

Ccnb1
160.8
1.25
1.37
1.44E−08
5.81E−07

Ccna2
1680.2
1.24
1.3
7E−15
1.41E−12

Anxa2
5777.8
1.24
1.34
3.39E−10
2.01E−08

Gzma
3261.2
1.24
1.86
0.000818
0.007121

Shcbp1
274.8
1.23
1.29
2.78E−13
3.78E−11

Cdca8
1251.5
1.23
1.3
2.54E−12
2.68E−10

2610318N02Rik
193.2
1.23
1.32
8.3E−10
4.48E−08

Bst1
74.2
1.23
1.33
7.18E−09
3.08E−07

Sgo2a
143.4
1.23
1.33
9.33E−09
3.88E−07

Lrr1
59
1.23
1.37
8.05E−07
1.99E−05

Epas1
1633
1.23
1.59
0.000164
0.001873

Plppr3
42.4
1.23
1.76
0.000686
0.006205

Dapk2
315.5
1.22
1.27
0
7.7E−14

Aurkb
806.6
1.22
1.28
4.93E−13
6.25E−11

Plk1
794.8
1.22
1.3
3.01E−11
2.38E−09

Heatr9
204.6
1.22
1.36
3.37E−07
9.5E−06

Npy
11.8
1.22
1.86
0.001046
0.008713

Myrf
11.2
1.22
2.86
0.001338
0.010572

Chsy1
2251.6
1.21
1.27
3E−15
6.66E−13

Mxd3
280.1
1.21
1.26
2.7E−14
4.71E−12

Sapcd2
275.5
1.21
1.3
1.06E−09
5.54E−08

Troap
236.4
1.21
1.31
6.8E−09
2.94E−07

Tigit
2942.9
1.21
1.33
3.76E−08
1.35E−06

Rtkn
29.5
1.21
1.48
5.85E−05
0.000791

Alox12
39.7
1.21
1.5
7.16E−05
0.000937

Il13
60.8
1.21
1.61
0.000618
0.005699

Hist1h3g
9.3
1.21
1.63
0.000849
0.007357

Cdca5
791.4
1.2
1.25
0
1.5E−14

Gimap7
2437.5
1.2
1.27
1.58E−11
1.35E−09

Ccnb2
1255.4
1.2
1.28
8.31E−11
5.8E−09

Fam78b
52.7
1.2
1.45
1.68E−05
0.000274

Gm9531
16
1.2
1.66
0.000496
0.004738

Tmem98
10.9
1.2
2.01
0.001307
0.010371

Eomes
4109.7
1.19
1.22
0
0

Penk
1018
1.19
1.26
1.11E−11
9.94E−10

Serpinb6b
1199
1.19
1.26
1.24E−11
1.08E−09

Prr11
288
1.19
1.32
2.76E−07
7.89E−06

Ica1
85.1
1.19
1.38
6.49E−06
0.000121

Gm4841
29.9
1.19
1.54
0.000218
0.002367

Fbxo41
15.3
1.19
1.51
0.00028
0.002912

Col6a6
6.3
1.19
3.11
0.001409
0.011028

Cst7
1893.9
1.18
1.22
0
1.5E−14

Top2a
4726.3
1.18
1.23
0
1.3E−13

Ncaph
822.4
1.18
1.24
3.2E−14
5.46E−12

Cdca2
444.5
1.18
1.24
1.09E−11
9.85E−10

Rrm2
1718.2
1.18
1.26
3.64E−10
2.15E−08

Rad54l
350.2
1.18
1.27
2.64E−09
1.27E−07

Ttk
211.5
1.18
1.27
4.53E−09
2.04E−07

BC030867
129.6
1.18
1.27
2.3E−08
8.82E−07

Cdc25c
143.9
1.18
1.3
5.8E−07
1.52E−05

Rai14
77.8
1.18
1.34
2.2E−06
4.79E−05

Hist1h3c
11.8
1.18
1.67
0.001203
0.009747

Gbp11
355.1
1.17
1.23
2.77E−11
2.23E−09

Foxd2os
95
1.17
1.24
8.95E−11
6.2E−09

Fignl1
145.7
1.17
1.23
1.17E−10
7.79E−09

Nek2
554.5
1.17
1.26
3.39E−09
1.56E−07

E2f8
535.1
1.17
1.27
3.38E−08
1.23E−06

C78197
19.1
1.17
1.65
0.00074
0.006571

Mki67
5899.1
1.16
1.24
4.42E−09
1.99E−07

E2f7
275.3
1.16
1.26
5.55E−08
1.9E−06

Spp1
51
1.16
1.36
4.36E−05
0.000612

Gp9
24.8
1.16
1.46
0.000374
0.003703

Vash1
14.6
1.16
1.66
0.001212
0.009798

Gm867
11.8
1.16
1.91
0.001595
0.012127

Nlrp6
5.5
1.16
3.11
0.002039
0.014755

Tyms
809.6
1.15
1.19
0
7.4E−14

Espl1
809.6
1.15
1.22
2.11E−11
1.75E−09

Ncapg
383.1
1.15
1.22
8.97E−10
4.77E−08

Ckap2l
455.4
1.15
1.24
2.19E−08
8.5E−07

Gm12250
177.3
1.15
1.28
5.22E−07
1.38E−05

Spns2
183.2
1.15
1.38
6.6E−05
0.000874

Il20ra
9.2
1.15
1.8
0.001826
0.01357

Nusap1
813.1
1.14
1.19
1.06E−12
1.21E−10

Serpinb9
1224.7
1.14
1.21
1.43E−11
1.24E−09

Spag5
1183.2
1.14
1.2
3.25E−11
2.54E−09

Cenpe
685.5
1.14
1.21
1.07E−10
7.2E−09

Bub1
362.3
1.14
1.22
3.72E−09
1.7E−07

Serpina3f
452
1.14
1.24
7.57E−08
2.52E−06

Ccl4
2257.9
1.14
1.43
0.000269
0.002817

Ckap2
394.5
1.13
1.17
9E−15
1.82E−12

Cdkn3
129.2
1.13
1.18
1.04E−10
7.09E−09

Sgo1
162.8
1.13
1.19
6.38E−10
3.57E−08

Serpina3g
2916.5
1.13
1.23
3.48E−08
1.25E−06

Ccl1
31
1.13
1.43
0.000689
0.006224

Prf1
1704.5
1.12
1.14
0
0

Spc24
506
1.12
1.16
1.96E−13
2.79E−11

Kif11
1109.9
1.12
1.17
2.93E−13
3.94E−11

Bub1b
1344.3
1.12
1.18
7.17E−11
5.17E−09

Lgals1
11014.9
1.12
1.18
2.68E−10
1.64E−08

Esco2
149.5
1.12
1.21
1.11E−07
3.55E−06

Tek
24.8
1.12
1.43
0.000437
0.004239

Msantd3
9.2
1.12
1.65
0.002189
0.015686

Acot7
2318.6
1.11
1.17
2.16E−11
1.77E−09

Ttn
292.1
1.11
1.21
8E−08
2.66E−06

Nuf2
279.6
1.11
1.21
5.67E−07
1.49E−05

Rgs18
52.3
1.11
1.38
0.00036
0.003592

Dio2
18.9
1.11
1.6
0.001465
0.011345

Pcdh7
8.7
1.11
1.82
0.002625
0.018256

Kif4
650.2
1.1
1.15
1.06E−11
9.7E−10

Clspn
637.1
1.1
1.17
1.4E−09
7.11E−08

Dyrk3
249.1
1.1
1.21
4.14E−07
1.12E−05

1700020L24Rik
90.2
1.1
1.23
4.95E−06
9.58E−05

Lmtk3
107.1
1.1
1.28
4.1E−05
0.00058

Ms4a3
31.1
1.1
1.55
0.002955
0.019997

Asf1b
1276.4
1.09
1.14
1.09E−12
1.23E−10

Pask
202.9
1.09
1.15
7.34E−10
4.02E−08

Cit
784.3
1.09
1.16
2.83E−09
1.35E−07

Pclaf
522.9
1.09
1.19
6.53E−07
1.68E−05

Klrc1
236.6
1.09
1.2
1.62E−06
3.64E−05

Rnase2a
5.3
1.09
1.76
0.003732
0.024243

6530402F18Rik
553.7
1.08
1.1
0
0

Cdc45
628.7
1.08
1.11
0
2E−15

Podnl1
2382.6
1.08
1.12
4.8E−14
7.96E−12

Lilr4b
1553.9
1.08
1.16
4.21E−08
1.49E−06

Perp
113.8
1.08
1.17
9.07E−08
2.95E−06

Aspm
319.4
1.08
1.17
2.91E−07
8.3E−06

Gzmc
56.6
1.08
1.32
0.000285
0.002956

Clstn3
34.6
1.08
1.39
0.000965
0.008168

Traj35
15.6
1.08
1.44
0.001132
0.009302

Pcp4l1
21
1.08
1.56
0.002232
0.015944

Serpina3n
16.7
1.08
1.62
0.002935
0.01989

Chil3
42.4
1.08
1.54
0.003165
0.021138

AC131739.1
7.2
1.08
1.94
0.003928
0.025182

Kif20a
898.5
1.07
1.13
3.27E−10
1.95E−08

Tjp2
411.7
1.07
1.14
2.03E−08
7.97E−07

Gm5391
5.3
1.07
2.14
0.004733
0.02915

Cdc20
1268.9
1.06
1.1
1.22E−13
1.87E−11

Kcnk5
399.5
1.06
1.1
6.1E−13
7.51E−11

Psrc1
122.1
1.06
1.12
2.48E−09
1.19E−07

Iqgap3
284.2
1.06
1.13
7.9E−09
3.37E−07

Cks1b
772.5
1.06
1.14
6.32E−08
2.16E−06

Grb10
61
1.06
1.32
0.000464
0.004477

Ptger3
20.8
1.06
1.62
0.004232
0.026687

Cdkn2c
421.3
1.05
1.08
8.39E−13
9.78E−11

Ndc80
345.5
1.05
1.1
1.74E−10
1.12E−08

S100a10
7676.1
1.05
1.1
5.19E−10
2.98E−08

Mcm10
477.4
1.05
1.11
1.26E−09
6.54E−08

Fut7
69.8
1.05
1.18
2.47E−05
0.000382

Tfr2
139.5
1.05
1.34
0.001188
0.009649

Hist1h2bm
8.5
1.05
1.8
0.005816
0.034129

Stmn1
257.1
1.04
1.1
2.99E−09
1.41E−07

Ska1
210.9
1.04
1.13
4.21E−06
8.29E−05

Klrc2
53.2
1.04
1.17
2.62E−05
0.000399

Cym
46.2
1.04
1.31
0.000743
0.006595

Hist1h2ai
9.8
1.04
1.51
0.004383
0.027452

Phlda3
37.7
1.03
1.2
0.000147
0.00171

Gm14148
21.4
1.03
1.25
0.000572
0.005313

Hist1h2bj
17.7
1.03
1.24
0.001234
0.009915

a
12.6
1.03
1.58
0.004957
0.030315

4930519L02Rik
9.9
1.03
1.87
0.006787
0.038499

Knstrn
855.1
1.02
1.05
5E−15
1.13E−12

Chst11
1091.1
1.02
1.05
1.6E−14
2.94E−12

Spdl1
204.5
1.02
1.07
3.91E−10
2.29E−08

Melk
329.8
1.02
1.07
7E−10
3.88E−08

Kifc1
310.5
1.02
1.09
8.1E−08
2.69E−06

Anln
217.2
1.02
1.1
1.27E−06
2.94E−05

Gm17745
84
1.02
1.11
1.34E−06
3.07E−05

Runx2os1
41.2
1.02
1.28
0.001157
0.009439

H1fx
65.9
1.02
1.27
0.001224
0.00986

Col1a1
89.2
1.02
1.37
0.002114
0.015218

Slc30a2
29
1.02
1.34
0.002562
0.017915

Mt3
18.3
1.02
1.35
0.003871
0.024928

Cxcr6
818
1.01
1.04
7.2E−14
1.15E−11

Kif15
595.3
1.01
1.07
1.32E−08
5.37E−07

Ect2
255.1
1.01
1.12
1.58E−05
0.00026

Tex15
58.4
1.01
1.12
4.2E−05
0.000591

Ddah2
90
1.01
1.15
6.54E−05
0.000868

G0s2
77.4
1.01
1.24
0.000666
0.006046

Hist1h3a
32.2
1.01
1.25
0.001415
0.011057

Pde10a
17
1.01
1.3
0.002501
0.017557

Morc1
8.4
1.01
2.01
0.007372
0.040918

Lmnb1
6788.3
1
1.03
5.3E−14
8.63E−12

Bard1
316.8
1
1.05
1.61E−10
1.06E−08

Ska3
252.1
1
1.05
4.68E−10
2.72E−08

Tk1
1004.9
1
1.05
5.56E−10
3.14E−08

Sytl3
850.3
1
1.09
2.44E−06
5.19E−05

Bag2
63
1
1.1
1.06E−05
0.000185

F10
33.6
1
1.38
0.004704
0.029055

C1s1
21.8
1
1.52
0.005149
0.031153

Usp50
16.7
1
1.55
0.006828
0.038667

Dlk2
11.4
1
1.71
0.0082
0.044224

Efna5
6.6
1
1.98
0.00842
0.045107

Ptk2
617.3
−1
−1.03
0
1.3E−13

Myo10
1090.4
−1
−1.06
3.72E−08
1.34E−06

Tmem8b
139.4
−1
−1.09
8.06E−07
1.99E−05

Tgm2
2908.8
−1
−1.09
1.15E−06
2.7E−05

Dysf
302
−1
−1.1
3.86E−06
7.73E−05

Mreg
104.8
−1
−1.11
7.36E−06
0.000136

Fam167a
155
−1
−1.11
1.28E−05
0.000218

Adam33
60.3
−1
−1.17
0.00027
0.002823

Eps8
43.8
−1
−1.19
0.000346
0.003473

Syt3
18.7
−1
−1.32
0.001772
0.013249

Rubcnl
142.1
−1
−1.34
0.001911
0.014015

Napb
20.4
−1
−1.57
0.004628
0.02868

Sat2
9
−1
−1.91
0.008166
0.044102

Tsix
8.2
−1
−2.12
0.008513
0.045495

Gm42870
22.1
−1
−2.22
0.008535
0.045552

Myo9a
1226.4
−1.01
−1.06
1.33E−09
6.81E−08

Tns3
884
−1.01
−1.1
6.53E−07
1.68E−05

Nat8l
123.4
−1.01
−1.11
2.77E−06
5.81E−05

Col17a1
97
−1.01
−1.13
1.15E−05
0.000198

Adrb1
207
−1.01
−1.15
3.23E−05
0.00047

Sh3tc1
174.9
−1.01
−1.16
6.02E−05
0.000809

Abca9
130.3
−1.01
−1.29
0.000973
0.008226

Igkv6-25
29.5
−1.01
−1.73
0.006776
0.038448

Gm36159
8.5
−1.01
−2.04
0.007309
0.040646

Gm8463
8.7
−1.01
−3.66
0.00768
0.042258

Ebf1
216.1
−1.01
−2.27
0.007802
0.042823

Tbc1d8
696.1
−1.02
−1.12
1.77E−06
3.92E−05

Adgrl2
148.4
−1.02
−1.13
3.22E−06
6.58E−05

Ppp1r14a
48.4
−1.02
−1.4
0.00191
0.014015

Padi4
34.4
−1.02
−1.28
0.002072
0.014949

Gm30292
19.5
−1.02
−1.37
0.00212
0.01525

Slc13a2
13.1
−1.02
−1.9
0.005131
0.031092

Epha4
19.9
−1.02
−1.81
0.005323
0.031904

Stfa3
14.7
−1.02
−1.71
0.005735
0.03378

Gm25776
9.5
−1.02
−1.77
0.00584
0.034194

Crisp3
7.4
−1.02
−4.01
0.00631
0.03631

Igha
3248.3
−1.02
−1.95
0.006417
0.036825

Igkv1-135
261.8
−1.02
−1.85
0.006595
0.037626

Pax5
1019.4
−1.02
−2.07
0.007319
0.040689

Plxna1
900.2
−1.03
−1.11
8.86E−08
2.89E−06

Cpq
349.9
−1.03
−1.12
6.17E−07
1.61E−05

Hck
1125
−1.03
−1.12
6.33E−07
1.64E−05

Pnck
68.8
−1.03
−1.16
6.44E−06
0.000121

Nectin4
97.5
−1.03
−1.18
5.48E−05
0.000746

Cd180
364.5
−1.03
−1.2
6.2E−05
0.000829

Btnl2
69.3
−1.03
−1.22
0.000154
0.001777

Zfyve9
64.7
−1.03
−1.3
0.000587
0.00543

Krt80
33
−1.03
−1.33
0.000948
0.00806

Cmtm8
27.8
−1.03
−1.34
0.000991
0.008347

Adgra2
49.3
−1.03
−1.44
0.001853
0.013683

Tnnc2
16.7
−1.03
−1.54
0.002356
0.016673

Klhl14
29.4
−1.03
−1.95
0.005716
0.033707

Rasgrp3
260.5
−1.03
−1.87
0.006222
0.035888

Tcn2
1368.6
−1.04
−1.08
1E−15
1.65E−13

Ppp1r32
136.8
−1.04
−1.13
2.11E−07
6.25E−06

Fcgrt
2555.6
−1.04
−1.13
5.87E−07
1.54E−05

Nphp3
103.3
−1.04
−1.16
1.69E−06
3.77E−05

Cd300lf
581.8
−1.04
−1.16
3.88E−06
7.77E−05

Ly86
762.3
−1.04
−1.17
1.71E−05
0.000278

Clec12a
552.3
−1.04
−1.2
3.22E−05
0.00047

Arhgap32
163.9
−1.04
−1.22
6.6E−05
0.000874

Eva1a
33
−1.04
−1.25
0.000199
0.002202

Wtip
28.7
−1.04
−1.44
0.00145
0.011259

B3gnt7
121.7
−1.04
−1.43
0.001841
0.013654

Npc1l1
18.3
−1.04
−1.68
0.003219
0.021411

Prex2
8.7
−1.04
−2.21
0.005599
0.03317

Cd209c
9.8
−1.04
−2.31
0.005938
0.034665

Zbtb10
1450.8
−1.05
−1.09
3.33E−12
3.42E−10

Pla2g7
1374.2
−1.05
−1.13
6.38E−08
2.18E−06

Fcgr1
319.2
−1.05
−1.15
3.57E−07
9.94E−06

Pla2g15
1269.3
−1.05
−1.15
5.99E−07
1.57E−05

B230303O12Rik
93.4
−1.05
−1.16
7.3E−07
1.84E−05

Nr3c2
42.8
−1.05
−1.28
0.000159
0.001828

Gpr4
64.4
−1.05
−1.29
0.00028
0.002912

Chn1
24
−1.05
−1.26
0.000645
0.005891

Bmf
141.4
−1.05
−1.4
0.000933
0.007945

Gm44860
11.3
−1.05
−2.25
0.005162
0.031194

Mcf2
7.3
−1.05
−3.2
0.005808
0.034089

Hebp1
934.9
−1.06
−1.19
4.49E−06
8.77E−05

Susd4
89.1
−1.06
−1.2
1.03E−05
0.000181

Tspan15
134
−1.06
−1.22
1.65E−05
0.00027

Fpr1
89.9
−1.06
−1.32
0.000293
0.003019

Ighv2-3
64.5
−1.06
−1.36
0.000905
0.007792

Alas2
140.4
−1.06
−1.75
0.004005
0.025599

Paqr6
11.7
−1.06
−1.97
0.004373
0.02741

Gm15929
8.8
−1.06
−1.92
0.004709
0.029072

Gm37829
7
−1.06
−2.2
0.004866
0.02984

Gm38160
15.5
−1.06
−2.07
0.004891
0.029962

Gm26575
11.1
−1.06
−2.25
0.004985
0.03041

Gm2814
9.6
−1.06
−2.68
0.005305
0.031827

Sort1
1028.3
−1.07
−1.19
9.44E−07
2.29E−05

Cdk14
85
−1.07
−1.26
6.08E−05
0.000815

Shank1
125.8
−1.07
−1.41
0.000743
0.006595

Ighv1-43
24.1
−1.07
−1.92
0.004221
0.026637

Ctsf
383.8
−1.08
−1.16
4.68E−09
2.1E−07

Tbxas1
561.4
−1.08
−1.17
1.11E−07
3.55E−06

Alpk1
263.7
−1.08
−1.18
2.53E−07
7.29E−06

Sccpdh
123.3
−1.08
−1.17
6.21E−07
1.61E−05

Lpl
1230.2
−1.08
−1.21
3.08E−06
6.34E−05

Stab2
1255.1
−1.08
−1.22
7.89E−06
0.000144

Apol9b
51.1
−1.08
−1.23
9.87E−06
0.000174

Gm4258
54.5
−1.08
−1.25
1.92E−05
0.000305

H2-Ea-ps
7790.9
−1.08
−1.36
0.000366
0.003633

Ciita
568.4
−1.08
−1.39
0.000641
0.005863

Igkv4-70
65.4
−1.08
−1.47
0.001124
0.009265

Ighv1-42
5.8
−1.08
−3.23
0.002259
0.016093

Igkv4-50
54.1
−1.08
−1.76
0.003011
0.020314

Cd86
1049.2
−1.09
−1.15
3.61E−11
2.81E−09

Fam43a
1414.6
−1.09
−1.18
3.94E−08
1.39E−06

Smagp
295.1
−1.09
−1.2
3.82E−07
1.05E−05

Zfhx3
113.3
−1.09
−1.24
6.09E−06
0.000115

Trim30b
52.4
−1.09
−1.36
0.000154
0.001778

Fst
29.8
−1.09
−1.52
0.000952
0.008075

Pigr
17.1
−1.09
−1.7
0.002701
0.018704

2010007H06Rik
20.7
−1.09
−1.99
0.003403
0.022397

Rgl1
1317.4
−1.1
−1.2
8.26E−08
2.73E−06

Clec4a2
262.2
−1.1
−1.26
1.09E−05
0.00019

Adhfe1
58
−1.1
−1.3
2.42E−05
0.000374

Iglv3
74.4
−1.1
−1.68
0.001978
0.014396

C130050O18Rik
95.9
−1.11
−1.26
1.45E−06
3.32E−05

Slpi
1443.3
−1.11
−1.38
0.00016
0.001838

Mybpc3
45.4
−1.11
−1.44
0.000231
0.002478

Adgrl3
33.2
−1.11
−1.64
0.001005
0.008438

Gm9530
13.7
−1.11
−1.85
0.001935
0.014156

Igkv17-127
57.8
−1.11
−2.86
0.003056
0.020527

4930426D05Rik
77.3
−1.11
−2.7
0.003313
0.021946

Fcer2a
783
−1.11
−2.3
0.003321
0.021992

Nfam1
713.9
−1.12
−1.19
1.76E−10
1.14E−08

Epb41l3
649.5
−1.12
−1.23
9.17E−08
2.97E−06

Naip5
181.7
−1.12
−1.27
2.06E−06
4.5E−05

Gas6
210.8
−1.12
−1.29
1.06E−05
0.000185

A530099J19Rik
38
−1.12
−1.84
0.001581
0.012052

0610040J01Rik
15.3
−1.12
−1.97
0.001998
0.014505

Gm17999
9.8
−1.12
−2.45
0.00274
0.018921

Gm14963
10.5
−1.12
−1.98
0.002748
0.018963

Tal2
7.8
−1.12
−2.87
0.002908
0.019726

Gm10605
75.3
−1.13
−1.35
2.32E−05
0.000361

Abcd2
85.6
−1.13
−1.35
3.96E−05
0.000563

Pcolce2
67.5
−1.13
−1.42
9.12E−05
0.001153

Pigz
58.4
−1.13
−1.4
0.000141
0.001652

Trf
681.7
−1.13
−1.49
0.000353
0.003532

A930030B08Rik
34.4
−1.13
−1.59
0.000525
0.004961

Mir5107
156.7
−1.13
−1.77
0.001511
0.011598

Kcnj2
25
−1.13
−2
0.001945
0.014221

Tlr7
305.4
−1.14
−1.22
3.03E−09
1.42E−07

Igsf6
661.8
−1.14
−1.24
3.88E−08
1.38E−06

Lrrc25
483.1
−1.14
−1.28
6.57E−07
1.69E−05

Ptpro
88.1
−1.14
−1.29
1.06E−06
2.52E−05

Tppp
109.8
−1.14
−1.29
1.47E−06
3.35E−05

Dll4
78
−1.14
−1.29
2.41E−06
5.14E−05

Snn
825.3
−1.14
−1.31
3.98E−06
7.95E−05

Pdzd2
59.3
−1.14
−1.35
1.39E−05
0.000234

Erbb2
135
−1.14
−1.33
1.55E−05
0.000255

Ctnnd2
131.9
−1.14
−1.44
0.000131
0.001557

Gm15930
22
−1.14
−1.73
0.001034
0.008627

Igkv8-30
318.2
−1.14
−1.89
0.002044
0.014786

Blk
523.7
−1.14
−1.98
0.002156
0.015487

Cyp4f37
6.6
−1.14
−2.97
0.002672
0.018535

Tcf4
708.9
−1.15
−1.26
2.31E−08
8.84E−07

C1qb
6167.6
−1.15
−1.28
3.36E−07
9.48E−06

Ccnd1
258
−1.15
−1.29
3.86E−07
1.05E−05

Cfap61
26
−1.15
−1.82
0.001029
0.008606

Hepacam2
34.3
−1.15
−2.23
0.00188
0.013838

Gm15880
16.2
−1.15
−2.16
0.002139
0.015378

Clec4a4
10.7
−1.15
−2.6
0.002248
0.01604

Fmnl2
329.1
−1.16
−1.29
1.74E−07
5.28E−06

Pdgfc
56.2
−1.16
−1.45
8.78E−05
0.001115

Pde8b
30.7
−1.16
−1.81
0.000914
0.007828

Gm5466
17.5
−1.16
−1.97
0.001427
0.011109

Lima1
405.9
−1.17
−1.27
4.02E−09
1.82E−07

Slc16a7
76.5
−1.17
−1.39
1.46E−05
0.000243

Snx24
100.2
−1.17
−1.4
1.79E−05
0.000288

Fcrl1
520.9
−1.17
−1.52
0.000191
0.002126

Plxnb3
46.1
−1.17
−1.66
0.000571
0.00531

Gm36937
12.9
−1.17
−2.24
0.001847
0.013668

Igkv5-39
123.1
−1.17
−2.24
0.00205
0.014817

Tnfrsf21
641.6
−1.18
−1.26
5.23E−11
3.91E−09

Syk
3325.1
−1.18
−1.29
1.05E−08
4.34E−07

Cmbl
316.4
−1.18
−1.31
3.55E−07
9.91E−06

AC166361.2
101
−1.18
−1.36
5.35E−07
1.42E−05

Stac2
76.6
−1.18
−1.55
0.000206
0.002258

Ighv2-6
141.1
−1.18
−1.53
0.000266
0.002791

Igkv4-63
73.9
−1.18
−1.74
0.000748
0.006628

Igkv9-124
50.6
−1.18
−2.27
0.001681
0.012661

Mpeg1
6302.5
−1.19
−1.28
3.51E−10
2.08E−08

Siglech
840.4
−1.19
−1.32
1.89E−07
5.64E−06

Ccdc148
86.4
−1.19
−1.48
4.04E−05
0.000573

Blnk
622
−1.19
−1.59
0.000221
0.002396

Ighv1-4
158
−1.19
−1.8
0.000906
0.007795

Siglecg
751.8
−1.19
−1.93
0.001034
0.008627

Slc6a1
17.6
−1.19
−2.21
0.001218
0.009819

Ighv9-2
13.7
−1.19
−2.05
0.001368
0.010761

Igkv4-58
47
−1.19
−2.02
0.001407
0.011025

Fam213b
477.2
−1.2
−1.33
6.6E−08
2.24E−06

Gm13710
186.2
−1.2
−1.39
3.19E−06
6.52E−05

S1pr3
58.4
−1.2
−1.43
8.88E−06
0.00016

Tecta
37.6
−1.2
−1.56
0.000104
0.001281

Fam135a
30.1
−1.2
−1.66
0.000222
0.002413

Gbgt1
24
−1.2
−1.71
0.000334
0.003374

AC153955.5
18.5
−1.2
−1.86
0.000622
0.005722

Trim7
268.2
−1.2
−1.88
0.000812
0.007083

Igkv6-23
42.9
−1.2
−1.99
0.001136
0.009317

Map3k9
162.8
−1.21
−1.3
9.73E−11
6.66E−09

Fndc7
27.5
−1.21
−1.78
0.00023
0.002473

Apoe
8629.4
−1.22
−1.32
4.19E−10
2.45E−08

Sirpa
4992.1
−1.22
−1.35
2.63E−08
9.78E−07

Cd300a
1109.4
−1.22
−1.35
3.42E−08
1.24E−06

Cystm1
143.7
−1.22
−1.4
5.65E−07
1.49E−05

Snta1
208.6
−1.22
−1.4
1.01E−06
2.43E−05

Trpv4
50.5
−1.22
−1.54
2.56E−05
0.000392

Lifr
153.4
−1.22
−1.49
2.67E−05
0.000404

Pcdhgb4
14.2
−1.22
−1.79
0.00053
0.005005

Sdc3
7230.4
−1.23
−1.34
1.71E−09
8.52E−08

Marcks
880
−1.23
−1.39
3.81E−07
1.04E−05

Flt3
441.7
−1.23
−1.43
2.6E−06
5.48E−05

Wdfy4
2179.7
−1.23
−1.51
1.94E−05
0.000307

Igkv5-45
19.6
−1.23
−2.23
0.001131
0.009302

H2-Ob
1767
−1.24
−1.32
1.21E−12
1.33E−10

Fgd2
1084.5
−1.24
−1.37
3.13E−08
1.14E−06

Dmpk
201.4
−1.24
−1.43
5.01E−07
1.34E−05

Ttc12
74.5
−1.24
−1.49
8.57E−06
0.000154

AC153955.2
13.5
−1.24
−3.15
0.000817
0.00712

Dennd5b
158.4
−1.24
−2.45
0.001058
0.008786

Ppfibp2
358.3
−1.25
−1.4
4.67E−08
1.62E−06

Gm15922
138.1
−1.25
−1.47
2E−06
4.38E−05

Slc1a3
115.6
−1.25
−1.56
1.64E−05
0.000269

Fam71a
57.8
−1.25
−1.63
5E−05
0.000691

Btnl4
51.4
−1.25
−1.87
0.000251
0.002666

Plxnb2
2017.4
−1.26
−1.36
2.1E−10
1.33E−08

Mafb
1846.3
−1.26
−1.39
6.8E−09
2.94E−07

Scamp5
313.9
−1.26
−1.38
8.04E−09
3.4E−07

Lpcat2
297.8
−1.26
−1.41
1.94E−08
7.66E−07

Prkcg
96.9
−1.26
−1.45
1.88E−07
5.63E−06

Cd300c2
673.6
−1.26
−1.44
3.62E−07
1E−05

Cecr6
48.6
−1.26
−1.51
3.93E−06
7.85E−05

Scn3a
17.9
−1.26
−1.92
0.000287
0.002968

A4galt
38.5
−1.26
−1.89
0.000339
0.003419

Unc5a
20.3
−1.26
−2.62
0.000665
0.006038

Dock4
301.1
−1.27
−1.39
2.01E−10
1.28E−08

C1qc
6828
−1.27
−1.42
4.33E−08
1.52E−06

Zfp366
189.3
−1.27
−1.44
6.67E−08
2.26E−06

Samd4
57.2
−1.27
−1.61
1.4E−05
0.000235

Cd209d
96.9
−1.27
−1.89
0.000206
0.002258

Ifi207
440.3
−1.28
−1.42
6.19E−09
2.71E−07

Glis3
183.7
−1.28
−1.48
3.46E−07
9.69E−06

Gpc4
55.1
−1.28
−1.58
6.38E−06
0.00012

Tgfb2
26.2
−1.28
−1.82
7.16E−05
0.000937

Ffar1
97.9
−1.28
−1.83
0.000194
0.002157

Epha2
215.5
−1.29
−1.42
1.19E−09
6.2E−08

Cd5l
2824.6
−1.29
−1.41
1.31E−09
6.73E−08

Nxpe5
131.7
−1.29
−1.49
3.09E−07
8.77E−06

Gm33280
43.4
−1.29
−1.78
7E−05
0.000918

Gm15448
29.6
−1.29
−1.86
0.000127
0.00152

Tlr13
195.6
−1.3
−1.4
5.26E−12
5.13E−10

Kcnk13
70
−1.3
−1.51
2.63E−07
7.54E−06

Rasal2
84.5
−1.3
−1.53
4.32E−07
1.17E−05

Cd300c
47.9
−1.3
−1.67
1.67E−05
0.000273

Gm14137
13.8
−1.3
−3.35
0.000624
0.005734

6430548M08Rik
386.7
−1.31
−1.4
5.27E−13
6.58E−11

C2
119.2
−1.31
−1.41
2.4E−11
1.95E−09

Fgd4
96.4
−1.31
−1.63
4.58E−06
8.9E−05

Gm14221
27.8
−1.31
−2.05
0.00018
0.002021

Gm28050
11.2
−1.31
−2.15
0.000277
0.002883

Il22ra2
7.5
−1.31
−3.06
0.000546
0.005125

Rims3
1029.1
−1.32
−1.41
7.3E−14
1.16E−11

I830077J02Rik
399
−1.32
−1.41
1.46E−12
1.59E−10

Cfp
5197
−1.32
−1.44
4.78E−10
2.76E−08

Sirpb1c
21.5
−1.32
−1.75
2.9E−05
0.000432

Adam11
386.6
−1.33
−1.39
0
1E−15

Hpgds
565.3
−1.33
−1.43
5.79E−12
5.61E−10

Hdac9
184.1
−1.33
−1.49
8.39E−09
3.53E−07

Clec4a1
389.4
−1.33
−1.52
6.73E−08
2.27E−06

Gm10552
10.1
−1.33
−2.86
0.000376
0.003714

Abcg3
1117.1
−1.34
−1.42
6E−15
1.18E−12

Slc7a7
700.4
−1.34
−1.45
1.16E−11
1.03E−09

Amz1
508.4
−1.34
−1.46
4.31E−11
3.29E−09

Pirb
1339.8
−1.34
−1.49
2.08E−09
1.01E−07

Cd14
1110.8
−1.34
−1.51
5.3E−09
2.36E−07

Cyp27a1
464.9
−1.34
−1.52
1.71E−08
6.82E−07

Clec4b1
78.8
−1.34
−1.73
1.22E−05
0.000208

Rasgef1b
1868
−1.35
−1.41
0
1E−15

Jup
1184.5
−1.35
−1.44
1.31E−13
1.98E−11

Grk3
1230.7
−1.35
−1.46
3.6E−12
3.63E−10

C1qa
7016.9
−1.35
−1.51
8.15E−09
3.43E−07

Dgki
117.3
−1.35
−1.56
3.91E−08
1.38E−06

Agap1
495.9
−1.36
−1.51
6.16E−10
3.46E−08

Timd4
240.3
−1.36
−1.53
7.81E−09
3.33E−07

Ighv1-7
114.3
−1.36
−1.76
1.71E−05
0.000278

Tlr8
86.3
−1.37
−1.62
2.21E−07
6.48E−06

Zfp608
304
−1.38
−1.52
1.86E−10
1.19E−08

Kif26a
36.7
−1.38
−2.16
8.08E−05
0.001037

Reln
18.1
−1.38
−2.59
0.000204
0.002246

Dhtkd1
7
−1.38
−3.09
0.000253
0.002684

Gm15848
16.2
−1.38
−3.38
0.00027
0.002823

Prkar1b
244.4
−1.39
−1.54
8.05E−11
5.67E−09

Slc16a9
181.6
−1.39
−1.59
1.74E−08
6.93E−07

Gfra4
95
−1.39
−1.61
2.35E−07
6.87E−06

Pid1
185.8
−1.39
−1.68
7.4E−07
1.86E−05

Apoc1
69.6
−1.39
−1.83
1.3E−05
0.00022

Scn4a
77.9
−1.39
−2.02
7.44E−05
0.000968

Ptgs1
1227.5
−1.4
−1.51
1.12E−12
1.26E−10

AC125351.1
47.4
−1.4
−1.8
2.46E−06
5.24E−05

Fstl4
14.3
−1.4
−2.76
0.000124
0.001486

Plbd1
1881.3
−1.41
−1.54
2.06E−11
1.72E−09

Gm15931
220.2
−1.41
−1.6
2.87E−09
1.36E−07

Tcf7l2
320.1
−1.41
−1.64
2.26E−08
8.71E−07

Capn9
80.5
−1.41
−1.69
1.32E−07
4.12E−06

March1
226
−1.41
−1.68
2.64E−07
7.58E−06

Mcc
21.2
−1.41
−2.32
6.53E−05
0.000866

Etv1
13.7
−1.41
−2.38
6.82E−05
0.000895

AC160028.2
11.6
−1.41
−3.13
0.000179
0.002013

Nav1
825.1
−1.42
−1.59
2.42E−10
1.5E−08

Igf1
580.6
−1.42
−1.61
3.17E−09
1.47E−07

Cpne8
29
−1.42
−1.91
1.06E−05
0.000185

Csf1r
9890.8
−1.43
−1.54
1.16E−13
1.79E−11

Hpgd
485.4
−1.43
−1.57
1.74E−11
1.47E−09

Tmem26
319.6
−1.43
−1.58
5.08E−11
3.83E−09

Prss29
62.2
−1.43
−1.75
1.22E−07
3.86E−06

Ighv5-2
17.8
−1.43
−3.08
0.000154
0.001777

Cd209b
9.6
−1.43
−3.28
0.00016
0.001835

Axl
7411.5
−1.44
−1.56
4.08E−13
5.33E−11

Tbc1d9
512.6
−1.44
−1.57
1.2E−12
1.33E−10

Plpp3
349.2
−1.44
−1.62
4.78E−10
2.76E−08

Clec4a3
317.3
−1.44
−1.61
4.9E−10
2.82E−08

Scnn1a
79.5
−1.44
−1.72
1.08E−07
3.49E−06

Jhy
47.7
−1.44
−2.65
9.59E−05
0.001207

Nr1d1
927.5
−1.45
−1.54
0
1.35E−13

Itga9
882.5
−1.45
−1.6
8.13E−12
7.66E−10

Slc45a3
1350.4
−1.45
−1.59
2.01E−11
1.68E−09

Clec4n
902.8
−1.45
−1.62
1.39E−10
9.13E−09

Lrp4
160.7
−1.45
−1.7
9.85E−09
4.07E−07

Cacna1e
284.9
−1.45
−1.73
1.14E−07
3.62E−06

Mmp12
54
−1.45
−2.08
1.42E−05
0.000237

P2ry13
230.8
−1.46
−1.59
5.57E−13
6.92E−11

Nuak1
190.3
−1.46
−1.6
6.64E−13
7.9E−11

Adap2
260.1
−1.46
−1.72
4.52E−08
1.58E−06

Adgrg6
43.3
−1.46
−1.97
4.05E−06
8.05E−05

Kcnj10
1374.3
−1.47
−1.58
4E−15
8.36E−13

Ppp1r9a
128.9
−1.47
−1.8
1.43E−07
4.44E−06

Rab30
116
−1.47
−2.11
1.86E−05
0.000297

Gfra2
1060.6
−1.48
−1.62
4.86E−12
4.82E−10

St6galnac2
280.3
−1.48
−1.68
7.6E−10
4.15E−08

2900052N01Rik
111.1
−1.48
−1.76
1.26E−08
5.15E−07

Slc8a1
117.6
−1.48
−1.88
7.25E−07
1.83E−05

Ighv1-39
28.6
−1.48
−2.7
9.84E−05
0.001231

Gpr137b
186
−1.49
−1.71
1.37E−09
7.01E−08

Trpm2
1260.6
−1.5
−1.66
3.19E−12
3.31E−10

Aatk
130.6
−1.5
−1.7
7.89E−11
5.6E−09

A530064D06Rik
30
−1.5
−1.98
7.1E−07
1.8E−05

Pyroxd2
242.8
−1.51
−1.68
9.76E−12
9.04E−10

Hcar2
808.2
−1.51
−1.74
8.34E−10
4.5E−08

Catsperg2
14.8
−1.51
−2.17
9.58E−06
0.00017

Slc40a1
4625.9
−1.53
−1.66
1E−14
1.89E−12

Mmp19
385.3
−1.53
−2.04
1.82E−06
4.01E−05

Mpzl1
469.8
−1.55
−1.7
2.45E−13
3.38E−11

Dnah2
262.4
−1.55
−1.85
3.81E−09
1.74E−07

Igkv6-15
223
−1.55
−2.65
3.22E−05
0.00047

Cadm1
1067.9
−1.56
−1.72
1.47E−13
2.19E−11

Ear2
200.6
−1.56
−1.76
4.64E−11
3.53E−09

Cd302
387
−1.57
−1.73
3.57E−13
4.7E−11

Lilra6
252.1
−1.58
−1.75
2.95E−13
3.94E−11

Cd163
1248.1
−1.58
−1.77
1.19E−11
1.05E−09

Lilra5
286.1
−1.58
−1.8
2.95E−11
2.34E−09

Treml4
1601.2
−1.59
−1.7
0
2E−15

Slc11a1
1813.6
−1.59
−1.72
4E−15
8.92E−13

Itgb5
955.2
−1.6
−1.73
1E−15
2.17E−13

Adgre1
1926.6
−1.6
−1.75
1.8E−14
3.26E−12

Rgl3
91.8
−1.6
−1.86
2.53E−10
1.57E−08

Ccr3
902.9
−1.61
−1.75
2E−15
5.62E−13

Siglec1
622.7
−1.61
−1.78
1.01E−12
1.15E−10

Slco2b1
818.9
−1.62
−1.75
1E−15
1.75E−13

Apod
9.3
−1.62
−3.32
2E−05
0.000315

C6
1221.3
−1.63
−1.78
1.5E−14
2.85E−12

Sirpb1b
32.1
−1.64
−2.12
2.61E−08
9.73E−07

Sirpb1a
105.6
−1.65
−1.88
3.33E−12
3.42E−10

Lhfp
173.9
−1.66
−1.92
3.96E−11
3.03E−09

Hmox1
16492.8
−1.66
−2.02
3.06E−09
1.43E−07

Gm2762
38.5
−1.66
−1.94
2.82E−08
1.04E−06

Pilra
854.5
−1.67
−1.82
1E−15
1.75E−13

Angptl7
118.5
−1.67
−1.89
2.22E−12
2.38E−10

Matn2
73.1
−1.67
−2
5.51E−10
3.12E−08

Mertk
1388.9
−1.67
−2.06
6.31E−09
2.76E−07

Cxcl1
159.6
−1.67
−2.1
1.42E−08
5.76E−07

Adamdec1
659.5
−1.68
−1.83
0
1.2E−13

Tenm4
307.5
−1.68
−1.89
1.99E−13
2.82E−11

Bank1
715.4
−1.68
−1.96
5.8E−11
4.31E−09

Gm5150
271.8
−1.69
−1.83
0
9E−15

Igkv3-7
151.3
−1.69
−3.52
8.26E−06
0.000149

Aspa
38.6
−1.7
−2.48
1.27E−07
3.99E−06

Gdf15
168.7
−1.71
−2.33
5.3E−08
1.82E−06

Adam22
142.7
−1.72
−2.24
2.59E−08
9.71E−07

Vcam1
9147.6
−1.73
−1.91
6E−15
1.2E−12

Fjx1
45.8
−1.73
−3.32
1.23E−06
2.86E−05

Paqr9
427.9
−1.74
−1.96
4E−14
6.63E−12

Slc22a23
202.4
−1.75
−1.94
0
7.9E−14

Dlc1
173.9
−1.75
−2.01
6.48E−13
7.83E−11

Sema6a
113.4
−1.75
−2.11
8.35E−11
5.81E−09

Snx31
36.9
−1.75
−2.37
2.42E−08
9.17E−07

Mrc1
3441.3
−1.76
−1.96
3E−15
6.66E−13

Pira2
53.5
−1.76
−2.34
7.75E−09
3.32E−07

Siglece
886.5
−1.77
−1.91
0
0

Il1a
348.3
−1.77
−2.22
7.34E−10
4.02E−08

Cecr2
73.9
−1.77
−2.86
9.93E−07
2.39E−05

Pilrb1
346.4
−1.78
−2.02
4E−14
6.68E−12

Stra6l
77.8
−1.78
−2.07
1.68E−12
1.8E−10

Sash1
878.3
−1.8
−2.01
1E−15
1.46E−13

Hfe
584
−1.81
−2.02
2E−15
5.33E−13

Kirrel3
96.4
−1.82
−2.06
1E−14
1.9E−12

Gm14548
229.3
−1.82
−2.13
2.57E−12
2.7E−10

Gpd1
933.9
−1.84
−2.05
1E−15
1.85E−13

Ace
93.4
−1.84
−2.73
5.03E−08
1.75E−06

Pilrb2
349.2
−1.85
−2.1
4E−15
9.43E−13

Spic
2050.5
−1.86
−2.04
0
1E−15

Lrp1
4560.8
−1.86
−2.08
0
6.6E−14

Slc15a2
279.3
−1.89
−2.18
1.7E−14
3.03E−12

Vstm4
221
−1.89
−2.18
2E−14
3.63E−12

Abcc3
2372
−1.9
−2.04
0
0

Cd209a
127.2
−1.9
−2.44
1.68E−10
1.09E−08

Cd300e
272.8
−1.91
−2.34
7.55E−12
7.15E−10

Sned1
230.5
−1.92
−2.29
9.22E−13
1.07E−10

Myo18b
126.8
−1.96
−2.34
1.63E−13
2.4E−11

Ptprm
389.7
−1.98
−2.21
0
0

Kcnj16
221.3
−1.98
−2.22
0
4E−15

Gm6377
147.2
−1.99
−2.51
8.92E−12
8.32E−10

Mybpc2
29.1
−1.99
−3.53
2.54E−08
9.54E−07

Sema6d
939.7
−2.01
−2.3
0
2.2E−14

Cbr2
76.5
−2.01
−2.9
8.13E−10
4.42E−08

Itgad
5960.5
−2.04
−2.25
0
0

Adam23
301.5
−2.04
−2.27
0
0

Postn
510.9
−2.04
−2.28
0
1E−15

Cbln1
33.6
−2.04
−2.89
1.25E−10
8.28E−09

Rnf150
123.1
−2.07
−2.32
0
0

Hs3st2
297.9
−2.13
−2.53
0
1.3E−13

Fcna
5828.3
−2.2
−2.39
0
0

Col14a1
699.7
−2.25
−2.55
0
0

Akr1b7
148.5
−2.26
−2.71
0
9E−15

Wisp2
41.1
−2.27
−3.56
1.66E−11
1.41E−09

Adgre4
851.8
−2.37
−2.61
0
0

Nfasc
204.4
−2.38
−2.98
0
1.5E−14

Next, NIH DAVID analysis using the upregulated gene list was run. In both comparisons, cell cycle genes upregulation is the highest enrichment cluster [although SV+α4-1BB mAb vs. SV has a lower enrichment score compared with SV plus α4-1BB mAb vs. untreated samples (FIGS. 23B and 25). This indicates that SV+α4-1BB mAb induced more potent T cell cycle progression compared with SV only. T cell proliferation is critical for an effective anti-tumor response against A20 lymphoma. The CD4/CD8 T cell ratio in untreated mice decreased markedly by day 28 after tumor inoculation (FIG. 26A-26B). In addition, Treg/CD8 T cell ratio increased by day 28, indicating impairment of T cell function (FIG. 26C-26D). In other groups the T cell ratio remained constant due to proliferation.

CD69 is the earliest marker of immune system activation. SV plus α4-1BB mAb treatment synergistically upregulated CD69 on day 2 (FIG. 23D). Additionally, KEGG GSEA indicates that T cell receptor signaling gene sets were enriched when comparing SV+α4-1BB vs untreated samples (enrichment score=0.35, Normalized Enrichment Score (NES)=1.56, FDR q value=0.17, nominal p value=0) (FIG. 23E).

SV Plus α4-1BB mAb Stimulated Cytotoxic T Cell Function

To investigate the antitumor cytotoxicity of SV/α4-1BB treated splenocytes, f-Luc A20 lymphoma cells were co-cultured with splenocytes on day 7. The ratios explored between splenocytes and tumor cell were 40:1, 20:1, 10:1. SV plus α4-1BB treated splenocytes demonstrated the highest cytotoxicity among all groups, as calculated by the reduction of f-Luc activity (FIG. 27A). To understand if this response is induced by TAA or anti-viral immunity, the same experiment was performed using mice under treatment but without tumor inoculation. We found that SV plus α4-1BB achieves the same effect as the combination treatment with tumor inoculation. This indicates that anti-tumor response on day 7 was not tumor specific. Accordingly, NKG2D, granzyme B and perforin were highly expressed in CD8 T cells from α4-1BB treated mice. In addition, SV plus α4-1BB in combination induced the highest expression of NKG2D and granzyme B in CD8 T cells. NKG2D, granzyme B and perforin upregulation was tumor independent because the same pattern was observed in all treatments without tumor inoculation (FIG. 27B-27C). Correspondingly, IPA indicates that gene sets of cytotoxic T cell development are significantly upregulated in SV plus α4-1BB mAb. These genes include Gzmb (granzyme B), Prfl (perforin) and Klrkl (NKG2D) (FIG. 27D). These data indicate that SV plus α4-1BB mAb markedly enhanced cytotoxic T cell activity.

SV Plus α4-1BB mAb Induced IFNγ Production from T Cells

Other upregulated genes in the SV plus α4-1BB mAb combined treatment include STAT4 (FIG. 27D) and IL12rbl (FIG. 28D), which are required for the development of Th1 cells from naïve CD4+ T cells and IFNγ production (FIG. 27D) in response to IL-12 [Jacobson N G et al., J Exp Med. 1995]. Consistent with this observation, splenocytes from SV plus α4-1BB mAb treatment produced significantly higher number of IFNγ spots compared with other groups, reaching peak production on day 7 (FIG. 28A, upper panel). After day 7, the response dampened but still remained at the highest level compared with other groups (FIG. 28A, lower panel). This is in line with increased IFNγ RNA levels. To identify if TAA or viral antigen induces IFNγ production on day 7, the same experiment was performed in mice not inoculated with tumor cells. For both SV or SV plus α4-1BB treatment, the presence or absence of tumor did not significantly affect IFNγ levels (FIG. 29), confirming that IFNγ production on day 7 was mainly an anti-viral response. To identify whether T cells or antigen presentation cells (APCs) play the major role in IFNγ production, we harvested SV treated splenic T cells and naive T cells respectively. T cells from SV treated mice were co-cultured with naive APCs. Conversely, APCs from SV treated mice were cultured with naive T cells. T cells from SV treated mice produced IFNγ when co-cultured with naive APC. Naive T cells produce much less IFNγ spots when cultured with SV infected APC. However, neither T cell nor APC alone could produce elevated numbers of IFNγ spots. These observations indicate that T cells play the dominant role in IFNγ production during SV infection (FIG. 30A). APCs are essential for helping T cells to produce IFNγ.

Next, to identify whether CD4 or CD8 T cells produce IFNγ, flow cytometric analysis was performed for cytokine analysis. Among splenocytes, 2-2.5% SV plus α4-1BB mAb treated CD4 T cells produced IFNγ, which is significantly higher than other groups. Very low percentages of CD8 T cells produced IFNγ in all groups (FIG. 28B). There were much less IFNγ producing T cells after removing APC (FIG. 28B). Also, there was no difference among all groups for IFNγ production. This suggests that T cell-APC interaction is essential for IFNγ production. To test the antitumor IFNγ production activity of the purified T cells, they were co-cultured for 5 h with A20 cells, which express major histocompatibility complex (MHC) I and II molecules [Pizzoferrato E et al., Int J Cancer, 2004]. Only CD4 T cells from the SV plus α4-1BB mAb group produced IFNγ after co-culture (FIGS. 28C and 30B). This indicates that SV plus α4-1BB mAb induces anti-tumor IFNγ production activity. Besides IFNγ, several Th1 associated genes were also upregulated in the T cells from SV plus α4-1BB mAb treated groups. These include Ccr5, Cxcr3, Havcr2 (Tim3), IL12rbl and Klrc1 (FIG. 4d). T-bet is the key transcription factor which is essential for type 1 immune response (IFNγ production, T cell cytotoxicity) and memory T cell differentiation. In correspondence with the IFNγ expression findings, it was observed that SV plus α4-1BB mAb coordinately upregulates T-bet in T cells on day 7 (FIG. 28E). This suggests that SV helps α4-1BB boost the type 1 immune response, which is critical for controlling tumor growth. SV or α4-1BB mAb alone could not induce high IFNγ production due to low T-bet upregulation. Eomesodermin (EOMES), another important transcription factor, is upregulated in activated T cells and is essential for memory CD8 T cell development. Both α4-1BB mAb and SV plus α4-1BB mAb induced high expression of EOMES on day 7 (FIG. 28F). The lack of both T-bet and EOMES results in a lower expression of CXCR3 in T cells and a drastic decrease in the number of tumor-infiltrating T cells [28]. The data disclosed herein are consistent with these observations. Elevated CXCR3 (FIG. 28D), T-bet and EOMES (FIGS. 28E and 28F) in T cells of the combined SV plus α4-1BB mAb treated animals, were found.

SV and α4-1BB mAb Stimulated Chemotaxis, Adhesion and Enhanced T Cell Infiltration and Activation in Tumor

Through RNA-Seq, a series of chemokines and chemokine receptors have been identified to be upregulated in SV plus α4-1BB mAb (FIG. 31A). Among those molecules, CCR5 upregulation was confirmed by flow cytometry (FIG. 31B). CCR5 potentiates CD4 T helper cell functions boosting overall anti-tumor responses [Gonzalez-Martin A et al., Oncoimmunology, 2012]. SV plus α4-1BB significantly was found to upregulate CD11a and ICAM-1(CD54). These two adhesion molecules are highly expressed on activated T cells. LFA-1 (CD11a/CD18)-ICAM-1 interaction is essential for the formation of immune synapses between T cell and APC [Walling B L et al., Front Immunol, 2018]. LFA-1 and ICAM-1 are also required for T cell-T cell homotypic aggregation and activation [Sabatos C A, et al., Immunity, 2008; Gerard A, et al., Nat Immunol. 2013]. α4-1BB mAb stimulation induced significant upregulation of CD11a and ICAM-1 in both CD4 and CD8 T cells whereas SV does not (FIGS. 31C-31E). In addition, T cell costimulatory molecule, OX40, was also significantly upregulated in T cells of mice treated with α4-1BB. (FIG. 31F, left). OX40 engagement promotes effector T cell function and survival [33 Croft M, et al., Immunol Rev. 2009]. ICOS, another CD4 T cell costimulatory molecule, was upregulated in SV or α4-1BB alone but upregulated most in the SV plus α4-1BB combination treatment, suggesting a synergistic effect exists (FIG. 31F, right).

TIL play a critical anti-tumor role and is an important marker for prognosis. Compared with untreated, the percentage of CD3 and CD8 T cells were increased about 2 fold after combination treatment (FIG. 31G). Ki67 were upregulated in those T cells which indicated active division (FIG. 32A). For untreated TIL, the frequency of Foxp3+ Treg cells was the highest (FIG. 32B) and CD8/Treg ratio was the lowest (FIG. 31H). Treatment enhanced the T-bet and EOMES expression in T cells (FIG. 32C-32D). NKG2D and granzyme B were highly upregulated in tumor infiltrating CD8 T cells (FIGS. 31I, and 32E). Overall, these data indicate that combination treatment enhanced T cell infiltration, division, activation, cytotoxicity and downregulated the inhibitory Treg population.

SV and α4-1BB mAb Synergistically Enhanced Oxidative Phosphorylation

T cell activation requires a quick consumption of energy through both enhanced glycolysis and oxidative phosphorylation [Wahl D R et al., Immunol Rev., 2012]. Metabolic switch is a major feature of T cell activation and memory T cell development [van der Windt G J et al., Immunol Rev., 2012]. GSEA KEGG analysis identified that the glycolysis gene set is upregulated in SV plus α4-1BB vs. untreated samples (FIG. 33A). This process quickly produces ATP and supports T cell migration and cytotoxicity in hypoxic or acidic microenvironments. IP A confirms that SV plus α4-1BB mAb synergistically enhanced oxidative phosphorylation (FIG. 33B).

Both oxygen consumption rate (OCR, represents oxidative phosphorylation) and extracellular acidification rate (ECAR, represents glycolysis) of all groups (FIG. 33C) was assessed. Compared with other groups, SV plus α4-1BB significantly increased both OCR and ECAR. This indicates that both glycolysis and oxidative phosphorylation are activated in T cells of animals treated with SV plus α4-1BB.

SV Plus Low Dose α4-1BB mAb Cured A20 Tumor Bearing Mice

To reduce the potential risk of cytotoxicity and expense of treatment with SV vectors plus α4-1BB, the study disclosed herein explored whether low doses of α4-1BB mAb and fewer injections would be as effective in curing tumor bearing mice as the higher doses and frequencies used in our initial studies. As demonstrated (FIGS. 34A and 34B), A20 tumor bearing mice can be completely cured by SV (3 times per week for 3 weeks) plus a low dose of α4-1BB mAb (50 g per week for 3 weeks). This reduces both the SV and α4-1BB mAb dosing requirements. The reduced dose of α4-1BB mAb would be helpful, as well, in preventing the α4-1BB mAb induced liver toxicity reported by some investigators [Bartkowiak T, et al., Clin Cancer Res., 2018].

All Tumor Cured Mice Acquired Long Lasting Antitumor Immunity

To investigate the memory response to A20 lymphoma, naive and tumor cured mice were inoculated with 3×10⁶A20 tumor cells. Only mice that had survived more than 4 months after 1st time of tumor challenge were chosen. In all tumor cured mice, we found that A20 lymphoma was completely rejected whereas naive mice were susceptible to A20 inoculation (FIG. 35A).

To confirm anti-tumor specificity has been elicited, IFNγ production of purified T cells in the presence or absence of tumor cells was measured by Elispot assay. T cells were isolated from naive and cured mice under SV plus α4-1BB treatment (4 months after treatment finished). Isolated T cells were co-cultured with A20 and CT26 tumor cells respectively. Co-culturing with A20 cells dramatically enhanced IFNγ production, whereas co-culturing with CT26 cells only slightly enhanced IFNγ production (FIG. 35B).

Next, cytotoxicity to both naive and cured mice under SV plus α4-1BB treatment (the same method as FIG. 27A) was measured. Compared with naive, cured mice had enhanced cytotoxicity to A20 lymphoma cells, but not to CT26 tumor cells. To confirm that this is mediated by T cells, the same experiment was done using purified T cells. Cured mice had enhanced cytotoxicity compared with naive mice (FIG. 35C).

To better understand differences between this memory T cell response and the initial treatment responses as observed on day 7, RNA-Seq was performed by using purified splenic T cells from all re-challenged groups. In T cells of these re-challenged mice we found only a few differentially expressed genes among the three treated groups (Table 5), indicating that tumor cured mice develop a very similar T cell gene expression profile regardless of treatment method. Compared with untreated, KEGG analysis indicates that TCR signaling is the highest upregulated pathway in SV plus α4-1BB group (FIG. 35D), indicating that continuously enhanced TCR signaling is critical for maintaining antitumor immunity.

TABLE 5

The SD expressed gene lists among all tumor cured mice groups.

gene
baseMean
log2FC
log2FCunshrunk
pvalue
padj

SV + α4-1BB recha vs SV rechal

Arl5c
1644.9
−0.7
−0.71
4.57E−07
0.014007

SV + α4-1BB rechal vs α4-1BB rechal

Prdm16
86.7
−1.85
−2.48
4.63E−07
0.001563

Scamp5
106.1
−1.59
−1.99
3.98E−06
0.010743

Klri1
117.3
−1.57
−1.98
5.79E−06
0.013016

Ighv2-2
457.3
−1.52
−1.94
1.05E−05
0.020265

Kcnj10
439.7
−1.45
−1.76
1.24E−05
0.020949

Slc40a1
1111.8
−1.41
−1.69
1.53E−05
0.022994

Trbv29
826.4
−1.3
−1.36
6.35E−12
8.57E−08

Tmcc3
416.3
−1.23
−1.35
1.28E−07
0.000575

Abcc3
1020.9
−1.2
−1.36
2.12E−05
0.02863

Arl5c
1644.9
−0.76
−0.77
2.43E−08
0.000164

SV rechal vs. α4-1BB rechal

Gzmk
1000.8
−1.78
−2.44
1.81E−06
0.033784

Penk
2127.9
−1.35
−1.6
6.37E−06
0.038894

Smoc2
154.2
−1.85
−2.98
8.65E−06
0.038894

Spag6
6.7
−1.59
−7.14
8.74E−06
0.038894

Ighv3-5
85.6
−1.86
−3.04
1.08E−05
0.038894

Wipf3
150.6
−1.84
−5.01
1.25E−05
0.038894

The conventional view of oncolytic virus therapy against tumors is that it requires selective infection of cancer cells resulting in the induction of cancer cell lysis and apoptosis. TAAs, released from dead tumor cells, attract and further stimulate an antitumor immune response. The study described herein found that encoding a TAA is not necessary for SV vectors plus α4-1BB mAb therapy to be fully successful. SV vectors lacking an A20 lymphoma TAA were able to treat A20 lymphoma and, in combination with α4-1BB mAb, eradicated the growing tumors. This is particularly important when effective immune reactive TAAs are unknown. It is possible that the immunotherapeutic response of SV vectors plus α4-1BB mAb is independent of whether a tumor is “cold” (i.e., having few TAAs or mutation-specific neoantigens capable of promoting robust T cell activation) or “hot.”

The study describe herein showed that both NKG2D (KLRK1) and granzyme B are highly expressed under combination treatment. This massive nonspecific activation is critical for controlling tumor growth at an early time point (day 7). This step is also important for inducing anti-tumor specificity that is mediated by TAAs released from dead tumor cells due to nonspecific killing. After tumor regression, T cells from treated animals were able maintain the ability to produce IFNγ and acquired immunological memory to rapidly reject A20 lymphoma rechallenges. IFNγ production from purified T cells of cured mice was significantly enhanced after encountering A20 tumor cells. This demonstrates that anti-tumor specificity is fully established in cured mice. Upregulated molecular pathways of responsive T cells induced by SV vectors and a 4-1 in mAbs alone and in combination were identified and compared in the study described herein. The combination of SV and α4-1BB mAb has a synergistic effect and represents a potent and robust therapeutic treatment able to cure B lymphomas and provide long term protection in a preclinical model.

In conclusion, SV vectors in combination with α4-1BB mAb completely eradicated a B-cell lymphoma in a preclinical mouse model, a result that could not be achieved with either treatment alone. Tumor elimination involves a synergistic effect of the combination that significantly boosts T cell cytotoxicity, IFN-γ production, migration, tumor infiltration and oxidative phosphorylation. In addition, all mice that survived after treatment developed long lasting antitumor immunity. The studies disclosed herein provides a novel, alternative method for B cell lymphoma treatment and describes a rationale to help translate SV vectors plus agonistic mAbs into clinical applications.

Example 4: Sindbis Viral Vector Expressed NY-ESO-1 and IL-12 Enhances Survival of Subjects with Established Tumors

The study described herein investigates the effect of administering a tumor associated antigen and an immunostimulatory molecule, as expressed by a Sindbis viral vector on anti-tumor response and survival in a subject with an established tumor. Previous studies, demonstrated vectors encoding TAAs, such as NY-ESO-I, could cure CT26-NY-ESO-1 tumors [Galon J, et al., Nature reviews Drug discovery 2019; Gupta S, et al., Frontiers in oncology 2017]. However, while this approach has been effective in enhancing the immune response to and clearance of established tumors of colon and prostate cancers, the efficacy in curing other cancers, e.g. ovarian cancer has been limited. Therefore, an approach of administering a combination of a SV expressed immunostimulatory molecule, IL-12 along with the SV-NY-ESO-1, to a subject with an established tumor was tested.

Combination of NY-ESO-1 and IL-12 Expressed by Separate Sindbis Viral Vectors Enhances Survival of Subjects with Established Tumors

The study described herein investigates the effect of administering IL-12 and NY-ESO-1, both expressed by separate Sindbis viral vectors, on established tumors. C57/B16 albino (female) mice re-injected with Alm5-2Fluc-17 ovarian cancer cells to establish a tumor (FIG. 19), and treated with either a SV vector expressing IL-12 (SV-IL-12), a SV vector expressing NY-ESO-1 (SVNYESO) or a 50% mix of a SV-IL-12 and a SVNYESO (SV-NYESO_SV-IL12).

A Sindbis replicon expressing NYESO-1 cDNA (SV-NYESO1) was made by PCR amplification of the NYESO-1 gene from the pReceiver-M02 plasmid. Expression of the NYESO-1 gene was confirmed by western blot. NYESO-1 was detected by western blot following standard protocol, using as a primary antibody the anti-NYESO-1 clone E978 (Upstate) at a dilution 1/5,000 in Tris-buffered saline-Tween (TBS-T) with 5% non-fat milk. SV.IL12 plasmid used in this study has been published in 2002 [Tseng J C et al., J Natl Cancer Inst. 2002]. To construct a Sindbis viral vector containing genes for interleukin 12 (IL-12), the Sindbis viral vector SinRep/2PSG was first constructed, which contains a secondary subgenomic promoter that is responsive to the Sindbis replicase. Two DNA oligonucleotide primers (sequence 5′ CGCGTAAAGCATCTCTACGGTGGTCCTAATAGTGCATG-3′; SEQ ID NO: 29) and its complementary strand 5′CACTATTAGGACCACCGTCGAGATGCTTTA-3′; SEQ ID NO: 30) containing the subgenomic promoter sequence were annealed and ligated into the MluI and SphI sites of the SinRep plasmid. The murine IL-12 α subunit gene (mp35; ATCC 87596) and the IL-12 β subunit gene (mp40; ATCC 87595) were subcloned into the MluI and the StuI sites of SinRep/2PSG, respectively, to produce the Sin-Rep/IL12 plasmid.

As expected the SV-IL-12 treatment group showed a better percentage survival of mice with tumor over the SVNYESO treatment group and the untreated (control) group. However, a synergistically higher showed enhanced percentage survival rate was observed in the SV-NYESO_SV-IL12 in comparison to the SV-IL-12 treatment group (FIG. 36). The results described herein clearly show the possibility of using a combination of SV vectors expressing IL-12 and NY-ESO-1, for treatment of cancers that may be resistant to treatment with a SV expressing a tumor associated antigen.

Combination of NY-ESO-1 and IL-12 Expressed by the Same Sindbis Viral Vectors Enhances Survival of Subjects with Established Tumors

The study described herein investigates the effect of administering IL-12 and NY-ESO-1, both expressed by the same Sindbis viral vector, on established tumors. C57/B16 albino (female) mice re-injected with Alm5-2Fluc-17 ovarian cancer cells to establish a tumor (FIG. 19), and treated with either a SV vector expressing IL-12 (SV-IL-12), a SV vector expressing NY-ESO-1 (SVNYESO) or a Sindbis viral vector that expresses both IL-12 and NYESO (SV-NYESO_SGP2_IL12). As shown in FIG. 36, the SV-IL-12 treatment group showed a better percentage survival of mice with tumor over the SVNYESO treatment group and the untreated (control) group. As expected, the SVNYESO treatment group and the untreated (control) group showed similar survival rate, thereby showing that certain tumors are resistant to treatment with a SV expressing a tumor associated antigen (TAA) like NY-ESO-1. A synergistically higher enhanced percentage survival rate was observed in the SV-NYESO_SGP2_IL12 treatment group, in comparison to the SV-IL-12 treatment group (FIG. 37).

The study described herein, provides plasmid constructs for expressing NY-ESO-1, IL-12 and anti-OX40 in a SV vector. The study described herein, provides plasmid constructs encoding IL-12 α and b subunits (FIG. 38), anti-OX40 IgG2a heavy and light chains (FIG. 39), a single chain antibody to OX40 (OX40 ScFv) (FIG. 40), a human NY-ESO-1 (FIG. 41) and an OX40 ligand fused to a Fc peptide (OX40L-Fc T2A) and a NY-ESO-1 with a termination peptide sequence T2A in between (FIG. 42).

In summary, the results of the study described herein clearly show the possibility of using a SV vectors expressing both IL-12 and NY-ESO-1, for treatment of cancers that may be resistant to treatment with a SV expressing a tumor associated antigen.

TUMOR IMMUNOTHERAPY USING SINDBIS VIRAL VECTORS AND AGONIST MONOCLONAL ANTIBODIES

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