US20190142967A1

US20190142967A1 - Immunomodulatory Oncolytic Adenoviral Vectors, and Methods of Production and Use Thereof for Treatment of Cancer

Info

Publication number: US20190142967A1
Application number: US16/253,056
Authority: US
Inventors: Daniel Hicklin; Kenneth Nelson Wills; Cynthia Seidel-Dugan; William Winston; Philipp Steiner
Original assignee: Trieza Therapeutics Inc
Current assignee: Epicentrx Inc
Priority date: 2016-12-30
Filing date: 2019-01-21
Publication date: 2019-05-16
Also published as: US20210393803A1; WO2018126282A1; EP3562516A1; CA3048998A1; US10232053B2; US20180185515A1; US20200014798A1; JP2020503342A

Abstract

Disclosed herein are compositions and methods for treating cancer in a subject. This involves administering an oncolytic virus containing a heterologous DNA sequence encoding one or more immunomodulatory and/or immunostimulatory polypeptide(s) of interest to the subject under conditions effective to enhance an anti-tumor immune response in the subject, and to treat cancer. It also relates to a method of enhancing the delivery to and distribution within a tumor mass of therapeutic viruses.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser. No. 15/834,690, filed Dec. 7, 2017, which claims the benefit of U.S. Provisional Application Nos. 62/572,206, filed Oct. 13, 2017; 62/440,646, filed Dec. 30, 2016; and 62/440,670, filed Dec. 30, 2016, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 21, 2017, is named 42022US_CRF_sequencelisting.txt and is 266,186 bytes in size.

FIELD

The invention described herein relates generally to the fields of immunology, virology, molecular biology, and more specifically to oncolytic adenoviruses having therapeutic applications.

BACKGROUND

Cancer is a leading cause of death in the United States and elsewhere. Depending on the type of cancer, it is typically treated with surgery, chemotherapy, and/or radiation. These treatments often fail, and it is clear that new therapies are necessary, to be used alone or in combination with current standards of care.
Originally conceived as solely tumor-lysing therapeutics, viruses that can preferentially target tumor cells for destruction are being used experimentally as vectors for the delivery of immune-stimulating cargo. The propagation of a lasting anti-tumor host immune response in combination with the destruction of tumor cells is described in, e.g., Lichty et al., 2014, Nature Reviews Cancer, 14: 559-567.
Clinical trials employing adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus and vaccinia as oncolytic viruses have suggested that these platforms may all be safe treatment approaches.
Adenoviruses are medium-sized (90-100 nm), non-enveloped icosahedral viruses, which have double stranded linear DNA of about 36 kilobase pairs in a protein capsid. The viral capsid has fiber structures that participate in attachment of the virus to the target cell. First, the knob domain of the fiber protein binds to the receptor of the target cell (e.g., CD46 or Coxsackie and adenovirus receptor (CAR)), secondly, the virus interacts with an integrin molecule and thirdly, the virus is endocytosed into the target cell. Next, the viral genome is transported from endosomes into the nucleus and the replication machinery of the target cell is utilized also for viral purposes.
The adenoviral genome has early (E1-E4), intermediate (IX and IVa2) and late genes (L1-L5), which are transcribed in sequential order. Early gene products affect defense mechanisms, cell cycle and cellular metabolism of the host cell. Intermediate and late genes encode structural viral proteins for production of new virions.
More than 60 different serotypes of adenoviruses have been found in humans. Serotypes are classified into six subgroups A-F and different serotypes are known to be associated with different conditions i.e. respiratory diseases, conjunctivitis and gastroenteritis. Adenovirus serotype 5 (Ad-5) is known to cause respiratory diseases and it is the most common serotype studied in the field of gene therapy. In the first Ad5 vectors E1 and/or E3 regions were deleted enabling insertion of foreign DNA to the vectors.
Furthermore, deletions of other regions as well as further mutations have provided extra properties to viral vectors. Indeed, various modifications of adenoviruses have been suggested for achieving efficient anti-tumor effects.
Adenoviral vectors mediate gene transfer at a high efficacy compared to other vector systems, and they are currently the most frequently used vectors for cancer gene therapy. A non-replicating p53 expressing adenoviral vector and a replication selective virus (H101) have received regulatory approval in China. Several attempts to achieve tumor-selective control through the insertion of tumor selective promoter elements upstream of the E1 or other adenovirus critical promoters have had variable levels of success, but ultimately were limited by “leaky” gene expression of viral proteins in non-tumor cells and by reduced ability to propagate and lyse tumor cells compared to wild-type virus infections.

SUMMARY

In a first aspect, a pharmaceutical composition is provided comprising an effective amount of a recombinant adenoviral vector comprising: a transgene insertion site located between the start site of adenoviral E1b-19K and the start site of adenoviral E1b-55K, wherein a first DNA sequence and a second DNA sequence are each inserted into the transgene insertion site; wherein the first DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, and wherein the second DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand; and wherein the adenoviral vector comprises a modified adenoviral E1a regulatory sequence wherein at least one Pea3 binding site, or a functional portion thereof, of the recombinant adenoviral vector is modified or deleted.
In one embodiment, the adenoviral vector comprises an IRES element or encodes a self-cleaving 2A peptide sequence between the first DNA sequence and the second DNA sequence. In another embodiment, the vector comprises a modified E3 region. In another embodiment, the vector comprises an intact E3 region. In another embodiment, the vector comprises a third DNA sequence inserted into the E3 region, wherein the third DNA sequence encodes a polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, or a human OX40 ligand. In one embodiment, the chimeric human IL-12 polypeptide comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide. In another embodiment, the chimeric human IL-12 polypeptide comprises a sequence as set forth in SEQ ID NO:46.
In one embodiment, the adenoviral vector comprises a nucleic acid sequence at least 95% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 85% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 75% identical in an E3 region to vector d1327.
In one embodiment, the pharmaceutical composition is formulated for systemic administration.
In another embodiment, the pharmaceutical composition is formulated for systemic administration.
In a second aspect is provided a pharmaceutical composition comprising an effective amount of a recombinant adenoviral vector comprising: a first transgene insertion site located between the start site of adenoviral E1b-19K and the start site of adenoviral E1b-55K; a second transgene insertion site located in adenoviral E3 region; a first DNA sequence, present in the first transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand; and a second DNA sequence, present in the second transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, wherein the adenoviral vector comprises a modified adenoviral E1a regulatory sequence. In one embodiment, the pharmaceutical composition further comprises a third DNA sequence inserted into a third transgene insertion site, encoding one or a plurality of polypeptides selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand.
In another embodiment, at least one of the first DNA sequence, the second DNA sequence, and the third DNA sequence independently comprises an IRES element and/or a self-cleaving 2A peptide. In one embodiment, at least one E1a regulatory sequence Pea3 binding site, or a functional portion thereof of the adenoviral vector, is modified or deleted. In another embodiment, a sequence between two Pea3 sites of the adenoviral vector is deleted. In one embodiment, the adenoviral vector comprises a modified E3 region. In another embodiment the adenoviral vector comprises a nucleic acid sequence at least 95% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 85% identical in an E3 region to vector d1327. In another embodiment, the adenoviral vector comprises a nucleic acid sequence at least 75% identical in an E3 region to vector d1327.
In one embodiment, the chimeric human IL-12 polypeptide comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide.
In another aspect is provided a method for treating a tumor in a human subject in need thereof, comprising administering to the human with the tumor a therapeutic amount of the pharmaceutical composition of the first aspect by systemic or intratumoral administration.
In another aspect is provided method for treating a tumor in a human subject in need thereof, comprising administering to the human with the tumor a therapeutic amount of the pharmaceutical composition of the second aspect by systemic or intratumoral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cartoon illustrating transcription factor binding regulation of conformational structure/activity of E1a enhancer region.

FIG. 2 is an illustration of a vector map demonstrating an adenoviral vector expressing two immunomodulatory polypeptides.

FIG. 3 is an illustration of a vector map demonstrating an adenoviral vector expressing at least one immunomodulatory polypeptide at an E4 site.

FIG. 4 is an illustration of the TRZ200 virus genome: an E1b 19k empty, E3-deleted virus.

FIG. 5 is an illustration of a vector map showing the plasmid comprising the TRZ6 hIL-12 plasmid.

FIG. 6A is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6A shows the activity of various oncolytic viruses compared to the empty virus (“d19k”), 38 days after cell implantation (primary tumor). Black bars represent virus alone and hatched bars represent virus+anti-PD-L1 antibody.

FIG. 6B is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6B is a graph comparing the average tumor size (primary tumor) over time of tumors injected with virus buffer (black circles), empty vector (blue solid squares), anti-PD-L1 alone (antibody given on days 16, 20, 24, and 28, right-hand arrow in each pair of arrows), each of five viruses alone (CTLA-4, IL-12, IL-7, CD70, and IL-10) or combined with anti-PD-L1 antibody.

FIG. 6C is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6C is a graph comparing the average tumor size (primary tumor) over time of tumors injected with virus buffer (black circles), empty vector (blue solid squares), anti-PD-L1 alone (antibody given on days 16, 20, 24, and 28, right-hand arrow in each pair of arrows), showing three viruses—OX40L, CD40L, and GM-CSF alone or combined with anti-PD-L1 antibody.

FIG. 6D is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6D shows virus buffer and control IgG only (left) and virus buffer and anti-PD-L1 antibody (right); the thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6E is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6E shows d19k (empty virus) and control IgG only (left) and d19k and anti-PD-L1 antibody (right); the thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6F is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6F shows CTLA-4 virus with control IgG (left) or anti-PD-L1 (right); the thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6G is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6G shows IL-12 virus with control IgG (left) or anti-PD-L1 antibody (right); the thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6H is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6H shows GM-C S F virus with control IgG (left) or anti-PD-L1 antibody (right); The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6I is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6I shows IL-7 virus with control IgG (left) or anti-PD-L1 antibody (right); The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6J is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6J shows CD40L virus with control IgG (left) or anti-PD-L1 antibody (right); The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6K is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6K shows L10 trap virus with control IgG (left) or anti-PD-L1 antibody (right); The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 6L is graph(s) showing results of treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising transgene(s), with or without anti-PD-L1. FIG. 6L shows OX40L virus with control IgG (left) or anti-PD-L1 antibody. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows).

FIG. 7A is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7A shows virus buffer only.

FIG. 7B is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7B shows empty virus only (TRZ000).

FIG. 7C is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7C shows TRZ010 (IL-10trap)+empty virus.

FIG. 7D is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7D shows TRZ011 (OX40 ligand)+empty virus.

FIG. 7E is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7E shows TRZ009 (CD70)+empty virus.

FIG. 7F is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7F shows TRZ007 (IL-7)+empty virus.

FIG. 7G is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7G shows TRZ002 (IL-12)+empty virus.

FIG. 7H is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7H shows TRZ004 (GM-CSF)+empty virus.

FIG. 7I is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7I shows TRZ003 (flagellin)+empty virus.

FIG. 7J is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7J shows TRZ002+TRZ010.

FIG. 7K is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7K shows TRZ002+TRZ007.

FIG. 7L is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7L shows TRZ007+TRZ010.

FIG. 7M is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7M shows TRZ011+TRZ004.

FIG. 7N is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7N shows TRZ009+TRZ003.

FIG. 7O is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7O shows TRZ002+TRZ009.

FIG. 7P is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7P shows TRZ007+TRZ009.

FIG. 7Q is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7Q shows TRZ007+TRZ004.

FIG. 7R is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7R shows TRZ002+TRZ011.

FIG. 7S is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7S shows TRZ010+TRZ004.

FIG. 7T is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7T shows TRZ002+TRZ004.

FIG. 7U is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7U shows virus buffer and anti-PD-L1.

FIG. 7V is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7V shows TRZ002+empty virus+anti-PD-L1.

FIG. 7W is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7W shows TRZ009+anti-PD-L1.

FIG. 7X is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7X shows TRZ007+empty virus+anti-PD-L1.

FIG. 7Y is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7Y shows TRZ002+TRZ007+anti-PD-L1.

FIG. 7Z is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7Z shows TRZ007+TRZ009+anti-PD-L1.

FIG. 8A is schematic representations of the rationale for modifications of the E1a Enhancer region of adenoviral vectors, in which Pea3 sites are altered, moved, or deleted in order to produce vectors with a variety of expression characteristics. FIG. 8A is an illustration of a wild-type adenoviral genome (top) and the TAV-255 adenoviral construct in which residues -305 to -255 are deleted (bottom).

FIG. 8B is schematic representations of the rationale for modifications of the E1a Enhancer region of adenoviral vectors, in which Pea3 sites are altered, moved, or deleted in order to produce vectors with a variety of expression characteristics. FIG. 8B is an illustration of the potential method of action of the TAV-255 construct, in which the deletion removes Pea3 sites II and III, which moves Pea3 sites IV and V closer to the promoter as distal control elements.

FIG. 9A is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with dual transgene (expressed from E1 and E3) oncolytic viruses comprising transgene(s), with either anti-PD-L1 antibody or anti-PD-1 antibody treatment. FIG. 9A is a cartoon of the contrast between the single transgene constructs (top) used in the virus mixing examples, and the dual transgene vectors used to make the E1/E3 transgene adenoviruses.

FIG. 9B is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with dual transgene (expressed from E1 and E3) oncolytic viruses comprising transgene(s), with either anti-PD-L1 antibody or anti-PD-1 antibody treatment. FIG. 9B shows the results of mice injected i.t. with Empty virus+/−anti-PD-L1 or with TRZ-409 (IL-12/IL-7 dual transgene)+/−anti-PD-L1. The tumor volume of the injected tumor is shown in the left panel, and the tumor volume of the contralateral tumor is shown in the right panel. For each pair of arrows, the left arrow indicates virus injection and the right arrow indicates anti-PD-L1 injection. As can be seen for both the primary and contralateral tumors, TRZ-409 injection reduced tumor volume significantly compared to empty virus. The combination with anti-PD-L1 was slightly more efficacious in this study.

FIG. 9C is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with dual transgene (expressed from E1 and E3) oncolytic viruses comprising transgene(s), with either anti-PD-L1 antibody or anti-PD-1 antibody treatment. FIG. 9C shows the results of a second experiment using the inverse of TRZ-409, TRZ-403, in which the IL-7 transgene occupies the E1 region and the IL-12 gene occupies the E3 region (which has a stronger promoter than the E1). As can be seen in the Figure, TRZ-403 strongly inhibits primary and distant tumor growth, with or without anti-PD-L1.

FIG. 9D is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with dual transgene (expressed from E1 and E3) oncolytic viruses comprising transgene(s), with either anti-PD-L1 antibody or anti-PD-1 antibody treatment. As can be seen in the Figure, TRZ-403 strongly inhibits primary and distant tumor growth, with or without anti-PD-L1. Plasma levels of IL-12 and IFN-γ (FIG. 9D) for all mice injected with TRZ-403 show that expression of the transgenes is well tolerated. The study was extended for all mice having tumors over 500 mm³.

FIG. 9E is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with dual transgene (expressed from E1 and E3) oncolytic viruses comprising transgene(s), with either anti-PD-L1 antibody or anti-PD-1 antibody treatment. As can be seen in the FIG. 9E, mice injected with TRZ-403 had a much higher percentage of survival over TRZ-409 at the time end of the study (70 days). Mice having primary tumors injected with TRZ-403 (IL-7+IL-12), TRZ-403+anti-PD-L1, TRZ-403+control IgG, or TRZ-409 (IL-12+1L-7), and controls including untreated mice and mice injected with empty vector TRZ-d19K with anti-PD-L1, anti-PD-1, or a control IgG. As can be seen in the Figure, mice injected with TRZ-403 had a much higher percentage of survival over TRZ-409 at the time end of the study (70 days). Both TRZ-403 and TRZ-409 showed efficacy over the controls; all mice receiving control injections were deceased by day 56 of the study.

FIG. 9F is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with dual transgene (expressed from E1 and E3) oncolytic viruses comprising transgene(s), with either anti-PD-L1 antibody or anti-PD-1 antibody treatment. FIG. 9F compares a mixture of two single transgene viruses with a dual transgene virus having the same transgenes. As can be seen in the primary tumor (left panel) TRZ-403 showed the most efficacy in reducing tumor volume, followed by the mixture of IL-7 and IL-12 single transgene viruses. The single transgene IL-12 virus showed a small amount of efficacy by comparison. In the contralateral tumor, however (right panel) only the dual transgene virus, TRZ-403, reduced the tumor volume, showing significant superiority over the mixture of viruses.

FIG. 10 is graph(s) showing treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising transgene(s), with or without anti-PD-L1 treatment. The left-hand panel of represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and antibody (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). FIG. 7AA shows TRZ002+TRZ009+anti-PD-L1.

DEFINITIONS

The term “replicating virus” is meant to include a virus that undergoes the process of intracellular viral multiplication, consisting of the synthesis of proteins, nucleic acids, and sometimes lipids, and their assembly into a new infectious particle.
As used herein, the term “adenovirus” refers to any of a group of DNA-containing viruses (small infectious agents) that cause conjunctivitis and upper respiratory tract infections in humans. Adenoviral vectors are described in Peng, Z., “Current Status of Gendicine in China: Recombinant Human Ad-p53 Agent for Treatment of Cancers,” Hum Gene Ther 16:1016-1027 (2005); No authors listed, “The End of the Beginning: Oncolytic Virotherapy Achieves Clinical Proof-of-concept,” Mol Ther 13:237-238 (2006); Vile et al., “The Oncolytic Virotherapy Treatment Platform for Cancer: Unique Biological and Biosafety Points to Consider,” Cancer Gene Ther 9:1062-1067 (2002); Harrison et al., “Wild-type Adenovirus Decreases Tumor Xenograft Growth, but Despite Viral Persistence Complete Tumor Responses are Rarely Achieved—Deletion of the Viral Elb-19-kD Gene Increases the Viral Oncolytic Effect,” Hum Gene Ther 12:1323-1332 (2001); Kim et al., “Clinical Research Results with d11520 (Onyx-015), a Replication-selective Adenovirus for the Treatment of Cancer: What Have We Learned?,” Gene Ther 8:89-98 (2001); and Thorne et al., “Oncolytic Virotherapy: Approaches to Tumor Targeting and Enhancing Antitumor Effects,” Semin Oncol 32:537-548 (2005), each of which is hereby incorporated by reference in their entirety. Adenoviral positions referenced herein are to positions in Adenovirus type 5 (GenBank 10 accession #M73260; the virus is available from the American Type Culture Collection, Rockville, Md., U.S.A., under accession number VR-5). It will be understood that corresponding positions can be identified in other adenovirus vectors by alignment using BLAST 2.0 under default settings (Altschul et al., J. Mol. Biol. 215:403-410 (1990)). Software for performing BLAST analyses is publicly available on the Web through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
Current research in the field of viral vectors is producing improved viral vectors with high-titer and high-efficiency of transduction in mammalian cells (see, e.g., U.S. Pat. No. 6,218,187 to Finer et al., which is hereby incorporated by reference in its entirety). Such vectors are suitable in the present invention, as is any viral vector that comprises a combination of desirable elements derived from one or more of the viral vectors described herein. It is not intended that the expression vector be limited to a particular viral vector.
Certain “control elements” or “regulatory sequences” can also be incorporated into the vector-construct. The term “control elements” refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence(s) in a recipient cell. Not all of these control elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed, and translated in an appropriate host cell.
The term “promoter region” is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3′-direction) coding sequence. Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources, including genes in yeast, insect, and mammalian cells, and viruses. Analogous control elements, i.e., promoters, are also found in prokaryotes. Such elements may vary in their strength and specificity. For example, promoters may be “constitutive” or “inducible.”
A constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism. Examples of some constitutive promoters that are widely used for inducing expression of transgenes include the cytomegalovirus (CMV) early promoter, those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Pat. No. 6,002,068 to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter, which is a gene product known to accumulate in many cell types.
To ensure efficient expression, 3′ polyadenylation regions can be present to provide for proper maturation of the mRNA transcripts. The 3′ polyadenylation region will preferably be from the adenovirus sequence downstream of the inserted transgene, but the native 3′-untranslated region of the immunomodulatory gene may be used, or an alternative polyadenylation signal from, for example, SV40, particularly including a splice site, which provides for more efficient expression, could also be used. Alternatively, the 3′-untranslated region derived from a gene highly expressed in a particular cell type could be fused with the immunomodulatory gene.
“Inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity of a described target protein, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term “modulator” includes inhibitors and activators. Inhibitors are agents that, e.g., inhibit expression or bind to, partially or totally block stimulation or protease inhibitor activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, e.g., antagonists. Activators are agents that, e.g., induce or activate the expression of a described target protein or bind to, stimulate, increase, open, activate, facilitate, enhance activation or protease inhibitor activity, sensitize or up regulate the activity of described target protein (or encoding polynucleotide), e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists and agonists (e.g., small chemical molecules, antibodies and the like that function as either agonists or antagonists). Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to cells expressing the described target protein and then determining the functional effects on the described target protein activity, as described above. Samples or assays comprising described target protein that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition of a described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of the described target protein is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or higher.
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain. Within the variable region of the light chain is a C-terminal portion known as the J region. Within the variable region of the heavy chain, there is a D region in addition to the J region. Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are directly involved in antigen binding. Proceeding from the amino-terminus, these regions are designated CDR1, CDR2 and CDR3, respectively. The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino-terminus, these regions are designated FR1, FR2, FR3, and FR4, respectively. The locations of CDR and FR regions and a numbering system have been defined by, e.g., Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office (1991)).
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (V_H) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to V_H-C_H1by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy (1985)). “Monoclonal” antibodies refer to antibodies derived from a single clone. Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)).
A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
A “humanized” antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for instance, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169-217 (1994).
As used herein, the terms “treat” and “treating” in the context of the administration of a therapy refers to a treatment/therapy from which a subject receives a beneficial effect, such as the reduction, decrease, attenuation, diminishment, stabilization, remission, suppression, inhibition or arrest of the development or progression of cancer, or a symptom thereof. In certain embodiments, the treatment/therapy that a subject receives results in at least one or more of the following effects: (i) the reduction or amelioration of the severity of cancer and/or a symptom associated therewith; (ii) the reduction in the duration of a symptom associated with cancer; (iii) the prevention in the recurrence of a symptom associated with cancer; (iv) the regression of cancer and/or a symptom associated therewith; (v) the reduction in hospitalization of a subject; (vi) the reduction in hospitalization length; (vii) the increase in the survival of a subject; (viii) the inhibition of the progression of cancer and/or a symptom associated therewith; (ix) the enhancement or improvement the therapeutic effect of another therapy; (x) a reduction or elimination in the cancer cell population; (xi) a reduction in the growth of a tumor or neoplasm; (xii) a decrease in tumor size; (xiii) a reduction in the formation of a tumor; (xiv) eradication, removal, or control of primary, regional and/or metastatic cancer; (xv) a decrease in the number or size of metastases; (xvi) a reduction in mortality; (xvii) an increase in cancer-free survival rate of patients; (xviii) an increase in relapse-free survival; (xix) an increase in the number of patients in remission; (xx) a decrease in hospitalization rate; (xxi) the size of the tumor is maintained and does not increase in size or increases the size of the tumor by less 5% or 10% after administration of a therapy as measured by conventional methods available to one of skill in the art, such as MRI, X-ray, and CAT Scan; (xxii) the prevention of the development or onset of cancer and/or a symptom associated therewith; (xxiii) an increase in the length of remission in patients; (xxiv) the reduction in the number of symptoms associated with cancer; (xxv) an increase in symptom-free survival of cancer patients; and/or (xxvi) limitation of or reduction in metastasis. In some embodiments, the treatment/therapy that a subject receives does not cure cancer, but prevents the progression or worsening of the disease. In certain embodiments, the treatment/therapy that a subject receives does not prevent the onset/development of cancer, but may prevent the onset of cancer symptoms.
As used herein, the term “in combination” in the context of the administration of (a) therapy(ies) to a subject, refers to the use of more than one therapy. The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. A first therapy can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy to a subject.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, e.g., of the entire polypeptide sequences or specific region, if indicated), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.”
For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full-length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison are conducted by a BLAST 2.0 algorithm, which is described in Altschul et al. (1990) J Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods of treating cancer in a subject. Provided are replicating viral vectors, specifically oncolytic adenoviral vectors, that contain one or more recombinant nucleic acid sequences encoding therapeutic polypeptides.
Oncolytic adenovirus (AV) expressing a transgene display highly oncolytic and immunogenic properties. Therefore, the recombinant adenoviral vectors provided herein have the potential to have broad activity against a primary tumor infected with adenovirus but also against tumors in the metastatic disease setting. In some embodiments, the metastatic tumors need not be directly injected with virus nor does the virus injected into the primary site travel to the metastatic setting (e.g. through the blood stream). However, due to expression of an immune stimulatory transgene from the adenovirus (for example, including but not limited to an E1b 19K deleted region in an exemplary vector such as TAV-255 (e.g., TAV-255 Δ19)), the immune system will be primed to fight cancer systemically in the entire body of the cancer patient as long as the metastatic tumor cells express the same tumor antigens as the primary tumor cells. Since metastases are derived from the primary tumors and genetically very similar to the primary tumor cells, metastatic tumor growth will be inhibited, in some embodiments, to the same extent as the primary tumor.
Provided are adenoviruses as described herein. In another aspect, a cell transformed with any one of the recombinant adenoviruses described herein is provided.
In another aspect, a method is provided of selectively expressing a peptide in a target cell comprises contacting the target cell with any one of the recombinant adenoviruses described herein. In one aspect, the recombinant adenovirus comprises a E1a regulatory sequence deletion mutant operably linked to a nucleotide sequence encoding a peptide, e.g., a peptide associated with viral replication or with cancer.
Also provided herein are methods of adenoviral therapy that utilize the oncolytic adenoviruses of the instant invention as adenoviral vectors that express one, two, or more recombinant immunomodulatory genes. The oncolytic adenovirus contains a heterologous gene that encodes a therapeutic protein, incorporated within the viral genome, such that the heterologous gene is expressed within an infected cell. A therapeutic protein, as used herein, refers to a protein that provides one or more therapeutic benefit when expressed in a given cell. In particular, the therapeutic benefit includes recruitment of the host immune system to the tumor.

Modified Regulatory Regions

E1a is the first protein produced by an adenovirus upon infection of a cell, activating other adenoviral promoters and facilitating infected cells to enter cell division. Rendering expression of this protein under tumor-selective control is an effective means of limiting expression of viral proteins and oncolysis to tumor cells.
Normal cells require mitogenic growth signals (GS) before they can move from a quiescent state into an active proliferative state. Tumor cells are able to generate many of their own growth signals or mimic normal growth signals, and transcription factors such as E2F1 and Pea3 are commonly overexpressed in tumor cells at levels that can cooperate in forming conformational structures optimal for driving E1a transcription during adenovirus infection and replication. (Hanahan D, Weinberg R A. The Hallmarks of Cancer. Cell 2000; 100: 57-70; de Launoit Y, Chotteau-Lelievre A, Beaudoin C, Coutte L, Netzer S, Brenner C et al., The PEA3 Group of ETS-Related Transcription Factors: Role in Breast Cancer Metastasis, Adv Exp Med Biol 2000; 480: 107-116; Bruder J T, Hearing P: Cooperative Binding of EF-1A to the E1A Enhancer Region Mediates Synergistic Effects on E1A Transcription During Adenovirus Infection. J Virol 1991; 65:5084-5087).
Small deletions selectively targeting the binding sites for E2F1 and Pea3 sites in the E1a enhancer region are an alternative and less disruptive method than complete replacement of the E1a enhancer region with a transcriptionally restricted promoter element. Cooperative binding and transcriptionally optimized conformation of the E1a enhancer region could still take place due to the over-abundance of transcription factors found in tumor cells, while in normal, non-dividing cells, the disruption of binding sites would further inhibit the ability to form optimized conformations in the limiting level of mitogenic growth signals.
Thus, disclosed herein are methods of engineering adenoviral vectors via the Pea3 binding sites. The five Pea3 transcription factor binding sites, also known as E1AF (or originally as EF-1A-enhancer binding factor to the E1a core motif) have differential effects on the production of E1a mRNA levels as demonstrated by specific deletions of individual and paired sites. The murine Pea3 sites are described herein as Pea3 sites I, II, III, IV, and V. The main binding sites for Pea3 are sites I and III, while sites II, IV, and V are slightly degenerate versions. Pea3 binds cooperatively between sites II and III, IV and V, and II and I, and this cooperative binding activates E1a transcription. (see, e.g., Hearing (J. Virol 65, 1991, Mol Cell Biol 9, 1989, and Nuc Acids Res 20, 1992). Pea3 itself is a dimer of both α
and β subunits, where the α subunit makes the primary DNA contact and the β subunit forms a heteromultimeric complex with the β subunit both in solution or on a dimeric binding site. During normal infection conditions, binding at sites I and III, followed by the cooperative binding at site II would cause conformational changes that serve to bring this protein/DNA complex closer to the activation transcription factor (ATF) binding site and TATA box for full activation of E1a mRNA expression. The two lower affinity sites, Pea3 IV and Pea3 5, do not appear to contribute much to activation under these normal circumstances, as they may be too far away or unoccupied. Deletion of site II causes the greatest reduction in transcriptional activity, pointing to the importance of the cooperative binding effect it has for sites III and I in activating transcription, presumably through a conformational change. Deletion of either site III or I had much less of a reduction, since presumably you could still have cooperative binding between site II and the remaining site III or I. Deletion of both sites I and III (but not II) reduces transcription to the levels seen with Pea3 site II deletion alone, and the combination of deleting both sites III and II, or sites II and I also results in similar (but not greater) levels of reduction.
Deletion of the region encompassing sites III and II removes this cooperative binding capacity derived from site II, but it also moves sites IV and V closer to site I. Although lower affinity, sites IV and V do show cooperative binding, and by moving them closer to site I, binding at these three sites may be able to mimic the conformation change normally occurring with binding of sites leading to transcriptional activation. Because sites IV and V are lower affinity sites, there may not be enough of this transcription factor around in normal cells to get full binding on the deleted construct for activation, but there are several publications reporting increased levels of E1AF/Pea3 with tumor progression and invasiveness (i.e., Horiuchi S. et. al., J Pathol 2003 August; 200(5): 568-76), indicating that tumor cells may have enough E1AF to bind the lower affinity binding sites IV and V, along with site I, and potentially lead to a conformational change needed to activate transcription of E1a in tumor but not normal cells with this deleted virus.
The present invention provides replicating adenoviruses. In some embodiments, the replicating vectors of the instant invention contain recombinant (e.g., exogenous) transgene(s) expressing immunomodulatory polypeptides that are controlled by endogenous adenovirus early promoters, thereby driving meaningfully higher expression levels than can be generally achieved in replication deficient viruses. In addition to the enhanced anti-tumor efficacy resulting from tumor-specific oncolysis from the replicating adenovirus, the higher expression levels from the recombinant transgenes in a replicating virus results in enhanced immunomodulatory effect(s) over the lower expression levels in replication deficient viruses.
In one aspect, a recombinant virus comprises a modified E1a regulatory sequence, wherein at least one Pea3 binding site, or a functional portion thereof, is deleted. In one aspect, a sufficient number of nucleotides in the range of −305 to -141 are retained to maintain functional activity of the Ad packaging signal function and (near) optimal transcription of the E1a protein in tumor but not growth arrested normal cells.
Additional deletions or modifications of individual or combinations of Pea3 V, Pea3IV, Pea3III, Pea3II and/or E2F1 binding sites between base pairs-394 and -218 designed to inhibit binding of Pea3 and/or E2F1 to these sites and take part in cooperative binding induced conformational changes in the E1a enhancer region are also encompassed by this description.
In one aspect, at least one of Pea3 II, Pea3 III, Pea3 IV, and Pea3 V, or a functional portion thereof, is deleted or modified (e.g., at least one nucleotide of the sequence is changed or an additional nucleotide is inserted into the sequence). In another aspect, at least one of Pea3 II and Pea3 III, or a functional portion thereof, is deleted or modified. In one aspect, Pea3 II or a functional portion thereof, and Pea3 III or a functional portion thereof, is deleted or modified. In another aspect, at least one of Pea3 IV and Pea3 V, or a functional portion thereof, is deleted or modified. In another aspect, Pea3 I, or a functional portion thereof, is retained. By “retained” is meant that the element is present in the recombinant adenoviral vector, preferably at the same location as a reference adenoviral vector. In one aspect, at least one E2F1 binding site, or a functional portion thereof, is retained.
In one aspect, the vector is d1309-6, TAV-255, d155, d1200, d1230, or d1200+230. In another aspect, the vector is TAV-255. In another aspect, the E1a deletions in d1309-6, TAV-255, d155, d1200, d1230, d1200+230, or other E1a modifications affecting Pea3 and/or E2F1 binding sites between -394 to -218, are paired with a non-d1309 based E3 deletion such as the E3 deletion found in pBHG10 (Microbix, Ad5 base pairs (bp) 28133-30818), d1327 (Ad5 bp 28593-30470) or a similar size E3 deletion such that >3 kb of exogenous DNA can be successfully packaged and expressed from a recombinant adenovirus with the E1a deletions listed above, in combination with a deletion between the start site of the E1b 19K protein and the start site of the E1b 55K protein of approximately 202 base pairs. In one aspect, the vector is a d1309 vector having one or more mutations in reference to the wild type sequence of Ad5 (see, e.g., Chroboczek et al., Virology (1992) January; 186(1):280-5, herein incorporated by reference), including a disruption in the coding sequences for one or more of the 10.4K, 14.5K, and 14.7K proteins in the E3 region.
In one aspect, a recombinant virus selectively expresses at least one E1a isoform, e.g., E1a-12S or E1a-13S. In one aspect, the sequence encoding the E1a isoform is operably linked to a modified E1a regulatory sequence, wherein at least one Pea3 binding site, or a functional portion thereof, is deleted or modified.
In one aspect, a recombinant virus comprises a DNA sequence, e.g., a transgene, inserted into an E1b-19K insertion site. In one aspect, the insertion site is located between the start site of E1b-19K and the start site of E1b 55K. In another aspect, the insertion site comprises a deletion of 202 base pairs following the start site of E1b-19K. A transgene (also, “insert”) may be a full natural sequence of the gene of interest or a fragment thereof. It may be modified too include a Kozak sequence, stop codon, or other regulatory elements. A transgene may include one or more endonuclease restriction sites.
In another aspect, expression the transgene is operably linked to a modified E1a regulatory sequence, wherein at least one Pea3 binding site, or a functional portion thereof, is modified or deleted. The transgene may be located in the E1, E2, E3 and/or E4 regions of the adenovirus but its expression is controlled by E1a mediated activation of the endogenous upstream adenovirus promoter, generating high levels of transgene expression only during E1a mediated viral replication.
In specific embodiments, the transgene is located in the E3 region. Exogenous transgenes can be inserted into one or more deleted regions of the adenovirus E3 region, generally such transgenes are inserted into the adenoviral vector as to have their expression controlled by the endogenous E3 promoter (Luo J et al, Clin Cancer Res 2008: 14 2450-2457). This results in high levels of transgene expression that are specifically expressed during periods of viral replication, as the E3 promoter only becomes transcriptionally active during these times. Using a modified adenovirus backbone, such as the E1a enhancer modification described herein, to limit viral replication to infected tumor cells, expression from the introduced transgene is also limited to infected tumor cells and, generally, not normal cells. In certain embodiments, the entire E3 region, or a substantial portion of the E3 region, is deleted.
In specific embodiments, the transgene is located in the E4 region. In a similar fashion to inserting exogenous transgenes into deleted regions in the E3 or E1 regions of adenovirus, there are regions in the E4 region of adenovirus that can be deleted without significant effect on viral growth characteristics (Gao, G P et al, J. Virol 1996: 70; 8934-8943). Generally, at least ORF3 and/or ORF6 are retained in the adenoviral vector in which the remainder of the E4 region. It should be possible to insert a foreign transgene into one of these deletions in the E4 region and drive expression of this gene with the endogenous E4 promoter of adenovirus, restricting high level expression of this gene to conditions where viral replication is expected to occur.

Tumor-Directed Recombinant Immunomodulatory Polypeptide Production.

The adenoviruses described herein can be engineered to express an immunomodulatory agent or immunomodulatory polypeptide, e.g., a polypeptide agonist of a co-stimulatory signal of an immune cell. In some embodiments, the polypeptide agonist is an agonist of a T effector cell and/or the polypeptide agonist functions as a polypeptide antagonist of an inhibitory signal of an immune cell such as a regulatory T cell. As provided herein, an “immunomodulatory protein” or an “immunomodulatory polypeptide” includes any polypeptide or set of polypeptides capable of modulating (e.g., stimulating) the anti-tumor immune response induced by the adenovirus. Generally, an “immunomodulatory polypeptide” includes a desired immunostimulatory activity. An immunomodulatory polypeptide can include a set of polypeptides, linked or unlinked, that can form a multimer (e.g., a dimer) capable of modulating the anti-tumor immune response induced by adenovirus, e.g., IL-12 dimer formed from p40 and p35, with or without a linker. Immunomodulatory polypeptides can be full length proteins as occur in nature or can be fusions, variants, or fragments thereof that retain at least about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the immunomodulatory activity of the full-length protein.
As used herein, the term “agonist(s)” refers to a molecule(s) that binds to another molecule and induces an increased biological reaction. In a specific embodiment, an agonist is a molecule that binds to a receptor on a cell and triggers or stimulates one or more signal transduction pathways. For example, an agonist can include an antibody or ligand that binds to a receptor on a cell and induces one or more signal transduction pathways. In other embodiments, the agonist facilitates the interaction of the native ligand with the native receptor. As used herein, the term “antagonist(s)” refers to a molecule(s) that inhibits the action of another molecule, optionally without provoking an independent biological response itself. In a specific embodiment, an antagonist is a molecule that binds to a receptor on a cell and blocks or dampens the biological activity of an agonist. For example, an antagonist can include an antibody or ligand that binds to a receptor on a cell and blocks or dampens binding of the native ligand to the cell, optionally without inducing one or more signal transduction pathways. Another example of an antagonist includes an antibody or soluble receptor that competes with the native receptor on cells for binding to the native ligand, and thus, blocks or dampens one or more signal transduction pathways induced when the native receptor binds to the native ligand. A further example of an antagonist includes an antibody or soluble receptor that competes with the native receptor on cells for binding to the native ligand or blocks receptor internalization, and thus, blocks or dampens one or more signal transduction pathways induced when the native receptor binds to the native ligand.
In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is a costimulatory ligand, e.g., GITR ligand (GITRL), OX40 ligand (OX40L), or CD40 ligand (CD40L). Expression of one or more costimulatory ligands in the tumor microenvironment and the specific binding to the cognate receptor results in an increase in activity of a tumor-infiltrating lymphocyte (TIL), such activity including TIL proliferation and cytokine release, thereby increasing the anti-tumor activity of the pharmaceutical composition.
In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is a pro-inflammatory cytokine, e.g., GMCSF, IL-7, IL-12, or an IL-15 hybrid (e.g., a hybrid of IL-15 and IL-15 receptor alpha). Expression of one or more pro-inflammatory cytokine in the tumor microenvironment results in a tumor-localized increase in the inflammatory milieu, thereby increasing the anti-tumor activity of the pharmaceutical composition, and also increasing safety of the composition by decreasing or eliminating undesired effects of systemic administration of a pro-inflammatory cytokine.
In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is an inhibitor (e.g., a “receptor trap” or a “trap”) of an inhibitory cytokine, e.g., IL-10 or IL-27. In other embodiments, the immunomodulatory agent/inhibitor of an inhibitory cytokine is an antibody against, e.g., TGFB or IL-10R. Such inhibitory cytokines decrease T effector cell function. Expression of one or more inhibitory cytokine receptor traps in the tumor microenvironment results in a tumor-localized binding to and neutralization of the inhibitory cytokine, thereby reducing or preventing its inhibitory activity and increasing the anti-tumor activity of the pharmaceutical composition, and also increasing safety of the composition by decreasing or eliminating undesired effects of systemic administration of a blockage of an inhibitory cytokine.
In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is an initiator of a localized immune response, e.g., a protein (e.g., secreted flagellin) that activates a toll-like receptor ligand such as TLR-5. Expression of one or more immune response initiators in the tumor microenvironment results in a tumor-localized immune response (e.g., infiltration of TILs and antigen presenting cells (APCs) and increasing the anti-tumor activity of the pharmaceutical composition.
In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is an inhibitor (e.g., an antibody antagonist) of a co-inhibitory checkpoint molecule, e.g., CTLA4. Such inhibitory checkpoint molecules decrease T effector cell function. Expression of one or more inhibitory checkpoint molecules at the surface of an activated T cell (e.g., an activated T effector cell) attenuates the functional activity of the T cell. Thus, expression of an antagonist of the co-inhibitory checkpoint molecule in the tumor microenvironment results in blocking the co-inhibitory activity and increasing the anti-tumor activity of the pharmaceutical composition, and also increasing safety of the composition by decreasing or eliminating undesired effects of systemic administration of a blockage of a co-inhibitory checkpoint molecule.
In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is a cluster of differentiation (CD) molecule or a ligand of a cluster of differentiation (CD) molecule such as CD27, e.g., CD70. Expression of a CD27 ligand in the tumor microenvironment results in a tumor-localized NK-mediated tumor clearance and promotes the adaptive immune response against the tumor, thereby increasing the anti-tumor activity of the pharmaceutical composition. Other suitable CD molecules include CD1d, CD2, CD3, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD21, CD25, CD37, CD40, CD49b, CD53, CD57, CD69, CD80, CD81, CD82, CD86, CD99, CD103, CD134, CD152, CD154, CD165, CD244, CD267, CD272, CD273, CD274, CD278, CD305, CD314, CD357, and CD360, or the ligands thereof, or any modulator (e.g., a stimulator or an inhibitor) thereof.
In specific embodiments, the immunomodulatory polypeptide expressed by the adenovirus is selected from a polypeptide provided in Table 1.

TABLE 1

Exemplary Immunomodulatory polypeptides

SEQ ID
NO:	Immunomodulatory Polypeptide	Accession Number(s)

1	GITR-Ligand (GITR-L; TNFSF18)	NP_005083
2	GITR-Ligand (GITR-L; TNFSF18)	NM_183391
3	CD28 ligand or agonist (CD80)	EAW79565
4	TNFα	CAA78745
5	Non-cleavable TNFα	NM_013693
6	GM-CSF	NM_009969
7	ICOS ligand or agonist	NP_056074
8	4-1BB ligand or agonist	AAA53134 (human)
9	OX40 ligand or agonist	CAE11757
10	OX40 ligand	NM_009452
11	CD40 ligand or agonist	NP_000065
12	CD40 ligand	NM_011616
13	CD27 ligand or agonist	AAA36175
14	CD70 ligand or agonist	NM_011617
15	Interleukin-2 (IL-2)	AAB46883
16	Interleukin-7 (IL-7)	AAH47698
17	Interleukin-7 (IL-7)	NM_00837
18	Interleukin-12 (IL-12) alpha subunit	AAD16432
19	Interleukin-12 (IL-12) beta subunit	NP_005526
20	Interleukin-12 fusion polypeptide	N/A
21	IL-15	CAA62616
22	IL-15 hybrid	N/A
23	IL-10R TRAP (IL-10 antagonist)	NP_001549
	[alpha]
24	IL-10R Trap	N/A
25	IL-27R TRAP (IL-27 antagonist)	NP_004834
26	IL-13	AAH96140
27	IL-17	AAC50341
28	IL-33 Isoform a	NP_001300974
29	IL-33 Isoform a	NP_001300973
30	IL-33 Isoform b	NP_001186569
31	IL-33 Isoform c	NP_001186570
32	IL-33 Isoform d	NP_001300975;
		NP_001300976
33	IL-33 Isoform e	NP_001300977
34	IFN-gamma	AAB59534
35	secreted flagellin	N/A
36	anti-CTLA4 (CTLA-4 antagonist	N/A
	antibody)
38	Interleukin-2 (IL-2) (murine)	NM_008366.3
39	Xc11 (murine)	GenBank: BC062249.1
42	4-1BBL (TNFSF9) (murine)	GenBank: BC138767.1
55	IL-15Rα	N/A
58	hαCTLA4	N/a com1-19
	Anti-PD-1 (PD-1 antagonist
	antibody)
	Anti-PD-L1 (PD-L1 antagonist
	antibody)
	Anti-TIGIT (TIGIT antagonist
	antibody)
	Anti-TGFβ (TGFβ antagonist
	antibody)
	Anti-IL6R (IL-6 receptor antibody)

Additional detail about the above-discussed agonists that can be expressed from an adenovirus vector as described herein is provided below:

GITR Ligand.

In one embodiment, the heterologous gene is a GITR ligand family gene, such as TNFSF18 (also known as GITRL) (See, e.g., Tone, M., Tone, Y., Adams, E., Yates, S. F., Frewin, M. R., Cobbold, S. P., & Waldmann, H. (2003). Mouse glucocorticoid-induced tumor necrosis factor receptor ligand is costimulatory for T cells. Proceedings of the National Academy of Sciences of the United States of America, 100(25), 15059-15064. doi:10.1073/pnas.2334901100). The GITR/GITRL signaling pathway is associated with activation of immune cells Nocentini, G., Ronchetti, S., Petrillo, M. G., & Riccardi, C. (2012). Pharmacological modulation of GITRL/GITR system: therapeutic perspectives. British Journal of Pharmacology, 165(7), 2089-99. doi:10.1111/j.1476-5381.2011.01753.x). Specifically, the GITRL protein has an immunomodulatory activity including inhibiting the suppressive activity of T regulatory cells and activation of T effector cells. The intratumoral localization of effective amounts of GITRL protein results in stimulation of the immune system and inhibition of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the costimulatory activity of GITRL protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation

IL-10 TRAP.

In one embodiment, the heterologous gene is an engineered IL-10 trap, such as IL-10 receptor fused to a human immunoglobulin Fc domain. Examples include IL10RA-Fc fusion protein or IL10RA-IL10RB-Fc fusion protein. (See, e.g., Economides, A. N., Carpenter, L. R., Rudge, J. S., Wong, V., Koehler-Stec, E. M., Hartnett, C., . . . Stahl, N. (2003). Cytokine traps: multi-component, high-affinity blockers of cytokine action. Nature Medicine, 9(1), 47-52. doi:10.1038/nm811). The IL-10 family is associated with inhibition of inflammatory response in immune cells through inhibition of the expression of proinflammatory cytokines and co-stimulatory molecules. Expression of IL-10 by T regulatory cells suppresses the activity of T effector cells (Mosser D M, Zhang X. Immunol Rev. 2008 December; 226:205-18. Interleukin-10: new perspectives on an old cytokine). Specifically, the IL-10 trap protein has an immunomodulatory activity by inhibiting the anti-inflammatory activity of IL-10. The intratumoral localization of effective amounts of IL-10 trap protein results in stimulation of the immune system and inhibition of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, IL-10 trap protein inhibition of IL-10's anti-inflammatory activity has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation. In one embodiment, the IL-10 Receptor Trap includes all or a portion of the extracellular domains of IL-10Ra and IL-10Ra.

ANTI-CTLA4.

In one embodiment, the heterologous gene is an antibody (or domain or fragment thereof) that inhibits the function of CTLA4 (See, e.g., Leach D R, Krummel M F, Allison J P. Enhancement of antitumor immunity by CTLA-4 blockade. Science. 1996 Mar. 22; 271(5256):1734-6.) The CTLA4 family is associated with inhibition of T cells through the interaction with ligands CD80 and CD86 (Krummel M F, Allison J P (1995). “CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation”. J. Exp. Med. 182 (2): 459-65.) Specifically, the anti-CTLA4 antibody has an immunomodulatory activity including blocking the inhibitory function of CTLA4 resulting in more efficient activation of T effector cells. The intratumoral localization of effective amounts of anti-CTLA4 antibody results in stimulation of the immune system and inhibition of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, anti-CTLA4 inhibition of CTLA4's T cell inhibitory activity has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation.

IL-12:

In one embodiment, the heterologous gene is a member of the Interleukin cytokine family such as IL-12. The IL-12 cytokine family is associated with induction of IFNγ and mediating T-cell dependent immunity. Specifically, IL-12 is an immunostimulatory cytokine with strong antiangiogenic effects. IL-12 has immunomodulatory activity including cell proliferation, lymphocyte differentiation and NK cell activation. The intratumoral localization of effective amounts of IL-12 results in the differentiation, proliferation, and maintenance of T helper 1 (Th1) responses that lead to IFNγ and IL-2 production that in turn, promote T cell responses and macrophage activation. The local expression of effective amounts of IL-12 from intratumoral injections may provide a safety benefit over systemic administration and side effects associated with high IL-12 serum levels. Moreover, the immunostimulatory activity and induction of cytotoxicity mediated by natural killer cells and T cells by IL-12 may have a synergistic effect with the tumor-directed cell-lytic and immune stimulating activity of our adenovirus providing a more effective viral-based therapeutic treatment of human subjects suffering from cancer. In one embodiment, the IL-12 polypeptide is a fusion of IL-12 subunits p35 and p40, linked by a 45 bp linker, including IL-12 β:p40 (NM_001303244) and IL-12 Alpha:p35 (NM_008351.1).

GM-CSF:

In one embodiment, the heterologous gene is a cytokine such as Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony stimulating factor 2 (CSF2). Cytokines are secreted proteins or peptides that mediate and regulate immunity and inflammation. Specifically, GM-CSF has an immunomodulatory activity of functioning as an immune adjuvant and facilitates development of the immune system, acting as a growth factor for DCs and APCs. The localized secretion of effective amounts of GM-CSF results in an increase in dendritic cell (DC) maturation and function as well as macrophage activity, recruiting immune cells to the inflammatory site of tumor treatment, resulting in more effective therapeutic treatment of human subjects suffering from cancer. Moreover, GM-CSF has been demonstrated to be capable of induced long-lasting, specific anti-tumor immunity when combined with cancer vaccines, potentially providing a synergistic effect with the tumor-directed cell-lytic activity of our adenovirus. In a recent phase III clinical trial, an oncolytic herpes simplex virus armed with GM-CSF (T-VEC) showed durable response rates in advanced melanoma patients compared with GM-CSF protein alone.

Secreted Flagellin:

In one embodiment, the heterologous gene comes from a gram-negative bacterium in the Salmonellae family, such as the gene encoding flagellin. Bacterial proteins, including flagellin, are associated with the activation of the innate immune response, leading to production of proinflammatory cytokines and the up-regulation of costimulatory molecules. Specifically, flagellin is a TLR5 agonist, and binding of secreted flagellin to TLR5 stimulates production of TNFα, and induces infiltration of APC's and TIL's to the local tumor environment. By acting as a strong adjuvant, flagellin is able to prime the immune system to elicit strong adaptive immune responses, resulting in enhanced and broadened immune response a more effective viral-based therapeutic treatment of human subjects suffering from cancer. In some embodiments, secreted Flagellin contains a murine IL-2 signal sequence (See NM_008366) and Salmonella Flagellin, GenBank: D13689.

TNFA AND NON-CLEAVABLE TNFA.

In one embodiment, the heterologous gene is an engineered TNFα ligand family gene, such as a non-cleavable (membrane-bound, transmembrane) form of TNFα (See, e.g., Li Q, Li L, Shi W, Jiang X, Xu Y, Gong F, Zhou M, Edwards C K 3rd, Li Z., Mechanism of action differences in the antitumor effects of transmembrane and secretory tumor necrosis factor-alpha in vitro and in vivo. Cancer Immunol Immunother. 2006: 55, 1470-9.). TNFα belongs to a family of pro-inflammatory cytokines (Calcinotto A, Grioni M, Jachetti E, Curnis F, Mondino A, Parmiani G, Corti A, Bellone M. Targeting TNF-α to neoangiogenic vessels enhances lymphocyte infiltration in tumors and increases the therapeutic potential of immunotherapy. J Immunol. 2012; 188: 2687-94.). Specifically, expression of TNFα in the tumor microenvironment is expected to increase the inflammatory milieu resulting in increased anti-tumor immune responses. Use of a non-cleavable TNFα results in a tethered form of TNFα which remains membrane-bound. The intratumoral localization of effective amounts of TNFα protein results in stimulation of the immune system and inhibition of growth of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. In addition, local expression may provide a safety benefit over systemic administration of TNFα. Moreover, the immunomodulatory activity of the membrane-bound TNFα protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation.

OX40L.

In one embodiment, the heterologous gene is an OX40L family gene, such as TNFSF4 (also called OX40L, CD252) (See, e.g., Dannull J, Nair S, Su Z, Boczkowski D, DeBeck C, Yang B, Gilboa E, Vieweg J. Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand. Blood. 2005, 105: 3206-13.). The TNFSF is associated with activation of immune cells (Croft M, So T, Duan W, Soroosh P. The significance of OX40 and OX40L to T-cell biology and immune disease. Immunol Rev. 2009; 229:173-91.). Specifically, OX40L is a co-stimulatory ligand for TNFRSF4 (OX40, CD134) resulting in activation of T cells. Expression of OX40L in the tumor microenvironment and binding to its cognate receptor (OX40) is expected to increase the activity (proliferation, cytokine release) of tumor infiltrating lymphocytes (TILs) resulting in antitumor activity. The intratumoral localization of effective amounts of OX40L protein results in stimulation of the immune system and inhibition of growth of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Local expression may provide a safety benefit over systemic administration of OX40L. Moreover, the immunomodulatory activity of the OX40L protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation IL-7.
In one embodiment, the heterologous gene is an IL-7 cytokine family gene, such as IL-7 (See, e.g., Gao J, Zhao L, Wan Y Y, Zhu B., Mechanism of Action of IL-7 and Its Potential Applications and Limitations in Cancer Immunotherapy. Int J Mol Sci. 2015, 16: 10267-80.). IL-7 belongs to a family of pro-inflammatory cytokines (Geiselhart L A, Humphries C A, Gregorio TA, Mou S, Subleski J, Komschlies KL. IL-7 administration alters the CD4:CD8 ratio, increases T cell numbers, and increases T cell function in the absence of activation. J Immunol. 2001; 166: 3019-27.). Specifically, expression of IL-7 in the tumor microenvironment is expected to increase the inflammatory milieu resulting in increased anti-tumor immune responses. The intratumoral localization of effective amounts of IL-7 protein results in stimulation of the immune system and inhibition of growth of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the immunomodulatory activity of the IL-7 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting from activation and/or recruitment of the immune system to the tumor and enhanced antigen presentation.

CD40L

In one embodiment, the heterologous gene is a member of the TNF superfamily, such as CD40L (also known as CD40LG or CD154) (See, e.g., Hassan G S, et al., 2015. “Role of CD154 in cancer pathogenesis and immunotherapy.” Cancer Treat Rev 4 1(5):431-40). The CD40L is the ligand for CD40 expressed on antigen presenting cells. Specifically, the CD40L protein has a costimulatory activity important for activation of T cell dependent immune responses (Sotomayor E M, et al., 1999. “Conversion of tumor-directed CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40.” Nat Med. 5:780-787; French R R, et al., 1999. “CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med. 5:548-553). The intratumoral localization of effective amounts of CD40 protein results in activation of the immune system and lysis of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the costimulatory immunomodulatory activity of CD40 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting in a local and systemic immune response against the tumor.

IL-15 AND IL-15 HYBRID

In one embodiment, the heterologous gene is a cytokine family gene, such as Interleukin 15 (IL-15) (See, e.g., Di Sabatino A, et. al., 2011 “Role of IL-15 in immune-mediated and infectious diseases”. Cytokine Growth Factor Rev. 22 (1): 19-33; Steel J C, et al., 2012, “Interleukin-15 biology and its therapeutic implications in cancer”. Trends Pharmacol. Sci. 33 (1): 35-41). IL-15 is a cytokine that regulates T cell and NK cell activation and proliferation. (Waldmann T A, et al., (1999). “The multifaceted regulation of interleukin-15 expression and the role of this cytokine in NK cell differentiation and host response to intracellular pathogens”. Annu. Rev. Immunol. 17: 19-49). Specifically, the IL-15 protein has an immunomodulatory activity by providing survival signals to maintain memory T cells in the absence of antigen. IL-15 has also been shown to enhance the anti-tumor immunity of CD8+ T cells (See, Klebanoff C A, et al., “IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T Cells” Proc. Natl. Acad. Sci. U.S.A. 101 (7): 1969-74; and Teague R M, et al., “Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors” Nat. Med. 12 (3): 335-41). Expression of an IL-15 hybrid molecule (IL-15 linked to the IL-15 Receptor alpha, see Tosic et al., PLos ONE 9(10): e109801 (2014)) leads to stabilization and increased bioactivity (Bergamaschi et al., 2008. “Intracellular Interaction of Interleukin-15 with Its Receptor a during Production Leads to Mutual Stabilization and Increased Bioactivity”, JBC, 283(7):4189-99; Bergamaschi et al., 2013. “Circulating IL-15 exists as heterodimeric complex with soluble IL-15Rα in human and mouse serum”, Blood 120(1):e1). The intratumoral localization of effective amounts of IL-15 protein results in activation of the immune system and lysis of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the survival signals provided by IL-15 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting in a local and systemic immune response against the tumor. In some embodiments, an IL-15 hybrid includes IL-15, P2A, and IL-15Rα, IL-15 (NM_008357), 68 bp P2A, and IL-15Rα (GenBank: BC132233.1).

CD70

In one embodiment, the heterologous gene is a member of the TNF superfamily, such as CD70 (also known as TNFSF7 or CD27L). (See, e.g., Denoeud J and Moser M., 2011. “Role of CD27/CD70 pathway of activation in immunity and tolerance.” J Leukoc Biol. 89(2):195-203). CD70 is expressed on activated T and B cells, as well as mature dendritic cells, and acts as a ligand for CD27. CD70 plays a costimulatory role in promoting T cell expansion and differentiation (Keller A M, et al., 2008. “Expression of costimulatory ligand CD70 on steady-state dendritic cells breaks CD8+ T cell tolerance and permits effective immunity,” Immunity 29(6):934-46; Bonehill A, et al., 2008. “Enhancing the T-cell stimulatory capacity of human dendritic cells by co-electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA,” Mol Ther. 16(6):1170-80). In addition, CD70 expression in the tumor microenvironment will increase NK-mediated tumor clearance and promote an adaptive immune response against the tumor (Kelly J M, et al., 2002. “Induction of tumor-directed T cell memory by NK cell-mediated tumor rejection,” Nat Immunol. 3(1):83-90). The intratumoral localization of effective amounts of CD70 protein results in activation of the immune system and lysis of the tumor, resulting in more effective viral-based therapeutic treatment of human subjects suffering from cancer. Moreover, the immunomodulatory activity of CD70 protein has a synergistic effect with the tumor-directed cell-lytic activity of the adenovirus, resulting in a local and systemic immune response against the tumor.
In a specific embodiment, the agonist of a co-stimulatory signal of an immune cell expressed by the adenovirus is an agonist of a co-stimulatory receptor expressed by an immune cell. Specific examples of co-stimulatory receptors that can be expressed by the adenovirus include glucocorticoid-induced tumor necrosis factor receptor (GITR), Inducible T-cell co-stimulator (ICOS or CD278), OX40 (CD134), CD27, CD28, 4-IBB(CD137), CD40, CD226, cytotoxic and regulatory T cell molecule (CRT AM), death receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML interactor (TACI), B cell-activating factor receptor (BAFFR), and B cell maturation protein (BCMA). In a specific embodiment, the agonist of a co-stimulatory receptor expressed by an immune cell is an antibody (or an antigen-binding fragment thereof) or ligand that specifically binds to the co-stimulatory receptor. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is an sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on an immune cell. In one embodiment, the bispecific antibody binds to a receptor on an immune cell and to another receptor on a cancer cell. In specific embodiments, the antibody is a human or humanized antibody. In certain embodiments, the ligand or antibody is a chimeric protein.
In a specific embodiment, the antagonist of an inhibitory signal of an immune cell is an antagonist of an inhibitory receptor expressed by an immune cell. Specific examples of inhibitory receptors include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD1 or CD279), B and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), T-cell membrane protein 3 (TIM3), adenosine A2a receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and CD160. In a specific embodiment, the antagonist of an inhibitory receptor expressed by an immune cell is an antibody (or an antigen-binding fragment thereof) that specifically binds to the co-stimulatory receptor.
Antibodies include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, single domain antibodies, camelid or camelized antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked bispecific Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies), and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In a specific embodiment, an antibody is a human or humanized antibody. In another specific embodiment, an antibody is a monoclonal antibody or scFv. In certain embodiments, an antibody is a human or humanized monoclonal antibody or scFv. In other specific embodiments, the antibody is a bispecific antibody. In certain embodiments, the bispecific antibody specifically binds to a co-stimulatory receptor of an immune cell or an inhibitory receptor of an immune, and a receptor on a cancer cell. In some embodiments, the bispecific antibody specifically binds to two receptors immune cells, e.g., two co-stimulatory receptors on immune cells, two inhibitory receptors on immune cells, or one co-stimulatory receptor on immune cells and one inhibitory receptor on immune cells.
The recombinant AVs described herein may be engineered to express any agonist of a co-stimulatory signal and/or any antagonist of an inhibitory signal of an immune cell, such as, e.g., a T-lymphocyte, NK cell or antigen-presenting cell (e.g., a dendritic cell or macrophage). In specific embodiments, the agonist and/or antagonist is an agonist of a human co-stimulatory signal of an immune cell and/or antagonist of a human inhibitory signal of an immune cell. In certain embodiments, the agonist of a co-stimulatory signal is an agonist of a co-stimulatory molecule (e.g., co-stimulatory receptor) found on immune cells, such as, e.g., T-lymphocytes (e.g., CD4+ or CD8+T-lymphocytes), NK cells and/or antigen-presenting cells (e.g., dendritic cells or macrophages). Specific examples of co-stimulatory molecules include glucocorticoid-induced tumor necrosis factor receptor (GITR), Inducible T-cell co-stimulator (ICOS or CD278), OX40 (CD134), CD27, CD28, 4-IBB (CD137), CD40, lymphotoxin alpha (LT alpha), LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), CD226, cytotoxic and regulatory T cell molecule (CRT AM), death receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML interactor (TACI), B cell-activating factor receptor (BAFFR), and B cell maturation protein (BCMA). In specific embodiments, the agonist is an agonist of a human co-stimulatory receptor of an immune cell. In certain embodiments, the agonist of a co-stimulatory receptor is not an agonist of ICOS. In some embodiments, the antagonist is an antagonist of an inhibitory molecule (e.g., inhibitory receptor) found on immune cells, such as, e.g., T-lymphocytes (e.g., CD4+ or CD8+T-lymphocytes), NK cells and/or antigen-presenting cells (e.g., dendritic cells or macrophages). Specific examples of inhibitory molecules include cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD1 or CD279), B and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), T-cell membrane protein 3 (TIM3), CD 160, adenosine A2a receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and CD 160. In specific embodiments, the antagonist is an antagonist of a human inhibitory receptor of an immune cell.
In a specific embodiment, the agonist of a co-stimulatory receptor is an antibody or antigen-binding fragment thereof that specifically binds to the co-stimulatory receptor. Specific examples of co-stimulatory receptors include GITR, ICOS, OX40, CD27, CD28, 4-1BB, CD40, LT alpha, LIGHT, CD226, CRT AM, DR3, LTBR, TACI, BAFFR, and BCMA. In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is an sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on an immune cell. In other embodiments, the bispecific antibody binds to a receptor on an immune cell and to another receptor on a cancer cell. In specific embodiments, the antibody is a human or humanized antibody.
In another embodiment, the agonist of a co-stimulatory receptor expressed by the adenovirus is a ligand of the co-stimulatory receptor. In certain embodiments, the ligand is fragment of a native ligand. Specific examples of native ligands include ICOSL, B7RP1, CD137L, OX40L, CD70, herpes virus entry mediator (HVEM), CD80, and CD86. The nucleotide sequences encoding native ligands as well as the amino acid sequences of native ligands are known in the art. For example, the nucleotide and amino acid sequences of B7RP1 (otherwise known as ICOSL; GenBank human: NM_015259.4, NP_056074.1 murine: NM_015790.3, NP_056605.1), CD137L(GenBank human: NM_003811.3, NP_003802.1, murine: NM_009404.3, NP_033430.1), OX40L(GenBank human: NM_003326.3, NP_003317.1, murine: NM_009452.2, NP_033478.1), CD70(GenBank human: NM_001252.3, NP_001243.1, murine: NM_011617.2, AAD00274.1), CD80(GenBank human: NM_005191.3, NP_005182.1, murine: NM_009855.2, NP_033985.3), and CD86 (GenBank human: NM_005191.3, CAG46642.1, murine: NM_019388.3, NP_062261.3) can be found in GenBank. In other embodiments, the ligand is a derivative (e.g., a fragment, domain, fusion, or other modification of a full-length polypeptide) a native ligand. In some embodiments, the ligand is a fusion protein comprising at least a portion of the native ligand or a derivative of the native ligand that specifically binds to the co-stimulatory receptor, and a heterologous amino acid sequence. In specific embodiments, the fusion protein comprises at least a portion of the native ligand or a derivative of the native ligand that specifically binds to the co-stimulatory receptor, and the Fc portion of an immunoglobulin or a fragment thereof. An example of a ligand fusion protein is a 4-IBB ligand fused to Fc portion of immunoglobulin (described by Meseck M et al., J Immunother. 2011 34: 175-82).
In another embodiment, the antagonist of an inhibitory receptor expressed by the adenovirus is an antibody (or an antigen-binding fragment) or a soluble receptor that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transducing an inhibitory signal(s). Specific examples of native ligands for inhibitory receptors include PDL-1, PDL-2, B7-H3, B7-H4, HVEM, Gal9 and adenosine. Specific examples of inhibitory receptors that bind to a native ligand include CTLA-4, PD-1, BTLA, KIR, LAG3, TIM3, and A2aR.
In specific embodiments, the antagonist of an inhibitory receptor expressed by the adenovirus is a soluble receptor that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transducing an inhibitory signal(s). In certain embodiments, the soluble receptor is a fragment of a native inhibitory receptor or a fragment of a derivative of a native inhibitory receptor that specifically binds to native ligand {e.g., the extracellular domain of a native inhibitory receptor or a derivative of an inhibitory receptor). In some embodiments, the soluble receptor is a fusion protein comprising at least a portion of the native inhibitory receptor or a derivative of the native inhibitory receptor (e.g., the extracellular domain of the native inhibitory receptor or a derivative of the native inhibitory receptor), and a heterologous amino acid sequence. In specific embodiments, the fusion protein comprises at least a portion of the native inhibitory receptor or a derivative of the native inhibitory receptor, and the Fc portion of an immunoglobulin or a fragment thereof. An example of a soluble receptor fusion protein is a LAG3-Ig fusion protein (described by Huard B et al, Eur J Immunol (1995) 25:2718-21).
In specific embodiments, the antagonist of an inhibitory receptor expressed by the adenovirus is an antibody (or an antigen-binding fragment) that specifically binds to the native ligand for the inhibitory receptor and blocks the native ligand from binding to the inhibitory receptor and transducing an inhibitory signal(s). In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is an scFv. In particular embodiments, the antibody is a human or humanized antibody. A specific example of an antibody to inhibitory ligand is anti-PD-L1 antibody (Iwai Y, et al. PNAS 2002; 99: 12293-12297).
In another embodiment, the antagonist of an inhibitory receptor expressed by the adenovirus is an antibody (or an antigen-binding fragment) or ligand that binds to the inhibitory receptor, but does not transduce an inhibitory signal(s). Specific examples of inhibitory receptors include CTLA-4, PD1, BTLA, TIGIT, KIR, LAG3, TIM3, and A2aR. In certain specific embodiments, the antibody is a monoclonal antibody. In other specific embodiments, the antibody is an scFv. In particular embodiments, the antibody is a human or humanized antibody. A specific example of an antibody to inhibitory receptor is anti-CTLA-4 antibody (Leach D R, et al. Science 1996; 271: 1734-1736). Another example of an antibody to inhibitory receptor is anti-PD-1 antibody (Topalian S L, NEJM 2012; 28:3167-75).
In certain embodiments, a chimeric adenovirus described herein is engineered to produce an antagonist of CTLA-4, such as, e.g., ipilimumab or tremelimumab. In certain embodiments, a chimeric adenovirus described herein is engineered to an antagonist of PD1, such as, e.g., MDX-1106 (BMS-936558), MK3475, CT-011, AMP-224, or MDX-1105. In certain embodiments, a chimeric adenovirus described herein is engineered to express an antagonist of LAG3, such as, e.g., IMP321. In certain embodiments, a chimeric adenovirus described herein is engineered to express an antibody (e.g., a monoclonal antibody or an antigen-binding fragment thereof, or scFv) that binds to B7-H3, such as, e.g., MGA271. In specific embodiments, a chimeric adenovirus described herein is engineered to express an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell. In specific embodiments, adenovirus described herein is engineered to express anti-CD28 scFv, ICOSL, CD40L, OX40L, CD137L, GITRL, and/or CD70.
In certain embodiments, an agonist of a co-stimulatory signal of an immune cell expressed by the adenovirus induces (e.g., selectively) induces one or more of the signal transduction pathways induced by the binding of a co-stimulatory receptor to its ligand. In specific embodiments, an agonist of a co-stimulatory receptor induces one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more of its ligands by at least 25%, 30%, 40%>, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more of its ligands in the absence of the agonist. In specific embodiments, an agonist of a co-stimulatory receptor: (i) induces one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to the particular ligand in the absence of the agonist; and (ii) does not induce, or induces one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more other ligands by less than 20%, 15%, 10%, 5%, or 2%, or in the range of between 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to such one or more other ligands in the absence of the agonist.
In certain embodiments, an agonist of a co-stimulatory signal of an immune cell activates or enhances (e.g., selectively activates or enhances) one or more of the signal transduction pathways induced by the binding of a co-stimulatory receptor to its ligand. In specific embodiments, an agonist of a co-stimulatory receptor activates or enhances one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more of its ligands by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%), or 75%) to 100% relative to the one or more signal transduction pathways induced by the binding of co-stimulatory receptor to one or more of its ligands in the absence of the agonist. In specific embodiments, an agonist of a co-stimulatory receptor: (i) an agonist of a co-stimulatory signal activates or enhances one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to the particular ligand in the absence of the agonist; and (ii) does not activate or enhance, or activates or enhances one or more of the signal transduction pathways induced by the binding of the co-stimulatory receptor to one or more other ligands by less than 20%, 15%, 10%, 5%, or 2%, or in the range of between 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% relative to the one or more signal transduction pathways induced by the binding of the co-stimulatory receptor to such one or more other ligands in the absence of the agonist.
In some embodiments, an antagonist of an inhibitory signal of an immune cell (e.g., selectively) inhibits or reduces one or more of the signal transduction pathways induced by the binding of an inhibitory receptor to its ligand. In specific embodiments, an antagonist of an inhibitory receptor inhibits or reduces one or more of the signal transduction pathways induced by the binding of the inhibitory receptor to one or more of its ligands by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the inhibitory receptor to one or more of its ligands in the absence of the antagonist. In specific embodiments, an antagonist of an inhibitory receptor: (i) inhibits or reduces one or more of the signal transduction pathways induced by the binding of the inhibitory receptor to one particular ligand by at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, or in the range of between 25% to 50%, 25% to 75%, 50% to 75%, 50% to 95%, 75% to 95%, or 75% to 100% relative to the one or more signal transduction pathways induced by the binding of the inhibitory receptor to the one particular ligand in the absence of the antagonist; and (ii) does not inhibit or reduce, or inhibits or reduces one or more of the signal transduction pathways induced by the binding of the inhibitory receptor to one or more other ligands by less than 20%, 15%, 10%, 5%, or 2%, or in the range of between 2% to 5%, 2% to 10%, 5% to 10%, 5% to 15%, 5% to 20%, 10% to 15%, or 15% to 20% relative to the one or more signal transduction pathways induced by the binding of inhibitory receptor to such one or more other ligands in the absence of the antagonist.
In specific embodiments, an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell induces, activates and/or enhances one or more immune activities, functions or responses. The one or more immune activities, functions or responses can be in the form of, e.g., an antibody response (humoral response) or a cellular immune response, e.g., cytokine secretion (e.g., interferon-gamma), helper activity or cellular cytotoxicity. In one embodiment, expression of an activation marker on immune cells (e.g., CD44, Granzyme, or Ki-67), expression of a co-stimulatory receptor on immune cells (e.g., ICOS, CD28, OX40, or CD27), expression of a ligand for a co-stimulatory receptor (e.g., B7HRP1, CD80, CD86, OX40L, or CD70), cytokine secretion, infiltration of immune cells (e.g., T-lymphocytes, B lymphocytes and/or NK cells) to a tumor, antibody production, effector function, T cell activation, T cell differentiation, T cell proliferation, B cell differentiation, B cell proliferation, and/or NK cell proliferation is induced, activated and/or enhanced following contact with an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell. In another embodiment, myeloid-derived suppressor cell (MDSC) tumor infiltration and proliferation, Treg tumor infiltration, activation and proliferation, peripheral blood MDSC and Treg counts are inhibited following contact with an agonist of a co-stimulatory signal of an immune cell and/or an antagonist of an inhibitory signal of an immune cell.
In certain embodiments, a chimeric adenovirus described herein is engineered to produce two or more immunomodulatory polypeptides. In some embodiments, the chimeric adenovirus produces a first immunomodulatory polypeptide and a second immunomodulatory polypeptide.
For example, a first immunomodulatory polypeptide is a costimulatory ligand, a proinflammatory cytokine, an inhibitor of an inhibitory cytokine, an initiator of a localized immune response, an inhibitor of a co-inhibitory checkpoint molecule, or a ligand of a cluster of differentiation (CD) molecule, and the second immunomodulatory polypeptide is a costimulatory ligand, a proinflammatory cytokine, an inhibitor of an inhibitory cytokine, an initiator of a localized immune response, an inhibitor of a co-inhibitory checkpoint molecule, or a ligand of a cluster of differentiation (CD) molecule.
For example, a first immunomodulatory polypeptide is a costimulatory ligand, and the second immunomodulatory polypeptide is a costimulatory ligand.
For another example, a first immunomodulatory polypeptide is a costimulatory ligand and the second immunomodulatory polypeptide is a pro-inflammatory cytokine.
In some embodiments, two or more immunomodulatory polypeptides are expressed from a single transcript. To express two or more proteins from a single transcript determined by a viral or non-viral vector, an internal ribosome entry site (IRES) sequence is commonly used to drive expression of the second, third, fourth coding sequence, etc. When two coding sequences are linked via an IRES, the translational expression level of the second coding sequence is often significantly reduced (Furler et al. 2001. Gene Therapy 8:864-873). In fact, the use of an IRES to control transcription of two or more coding sequences operably linked to the same promoter can result in lower level expression of the second, third, etc. coding sequence relative to the coding sequence adjacent the promoter. In addition, an IRES sequence may be sufficiently long to impact complete packaging of the vector, e.g., the eCMV IRES has a length of 507 base pairs.
Internal ribosome entry site (IRES) elements were first discovered in picornavirus mRNAs (Jackson et al. 1990. Trends Biochem. Sci. 15:477-83) and Jackson and Kaminski, RNA (1995) 1:985-1000). Examples of IRES generally employed by those of skill in the art include those referenced in Table I and Appendix A, as well as those described in U.S. Pat. No. 6,692,736. Examples of “IRES” known in the art include, but are not limited to IRES obtainable from picornavirus (Jackson et al., 1990) and IRES obtainable from viral or cellular mRNA sources, such as for example, immunoglobulin heavy-chain binding protein (BiP), the vascular endothelial growth factor (VEGF) (Huez et al. 1998, Mol. Cell. Biol. 18:6178-6190), the fibroblast growth factor 2 (FGF-2), and insulin-like growth factor (IGFII), the translational initiation factor eIF4G and yeast transcription factors TFIID and HAP4, the encephelomyocarditis virus (EMCV) which is commercially available from Novagen (Duke et al. 1992. J. Virol 66:1602-9) and the VEGF IRES (Huez et al. 1998. Mol. Cell. Biol. 18:6178-90). IRES have also been reported in different viruses such as cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). As used herein, “IRES” encompasses functional variations of IRES sequences as long as the variation is able to promote direct internal ribosome entry to the initiation codon of a cistron. An IRES may be mammalian, viral or protozoan. The IRES promotes direct internal ribosome entry to the initiation codon of a downstream cistron, leading to cap-independent translation. Thus, the product of a downstream cistron can be expressed from a bicistronic (or multicistronic) mRNA, without requiring either cleavage of a polyprotein or generation of a monocistronic mRNA. Internal ribosome entry sites are approximately 450 nucleotides in length and are characterized by moderate conservation of primary sequence and strong conservation of secondary structure. The most significant primary sequence feature of the IRES is a pyrimidine-rich site whose start is located approximately 25 nucleotides upstream of the 3′ end of the IRES. See Jackson et al. (1990). Three major classes of picornavirus IRES have been identified and characterized: the cardio- and aphthovirus class (for example, the encephalomyocarditis virus, Jang et al. 1990. Gene Dev 4:1560-1572); the entero- and rhinovirus class (for example, polioviruses, Borman et al. 1994. EMBO J. 13:3149-3157); and the hepatitis A virus (HAV) class, Glass et al. 1993. Virol 193:842-852). For the first two classes, two general principles apply. First, most of the 450-nucleotide sequence of the IRES functions to maintain particular secondary and tertiary structures conducive to ribosome binding and translational initiation. Second, the ribosome entry site is an AUG triplet located at the 3′ end of the IRES, approximately 25 nucleotides downstream of a conserved oligopyrimidine tract. Translation initiation can occur either at the ribosome entry site (cardioviruses) or at the next downstream AUG (entero/rhinovirus class). Initiation occurs at both sites in aphthoviruses. HCV and pestiviruses such as bovine viral diarrhea virus (BVDV) or classical swine fever virus (CSFV) have 341 nt and 370 nt long 5′-UTR respectively. These 5′-UTR fragments form similar RNA secondary structures and can have moderately efficient IRES function (Tsukiyama-Kohara et al. 1992. J. Virol. 66:1476-1483; Frolov et al. 1998. RNA 4:1418-1435). Recent studies showed that both Friend-murine leukemia virus (MLV) 5′-UTR and rat retrotransposon virus-like 30S VL30) sequences contain IRES structure of retroviral origin (Torrent et al. 1996. Hum. Gene Ther 7:603-612). In eukaryotic cells, translation is normally initiated by the ribosome scanning from the capped mRNA 5′ end, under the control of initiation factors. However, several cellular mRNAs have been found to have IRES structure to mediate the cap-independent translation (van der Velde, et al. 1999. Int Biochem Cell Biol. 31:87-106). Examples of IRES elements include, without limitation, immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. 1991. Nature 353:90-94), antennapedia mRNA of Drosophila (Oh et al. 1992. Gene and Dev 6:1643-1653), fibroblast growth factor-2 (FGF-2) (Vagner et al. 1995. Mol. Cell. Biol. 15:35-44), platelet-derived growth factor B (PDGF-B) (Bernstein et al. 1997. J. Biol. Chem. 272:9356-9362), insulin-like growth factor II (Teerink et al. (1995) Biochim. Biophys. Acta 1264:403-408), and the translation initiation factor eIF4G (Gan et al. 1996. J. Biol. Chem. 271:623-626). Recently, vascular endothelial growth factor (VEGF) was also found to have IRES element (Stein et al. 1998. Mol. Cell. Biol. 18:3112-3119; Huez et al. 1998. Mol. Cell. Biol. 18:6178-6190). Further examples of IRES sequences include Picornavirus HAV (Glass et al. 1993. Virology 193:842-852); EMCV (Jang and Wimmer. 1990. Gene Dev. 4:1560-1572); Poliovirus (Borman et al. 1994. EMBO J. 13:3149-3157); HCV (Tsukiyama-Kohara et al. 1992. J. Virol. 66:1476-1483); pestivirus BVDV (Frolov et al. 1998. RNA. 4:1418-1435); Leishmania LRV-1 (Maga et al. 1995. Mol. Cell. Biol. 15:4884-4889); Retroviruses: MoMLV (Torrent et al. 1996. Hum. Gene Ther. 7:603-612). VL30, Harvey murine sarcoma virus, REV (Lopez-Lastra et al. 1997. Hum. Gene Ther. 8:1855-1865). IRES may be prepared using standard recombinant and synthetic methods known in the art. For cloning convenience, restriction sites may be engineered into the ends of the IRES fragments to be used.
In some embodiments, two immunomodulatory polypeptides are expressed from separate transcripts, i.e., a first transcript and a second transcript. In some embodiments, the two transcripts are encoded by a DNA insertion at the same location in the adenovirus, e.g., both inserted in E1b, E3, or E4. In some embodiments, the two transcripts are encoded by a DNA insertion at the different locations in the adenovirus, e.g., a first transcript DNA inserted in E1b and a second transcript DNA inserted in E3 or E4, or alternatively, a first transcript DNA inserted in E3 and a second transcript DNA inserted in E4.
Another aspect of the invention provides a virus for causing expression in a target cell of a plurality of recombinant immunomodulatory polypeptides or other protein(s) or polypeptides of interest, wherein the vector also includes a promoter operably linked to a first coding sequence for a first recombinant immunomodulatory polypeptide, a self-processing or other cleavage coding sequence, such as a 2A or 2A-like sequence or a protease recognition site, and a second coding sequence for a second recombinant immunomodulatory polypeptide, wherein the self-processing cleavage sequence or protease recognition site coding sequence is inserted between the first and the second coding sequences. In a related embodiment, the viral vector comprises an expression vector as described above wherein the expression vector further comprises an additional proteolytic cleavage site between the first and second recombinant immunomodulatory polypeptides. A preferred additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RXR/K-R.

Interaction of Oncolytic Activity and Immunomodulatory Activity.

Oncolytic viruses (OVs) were originally conceived as simply a means of targeted destruction of cancer cells. However, it is now thought that the most effective OV therapies will be those that combine tumor cell death with the stimulation of a host anti-tumor immune response. OVs engineered to express particular immunomodulatory cytokines in tumor cells will be able to specifically guide the immune system toward combating cancer cells. Combining the expression of these cytokines with the release of tumor-associated antigens (TAAs, i.e. tumor cell debris) upon viral lysis of cancer cells will allow for the development of cellular or antibody-mediated anti-tumor immune responses (Lichty et al., 2014, Nature Reviews Cancer, 14: 559-567).

Pharmaceutical Compositions.

A pharmaceutical composition of the invention comprises at least one of the vectors of the invention as described herein. Furthermore, the composition may comprise at least two, three or four different (i.e., expressing different transgenes) vectors of the invention. In addition to the vector, a pharmaceutical composition may also comprise any other vectors, such as other adenoviral vectors, other therapeutically effective agents, any other agents such as pharmaceutically acceptable carriers, buffers, excipients, adjuvants, antiseptics, filling, stabilizing or thickening agents, and/or any components, e.g., such as components found in corresponding viral or pharmaceutical products.
The vector(s) described herein can be administered (e.g., in a pharmaceutical composition) to any human or animal, including but not limited to a human or non-human animal having or diagnosed with cancer. According to one embodiment, the cancer is nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, brain cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, bone cancer, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer, tonsil cancer. The vector or pharmaceutical composition of the invention may be administered to any eukaryotic subject selected from a group consisting of animals and human beings, in a preferred embodiment of the invention, the subject is a human or a non-human animal. An animal may be selected from a group consisting of pets, domestic animals and production animals.
The adenoviral vector(s) of the present invention may be administered to a subject, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.
The therapeutic of the present invention may also be administered directly to the tumor in the form of a liquid, gel or suspension introduced by intratumoral injection. A study examining the treatment of mice bearing subcutaneous human pancreatic adenocarcinoma xenografts with recombinant Newcastle disease virus (rNDV) showed that intratumoral injection yielded better tumor regression than intravenous injection. In this study, animals were injected intratumorally every other day for a total of 4 injections, each containing 5×10′ 50% Tissue Culture Infective Dose (TCID50) rNDV in 50 μl (Buijs et al., 2015, Viruses, 6: 2980-2998). Similarly, another study examining the treatment of mice bearing subcutaneous bladder cancer xenografts with modified oncolytic adenovirus found that intratumoral injection significantly suppressed tumor growth. In this study, animals were injected intratumorally twice at a 1-day interval with 5×10⁸infectious unit (IFU) viruses in 100 μl (Yang et al., 2015, Cell Death and Disease, e1760). Shown to be well-tolerated in Phase 1 trials, Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus has been examined as an intratumorally-administered treatment for patients with advanced hepatocellular carcinoma (HCC). In this study design, patients were to be injected intratumorally 3 times every 2 weeks at one of 2 dose levels: 1×10⁸plaque forming units (pfu), or 1×10⁹pfu (Walther and Stein, 2015, Methods in Molecular Biology, 1317: 343-357).
A single administration of oncolytic adenoviral vectors of the invention may have therapeutic effects. However, in some embodiments of the invention, oncolytic adenoviral vectors or pharmaceutical compositions are administered several times during the treatment period. Oncolytic adenoviral vectors or pharmaceutical compositions may be administered for example from 1 to 10 times in the first 2 weeks, 4 weeks, monthly or during the treatment period. In one embodiment of the invention, administration is done three to seven times in the first 2 weeks, then at 4 weeks and then monthly, in a specific embodiment of the invention, administration is done four times in the first 2 weeks, then at 4 weeks and then monthly. The length of the treatment period may vary, and for example may last from two to 12 months or more.
To improve the efficacy of the present invention, in some embodiments, the therapeutic of the present invention is administered with an adjuvant. Suitable adjuvants include aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate, Incomplete Freund's Adjuvant (IFA), and monophosphoryl lipid A (MPL). These adjuvants are suitable for human administration, either alone or optionally all combinations thereof (Chang et al., “Adjuvant Activity of Incomplete Freund's Adjuvant,” Adv Drug Deliv Rev 32:173-186 (1998), which is hereby incorporated by reference in its entirety). Other adjuvants include cytokines, such as interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), di- and tri-palmitoyl-S-glyceryl cysteine (Pam2Cys and Pam3Cys, respectively), a TLR2 agonist, an anti-granulocyte macrophage colony-stimulating factor (GM-CSF) antibody, RR-XS15, Montanide®, and MALP-2.
The adenoviral vector of the present invention can be administered orally, parenterally, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, or by application to mucous membranes, such as, that of the nose, throat, and bronchial tubes. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid (e.g., aqueous) form such as, tablets, capsules, powders, solutions, suspensions, or emulsions.
The therapeutic of the present invention may be orally administered, for example, with an inert diluent, or with a suitable edible carrier, or they may be enclosed in hard or soft-shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the therapeutic may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Various other materials may be present as coatings or to modify the physical form of the dosage unit.
The therapeutic may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
The therapeutic of the present invention may also be administered directly to the airways in the form of an aerosol. For use as aerosols, the compounds of the present invention in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The materials of the present invention also may be administered in a non-pressurized form such as in a nebulizer or atomizer.
The therapeutic of this invention may be administered in sufficient amounts to transfect the desired cells and provide sufficient levels of integration and expression of the replicating virus to provide a therapeutic benefit without undue adverse effects or with medically acceptable physiological effects which can be determined by those skilled in the medical arts.
Dosages of the therapeutic will depend primarily on factors, such as the condition being treated, the age, weight, and health of the patient, and may thus vary among patients. The dosage will be adjusted to balance the therapeutic benefit against any viral toxicity or side effects.
The present invention also relates to a method of enhancing the delivery to and distribution within a tumor mass of therapeutic proteins expressed from viruses. For example, an adenovirus as described herein, optionally in a pharmaceutical composition as described herein, can be injected into a tumor mass such that the virus infects and lyses one or more tumor cell. Combination Therapy.
The viral immunotherapy of the invention is effective alone, but combination of multiple adenoviral immunotherapies, or one or more adenoviral immunotherapies with any other therapies, such as traditional therapy, may be more effective than either one alone. For example, each agent of the combination therapy may work independently in the tumor tissue, the adenoviral vectors may sensitize cells to chemotherapy or radiotherapy and/or chemotherapeutic agents may enhance the level of virus replication or effect the receptor status of the target cells. The agents of combination therapy may be administered simultaneously or sequentially.
In a preferred embodiment of the invention, the method or use further comprises administration of concurrent radiotherapy to a subject. In another preferred embodiment of the invention, the method or use further comprises administration of concurrent chemotherapy to a subject. As used herein “concurrent” refers to a therapy, which has been administered before, after or simultaneously with the gene therapy of the invention. The period for a concurrent therapy may vary from minutes to several weeks. In some embodiments, the concurrent therapy lasts for some hours.
Agents suitable for combination therapy include but are not limited to afatinib, all-trans retinoid acid, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, 5-fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, temozolomide, teniposide, tioguanine, valrubicin, vinblastine, vincristine, vindesine and vinorelbine.
In some embodiments, the method or use further comprises administration of verapamil or another calcium channel blocker to a subject. “Calcium channel blocker” refers to a class of drugs and natural substances which disrupt the conduction of calcium channels, and if may be selected from a group consisting of verapamil, dihydropyridines, gallopamil, diltiazem, mibefradil, bepridil, fluspirilene and fendiline.
In some embodiments, the method or use further comprises administration of autophagy inducing agents to a subject. Autophagy refers to a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. “Autophagy inducing agents” refer to agents capable of inducing autophagy and may be selected from a group consisting of, but not limited to, mTOR inhibitors (e.g., temsirolimus, sirolimus, everolimus, and ridaforolimus), P13K inhibitors (e.g., wortmannin, lithium, tamoxifen, chloroquine, bafilomycin, and temozolomide. In a specific embodiment of the invention, the method further comprises administration of temozolomide to a subject. Temozolomide may be either oral or intravenous temozolomide.
In some embodiments, the method or use further comprises administration of chemotherapy or anti-CD20 therapy or other approaches for blocking of neutralizing antibodies. “Anti-CD20 therapy” refers to agents capable of killing CD20 positive cells, and may be selected from a group consisting of rituximab and other anti-CD20 monoclonal antibodies. “Approaches for blocking of neutralizing antibodies” refers to agents capable of inhibiting the generation of anti-viral antibodies that normally result from infection and may be selected from a group consisting of different chemotherapeutics, immunomodulatory substances, corticoids and other drugs. These substances may be selected from a group consisting of, but not limited to, cyclophosphamide, ciclosporin, azathioprine, methylprednisolone, etoposide, CD40L, CTLA4, FK506 (tacrolimus), IL-12, IFN-γ, interleukin 10, anti-CD8, anti-CD4 antibodies, hematopoietic stem cell transplantation (HSCT) and oral adenoviral proteins.
In some embodiments, the oncolytic adenoviral vector of the invention induces virion-mediated oncolysis of tumor cells and activates human immune response against tumor cells. In some embodiments, the method or use further comprises administration of substances capable to downregulating regulatory T-cells in a subject in an amount to downregulate (e.g., by at least 10%, 20%, 50%, 70%, 90% or more) regulatory T-cells in the subject. “Substances capable to downregulating regulatory T-cells” refers to agents that reduce the numbers of cells identified as T-suppressor or Regulatory T-cells. These cells have been identified as consisting one or many of the following immunophenotypic markers: CD4+, CD25+, FoxP3+, CD127- and GITR+. Such agents reducing T-suppressor or Regulatory T-cells may be selected from a group consisting of anti-CD25 antibodies or chemotherapeutics.
In some embodiments, the method or use further comprises administration of cyclophosphamide to a subject. Cyclophosphamide is a common chemotherapeutic agent, which has also been used in some autoimmune disorders. In the present invention, cyclophosphamide can be used to enhance viral replication and the effects of GM-CSF induced stimulation of NK and cytotoxic T-cells for enhanced immune response against the tumor. It can be used as intravenous bolus doses or low-dose oral metronomic administration.

Kits

The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

EXAMPLES

The present invention is further described by the following examples, which are illustrative of specific embodiments of the invention, and various uses thereof. These exemplifications, which illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.
Unless otherwise indicated, the practice of the present invention employs conventional techniques of cell culture, molecular biology, microbiology, recombinant DNA manipulation, immunology science, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g. Cell Biology: a Laboratory Handbook: J. Cells (Ed). Academic Press. N.Y. (1996); Graham, F. L. and Prevec, L. Adenovirus-based expression vectors and recombinant vaccines. In: Vaccines: New Approaches to Immunological Problems. R. W. Ellis (ed) Butterworth. Pp 363-390; Grahan and Prevec Manipulation of adenovirus vectors. In: Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Techniques. E. J. Murray and J. M. Walker (eds) Humana Press Inc., Clifton, N.J. pp 109-128, 1991; Sambrook et al. (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), and Ausubel et al. (1995), Short Protocols in Molecular Biology, John Wiley and Sons.

Example 1. Virology and Recombinant Nucleic Acids

Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, organic synthetic chemistry, and pharmaceutical formulation and delivery, and treatment of patients. Methods for the construction of adenoviral mutants are generally known in the art. See Bett, A. J. et al, PNAS 1994 vol: 91, pages 8802-8806, Mittal, S. K., Virus Res., 1993, vol: 28, pages 67-90; and Hermiston, T. et al., Methods in Molecular Medicine: Adenovirus Methods and Protocols, W. S. M. Wold, ed, Humana Press, 1999. Further, the adenovirus 5 genome is registered as GenBank 10 accession #M73260, and the virus is available from the American Type Culture Collection, Rockville, Md., U.S.A., under accession number VR-5.

Viruses and Cell Lines.

Adenoviruses and cell lines were generally obtained from American Type Culture Collection (ATCC), Manassas, Va.) unless otherwise noted. Cell lines used in the Examples below may include one or more of the cell lines listed in Table 2.

TABLE 2

Viruses and Cell Lines

Cell Line	Cell/Tissue Type	Catalog #

HEK-293	Normal human embryonic kidney	ATCC ® CRL-1573 ™
MRC-5	Normal human lung	ATCC ® CCL-171 ™
HMEC-1	Normal human endothelium	ATCC ® CRL3243 ™
MCF 10A	Normal human mammary epithelial	ATCC ® CRL-10317 ™
NHLF	Normal human lung fibroblasts	Lonza CC-2512
HUVEC	Normal human umbilical	ATCC ® CRL-1730 ™
	endothelium
HCC827	lung adenocarcinoma	ATCC ® CRL-2868 ™
A549	human lung carcinoma	ATCC ® CCL-185 ™
ADS-12		a derivative from a murine
		KRAS mutant lung
		adenocarcinoma cell line (LKR-
		13)
NCI-H1734	NSCLC adenocarcinoma	ATCC ® CRL-5891 ™
NCI-H2110	NSCLC metastatic carcinoma	ATCC ® CRL-5924 ™
HT-1080	Connective tissue fibrosarcoma	ATCC ® CCL121 ™
PC-3	Prostate adenocarcinoma	ATCC ® CRL1435 ™,
SNU-449	Hepatocellular carcinoma	ATCC ® CRL-2234 ™
HepG2	Hepatocellular carcinoma	ATCC ® HB-8065 ™
MDA-MB-231	Breast adenocarcinoma	ATCC ® HTB-26 ™
PANC-1	Pancreatic carcinoma	ATCC ® CRL-1469 ™
SW780	Bladder carcinoma	ATCC ® CRL-2169 ™
FaDu	Head and neck squamous cell	ATCC ® HTB43 ™
	carcinoma
DLD-1	Colorectal adenocarcinoma	ATCC ® CCL-221 ™
CT26	Murine colon carcinoma	ATCC ® CRL-2638 ™
U-87	brain glioblastoma	ATCC ® HTB14 ™
MBT-2	Murine bladder cancer	described, e.g., in Takahashi et
		al., J Urol, 166(6), 2506-2511
B16F10	Murine skin melanoma	ATCC ® CRL-6475 ™
TAV-255 D19	Adenovirus	described in, e.g., Zhang et al.,
		Cancer Gene Ther. (2015)
		22(1): 17-22.
Ad5	Adenoid 75 strain	ATCC ® VR-5 ™

Viral Purification and Quantitation

Viral stocks were propagated on HEK-293 cells and purified by standard methods such as column purification kits (Virapure, Millipore) or CsCl gradient centrifugation (as described in Tollefson, A., Hermiston, T. W., and Wold, W. S. M.; “Preparation and Titration of CsCl-banded Adenovirus Stock” in Adenovirus Methods and Protocols, Humana Press, 1999, pp 1-10, W. S. M. Wold, Ed.). The method used to quantitate viral particles is based on simple OD 260/280 readings, e.g., using the method of Lehmberg et al. (1999) J. Chrom. B, 732:411-423. In the viral concentration range used, the A260 nm peak area of each sample is directly proportional to the number of viral particles in the sample. The number of viral particles per ml in each test sample was calculated by multiplying the known number of viral particles per ml in the standard by the ratio of the A260 nm viral peak area of the sample to the A260 nm viral peak area of the standard. One A260 unit contains approximately 1×10¹²viral particles. Virus Infectious Units/ml (IU/ml) were determined by hexon staining of infected cells (e.g., using Adeno-X™ Rapid titer kit from Takara Bio USA, Inc. (TBUSA, formerly known as Clontech Laboratories, Inc.).

Example 2. Bio Selection

Initial screening of recombinant virus was based on isolated viral DNA rescued from transfected 293 cells where viral propagation based cytopathic effects (CPE) were observed. Viral DNA was used as a template for PCR based detection of sequences flanking the site of the TAV-255 E1a enhancer deletion region. Wild-type sequence would produce a band of 350 bp, while DNA which had the E1a enhancer deletion would only generate a clearly distinguishable 300 bp band. In addition, primers specific for internal sequences of the IL-12 transgene and for Ad hexon sequences were used to verify 400 bp and 3 kb PCR amplified bands respectively that would only be present in viral DNA generated by recombination between the two parental plasmids containing either the E1 region and transgene insert or the late Ad structural proteins derived solely from the pAdEasy™ plasmid. Individual constructs were isolated by two rounds of plaque purification on A549 or 293 cells using standard methods (Tollefson, A., Hermiston, T. W., and Wold, W. S. M.; “Preparation and Titration of CsCl-banded Adenovirus Stock” in Adenovirus Methods and Protocols, Humana Press, 1999, pp 1-10, W. S. M. Wold, Ed). Dilutions of adenoviral lysates were used to infect A549 or 293 cells in a standard plaque assay. Well-individuated plaques were harvested, and the same plaque assay method was used to generate a second round of individual plaques from these harvests. Well isolated plaques from the second round of plaque purification were deemed pure, infected cultures are prepared using these purified plaques, and the oncolytic potency and selectivity of these culture supernatants was determined.

Example 3. Cytolytic Assay

Tumor specific viral lysis was evaluated in both tumor and non-tumor cell lines of murine and human origin by infection of the cells in vitro with the viruses, followed by standard crystal violet for cell viability over time as instructed in the kit manuals.

Example 4. DNA Sequencing

DNA sequencing of the human Ad5-based recombinant adenovirus genomic DNAs was performed as follows. Viral DNA was purified from recombinant adenoviruses such as TAV-255 or other modifications of these constructs by standard column purification methods such as the Qia-Amp® blood DNA purification kit from Qiagen®. PCR primers were used to amplify and isolate regions covering the E1a modified regions and the regions containing the transgene insert and sent out for sequencing at a CRO. Isolated DNA was also analyzed by standard restriction digestion and SDS PAGE analysis for verification of appropriate sized bands of digested DNA. Sequence information was analyzed using the Vector NTI program (Informatix).

Example 5. Construction of Recombinant Viruses

The base shuttle transfer vector includes pXC1 TAV 255 d19k (Zhang et al., Cancer Gene Ther. (2015) 22(1):17-22). This plasmid has a deletion between bp-305 to -255 of the E1a enhancer region, removing two Pea3 and one E2F binding sites which restrict replication and oncolysis of an adenovirus with this deletion to infected tumor cells (Hedrun, F. H., Shantanu K., and Reid, T. (2011) Cancer Gene Therapy 18; 717-723.) It also contains sites allowing deletion of the E1b 19k region and exogenous transgene insert by a Sal I/Xho I digest. Without the digest, the majority of the E1b 19k region is intact, but non-functional. cDNAs for each transgene of interest were synthesized (GeneArt™) or isolated from commercial plasmid sources (GE Lifesciences or GeneCopoeia™) by PCR using primers which added on 5′ SalI and 3′ XhoI restriction sequences for insertion into the SalI/XhoI digested pXC1 TAV 255 plasmids. The modified pXC1 TAV 255 gene insert plasmids were amplified in E. coli and purified using Qiagen® Maxi-prep plasmid kits.
To obtain recombinant adenoviruses, the pXC1 TAV 255 gene insert-containing plasmids were co-transfected into HEK-293 cells (ATCC) with pBHG10 (Microbix) as described (Bett, A. J., Haddara, W., Prevec, L., and Graham, F. (1994) PNAS 91; 8802-8806), using the calcium phosphate transfection protocol from Molecular Cloning: A laboratory manual (Maniatis Vol. 3; 16.30-16.36) for 2-5 μg plasmid DNA (for both plasmids so the pXC1 will be in molar excess) per 60 mm dish of cells. Recombination between homologous adenovirus sequences from each plasmid generates a full length, replication competent adenovirus containing the E1 modifications described earlier and a specific transgene inserted into the E1b 19k deletion site and the E3 deletion supplied by the pBHG10 plasmid. Optionally, the pBHG10 is provided as the adenoviral genome source, having substantial additional utility over vectors such as pJM17 (See, e.g., Hedrun et al, (2011)). Preferentially, an E3 deletion and/or other modifications allow increased packaging capacity for exogenous genes in excess of pJM17 capacity.
In another embodiment, the human IL-12 virus, TRZ627, was constructed using a modification of the pAdEasy™ Adenoviral Vector System (Agilent Technologies). First, sequences from pXCI-TAV d19K plasmid (Hedjran F et al., Cancer Gene Therapy (2011) 18, 717-723) which included the 50-nucleotide deletion in the enhancer of E1A which restricts viral propagation to tumor vs. non-tumor cells, were subcloned into the pShuttle™ vector supplied in the pAdEasy™ kit to create the TAV-255 Shuttle E1 cloning plasmid. Sequences between the first Pac I site (6) and the single Mfe I site (807) in pShuttle were replaced by pXC1 TAV d19K sequences from the beginning of the 5′ ITR sequence (21) to the Mfe I site (3874), corresponding to the same Mfe I site in pShuttle. A Pac I cloning site was added by PCR onto the 5′ end of this fragment, which brought in Ad E1a and E1b sequence not found in the original pShuttle plasmid. The TAV-255 deletion of 50 bp in the E1a enhancer region removes the Pea3 III, Pea3 II, and an E2F transcription factor binding sites and corresponds to human wild-type Ad5 sequence of bases 194-244. The added sequences also included a modification introducing Sal I/Xho I cloning sites into the E1b 19k coding region which can be used to replace the E1b 19k ORF with an exogenous transgene insert whose expression would be driven by the Ad E1b promoter during viral replication. Recombination between Pme I linearized pTAV-255 Shuttle E1 containing hIL-12 cloned in at the E1b 19k site and the pAdEasy vector took place in the recA proficient BJ5183 bacterial strain, which had been modified to already contain the pAdEasy plasmid. DNA isolated from Kanamycin resistant plated colonies was screened for full-length viral DNA recombinants by restriction digest. Positive clones (TRZ-627, hIL-12) were subsequently digested with Pac I to free up the Ad ITRs and then transfected into 293 cells to amplify the virus.
Infected HEK-293 cells were collected when visible sign of cytopathic effects due to viral replication were observed up to 2 weeks post-infection and resuspended in their media and lysed by 3 rounds of freeze/thaw. Virus can be purified from the lysate by several methods, including Anion-exchange HPLC (Shabram, P. W., et al (1997) Human Gene Therapy 8; 453-465) or several commercially available kits based on affinity chromatography or size exclusion membranes or columns (e.g., Adeno-X™ Maxi Purification Kit, Clontech® (Takara), Adenovirus Purification Virakit®, Virapur®). Purified virus can also undergo clonal isolation by standard plaque purification methods, followed by re-amplification and purification of the plaque purified viral clone.

Example 6. In Vivo Demonstration of Reduction in Tumor Volume with Oncolytic Adenoviral Vectors Encoding Immunomodulatory Polypeptides: Single Viruses with and without Antibody Treatment

Materials and Methods
An anti-mPD-L1 antibody is, e.g., from BioXCell®, Catalog# BE0101 (Rat IgG2b). This antibody was used in the below experiments.
Virus samples were stored in 25 mM NaCl, 10 mM Tris Tris(hydroxymethyl)aminomethane), and 5% glycerol with a pH value of 8.0. Vials were stored protected from light at −80° C. On each day of dosing, one vial was thawed at room temperature for approximately 20 minutes. A single dose is 1×10⁹pfu.
The mouse tumor model in this Example uses syngeneic immunocompetent mice. Female Jackson 129S1 (129S1/Sv1mJ) mice were used in this study. They were 6-7 weeks old on Day 1 of the experiment. The animals were fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum.
Animals were housed in static cages with Bed-O'Cobs™ bedding inside bioBubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complete air changes per hour.
All treatments, body weight determinations, and tumor measurements were carried out in the bubble environment. The environment was controlled to a temperature range of 70°±2° F. and a humidity range of 30-70%.
Cell Preparation
ADS-12 cells were grown in RPMI 1640 medium which was modified with 1% 100 mM Na pyruvate, 1% 200 mM L-glutamine, 1% 1M HEPES buffer, 1% of a 45% glucose solution and supplemented with 10% non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100× Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment was maintained in an incubator with a 5% CO₂atmosphere at 37° C. When expansion was complete, the cells (passage 7) were trypsinized using 0.25% trypsin/2.21 mM EDTA in HBSS solution. Following cell detachment, the trypsin was inactivated by dilution with complete growth medium and any clumps of cells were separated by pipetting. The cells were centrifuged at 200rcf for 8 minutes at 4° C., the supernatant was aspirated, and the pellet was re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) by pipetting. An aliquot of the homogeneous cell suspension was diluted in a trypan blue solution and counted using a Luna automated cell counter. The cell suspension was centrifuged at 200rcf for 8 minutes at 4° C. The supernatant was aspirated and the cell pellet was re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) to generate a final concentration of 1×10⁷trypan-excluding cells/ml. The cell suspension was maintained on wet ice during implantation. Following implantation, an aliquot of the remaining cells was diluted with a trypan blue solution and counted to determine the post-implantation cell viability. The cell viabilities of the suspensions used for implantation (two preps) are listed in Table 3.

TABLE 3

Implantation Cell Viability

	Pre-Implant Viability (%)	Post-Implant Viability (%)

Cell Prep 1	95	89
Cell Prep 2	95	89

Test animals were implanted subcutaneously on both flanks (on the back between the spine and the hip), the right flank on Day 0 and the left flank on Day 8, with 1×10⁶cells in 0.1 ml of serum-free medium using a 28-gauge insulin syringe with a fixed needle.
All mice were sorted into study groups based on caliper measurement estimation of tumor burden on Day 15 when the mean tumor burden for all animals on the right flank was approximately 82 mm³(range of group means, 75-90 mm³). The mice were distributed to ensure that the mean tumor burden on the right flank for all groups was within 10% of the overall mean tumor burden for the study population.
Results
The mean estimated right side tumor burden for all groups in the experiment on the first day of treatment was 82 mm3 and all of the groups in the experiment were well-matched (range of group means, 75-90 mm3). All animals weighed at least 13.3 g at the initiation of therapy. Mean group body weights at first treatment were also well-matched (range, 15.4-18.3 g). A tumor burden of 500 mm3 was chosen for evaluation of efficacy by tumor growth delay for the right and left tumors. The median Control Group (FIG. 6A) tumor burdens reached 500 mm3 on Day 47 for right tumors and Day 43 for left tumors. The median tumor volume doubling times for the Control Group were 12.1 and 10.1 days for the right and left tumors, respectively. Control animals experienced a 7.3% mean weight gain during the treatment regimen. There were no spontaneous regressions in the Control Group for either right or left side tumors, however, 50% of the tumors on the left side never reached the palpation limit. In some embodiments, mice with palpable left side tumors were put on study. Since in this experiment the implant was delayed 8 days, the left side tumors were not palpable at staging. These mice that had a left side tumor that remained a 0 throughout the study could have been triaged out of the study if implanted on the same day as the right side.
Results of mouse inoculation and tumor growth are shown in FIG. 6. The series of graphs shows treatment of tumor bearing mice (n=8 in each group) with oncolytic viruses comprising various transgenes, with or without anti-PD-L1.
FIG. 6A is a graph showing the activity of various oncolytic viruses compared to the empty virus (“d19k”), 38 days after cell implantation (primary tumor) as a function of tumor growth inhibition. Black bars represent virus alone and hatched bars represent virus+anti-PD-L1 antibody. FIG. 6B is a graph comparing the average tumor size (primary tumor) over time of tumors injected with virus buffer (black circles), empty vector (blue solid squares), anti-PD-L1 alone (antibody given on days 16, 20, 24, and 28, right-hand arrow in each pair of arrows), each of five viruses alone (viruses carrying CTLA-4, IL-12, IL-7, CD70, and IL-10 transgenes) or combined with anti-PD-L1 antibody. FIG. 6C shows similar data for three more viruses (viruses carrying OX40L, CD40L, and GM-CSF transgenes). As shown in the Figures, the IL-12 oncolytic virus treatment, alone or in combination with an anti-PD-L1 antibody, were the most effective at reducing tumor growth. CD70, IL-7, and CTLA-4 viruses were also able to reduce tumor volume significantly when combined with the anti-PD-L1 antibody.
The following Figures demonstrate efficacy (or lack thereof) of various viruses with transgenes with or without PD-L1 on the primary tumor only; the thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows). Treatments are shown for the primary tumor (receiving the oncolytic virus injection) and are as follows: 6D, virus buffer and control IgG only (left) and virus buffer and anti-PD-L1 antibody (right); 6E, d19k (empty virus) and control IgG only (left) and d19k and anti-PD-L1 antibody (right); 6F, CTLA-4 virus with control IgG (left) or anti-PD-L1 (right); 6G, IL-12 virus with control IgG (left) or anti-PD-L1 antibody (right); 6H, GM-CSF virus with control IgG (left) or anti-PD-L1 antibody (right); 6I, IL-7 virus with control IgG (left) or anti-PD-L1 antibody (right); 6J, CD40L virus with control IgG (left) or anti-PD-L1 antibody (right); 6K, L10 trap virus with control IgG (left) or anti-PD-L1 antibody (right); and 6L, OX40L virus with control IgG (left) or anti-PD-L1 antibody.
As shown in the Figures, combinations with the IL-12 adenovirus (FIG. 6G), showed the greatest ability to reduce tumor volume. As in FIG. 6A, the IL-7 (FIG. 6I) and CTLA-4 (FIG. 6F) oncolytic viruses also showed activity when combined with anti-PD-L1 antibody. Treatment with the oncolytic virus encoding the CD40 ligand also showed some activity when combined with the anti-PD-L1 antibody (FIG. 6J).

Example 7. In Vivo Demonstration of Reduction in Tumor Volume with Oncolytic Adenoviral Vectors Encoding Immunomodulatory Polypeptides: Virus Mixing Study

The mouse tumor model in this Example uses syngeneic immunocompetent mice. Animals were injected with approximately 1,000,000 cells subcutaneously (s.c.) into the right hind flank of the mouse. At the same time, the mice were also injected with 1,000,000 cells s.c. into the left hind flank of the mouse to create a bilateral tumor model. When the right (primary) tumors reached 63-80 mm³in size and the left tumor was palpable, they were injected with 25 μl of virus buffer or 25 μl of virus at 4×10⁹pfu/ml (plaque forming units per ml) directly into the center of the primary tumor every fourth day for a total of three doses. The mice were also treated intraperitoneally with 250 μl of an anti-PD-L1 antibody at 2 mg/ml every fourth day for a total of three doses. The antibody was administered 24 hours after administration of the virus. A reduction in the size of the primary and distal (contralateral) tumor would be noted relative to the virus buffer control and additional controls such as the wild-type virus (not expressing a transgene) with or without administration of the anti-PD-L1 antibody. A specific example of the treatment with oncolytic adenoviral vectors is described in more detail below for a panel of such vectors.
Evaluation of the primary tumor (right flank) was used to determine the direct effect of the oncolytic viruses whereas evaluation of the contralateral tumor (left flank) was used to see the systemic effects of the oncolytic viruses. ADS-12 (a murine KRAS-mutant lung adenocarcinoma cell line) grown in its syngeneic mouse strain is a tumor model known to support adenoviral infection and replication and is useful in the evaluation of host immune responses to oncolytic human adenoviruses.

Materials and Methods

An anti-mPD-L1 antibody is, e.g., from BioXCell®, Catalog# BE0101 (Rat IgG2b). This antibody was used in the below experiments.
Virus samples were stored in 25 mM NaCl, 10 mM Tris Tris(hydroxymethyl)aminomethane), and 5% glycerol with a pH value of 8.0. Vials were stored protected from light at −80° C. On each day of dosing, one vial was thawed at room temperature for approximately 20 minutes.

Animal Studies

Female 12951 (129S1/SvImJ) mice from The Jackson Laboratory were used in this study. They were approximately 7-8 weeks old on Day 14 of the experiment. The animals were fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum. Animals were housed in static cages with Bed-O'Cobs™ bedding inside Biobubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complete air changes per hour. All treatments, body weight determinations, and tumor measurements were carried out in the bubble environment. The environment was controlled to a temperature range of 70°±2° F. and a humidity range of 30-70%. All procedures carried out in this experiment were conducted in compliance with all the laws, regulations and guidelines of the National Institutes of Health (NIH) and with the approval of Molecular Imaging, Inc.'s Animal Care and Use Committee. Molecular Imaging, Inc. is an AAALAC accredited facility.

Cell Preparation

ADS-12 cells (murine KRAS-mutant lung adenocarcinoma) were grown in RPMI 1640 medium which is modified with 1% 100 mM Na pyruvate, 1% 200 mM L-glutamine, 1% 1M HEPES buffer, 1% of a 45% glucose solution and supplemented with 10% non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100×Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment was maintained in an incubator with a 5% CO₂atmosphere at 37° C. When expansion as complete, the cells are trypsinized using 0.25% trypsin/2.21 mM EDTA in HBSS solution. Following the cell viabilities of the suspensions used for implantation (two preps) are listed in the table below.

TABLE 4

Implantation Cell Viability

	Pre-Implant Viability (%)	Post-Implant Viability (%)

Cell Prep 1	94	92
Cell Prep 2	97	92

Test animals were implanted subcutaneously, on both flanks (on the back between the spine and the hip) on Day 0 with 1.00×10⁶cells in 0.1 ml of serum-free medium using a 28-gauge insulin syringe with a fixed needle.

Treatment

All mice were sorted into study groups based on caliper measurement estimation of tumor burden on Day 14 when the mean tumor burden for all animals on the right flank is approximately 68 mm³(range of group means, 65-71 mm³). The mice were distributed to ensure that the mean tumor burden for all groups was within 10% of the overall mean tumor burden for the study population.

Measurement and Endpoints

Tumor burden (mm³) was estimated from caliper measurements by the formula for the volume of a prolate ellipsoid assuming unit density as: Tumor burden (mm³)=(L× W²)/2, where L and W are the respective orthogonal tumor length and width measurements (mm). All groups were compared to the virus buffer control group.
The primary endpoints used to evaluate efficacy were: tumor growth delay, complete and partial tumor response, and the number of tumor-free survivors at the end of the study for both left and right tumors. A complete response (CR) is defined as a decrease in tumor mass to an undetectable size (<63 mm³), and a partial response (PR) is defined as a smaller tumor mass at the last measurement compared to at the first treatment. PRs are exclusive of CRs.
All animals were observed for clinical signs at least once daily. Animals were weighed on each day of treatment. Individual body weights were recorded three times weekly. Animals with combined tumor burdens in excess of 2000 mm³were euthanized, as were those found in obvious distress or in a moribund condition. Treatment-related weight loss in excess of 20% is generally considered unacceptably toxic. In this Example, a dosage level was determined to be tolerated if treatment-related weight loss (during and two weeks after treatment) was <20% and mortality during this period in the absence of potentially lethal tumor burdens was <10%.

Results

Results of mouse inoculation and tumor growth are shown in FIG. 7. The series of graphs shows treatment of tumor bearing mice (n=8 in each group) with single and combinations of oncolytic viruses comprising various transgenes, with or without anti-PD-L1. The left-hand panel of each Figure represents the primary tumor into which the virus was injected; the right-hand panel represents the contralateral tumor. The thick line in each graph shows the average tumor volume in mm³. The pairs of arrows on the x-axis represent day of treatment with virus (intratumoral, left arrows) and the anti-PD-L1 antibody, if used (intraperitoneal, right arrows). A summary of the responses of the 8 mice is shown in a table at the bottom of each graph, wherein CR=Complete Response (tumor volume=0) and PR=Partial Response (tumor volume on last day of measurements is smaller than tumor volume on the first day of measurements). Treatments shown are as follows: 7A, virus buffer only; 7B, empty virus only (TRZ000); 7C, TRZ010 (IL-10trap)+empty virus; 7D, TRZ011 (OX40 ligand)+empty virus; 7E, TRZ009 (CD70)+empty virus; 7F, TRZ007 (IL-7)+empty virus, 7G, TRZ002 (IL-12)+empty virus; 7H, TRZ004 (GM-CSF)+empty virus; 7I, TRZ003 (flagellin)+empty virus; 7J, TRZ002+TRZ010; 7K, TRZ002+TRZ007; 7L, TRZ007+TRZ010; 7M, TRZ011+TRZ004; 7N, TRZ009+TRZ003; 7O, TRZ002+TRZ009; 7P, TRZ007+TRZ009; 7Q, TRZ007+TRZ004; 7R, TRZ002+TRZ011; 7S, TRZ010+TRZ004; 7T, TRZ002+TRZ004; 7U, virus buffer and anti-PD-L1; 7V, TRZ002+empty virus+anti-PD-L1; 7W, TRZ009+anti-PD-L1; 7X, TRZ007+empty virus+anti-PD-L1; 7Y, TRZ002+TRZ007+anti-PD-L1; 7Z, TRZ007+TRZ009+anti-PD-L1; 7AA, TRZ002+TRZ009+anti-PD-L1. All viruses were administered at 1×10⁸pfu/dose for each virus, resulting in 2×10⁸pfu total in virus combinations.
As can be seen in the Figure, certain combinations of virus transgenes were extremely successful in lysing tumor cells, including a number of examples of complete response. For example, FIG. 7G shows tumor volume in mice treated with an IL-12 adenovirus. 5 out of 8 mice in this Figure had a complete response, and one mouse had a partial response. In the contralateral tumor, however, no complete or partial responses were seen, indicating that there were no systemic immune effects from treatment with the IL-12 virus alone. A better result is seen in FIG. 7K, which shows mice treated with the same IL-12 adenovirus in combination with an IL-7 adenovirus. In this group of mice, 7 out of 8 had a complete response in the primary tumor (into which the virus was injected) and in the contralateral tumor two mice showed a complete response and one a partial response. In FIGS. 7V (IL-12 adenovirus+anti-PD-L1 antibody) and 7AA (IL-12 adenovirus+CD70 adenovirus+anti-PD-L1 antibody), each group of 8 mice had 8 complete responses in the primary tumor and several in the contralateral tumor. These data demonstrate that certain combination therapies comprising oncolytic adenoviruses can be useful in treating primary and metastatic tumors.

Example 8. Adenoviral Vectors Expressing Multiple Immunomodulatory Polypeptides

If deletions in the adenovirus backbone are sufficiently large enough to allow packaging of viral DNA containing two exogenously added transgenes into the viral capsids, the two genes can be co-expressed by several methods from a single deletion site in adenovirus. Both added genes can be linked to each other by methods described below and have their expression controlled by an endogenous adenovirus promoter, not an exogenously added promoter, so that high expression will only occur during conditions of viral replication. Control of restricting viral replication to certain conditions such as after infection of tumor cells, is described elsewhere in this document. Co-expression of two proteins from a single transcript can be achieved through the use of virus components such as internal ribosome entry site (IRES) elements (Renaud-Gabardos E et al, World J Exp Med 2015, 5: 11-20), insertion of self-cleaving 2A peptide sequences derived from viruses such as Foot and Mouth Disease virus (FMDV) (Garry A. Luke (2012), Translating 2A Research into Practice, Innovations in Biotechnology, Dr. Eddy C. Agbo (Ed.), ISBN: 978-953-51-0096-6, InTech), or by combining the sequences of the two transgenes into a single fusion protein. An exemplary method would use one of the 2A sequences to direct more equal level of expression from both transgenes as opposed to lower expression levels typically seen from the second transgene when using IRES elements.
Descriptions of exemplary dual transgene constructs shown in Table 5 in this Example and Table 6 in Example 13. As can be seen in the table, any of the transgenes may be inserted into the E1 or E3 region. For example, in one embodiment a dual transgene construct may have IL-12 inserted into the E1 region and IL-2 in the E3 region. In another embodiment, a dual transgene construct may have IL-2 inserted into the E1 region and IL-12 in the E3 region. Use of the dual transgene constructs in therapeutic oncolytic adenoviruses is described in Examples 9 and 13.

TABLE 5

Exemplary dual transgene constructs.

TRZ Number	Brief Description

TRZ402*	TAV-255-d19kE1-mIL-10T-E3-mIL-12
TRZ403*	TAV-255-d19kE1-mIL-7-E3-mIL-12
TRZ404*	TAV-255-d19kE1-mCD70-E3-IL-12
TRZ405	TAV-255-d19kE1-mOX40L-E3-mGM-CSF
TRZ406	TAV-255-d19kE1-mIL-10T-E3-mIL-7
TRZ407	TAV-255-d19kE1-mIL-12-E3-mIL-10T
TRZ408	TAV-255-d19k-E3-mIL-12
TRZ409	E1-mIL-12/E3-mIL-7
TRZ413	E1-mIL-7/E3-mIL-12-P2A-ADP
TRZ418*	E1-trimeric mCD70/E3-mIL-12
TRZ421*	E1-mIL-2/E3-mIL-12
TRZ501	TAV-255-d19K Dual mIL-10T-P2A-mIL-12, E3 deleted
TRZ510	E1-mIL-7-P2A-mIL-12
TRZ512	E1-mIL-12-P2A-mIL-7

m = murine;
P2A = cleavage site
*See also Table 6

Example 9. Adenoviral Vectors Expressing Multiple Immunomodulatory Polypeptides

To maintain co-expression from two single gene inserts at different sites, the cDNA for each transgene can be inserted into separate deleted regions of adenovirus so that expression of each would be controlled separately by the endogenous upstream adenovirus promoter. No exogenous promoter would be added with the exogenous transgene sequence. Expression of the E1a proteins leads to the activation of the other adenovirus promoters and viral replication, so expression from each endogenous adenovirus promoter is linked to viral replication. Exogenous transgenes inserted behind different adenovirus promoters, such as the E1b promoter, the E3 promoter, and the E4 promoter, in place of deletions in these regions, leads to a construct where co-expression from each inserted transgene is limited to conditions of where viral replication occurs. Combined with modifications in the E1a enhancer region as described previously in this document to restrict viral replication to tumor cells, co-expression of both exogenous transgenes is restricted to tumor cells.
As shown schematically in FIG. 2, provided is an ΔE1b19K site that includes a deletion of bp 1714-1916 (numbered according to hAd5 vector sequence), which increases packaging capacity by approximately 200 bp. Also provided is a deletion at the E3 site (ΔE3 site as shown in FIG. 2); this deletion can be any deletion in the E3 region, generally at or about bp 27,900-30,800 bp (numbered according to hAd5 vector sequence), which increases packaging capacity up to an additional approximately 2400 bp, as compared to most wild type adenoviruses, which are typically limited to containing (i.e., packaging) approximately 1800 bp of exogenous sequences.
Provided herein are deletions in E3 open reading frames that are suitable for modification (e.g., truncation or deletion) without substantially decreasing viral propagation. By way of non-limiting example, provided are adenoviral vectors wherein the E3 12.5K coding region (27,852-28,175 bp) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 320 or greater than 320 bases are deleted from one or more truncation sites within the region) or entirely deleted. Also provided are adenoviral vectors wherein the E3 7.1K coding region (28,541-28,732) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or greater than 190 bases are deleted from one or more truncation sites within the region) or entirely deleted. The 7.1K sequence is associated with inhibition of TRAIL apoptosis and associated with one or more RID proteins. Also provided are adenoviral vectors wherein the E3 gp19K (28,729-29211) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450 or greater than 450 bases are deleted from one or more truncation sites within the region) or entirely deleted. The gp19K sequence is associated with inhibition of CTL killing. Also provided are adenoviral vectors wherein the E3 10.5 (also called E3 11.6 (ADP)) (29, 485-29,766) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, or greater than 250 bases are deleted from one or more truncation sites within the region) or entirely deleted. The 10.5 sequence is associated with promotion of virus release. Also provided are adenoviral vectors wherein the E3 (RIDα) (29,778-29,969) and/or the E3 (RIDβ) (30,057-30,455) is truncated (e.g., with respect to E3 (RIDα) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or greater than 190 bases are deleted from one or more truncation sites within the region) or entirely deleted, or truncated (e.g., with respect to E3 (RIDβ) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 390, or greater than 390 bases are deleted from one or more truncation sites within the region) or entirely deleted. These RID sequences are associated with inhibition of TNF, FasL, and TRAIL apoptosis and degrade EGFR. Any combination of the above-referenced deletions is also provided.
Also provided are adenoviral vectors wherein the E3 14.7K (30,488-30,834) is truncated (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 340, or greater than 340 bases are deleted from one or more truncation sites within the region) or entirely deleted. The 14.7K sequence is associated with inhibition of TNF, FasL, and TRAIL apoptosis.

Example 10. Adenoviral Vectors Expressing an Immunomodulatory Polypeptide from the E4 Region

A nucleic acid sequence encoding one or more immunomodulatory polypeptides is inserted into the adenoviral genome by truncation or deletion of a portion of the E4 region of the viral genome. An E4 deletion and exogenous insertion can be utilized in combination with any other viral modification provided herein or otherwise known in the art, or alternatively, without any other viral modification. Exemplary E4 regions useful as insertion sites include truncation or deletion of E4 ORF1 (35,136-35,522 bp) and/or E4 ORF2 (34,696-35,106 bp) (each numbered according to hAd5 vector sequence). The expression of the one or more immunomodulatory polypeptides is controlled by the endogenous E4 promoter. A schematic illustration of this embodiment is provided in FIG. 3.

Example 11. Adenoviral Vectors Comprising Altered Configuration of Pea3 Sites

Adenoviral vectors are designed in which sequences in and around Pea3 sites I-V near the E1a Enhancer region of adenoviral vectors, in which sequences in and around Pea3 sites are altered, moved, or deleted in order to produce vectors with a variety of expression characteristics. In one embodiment, adenovirus vectors are engineered which have lower affinity Pea3 sites compared to wild-type adenoviral vectors. Such vectors are designed to be efficient in cells in the tumor microenvironment where the concentration of transcription factors is high, but to be relatively inactive in normal (e.g., non-neoplastic) cells, thus reducing the possibility of side effects caused by damage to normal tissue cells. An illustration of the E1a area of wild-type and TAV-255 constructs is shown in FIG. 9A. A cartoon illustration of the proposed method of action of the E1a enhancer region mutants is shown in FIG. 9B. Constructs NV1 to NV7 are based in part on the TAV-255 construct.
In one embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the sequence is removed between Pea3 IV and Pea3 III sites, a single mutation is made in Pea3 III, the sequence between Pea3 III and Pea3 II, and the Pea3 II site is mutated. The sequence of the E1a enhancer region of the exemplary construct NV1 is set forth in SEQ ID NO:99.
In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the sequence is removed between Pea3 IV and Pea3 III sites, both Pea3 III and Pea3 II are mutated, and Pea3 V flanking sites are mutated such that the resultant Pea3 V site has an affinity more similar to Pea3 III and enhancer 1. The sequence of the E1a enhancer region of the exemplary construct NV2 is set forth in SEQ ID NO:100.
In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the sequence between Pea3 V and Pea3 IV sites is replaced with the sequence between the Pea3 III and Pea3 II sites; the sequence between the Pea3 IV and Pea3 III is deleted; the sequence between the Pea3 III and Pea3 II is deleted; and the Pea3 III and Pea3 II sites are both mutated, as well as the residues 3 bp that are immediately 5′ of the Pea3 V site. The sequence of the E1a enhancer region of the exemplary construct NV3 is set forth in SEQ ID NO:101.
In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the flanking sequences around the Pea3 III and Pea3 II sites are altered to mimic the lower affinity Pea3 V and Pea3 IV sites (thus engineering a vector in which all Pea3 sites except Pea3 I are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV4 is set forth in SEQ ID NO:102.
In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the flanking sequences around the Pea3 III and Pea3 II sites are altered to mimic the lower affinity Pea3 V and Pea3 IV sites, and a single point mutation is introduced in the Pea3 I site, rendering it a lower affinity Pea3 I site (thus engineering a vector in which all Pea3 sites are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV5 is set forth in SEQ ID NO:103.
In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence), the flanking sequences around the Pea3 III and Pea3 II sites are altered to mimic the lower affinity Pea3 V and Pea3 IV sites, and the flanking regions of the Pea3 I site are altered, rendering it a lower affinity Pea3 I site (thus engineering a vector in which all Pea3 sites are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV6 is set forth in SEQ ID NO:104.
In another embodiment, a vector is provided wherein, (in relation to the wild-type sequence) the flanking sequences around the Pea3 I site are altered to produce a lower-affinity Pea3 I site, and the flanking sequences around the Pea3 II site is altered to mimic the lower affinity Pea3 IV site (thus engineering a vector in which all Pea3 sites except Pea3 III are lower affinity). The sequence of the E1a enhancer region of the exemplary construct NV7 is set forth in SEQ ID NO:105.

Example 12. In Vivo Demonstration of Reduction in Tumor Volume with Oncolytic Adenoviral Vectors Comprising E1a Enhancer Region Alterations and Encoding Immunomodulatory Polypeptides: Single Viruses with and without Antibody Treatment

Materials and Methods

An anti-mPD-L1 antibody is, e.g., from BioXCell®, Catalog# BE0101 (Rat IgG2b). This antibody was used in the below experiments.
Virus samples are stored in 25 mM NaCl, 10 mM Tris Tris(hydroxymethyl)aminomethane), and 5% glycerol with a pH value of 8.0. Vials are stored protected from light at −80° C. On each day of dosing, one vial is thawed at room temperature for approximately 20 minutes. A single dose is 1×10⁹pfu. Viruses to be combined with anti-PD-L1 in this Example include NV1-NV7 (having E1a enhancer region sequences set forth in SEQ ID Nos:99-105).
The mouse tumor model in this Example uses syngeneic immunocompetent mice. Female Jackson 129S1 (12951/Sv1mJ) mice are used in this study. Mice are 6-7 weeks old on Day 1 of the experiment. The animals are fed irradiated Harlan 2918.15 Rodent Diet and water ad libitum. Animals are housed in static cages with Bed-O'Cobs™ bedding inside bioBubble® Clean Rooms that provide H.E.P.A filtered air into the bubble environment at 100 complete air changes per hour. All treatments, body weight determinations, and tumor measurements are carried out in the bubble environment. The environment is controlled to a temperature range of 70°±2° F. and a humidity range of 30-70%.

Cell Preparation

ADS-12 cells are grown in RPMI 1640 medium which is modified with 1% 100 mM Na pyruvate, 1% 200 mM L-glutamine, 1% 1M HEPES buffer, 1% of a 45% glucose solution and supplemented with 10% non-heat-inactivated Fetal Bovine Serum (FBS) and 1% 100× Penicillin/Streptomycin/L-Glutamine (PSG). The growth environment is maintained in an incubator with a 5% CO₂atmosphere at 37° C. When expansion is complete, the cells (passage 7) are trypsinized using 0.25% trypsin/2.21 mM EDTA in HBSS solution. Following cell detachment, the trypsin is inactivated by dilution with complete growth medium and any clumps of cells are separated by pipetting. The cells are centrifuged at 200rcf for 8 minutes at 4° C., the supernatant is aspirated, and the pellet is re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) by pipetting. An aliquot of the homogeneous cell suspension is diluted in a trypan blue solution and counted using a Luna automated cell counter. The cell suspension is centrifuged at 200rcf for 8 minutes at 4° C. The supernatant is aspirated and the cell pellet is re-suspended in cold Dulbecco's Phosphate Buffered Saline (DPBS) to generate a final concentration of 1×10⁷trypan-excluding cells/ml. The cell suspension is maintained on wet ice during implantation. Following implantation, an aliquot of the remaining cells is diluted with a trypan blue solution and counted to determine the post-implantation cell viability.
Test animals are implanted subcutaneously on both flanks (on the back between the spine and the hip), the right flank on Day 0 and the left flank on Day 8, with 1×10⁶cells in 0.1 ml of serum-free medium using a 28-gauge insulin syringe with a fixed needle.
All mice are sorted into study groups based on caliper measurement estimation of tumor burden on Day 15 when the mean tumor burden for all animals on the right flank is approximately 82 mm³(range of group means, 75-90 mm³). The mice are distributed to ensure that the mean tumor burden on the right flank for all groups is within 10% of the overall mean tumor burden for the study population.

Results

The mean estimated right side tumor burden for all groups in the experiment on the first day of treatment is approximately 82 mm³and all of the groups in the experiment are well-matched (range of group means, 75-90 mm³). All animals weigh at least 13.3 g at the initiation of therapy. Mean group body weights at first treatment are also well-matched (range, approximately 15.4-18.3 g). A tumor burden of 500 mm³is chosen for evaluation of efficacy by tumor growth delay for the right and left tumors. The median Control Group tumor burdens will reach 500 mm³on or about Day 47 for right tumors and on or about Day 43 for left tumors. The median tumor volume doubling times for the Control Group will be approximately 12 and 10 days for the right and left tumors, respectively.
Results of mouse inoculation and tumor growth will show that treatment of tumor bearing mice with oncolytic viruses comprising altered E1a regions and encoding various transgenes, with or without anti-PD-L1, show efficacy in reducing tumor volume.

Example 13. In Vivo Efficacy of Dual-Specificity Oncolytic Adenoviral Vectors in a Bilateral Tumor Model

Cancer immunotherapy is moving toward use of combinations to increase efficacy. Combining cancer immunotherapies can expand clinical benefits of existing approved monotherapies; however, systemically-administered combinations can produce excessive toxicity. Therefore, novel dual specificity oncolytic adenoviral vectors were developed having a transgene at both the E1 and the E3 regions in order to evaluate combinations of adenoviral-delivered immunomodulators to enhance systemic antitumor immunity. A summary of exemplary constructs contemplated for use by the methods disclosed herein (with additions to those disclosed in Table 5) is listed in Table 6.

TABLE 6

Exemplary E1/E3 Dual Transgene Constructs and Single
Transgene Comparators (*)

Name	E1	E3

TRZ-001*	CTLA-4	Del
TRZ-009*	CD70	intact
TRZ-018*	CD70 trimer	Del
TRZ-021*	Empty	IL-2
TRZ-032*	IL-2	Empty (has 12.5K protein)
TRZ-401	CTLA4	IL-12
TRZ-402	IL-10 Trap	IL-12
TRZ-403	IL-7	IL-12 (has 12.5K protein)
TRZ-404	CD70	IL-12
TRZ-408*	Empty	IL-12
TRZ-409	IL-12	IL-7
TRZ-411	OX40L	IL-12
TRZ-412	IL-2	IL-12
TRZ-414	CD40L	IL-12
TRZ-415	IL-12	CTLA4
TRZ-416	OX40L (trimer)	IL-12
TRZ-417	CD40L (trimer)	IL-12
TRZ-418	CD70 (trimer)	IL-12
TRZ-421	IL-12	IL-2
TRZ-510	IL-7-P2A-IL-12	TRZ-202-like (no 12.5)
TRZ-TBD	IL-7-P2A-IL-12	IL-2

A bilateral tumor model was prepared using ADS-12 tumor cells as described in the Examples above (e.g., Example 7). 1×10⁶ADS-12 tumor cells injected into primary (−2 days) and contralateral (0 days) flanks. At staging, mice are randomized based on contralateral tumors (88-150 mm³) and primary tumors (88-250 mm³). There are 8 mice/group, with tumors and body weight measured 3 times per week. An anti-PD-L1 (500 μg/dose) or an anti-PD-1 antibody (250 μg/dose) is administered i.p. to induce systemic T cell activation.
In this model, the virus is injected intratumorally (i.t.) into primary tumors only; such injection leads to oncolysis, immune infiltration, and tumor shrinkage. The contralateral tumor is not injected, and no oncolysis occurs. Rather, tumor shrinkage is solely due to antigen-specific activated tumor infiltrating lymphocytes.
Results are shown in FIG. 9. FIG. 9A is a cartoon of the contrast between the single transgene constructs (top) used in the virus mixing examples, and the dual transgene vectors used to make the E1/E3 transgene adenoviruses. FIG. 9B shows the results of mice injected i.t. with Empty virus+/−anti-PD-L1 or with TRZ-409 (IL-12/IL-7 dual transgene)+/−anti-PD-L1. The tumor volume of the injected tumor is shown in the left panel, and the tumor volume of the contralateral tumor is shown in the right panel. For each pair of arrows, the left arrow indicates virus injection and the right arrow indicates anti-PD-L1 injection. As can be seen for both the primary and contralateral tumors, TRZ-409 injection reduced tumor volume significantly compared to empty virus. The combination with anti-PD-L1 was slightly more efficacious in this study.
FIG. 9C shows the results of a second experiment using the inverse of TRZ-409, TRZ-403, in which the IL-7 transgene occupies the E1 region and the IL-12 gene occupies the E3 region (which has a stronger promoter than the E1). As can be seen in the Figure, TRZ-403 strongly inhibits primary and distant tumor growth, with or without anti-PD-L1. Plasma levels of IL-12 and IFN-γ (FIG. 9D) for all mice injected with TRZ-403 show that expression of the transgenes is well tolerated.
The study was extended for all mice having tumors over 500 mm³. Mice having primary tumors injected with TRZ-403 (IL-7+IL-12), TRZ-403+anti-PD-L1, TRZ-403+control IgG, or TRZ-409 (IL-12+1L-7), and controls including untreated mice and mice injected with empty vector TRZ-d19K with anti-PD-L1, anti-PD-1, or a control IgG are shown in FIG. 9E, which illustrates the survival as a percentage of mice still on study. As can be seen in the Figure, mice injected with TRZ-403 had a much higher percentage of survival over TRZ-409 at the time end of the study (70 days). Both TRZ-403 and TRZ-409 showed efficacy over the controls; all mice receiving control injections were deceased by day 56 of the study.
Next, a direct comparison was made between the dual transgene virus TRZ-403 (11-7+IL-12) and a mixture of IL-7 and IL-12 single transgene viruses. As can be seen in FIG. 9F, in the primary tumor (left panel) TRZ-403 showed the most efficacy in reducing tumor volume, followed by the mixture of viruses. The single transgene IL-12 virus showed a small amount of efficacy by comparison. In the contralateral tumor, however (right panel) only the dual transgene virus, TRZ-403, reduced the tumor volume, showing significant superiority over the mixture of viruses.

Endnotes

Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each specifically and individually indicated to be incorporated by reference.

APPENDIX A

SEQUENCE REFERENCE

		Accession
SEQ		Number(s)
ID		or
NO:	Molecule	description	Sequence

1	hGITR-	NP_005083	MTLHPSPITCEFLFSTALISPKMCLSHLENMPL
	Ligand		SHSRTQGAQRSSWKLWLFCSIVMLLFLCSFS
	(GITR-L;		WLIFIFLQLETAKEPCMAKFGPLPSKWQMASS
	TNFSF18)		EPPCVNKVSDWKLEILQNGLYLIY
			GQVAPNANYNDVAPFEVRLYKNKDMIQTLT
			NKSKIQNVGGTYELHVGDTIDLIFNSEHQVLK
			NNTYWGIILLANPQFIS

2	mGITR-	NM_183391	MEEMPLRESSPQRAERCKKSWLLCIVALLLM
	Ligand		LLCSLGTLIYTSLKPTAIESCMVKFELSSSKWH
	(GITR-L;		MTSPKPHCVNTTSDGKLKILQSGTYLIYGQVI
	TNFSF18)		PVDKKYIKDNAPFVVQIYKKNDVLQTLMNDF
			QILPIGGVYELHAGDNIYLKFNSKDHIQKTNT
			YWGIILMPDLPFIS

3	hCD28 ligand	EAW79565	MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHF
	or agonist		CSGVIHVTKEVKEVATLSCGHNVSVEELAQT
	(CD80)		RIYWQKEKKMVLTMMSGDMNIWPEYKNRTI
			FDITNNLSIVILALRPSDEGTYECVVLK
			YEKDAFKREHLAEVTLSVKADFPTPSISDFEIP
			TSNIRRIICSTSGGFPEPHLSWLENGEELNAINT
			TVSQDPETELYAVSSKLDFNMTTNHSFMCLIK
			YGHLRVNQTFNWNTTKQEHFP
			DNLLPSWAITLISVNGIFVICCLTYCFAPRCRE
			RRRNERLRRESVRPV

4	hTNFα	CAA78745	MSTESMIRDVELAEEALPKKTGGPQGSRRCLF
			LSLFSFLIVAGATTLFCLLHFGVIGPQREEFPR
			DLSLISPLAQAVRSSSRTPSDKPVAHVVANPQ
			AEGQLQWLNRRANALLANGVELR
			DNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLL
			THTISRIAVSYQTKVNLLSAIKSPCQRETPEGA
			EAKPWYEPIYLGGVFQLEKGDRLSAEINRPDY
			LDFAESGQVYFGIIAL

5	Non-cleavable	NM_013693	MIETYSQPSPRSVATGLPASMKIFMYLLTVFLI
	mTNFα		TQMIGSVLFAVYLHRRLDKVEEEVNLHEDFV
			FIKKLKRCNKGEGSLSLLNCEEMRRQFEDLV
			KDITLNKEEKKEDEDPVAHVVANHQVEEQLE
			WLSQRANALLANGMDLKDNQLVVPADGLY
			LVYSQVLFKGQGCPDYVLLTHTVSRFAISYQE
			KVNLLSAVKSPCPKDTPEGAELKPWYEPIYLG
			GVFQLEKGDQLSAEVNLPKYLDFAESGQVYF
			GIIAL

6	mGM-CSF	NM_009969	MWLQNLLFLGIVVYSLSAPTRSPITVTRPWKH
			VEAIKEALNLLDDMPVTLNEEVEVVSNEFSFK
			KLTCVQTRLKIFEQGLRGNFTKLKGALNMTA
			SYYQTYCPPTPETDCETQVTTYADFIDSLKTF
			LTDIPFECKKPGQK

7	hICOS ligand	NP_056074	MRLGSPGLLFLLFSSLRADTQEKEVRAMVGS
	or agonist		DVELSCACPEGSRFDLNDVYVYWQTSESKTV
			VTYHIPQNSSLENVDSRYRNRALMSPAGMLR
			GDFSLRLFNVTPQDEQKFHCLVLSQSL
			GFQEVLSVEVTLHVAANFSVPVVSAPHSPSQ
			DELTFTCTSINGYPRPNVYWINKTDNSLLDQA
			LQNDTVFLNMRGLYDVVSVLRIARTPSVNIG
			CCIENVLLQQNLTVGSQTGNDIGERD
			KITENPVSTGEKNAATWSILAVLCLLVVVAV
			AIGWVCRDRCLQHSYAGAWAVSPETELTGH
			V

8	h4-1BB	AAA53134	MEYASDASLDPEAPWPPAPRARACRVLPWA
	ligand or		LVAGLLLLLLLAAACAVFLACPWAVSGARAS
	agonist		PGSAASPRLREGPELSPDDPAGLLDLRQGMFA
			QLVAQNVLLIDGPLSWYSDPGLAGVSL
			TGGLSYKEDTKELVVAKAGVYYVFFQLELRR
			VVAGEGSGSVSLALHLQPLRSAAGAAALALT
			VDLPPASSEARNSAFGFQGRLLHLSAGQRLG
			VHLHTEARARHAWQLTQGATVLGLFRV
			TPEIPAGLPSPRSE

9	hOX40 ligand	CAE11757	MCVGARRLGRGPCAALLLLGLGLSTVTGLHC
	or agonist		VGDTYPSNDRCCHECRPGNGMVSRCSRSQNT
			VCRPCGPGFYNDVVSSKPCKPCTWCNLRSGS
			ERKQLCTATQDTVCRCRAGTQPLDSYK
			PGVDCAPCPPGHFSPGDNQACKPWTNCTLAG
			KHTLQPASNSSDAICEDRDPPATQPQETQGPP
			ARPITVQPTEAWPRTSQGPSTRPVEVPGGRAV
			AAILGLGLVLGLLGPLAILLALYLL
			RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHS
			TLAKI

10	mOX40	NM_009452	MEGEGVQPLDENLENGSRPRFKWKKTLRLV
	ligand		VSGIKGAGMLLCFIYVCLQLSSSPAKDPPIQRL
			RGAVTRCEDGQLFISSYKNEYQTMEVQNNSV
			VIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPI
			SIPMLNDGRRIVFTVVASLAFKDKVYLTVNAP
			DTLCEHLQINDGELIVVQLTPGYCAPEGSYHS
			TVNQVPL

11	hCD40 ligand	NP_000065	MIETYNQTSPRSAATGLPISMKIFMYLLTVFLI
	or agonist		TQMIGSALFAVYLHRRLDKIEDERNLHEDFVF
			MKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDI
			MLNKEETKKENSFEMQKGDQNP
			QIAAHVISEASSKTTSVLQWAEKGYYTMSNN
			LVTLENGKQLTVKRQGLYYIYAQVTFCSNRE
			ASSQAPFIASLCLKSPGRFERILLRAANTHSSA
			KPCGQQSIHLGGVFELQPGASVFVN
			VTDPSQVSHGTGFTSFGLLKL

12	mCD40	NM_011616	MIETYSQPSPRSVATGLPASMKIFMYLLTVFLI
	ligand		TQMIGSVLFAVYLHRRLDKVEEEVNLHEDFV
			FIKKLKRCNKGEGSLSLLNCEEMRRQFEDLV
			KDITLNKEEKKENSFEMQRGDEDPQIAAHVV
			SEANSNAASVLQWAKKGYYTMKSNLVMLE
			NGKQLTVKREGLYYVYTQVTFCSNREPSSQR
			PFIVGLWLKPSSGSERILLKAANTHSSSQLCEQ
			QSVHLGGVFELQAGASVFVNVTEASQVIHRV
			GFSSFGLLKL

13	hCD27 ligand	AAA36175	MPEEGSGCSVRRRPYGCVLRAALVPLVAGLV
	or agonist		ICLVVCIQRFAQAQQQLPLESLGWDVAELQL
			NHTGPQQDPRLYWQGGPALGRSFLHGPELDK
			GQLRIHRDGIYMVHIQVTLAICSSTTA
			SRHHPTTLAVGICSPASRSISLLRLSFHQGCTIV
			SQRLTPLARGDTLCTNLTGTLLPSRNTDETFF
			GVQWVRP

14	mCD70	NM_011617	MPEEGRPCPWVRWSGTAFQRQWPWLLLVVF
	ligand or		ITVFCCWFHCSGLLSKQQQRLLEHPEPHTAEL
	agonist		QLNLTVPRKDPTLRWGAGPALGRSFTHGPEL
			EEGHLRIHQDGLYRLHIQVTLANCSSPGSTLQ
			HRATLAVGICSPAAHGISLLRGRFGQDCTVAL
			QRLTYLVHGDVLCTNLTLPLLPSRNADETFFG
			VQWICP

15	hInterleukin-2	AAB46883	MYRMQLLSCIALSLALVTNSAPTSSSTKKTQL
	(IL-2)		QLEHLLLDLQMILNGINNYKNPKLTRMLTFK
			FYMPKKATELKHLQCLEEELKPLEEVLNLAQ
			SKNFHLRPRDLISNINVIVLELKGSE
			TTFMCEYADETATIVEFLNRWITFCQSIISTLT

16	hInterleukin-7	AAH47698	MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKD
	(IL-7)		GKQYESVLMVSIDQLLDSMKEIGSNCLNNEF
			NFFKRHICDANKEGMFLFRAARKLRQFLKMN
			STGDFDLHLLKVSEGTTILLNCTGQ
			VKGRKPAALGEAQPTKSLEENKSLKEQKKLN
			DLCFLKRLLQEIKTCWNKILMGTKEH

17	murine	MM_008371.5	MFHVSFRYIFGIPPLILVLLPVTSSECHIKDKEG
	Interleukin-7		KAYESVLMISIDELDKMTGTDSNCPNNEPNFF
	(IL-7)		RKHVCDDTKEAAFLNRAARKLKQFLKMNISE
			EFNVHLLTVSQGTQTLVNCTSKEEKNVKEQK
			KNDACFLKRLLREIKTCWNKILKGSI

18	Interleukin-12	AAD16432	MWPPGSASQPPPSPAAATGLHPAARPVSLQC
	(IL-12) alpha		RLSMCPARSLLLVATLVLLDHLSLARNLPVA
	subunit		TPDPGMFPCLHHSQNLLRAVSNMLQKARQTL
			EFYPCTSEEIDHEDITKDKTSTVEACL
			PLELTKNESCLNSRETSFITNGSCLASRKTSFM
			MALCLSSIYEDLKMYQVEFKTMNAKLLMDP
			KRQIFLDQNMLAVIDELMQALNFNSETVPQK
			SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVM
			SYLNAS

19	hInterleukin-	NP_005526	MEPLVTWVVPLLFLFLLSRQGAACRTSECCF
	12 (IL-12)		QDPPYPDADSGSASGPRDLRCYRISSDRYECS
	receptor		WQYEGPTAGVSHFLRCCLSSGRCCYFAAGSA
	subunit beta-1		TRLQFSDQAGVSVLYTVTLWVESWAR
	isoform		NQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLA
	precursor		GQLRMEWETPDNQVGAEVQFRHRTPSSPWK
			LGDCGPQDDDTESCLCPLEMNVAQEFQLRRR
			QLGSQGSSWSKWSSPVCVPPENPPQPQ
			VRFSVEQLGQDGRRRLTLKEQPTQLELPEGC
			QGLAPGTEVTYRLQLHMLSCPCKAKATRTLH
			LGKMPYLSGAAYNVAVISSNQFGPGLNQTW
			HIPADTHTEPVALNISVGTNGTTMYWPA
			RAQSMTYCIEWQPVGQDGGLATCSLTAPQDP
			DPAGMATYSWSRESGAMGQEKCYYITIFASA
			HPEKLTLWSTVLSTYHFGGNASAAGTPHHVS
			VKNHSLDSVSVDWAPSLLSTCPGVLKE
			YVVRCRDEDSKQVSEHPVQPTETQVTLSGLR
			AGVAYTVQVRADTAWLRGVWSQPQRFSIEV
			QVSDWLIFFASLGSFLSILLVGVLGYLGLNRA
			ARHLCPPLPTPCASSAIEFPGGKETWQ
			WINPVDFQEEASLQEALVVEMSWDKGERTEP
			LEKTELPEGAPELALDTELSLEDGDRCKAKM

20	murine	N/A	MCPQKLTISWFAIVLLVSPLMAMWELEKDVY
	Interleukin-12		VVEVDWTPDAPGETVNLTCDTPEEDDITWTS
	fusion		DQRHGVIGSGKTLTITVKEFLDAGQYTCHKG
	polypeptide		GETLSHSHLLLHKKENGIWSTEILKNFKNKTF
			LKCEAPNYSGRFTCSWLVQRNMDLKFNIKSS
			SSSPDSRAVTCGMASLSAEKVTLDQRDYEKY
			SVSCQEDVTCPTAEETLPIELALEARQQNKYE
			NYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVS
			WEYPDSWSTPHSYFSLKFFVRIQRKKEKMKE
			TEEGCNQKGAFLVEKTSTEVQCKGGNVCVQ
			AQDRYYNSSCSKWACVPCRVRSGGGGSGGG
			GSGGGGSRVIPVSGPARCLSQSRNLLKTTDD
			MVKTAREKLKHYSCTAEDIDHEDITRDQTST
			LKTCLPLELHKNESCLATRETSSTTRGSCLPPQ
			KTSLMMTLCLGSIYEDLKMYQTEFQAINAAL
			QNHNHQQIILDKGMLVAIDELMQSLNHNGET
			LRQKPPVGEADPYRVKMKLCILLHAFSTRVV
			TINRVMGYLSSA

21	hIL-15	CAA62616	MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHV
			FILGCFSAGLPKTEANWVNVISDLKKIEDLIQS
			MHIDATLYTESDVHPSCKVTAMKCFLLELQV
			ISLESGDASIHDTVENLIILANNSLSSNGNVTES
			GCKECEELEEKNIKEFLQSFVHIVQMFINTS

22	mIL-15	N/A	MKILKPYMRNTSISCYLCFLLNSHFLTEAGIH
	hybrid		VFILGCVSVGLPKTEANWIDVRYDLEKIESLIQ
			SIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVI
			LHEYSNMTLNETVRNVLYLANSTLSSNKNVA
			ESGCKECEELEEKTFTEFLQSFIRIVQMFINTS
			GSGATNFSLLKQAGDVEENPGPGTTCPPPVSI
			EHADIRVKNYSVNSRERYVCNSGFKRKAGTS
			TLIECVINKNTNVAHWTTPSLKCIRDPSLAHY
			SPVPTVVTPKVTSQPESPSPSAKEPEAFSPKSD
			TAMTTETAIMPGSRLTPSQTTSAGTTGTGSHK
			SSRAPSLAATMTLEPTASTSLRITEISPHSSKM
			TK

23	hIL-10	NP_001549	MLPCLVVLLAALLSLRLGSDAHGTELPSPPSV
	receptor		WFEAEFFHHILHWTPIPNQSESTCYEVALLRY
	subunit alpha		GIESWNSISNCSQTLSYDLTAVTLDLYHSNGY
	precursor		RARVRAVDGSRHSNWTVTNTRFSV
			DEVTLTVGSVNLEIHNGFILGKIQLPRPKMAP
			ANDTYESIFSHFREYEIAIRKVPGNFTFTHKKV
			KHENFSLLTSGEVGEFCVQVKPSVASRSNKG
			MWSKEECISLTRQYFTVTNVIIFF
			AFVLLLSGALAYCLALQLYVRRRKKLPSVLL
			FKKPSPFIFISQRPSPETQDTIHPLDEEAFLKVS
			PELKNLDLHGSTDSGFGSTKPSLQTEEPQFLLP
			DPHPQADRTLGNREPPVLGDSC
			SSGSSNSTDSGICLQEPSLSPSTGPTWEQQVGS
			NSRGQDDSGIDLVQNSEGRAGDTQGGSALGH
			HSPPEPEVPGEEDPAAVAFQGYLRQTRCAEE
			KATKTGCLEEESPLTDGLGPKFGRC
			LVDEAGLHPPALAKGYLKQDPLEMTLASSGA
			PTGQWNQPTEEWSLLALSSCSDLGISDWSFA
			HDLAPLGCVAAPGGLLGSFNSDLVTLPLISSL
			QSSE

24	mIL-10R	N/A	MLSRLLPFLVTISSLSLEFIAYGTELPSPSYVW
	Trap		FEARFFQHILHWKPIPNQSESTYYEVALKQYG
			NSTWNDIHICRKAQALSCDLTTFTLDLYHRSY
			GYRARVRAVDNSQYSNWTTTETRFTVDEVIL
			TVDSVTLKAMDGIIYGTIHPPRPTITPAGDEYE
			QVFKDLRVYKISIRKFSELKNATKRVKQETFT
			LTVPIGVRKFCVKVLPRLESRINKAEWSEEQC
			LLITTEQYFTVTNLSHPASSTKVDKKIVPRDC
			GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKV
			TCVVVDISKDDPEVQFSWFVDDVEVHTAQTQ
			PREEQFNSTFRSVSELPIMHQDWLNGKEFKCR
			VNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK
			EQMAKDKVSLTCMITDFFPEDITVEWQWNG
			QPAENYKNTQPIMDTDGSYFVYSKLNVQKSN
			WEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

25	hIL-27	NP_004834	MRGGRGAPFWLWPLPKLALLPLLWVLFQRT
	Receptor		RPQGSAGPLQCYGVGPLGDLNCSWEPLGDLG
	subunit alpha		APSELHLQSQKYRSNKTQTVAVAAGRSWVAI
	precursor		PREQLTMSDKLLVWGTKAGQPLWPPVFV
			NLETQMKPNAPRLGPDVDFSEDDPLEATVHW
			APPTWPSHKVLICQFHYRRCQEAAWTLLEPE
			LKTIPLTPVEIQDLELATGYKVYGRCRMEKEE
			DLWGEWSPILSFQTPPSAPKDVWVSG
			NLCGTPGGEEPLLLWKAPGPCVQVSYKVWF
			WVGGRELSPEGITCCCSLIPSGAEWARVSAVN
			ATSWEPLTNLSLVCLDSASAPRSVAVSSIAGS
			TELLVTWQPGPGEPLEHVVDWARDGD
			PLEKLNWVRLPPGNLSALLPGNFTVGVPYRIT
			VTAVSASGLASASSVWGFREELAPLVGPTLW
			RLQDAPPGTPAIAWGEVPRHQLRGHLTHYTL
			CAQSGTSPSVCMNVSGNTQSVTLPDL
			PWGPCELWVTASTIAGQGPPGPILRLHLPDNT
			LRWKVLPGILFLWGLFLLGCGLSLATSGRCY
			HLRHKVLPRWVWEKVPDPANSSSGQPHMEQ
			VPEAQPLGDLPILEVEEMEPPPVMESS
			QPAQATAPLDSGYEKHFLPTPEELGLLGPPRP
			QVLA

26	hIL-13	AAH96140	MVWSINLTAGMYCAALESLINVSGCSAIEKT
			QRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQ
			FVKDLLLHLKKLFREGRFN

27	hIL-17	AAC50341	MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGC
			PNSEDKNFPRTVMVNLNIHNRNTNTNPKRSS
			DYYNRSTSPWNLHRNEDPERYPSVIWEAKCR
			HLGCINADGNVDYHMNSVPIQQEIL
			VLRREPPHCPNSFRLEKILVSVGCTCVTPIVHH
			VA

28	hIL-33	NP_001300974	MKPKMKYSTNKISTAKWKNTASKALCFKLG
	Isoform a		KSQQKAKEVCPMYFMKLRSGLMIKKEACYF
			RRETTKRPSLKTGRKHKRHLVLAACQQQSTV
			ECFAFGISGVQKYTRALHDSSITGISPIT
			EYLASLSTYNDQSITFALEDESYEIYVEDLKK
			DEKKDKVLLSYYESQHPSNESGDGVDGKML
			MVTLSPTKDFWLHANNKEHSVELHKCEKPLP
			DQAFFVLHNMHSNCVSFECKTDPGVFI
			GVKDNHLALIKVDSSENLCTENILFKLSET

29	hIL-33	NP_001300973	MKPKMKYSTNKISTAKWKNTASKALCFKLG
	Isoform a		KSQQKAKEVCPMYFMKLRSGLMIKKEACYF
			RRETTKRPSLKTGRKHKRHLVLAACQQQSTV
			ECFAFGISGVQKYTRALHDSSITGISPIT
			EYLASLSTYNDQSITFALEDESYEIYVEDLKK
			DEKKDKVLLSYYESQHPSNESGDGVDGKML
			MVTLSPTKDFWLHANNKEHSVELHKCEKPLP
			DQAFFVLHNMHSNCVSFECKTDPGVFI
			GVKDNHLALIKVDSSENLCTENILFKLSET

30	hIL-33	NP_001186569	MKPKMKYSTNKISTAKWKNTASKALCFKLG
	Isoform b		KSQQKAKEVCPMYFMKLRSGLMIKKEACYF
			RRETTKRPSLKTGRKHKRHLVLAACQQQSTV
			ECFAFGISGVQKYTRALHDSSITDKVLLS
			YYESQHPSNESGDGVDGKMLMVTLSPTKDF
			WLHANNKEHSVELHKCEKPLPDQAFFVLHN
			MHSNCVSFECKTDPGVFIGVKDNHLALIKVD
			SSENLCTENILFKLSET

31	hIL-33	NP_001186570	MKPKMKYSTNKISTAKWKNTASKALCFKLG
	Isoform c		NKVLLSYYESQHPSNESGDGVDGKMLMVTL
			SPTKDFWLHANNKEHSVELHKCEKPLPDQAF
			FVLHNMHSNCVSFECKTDPGVFIGVKDNH
			LALIKVDSSENLCTENILFKLSET

32	hIL-33	NP 001300975;	MKPKMKYSTNKISTAKWKNTASKALCFKLG
	Isoform d	NP_001300976	KSQQKAKEVCPMYFMKLRSGLMIKKEACYF
			RRETTKRPSLKTGRKHKRHLVLAACQQQSTV
			ECFAFGISGVQKYTRALHDSSITEYLASL
			STYNDQSITFALEDESYEIYVEDLKKDEKKDK
			VLLSYYESQHPSNESGDGVDGKMLMVTLSPT
			KDFWLHANNKEHSVELHKCEKPLPDQAFFVL
			HNMHSNCVSFECKTDPGVFIGVKDNH
			LALIKVDSSENLCTENILFKLSET

33	hIL-33	NP_001300977	MKPKMKYSTNKISTAKWKNTASKALCFKLG
	Isoform e		KSQQKAKEVCPMYFMKLRSGLMIKKEACYF
			RRETTKRPSLKTGISPITEYLASLSTYNDQSITF
			ALEDESYEIYVEDLKKDEKKDKVLLS
			YYESQHPSNESGDGVDGKMLMVTLSPTKDF
			WLHANNKEHSVELHKCEKPLPDQAFFVLHN
			MHSNCVSFECKTDPGVFIGVKDNHLALIKVD
			SSENLCTENILFKLSET

34	hIFN-γ	AAB59534	MKYTSYILAFQLCIVLGSLGCYCQDPYVKEA
			ENLKKYFNAGHSDVADNGTLFLGILKNWKEE
			SDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVE
			TIKEDMNVKFFNSNKKKRDDFEKLTN
			YSVTDLNVQRKAIHELIQVMAELSPAAKTGK
			RKRSQMLFRGRRASQ

35	Flagellin	p06179	MAQVINTNSLSLLTQNNLNKSQSALGTAIERL
			SSGLRINSAKDDAAGQAIANRFTANIKGLTQA
			SRNANDGISIAQTTEGALNEINNNLQRVRELA
			VQSANSTNSQSDLDSIQAEITQRLNEIDRVSG
			QTQFNGVKVLAQDNTLTIQVGANDGETIDID
			LKQINSQTLGLDTLNVQQKYKVSDTAATVTG
			YADTTIALDNSTFKASATGLGGTDQKIDGDL
			KFDDTTGKYYAKVTVTGGTGKDGYYEVSVD
			KTNGEVTLAGGATSPLTGGLPATATEDVKNV
			QVANADLTEAKAALTAAGVTGTASVVKMSY
			TDNNGKTIDGGLAVKVGDDYYSATQNKDGSI
			SINTTKYTADDGTSKTALNKLGGADGKTEVV
			SIGGKTYAASKAEGHNFKAQPDLAEAAATTT
			ENPLQKIDAALAQVDTLRSDLGAVQNRFNSA
			ITNLGNTVNNLTSARSRIEDSDYATEVSNMSR
			AQILQQAGTSVLAQANQVPQNVLSLLR

36	anti-CTLA4	N/A	MDMRVPAQLLGLLLLWLRGARCDIVMTQTT
	(CTLA-4		LSLPVSLGDQASISCRSSQSIVHSNGNTYLEW
	antagonist		YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS
	antibody)		GTDFTLKISRVEAEDLGVYYCFQGSHVPYTFG
			GGTKLEIKRADAAPTVSGSGGGSGGGSGGGS
			EAKLQESGPVLVKPGASVKMSCKASGYTFTD
			YYMNWVKQSHGKSLEWIGVINPYNGDTSYN
			QKFKGKATLTVDKSSSTAYMELNSLTSEDSA
			VYYCARYYGSWFAYWGQGTLITVSTEPRGPT
			IKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMIS
			LSPIVTCVVVDVSEDDPDVQISWFVNNVEVH
			TAQTQTHREDYNSTLRVVSALPIQHQDWMSG
			KEFKCKVNNKDLPAPIERTISKPKGSVRAPQV
			YVLPPPEEEMTKKQVTLTCMVTDFMPEDIYV
			EWTNNGKTELNYKNTEPVLDSDGSYFMYSK
			LRVEKKNWVERNSYSCSVVHEGLHNHHTTK
			SFSRTPGK

37	murine IL-2	DNA	TATCACCCTTGCTAATCACTCCTCACAGTGA
		(NM_008366.3)	CCTCAAGTCCTGCAGGCATGTACAGCATGC
			AGCTCGCATCCTGTGTCACATTGACACTTGT
			GCTCCTTGTCAACAGCGCACCCACTTCAAG
			CTCCACTTCAAGCTCTACAGCGGAAGCACA
			GCAGCAGCAGCAGCAGCAGCAGCAGCAGC
			AGCAGCACCTGGAGCAGCTGTTGATGGACC
			TACAGGAGCTCCTGAGCAGGATGGAGAATT
			ACAGGAACCTGAAACTCCCCAGGATGCTCA
			CCTTCAAATTTTACTTGCCCAAGCAGGCCA
			CAGAATTGAAAGATCTTCAGTGCCTAGAAG
			ATGAACTTGGACCTCTGCGGCATGTTCTGG
			ATTTGACTCAAAGCAAAAGCTTTCAATTGG
			AAGATGCTGAGAATTTCATCAGCAATATCA
			GAGTAACTGTTGTAAAACTAAAGGGCTCTG
			ACAACACATTTGAGTGCCAATTCGATGATG
			AGTCAGCAACTGTGGTGGACTTTCTGAGGA
			GATGGATAGCCTTCTGTCAAAGCATCATCT
			CAACAAGCCCTCAATAACTATGTACCTCCT
			GCTTACAACACATAAGGCTCTCTATTTATTT
			AAATATTTAACTTTAATTTATTTTTGGATGT
			ATTGTTTACTATCTTTTGTAACTACTAGTCT
			TCAGATGATAAATATGGATCTTTAAAGATT
			CTTTTTGTAAGCCCCAAGGGCTCAAAAATG
			TTTTAAACTATTTATCTGAAATTATTTATTA
			TATTGAATTGTTAAATATCATGTGTAGGTA
			GACTCATTAATAAAAGTATTTAGATGATTC
			AAATATAAATAAGCTCAGATGTCTGTCATT
			TTTAGGACAGCACAAAGTAAGCGCTAAAAT
			AACTTCTCAGTTATTCCTGTGAACTCTATGT
			TAATCAGTGTTTTCAAGAAATAAAGCTCTC
			CTCTAAAAAAAAAAAAAAA

38	murine IL-2	amino acid	MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTA
		CAA25909	EAQQQQQQQQQQQQHLEQLLMDLQELLSRM
			ENYRNLKLPRMLTFKFYLPKQATELKDLQCL
			EDELGPLRHVLDLTQSKSFQLEDAENFISNIRV
			TVVKLKGSDNTFECQFDDESATVVDFLRRWI
			AFCQSIISTSPQ

39	murine Xc11	DNA	AGCCCAGCAAGACCTCAGCCATGAGACTTC
		(GenBank:	TCCTCCTGACTTTCCTGGGAGTCTGCTGCCT
		BC062249.1)	CACCCCATGGGTTGTGGAAGGTGTGGGGAC
			TGAAGTCCTAGAAGAGAGTAGCTGTGTGAA
			CTTACAAACCCAGCGGCTGCCAGTTCAAAA
			AATCAAGACCTATATCATCTGGGAGGGGGC
			CATGAGAGCTGTAATTTTTGTCACCAAACG
			AGGACTAAAAATTTGTGCTGATCCAGAAGC
			CAAATGGGTGAAAGCAGCGATCAAGACTGT
			GGATGGCAGGGCCAGTACCAGAAAGAACA
			TGGCTGAAACTGTTCCCACAGGAGCCCAGA
			GGTCCACCAGCACAGCGATAACCCTGACTG
			GGTAACAGCCTCCAGGACAATGTTTCCTCA
			CTCGTTAAGCAGCTCATCTCAGTTCCCAAA
			CCCATTGCACAAATACTTATTTTTATTTTTA
			ACGACATTCACATTCATTTCAAATGTTATAA
			GTAATAAATATTTATTATTGATGAAAAAAA
			AAAAAAAAAAAAA

40	murine Xc11	amino acid	MRLLLLTFLGVCCLTPWVVEGVGTEVLEESS
		(GenBank:	CVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTK
		BC062249.1)	RGLKICADPEAKWVKAAIKTVDGRASTRKN
			MAETVPTGAQRSTSTAITLTG

41	Murine 4-	(DNA)	GAGACGTGCACTGACCGACCGTGGTAATGG
	1BBL	GenBank:	ACCAGCACACACTTGATGTGGAGGATACCG
	(TNFSF9)	BC138767.1	CGGATGCCAGACATCCAGCAGGTACTTCGT
			GCCCCTCGGATGCGGCGCTCCTCAGAGATA
			CCGGGCTCCTCGCGGACGCTGCGCTCCTCT
			CAGATACTGTGCGCCCCACAAATGCCGCGC
			TCCCCACGGATGCTGCCTACCCTGCGGTTA
			ATGTTCGGGATCGCGAGGCCGCGTGGCCGC
			CTGCACTGAACTTCTGTTCCCGCCACCCAA
			AGCTCTATGGCCTAGTCGCTTTGGTTTTGCT
			GCTTCTGATCGCCGCCTGTGTTCCTATCTTC
			ACCCGCACCGAGCCTCGGCCAGCGCTCACA
			ATCACCACCTCGCCCAACCTGGGTACCCGA
			GAGAATAATGCAGACCAGGTCACCCCTGTT
			TCCCACATTGGCTGCCCCAACACTACACAA
			CAGGGCTCTCCTGTGTTCGCCAAGCTACTG
			GCTAAAAACCAAGCATCGTTGTGCAATACA
			ACTCTGAACTGGCACAGCCAAGATGGAGCT
			GGGAGCTCATACCTATCTCAAGGTCTGAGG
			TACGAAGAAGACAAAAAGGAGTTGGTGGT
			AGACAGTCCCGGGCTCTACTACGTATTTTTG
			GAACTGAAGCTCAGTCCAACATTCACAAAC
			ACAGGCCACAAGGTGCAGGGCTGGGTCTCT
			CTTGTTTTGCAAGCAAAGCCTCAGGTAGAT
			GACTTTGACAACTTGGCCCTGACAGTGGAA
			CTGTTCCCTTGCTCCATGGAGAACAAGTTA
			GTGGACCGTTCCTGGAGTCAACTGTTGCTC
			CTGAAGGCTGGCCACCGCCTCAGTGTGGGT
			CTGAGGGCTTATCTGCATGGAGCCCAGGAT
			GCATACAGAGACTGGGAGCTGTCTTATCCC
			AACACCACCAGCTTTGGACTCTTTCTTGTGA
			AACCCGACAACCCATGGGAATGAGAACTAT
			CCTTCTTGTGACTCCTAGTTGCTAAGTCCTC
			AAGCTGCTATGTTTTATGGGGTCTGAGCAG
			GGGT

42	Murine 4-	(protein)	MDQHTLDVEDTADARHPAGTSCPSDAALLR
	1BBL	GenBank:	DTGLLADAALLSDTVRPTNAALPTDAAYPAV
	(TNFSF9)	BC138767.1	NVRDREAAWPPALNFCSRHPKLYGLVALVLL
			LLIAACVPIFTRTEPRPALTITTSPNLGTRENNA
			DQVTPVSHIGCPNTTQQGSPVFAKLLAKNQA
			SLCNTTLNWHSQDGAGSSYLSQGLRYEEDKK
			ELVVDSPGLYYVFLELKLSPTFTNTGHKVQG
			WVSLVLQAKPQVDDFDNLALTVELFPCSMEN
			KLVDRSWSQLLLLKAGHRLSVGLRAYLHGA
			QDAYRDWELSYPNTTSFGLFLVKPDNPWE

43	Mouse 4-	with human	MDMRVPAQLLGLLLLWLRGARCRMKQIEDK
	1BBL	IGKV1-39	IEEILSKIYHIENEIARIKKLIGERGGGSGGGSG
	trimeric	kappa LC	GGSRTEPRPALTITTSPNLGTRENNADQVTPV
	version	signal	SHIGCPNTTQQGSPVFAKLLAKNQASLCNTTL
		sequence,	NWHSQDGAGSSYLSQGLRYEEDKKELVVDS
		trimerization	PGLYYVFLELKLSPTFTNTGHKVQGWVSLVL
		domain	QAKPQVDDFDNLALTVELFPCSMENKLVDRS
		from yeast,	WSQLLLLKAGHRLSVGLRAYLHGAQDAYRD
		and Linker	WELSYPNTTSFGLFLVKPDNPWE**
		1, followed
		by 4-1BBL
		(TNFSF9,
		Acc#
		P41274)
		residues
		104-309
		(minus ECD
		and
		transmembrane
		domain)

44	murine 4-	residues	RTEPRPALTITTSPNLGTRENNADQVTPVSHIG
	1BBL	104-209	CPNTTQQGSPVFAKLLAKNQASLCNTTLNWH
	(TNFSF9,	(minus ECD	SQDGAGSSYLSQGLRYEEDKKELVVDSPGLY
	p41274)	and	YVFLELKLSPTFTNTGHKVQGWVSLVLQAKP
		transmem-	QVDDFDNLALTVELFPCSMENKLVDRSWSQL
		brane	LLLKAGHRLSVGLRAYLHGAQDAYRDWELS
		domain)	YPNTTSFGLFLVKPDNPWE**

45	Linker 1	15-mer	GGGGSGGGGSGGGGS

46	TRZ202	AA	MCHQQLVISWFSLVFLASPLVAIWELKKDVY
	(hIL-12	Sequence	VVELDWYPDAPGEMVVLTCDTPEEDGITWTL
	Insert)		DQSSEVLGSGKTLTIQVKEFGDAGQYTCHKG
			GEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN
			KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVK
			SSRGSSDPQGVTCGAATLSAERVRGDNKEYE
			YSVECQEDSACPAAEESLPIEVMVDAVHKLK
			YENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQV
			EVSWEYPDTWSTPHSYFSLTFCVQVQGKSKR
			EKKDRVFTDKTSATVICRKNASISVRAQDRY
			YSSSWSEWASVPCSGGGGSGGGGSGGGGSR
			NLPVATPDPGMFPCLHHSQNLLRAVSNMLQK
			ARQTLEFYPCTSEEIDHEDITKDKTSTVEACLP
			LELTKNESCLNSRETSFITNGSCLASRKTSFM
			MALCLSSIYEDLKMYQVEFKTMNAKLLMDP
			KRQIFLDQNMLAVIDELMQALNFNSETVPQK
			SSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVM
			SYLNAS**

47	hIL-12 p40β	AF180563	MCHQQLVISWFSLVFLASPLVAIWELKKDVY
			VVELDWYPDAPGEMVVLTCDTPEEDGITWTL
			DQSSEVLGSGKTLTIQVKEFGDAGQYTCHKG
			GEVLSHSLLLLHKKEDGIWSTDILKDQKEPKN
			KTFLRCEAKNYSGRFTCWWLTTISTDLTFSVK
			SSRGSSDPQGVTCGAATLSAERVRGDNKEYE
			YSVECQEDSACPAAEESLPIEVMVDAVHKLK
			YENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQV
			EVSWEYPDTWSTPHSYFSLTFCVQVQGKSKR
			EKKDRVFTDKTSATVICRKNASISVRAQDRY
			YSSSWSEWASVPCS

48	hIL-12 p35α	AA	RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQ
		Sequence of	KARQTLEFYPCTSEEIDHEDITKDKTSTVEAC
		Accession	LPLELTKNESCLNSRETSFITNGSCLASRKTSF
		#AF101062.1	MMALCLSSIYEDLKMYQVEFKTMNAKLLMD
		(native	PKRQIFLDQNMLAVIDELMQALNFNSETVPQ
		start codon	KSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRV
		and signal	MSYLNAS**
		sequence
		removed)

SEQ	(TRZ015)	AA	GTCGACGCCACCMDMRVPAQLLGLLLLWL
ID	hGITRL	Sequence,	RGARCRMKQIEDKIEEILSKIYHIENEIARIKKL
NOS	insert,	including	IGERGGGSGGGSGGGSETAKEPCMAKFGPLP
49	Trimeric	Kozak	SKWQMASSEPPCVNKVSDWKLEILQNGLYLI
and	version	sequences,	YGQVAPNANYNDVAPFEVRLYKNKDMIQTL
107		RE sites,	TNKSKIQNVGGTYELHVGDTIDLIFNSEHQVL
		and stop	KNNTYWGIILLANPQFIS CTCGAG**
		codons

50	hIGKV1-39	AA	MDMRVPAQLLGLLLLWLRGARC
	kappa LC	Sequence
	signal
	sequence

51	Trimerization	AA	RMKQIEDKIEEILSKIYHIENEIARIKKLIGER
	domain	Sequence
	(yeast)

52	GITRL minus	AA	ETAKEPCMAKFGPLPSKWQMASSEPPCVNKV
	ECD and	Sequence	SDWKLEILQNGLYLIYGQVAPNANYNDVAPF
	transmembrane		EVRLYKNKDMIQTLTNKSKIQNVGGTYELHV
	domains		GDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS**

53	Linker 2	12-mer	GGGSGGGSGGGS

54	TRZ006 : hIL-	hIL-15Rα-	MAPRRARGCRTLGLPALLLLLLLRPPATRGIT
	15 hybrid	Linker-hIL-	CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
		15	RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
			DPALVHQRPAPPSTVTTASGGSGGGGSGGGS
			GGGGSNWVNVISDLKKIEDLIQSMHIDATLYT
			ESDVHPSCKVTAMKCFLLELQVISLESGDASI
			HDTVENLIILANNSLSSNGNVTESGCKECEEL
			EEKNIKEFLQSFVHIVQMFINTS**

55	hIL-15Rα	AA	MAPRRARGCRTLGLPALLLLLLLRPPATRG
	endogenous	Sequence
	signal peptide

56	Sushi domain	AA	MSVEHADIWVKSYSLYSRERYICNSGFKRKA
	from IL-15Rα	Sequence	GTSSLTECVLNKATNVAHWTTPSLKCIRDPAL
			VHQRPAPPSTVTTA

57	mature hIL-15	AA	NWVNVISDLKKIEDLIQSMHIDATLYTESDVH
		Sequence	PSCKVTAMKCFLLELQVISLESGDASIHDTVE
			NLIILANNSLSSNGNVTESGCKECEELEEKNIK
			EFLQSFVHIVQMFINTS

58	TRZ201:	Orientation	MDMRVPAQLLGLLLLWLRGARCEIVLTQSPA
	hαCTLA4	2 ANT2054	TLSLSPGERATLSCSASSSISYMHWFQQRPGQ
		scFv-IgG1	SPRRWIYDTSKLASGVPARFSGSGSGTDYTLT
		Fc	ISSLEPEDFATYYCHQRTSYPLTFGQGTKLEIK
			GGGGSGGGGSGGGGSQVQLVQSGAELKKPG
			ASVKVSCKASGYTFTSYWINWIRQAPGQGLE
			WIGRIAPGSGTTYYNEVFKGRVTITVDKSTST
			AYMELSSLRSEDTAVYFCARGDYGSYWGQG
			TLVTVSSDKTHTCPPCPAPELLGGPSVFLFPPK
			PKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
			YVDGVEVHNAKTKPREEQYNSTYRVVSVLT
			VLHQDWLNGKEYKCKVSNKALPAPIEKTISK
			AKGQPREPQVYTLPPSREEMTKNQVSLTCLV
			KGFYPSDIAVEWESNGQPENNYKTTPPVLDS
			DGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
			ALHNHYTQKSLSLSPGK**

59	Kappa	AA	EIVLTQSPATLSLSPGERATLSCSASSSISYMH
	variable	Sequence	WFQQRPGQSPRRWIYDTSKLASGVPARFSGS
			GSGTDYTLTISSLEPEDFATYYCHQRTSYPLTF
			GQGTKLEIK

60	Heavy	AA	QVQLVQSGAELKKPGASVKVSCKASGYTFTS
	variable	Sequence	YWINWIRQAPGQGLEWIGRIAPGSGTTYYNE
			VFKGRVTITVDKSTSTAYMELSSLRSEDTAVY
			FCARGDYGSYWGQGTLVTVSS

61	human Fc	AA	DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
	IgG1	Sequence	SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
			VHNAKTKPREEQYNSTYRVVSVLTVLHQDW
			LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
			PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
			AVEWESNGQPENNYKTTPPVLDSDGSFFLYS
			KLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
			KSLSLSPGK**

62	TRZ106	hIL-15	MAPRRARGCRTLGLPALLLLLLLRPPATRGIT
		hybrid (hIL-	CPPPMSVEHADIWVKSYSLYSRERYICNSGFK
		15Rα-	RKAGTSSLTECVLNKATNVAHWTTPSLKCIR
		Linker 2-	DPALVHQRPAPPSTVTTASGGSGGGGSGGGS
		hIL-15)	GGGGSNWVNVISDLKKIEDLIQSMHIDATLYT
		(hIL-15Rα	ESDVHPSCKVTAMKCFLLELQVISLESGDASI
		endogenous	HDTVENLIILANNSLSSNGNVTESGCKECEEL
		signal	EEKNIKEFLQSFVHIVQMFINTS**
		peptide,
		Sushi
		domain
		from IL-
		15Rα,
		linker,
		mature hIL-
		15)

63	hIL-15Ra		MAPRRARGCRTLGLPALLLLLLLRPPATRG
	endogenous
	signal peptide

64	mature hIL-15		NWVNVISDLKKIEDLIQSMHIDATLYTESDVH
			PSCKVTAMKCFLLELQVISLESGDASIHDTVE
			NLIILANNSLSSNGNVTESGCKECEELEEKNIK
			EFLQSFVHIVQMFINTS

65	TRZ108:	full length	MIETYNQTSPRSAATGLPISMKIFMYLLTVFLI
	hCD40	monomeric	TQMIGSALFAVYLHRRLDKIEDERNLHEDFVF
		version,	MKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDI
		membrane	MLNKEETKKENSFEMQKGDQNPQIAAHVISE
		bound	ASSKTTSVLQWAEKGYYTMSNNLVTLENGK
			QLTVKRQGLYYIYAQVTFCSNREASSQAPFIA
			SLCLKSPGRFERILLRAANTHSSAKPCGQQSIH
			LGGVFELQPGASVFVNVTDPSQVSHGTGFTSF
			GLLKL**

66	TRZ111:	full length	MERVQPLEENVGNAARPRFERNKLLLVASVI
	hOX40L	monomeric	QGLGLLLCFTYICLHFSALQVSHRYPRIQSIKV
	variant 1	version,	QFTEYKKEKGFILTSQKEDEIMKVQNNSVIIN
		membrane	CDGFYLISLKGYFSQEVNISLHYQKDEEPLFQ
		bound	LKKVRSVNSLMVASLTYKDKVYLNVTTDNT
			SLDDFHVNGGELILIHQNPGEFCVL**

67	TRZ114: hIL-	Extracellular	MLPCLVVLLAALLSLRLGSDAHGTELPSPPSV
	10RTrap	domain of	WFEAEFFHHILHWTPIPNQSESTCYEVALLRY
	Version2	hIL-10Rα	GIESWNSISNCSQTLSYDLTAVTLDLYHSNGY
		with	RARVRAVDGSRHSNWTVTNTRFSVDEVTLT
		endogenous	VGSVNLEIHNGFILGKIQLPRPKMAPANDTYE
		signal	SIFSHFREYEIAIRKVPGNFTFTHKKVKHENFS
		sequence,	LLTSGEVGEFCVQVKPSVASRSNKGMWSKEE
		without stop	CISLTRQYFTVTNDKTHTCPPCPAPELLGGPS
		and human	VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
		Fc IgG1	EVKFNWYVDGVEVHNAKTKPREEQYNSTYR
			VVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
			EKTISKAKGQPREPQVYTLPPSREEMTKNQVS
			LTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
			VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
			MHEALHNHYTQKSLSLSPGK**

68	Extracellular		HGTELPSPPSVWFEAEFFHHILHWTPIPNQSES
	domain of		TCYEVALLRYGIESWNSISNCSQTLSYDLTAV
	hIL-10Rα		TLDLYHSNGYRARVRAVDGSRHSNWTVTNT
			RFSVDEVTLTVGSVNLEIHNGFILGKIQLPRPK
			MAPANDTYESIFSHFREYEIAIRKVPGNFTFTH
			KKVKHENFSLLTSGEVGEFCVQVKPSVASRS
			NKGMWSKEECISLTRQYFTVTN

69	endogenous		MLPCLVVLLAALLSLRLGSDA
	signal
	sequence in
	TRZ114

70	human Fc		DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI
	IgG1		SRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
			VHNAKTKPREEQYNSTYRVVSVLTVLHQDW
			LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
			PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI
			AVEWESNGQPENNYKTTPPVLDSDGSFFLYS
			KLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
			KSLSLSPGK**

71	TRZ116:	Trimeric	MDMRVPAQLLGLLLLWLRGARCRMKQIEDK
	hOX40L	version	IEEILSKIYHIENEIARIKKLIGERGGGSGGGSG
		w/human	GGSETAKEPCMAKFGPLPSKWQMASSEPPCV
		IGKV1-39	NKVSDWKLEILQNGLYLIYGQVAPNANYND
		kappa LC	VAPFEVRLYKNKDMIQTLTNKSKIQNVGGTY
		signal	ELHVGDTIDLIFNSEHQVLKNNTYWGIILLAN
		sequence,	PQFIS**
		trimerization
		domain
		from yeast,
		and Linker
		1, followed
		by OX4OL
		minus ECD
		and
		transmem-
		brane
		domains

72	hOX4OL		QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKE
	minus ECD		DEIMKVQNNSVIINCDGFYLISLKGYFSQEVNI
	and		SLHYQKDEEPLFQLKKVRSVNSLMVASLTYK
	transmembrane		DKVYLNVTTDNTSLDDFHVNGGELILIHQNP
	domains		GEFCVL**

73	TRZ117:	Trimeric	MDMRVPAQLLGLLLLWLRGARCRMKQIEDK
	hCD4OL	version	IEEILSKIYHIENEIARIKKLIGERGGGSGGGSG
		w/human	GGSMQKGDQNPQIAAHVISEASSKTTSVLQW
		IGKV1-39	AEKGYYTMSNNLVTLENGKQLTVKRQGLYY
		kappa LC	IYAQVTFCSNREASSQAPFIASLCLKSPGREER
		signal	ILLRAANTHSSAKPCGQQSIHLGGVFELQPGA
		sequence,	SVFVNVTDPSQVSHGTGFTSFGLLKL**
		trimerization
		domain
		from yeast,
		and Linker
		1, followed
		by CD40L
		minus ECD
		and
		transmem-
		brane
		domains

76	hCD4OL		MQKGDQNPQIAAHVISEASSKTTSVLQWAEK
	minus ECD		GYYTMSNNLVTLENGKQLTVKRQGLYYIYA
	and		QVTFCSNREASSQAPFIASLCLKSPGRFERILL
	transmem-		RAANTHSSAKPCGQQSIHLGGVFELQPGASVF
	brane		VNVTDPSQVSHGTGFTSFGLLKL**
	domains

77	TRZ307: hIL-		MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKD
	7 variant 1		GKQYESVLMVSIDQLLDSMKEIGSNCLNNEF
			NFFKRHICDANKEGMFLFRAARKLRQFLKMN
			STGDFDLHLLKVSEGTTILLNCTGQVKGRKPA
			ALGEAQPTKSLEENKSLKEQKKLNDLCFLKR
			LLQEIKTCWNKILMGTKEH**

106	hIL-7		MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKD
			GKQYESVLMVSIDQLLDSMKEIGSNCLNNEF
			NFFKRHICDANKEGMFLFRAARKLRQFLKMN
			STGDFDLHLLKVSEGTTILLNCTGQVKGRKPA
			ALGEAQPTKSLEENKSLKEQKKLNDLCFLKR
			LLQEIKTCWNKILMGTKEH**

78	TRZ304:		MWLQSLLLLGTVACSISAPARSPSPSTQPWEH
	hGM-CSF		VNAIQEARRLLNLSRDTAAEMNETVEVISEM
			FDLQEPTCLQTRLELYKQGLRGSLTKLKGPLT
			MMASHYKQHCPPTPETSCATQIITFESFKENL
			KDFLLVIPFDCWEPVQE**

79	hGM-CSF		MWLQSLLLLGTVACSISAPARSPSPSTQPWEH
			VNAIQEARRLLNLSRDTAAEMNETVEVISEM
			FDLQEPTCLQTRLELYKQGLRGSLTKLKGPLT
			MMASHYKQHCPPTPETSCATQIITFESFKENL
			KDFLLVIPFDCWEPVQE**

80	TRZ309:	(residue in	MPEEGSGCSVRRRPYGCVLRAALVPLVAGLV
	hCD70	bold to be	ICLVVCIQRFAQAQQQLPLESLGWDVAELQL
		removed)	NHTGPQQDPRLYWQGGPALGRSFLHGPELDK
			GQLRIHRDGIYMVHIQVTLAICSSTTASRHHP
			TTLAVGICSPASRSISLLRLSFHQGCTIASQRLT
			PLARGDTLCTNLTGTLLPSRNTDETFFGVQW
			VRP**

81	hCD70	(residue in	MPEEGSGCSVRRRPYGCVLRAALVPLVAGLV
		bold to be	ICLVVCIQRFAQAQQQLPLESLGWDVAELQL
		modified to	NHTGPQQDPRLYWQGGPALGRSFLHGPELDK
		avoid	GQLRIHRDGIYMVHIQVTLAICSSTTASRHHP
		introducing	TTLAVGICSPASRSISLLRLSFHQGCTIASQRLT
		new Xho I	PLARGDTLCTNLTGTLLPSRNTDETFFGVQW
		site)	VRP**

82	E3 14.7k	Region	ATGACTGACACCCTAGATCTAGAAATGGA
		overlapping	CGGAATTATTACAGAGCAGCGCCTGCTAGA
		with E3	AAGACGCAGGGCAGCGGCCGAGCAACAGC
		14.5k ORF	GCATGAATCAAGAGCTCCAAGACATGGTTA
		in bold	ACTTGCACCAGTGCAAAAGGGGTATCTTTT
			GTCTGGTAAAGCAGGCCAAAGTCACCTACG
			ACAGTAATACCACCGGACACCGCCTTAGCT
			ACAAGTTGCCAACCAAGCGTCAGAAATTGG
			TGGTCATGGTGGGAGAAAAGCCCATTACCA
			TAACTCAGCACTCGGTAGAAACCGAAGGCT
			GCATTCACTCACCTTGTCAAGGACCTGAGG
			ATCTCTGCACCCTTATTAAGACCCTGTGCGG
			TCTCAAAGATCTTATTCCCTTTAACTAA

83	E3 14.5k	Region	ATGAAATTTACTGTGACTTTTCTGCTGATTA
		overlapping	TTTGCACCCTATCTGCGTTTTGTTCCCCGAC
		with E3	CTCCAAGCCTCAAAGACATATATCATGCAG
		14.7k in	ATTCACTCGTATATGGAATATTCCAAGTTGC
		bold	TACAATGAAAAAAGCGATCTTTCCGAAGCC
			TGGTTATATGCAATCATCTCTGTTATGGTGT
			TCTGCAGTACCATCTTAGCCCTAGCTATATA
			TCCCTACCTTGACATTGGCTGGAACGCAAT
			AGATGCCATGAACCACCCAACTTTCCCCGC
			GCCCGCTATGCTTCCACTGCAACAAGTTGTT
			GCCGGCGGCTTTGTCCCAGCCAATCAGCCT
			CGCCCACCTTCTCCCACCCCCACTGAAATC
			AGCTACTTTAATCTAACAGGAGGAGATGAC
			TGA

84	E3 10.4K		ATGATTCCTCGAGTTTTTATATTACTGACCC
			TTGTTGCGCTTTTTTTGTGCGTGCTCCACAT
			TGGCTGCGGTTTCTCACATCGAAGTAGACT
			GCATTCCAGCCTTCACAGTCTATTTGCTTTA
			CGGATTTGTCACCCTCACGCTCATCTGCAGC
			CTCATCACTGTGGTCATCGCCTTTATCCAGT
			GCATTGA

85	E3 10.5k		ATGACCAACACAACCAACGCGGCCGCCGCT
	(ADP)		ACCGGACTTACATCTACCACAAATACACCC
			CAAGTTTCTGCCTTTGTCAATAACTGGGATA
			ACTTGGGCATGTGGTGGTTCTCCATAGCGC
			TTATGTTTGTATGCCTTATTATTATGTGGCT
			CATCTGCTGCCTAAAGCGCAAACGCGCCCG
			ACCACCCATCTATAGTCCCATCATTGTGCTA
			CACCCAAACAATGATGGAATCCATAGATTG
			GACGGACTGAAACACATGTTCTTTTCTCTTA
			CAGTATGA

86	TRZ000	Region	ATGATTAGGTACATAATCCTAGGTTTACTC
	intact E3	overlapping	ACCCTTGCGTCAGCCCACGGTACCACCCAA
	gp19k ORF	with E3	AAGGTGGATTTTAAGGAGCCAGCCTGTAAT
		gp19k ORF	GTTACATTCGCAGCTGAAGCTAATGAGTGC
		in bold	ACCACTCTTATAAAATGCACCACAGAACAT
			GAAAAGCTGCTTATTCGCCACAAAAACAAA
			ATTGGCAAGTATGCTGTTTATGCTATTTGGC
			AGCCAGGTGACACTACAGAGTATAATGTTA
			CAGTTTTCCAGGGTAAAAGTCATAAAACTT
			TTATGTATACTTTTCCATTTTATGAAATGTG
			CGACATTACCATGTACATGAGCAAACAGTA
			TAAGTTGTGGCCCCCACAAAATTGTGTGGA
			AAACACTGGCACTTTCTGCTGCACTGCTAT
			GCTAATTACAGTGCTCGCTTTGGTCTGTACC
			CTACTCTATATTAAATACAAAAGCAGACGC
			AGCTTTATTGAGGAAAAGAAAATGCCTTAA

87	TRZ000	Region	ATGAACAATTCAAGCAACTCTACGGGCTAT
	intact E3 7.1K	overlapping	TCTAATTCAGGTTTCTCTAGAATCGGGGTTG
		with E3	GGGTTATTCTCTGTCTTGTGATTCTCTTTATT
		gp19k ORF	CTTATACTAACGCTTCTCTGCCTAAGGCTCG
		in bold	CCGCCTGCTGTGTGCACATTTGCATTTATTG
			TCAGCTTTTTAAACGCTGGGGTCGCCACCC
			AAGATGA

88	TRZ200		ATGTTAAGTGGAGAGGCAGAGCAACTGCGC
	intact E3		CTGAAACACCTGGTCCACTGTCGCCGCCAC
	12.5k		AAGTGCTTTGCCCGCGACTCCGGTGAGTTTT
			GCTACTTTGAATTGCCCGAGGATCATATCG
			AGGGCCCGGCGCACGGCGTCCGGCTTACCG
			CCCAGGGAGAGCTTGCCCGTAGCCTGATTC
			GGGAGTTTACCCAGCGCCCCCTGCTAGTTG
			AGCGGGACAGGGGACCCTGTGTTCTCACTG
			TGATTTGCAACTGTCCTAACCCTGGATTACA
			TCAAGATCTTTGTTGCCATCTCTGTGCTGAG
			TATAATAAATACAGAAATTAA

89	TRZ000		CACCCTAGATCTAGAAATGGACGGAATTAT
	intact E3		TACAGAGCAGCGCCTGCTAGAAAGACGCA
	14.7k ORF		GGGCAGCGGCCGAGCAACAGCGCATGAAT
			CAAGAGCTCCAAGACATGGTTAACTTGCAC
			CAGTGCAAAAGGGGTATCTTTTGTCTGGTA
			AAGCAGGCCAAAGTCACCTACGACAGTAAT
			ACCACCGGACACCGCCTTAGCTACAAGTTG
			CCAACCAAGCGTCAGAAATTGGTGGTCATG
			GTGGGAGAAAAGCCCATTACCATAACTCAG
			CACTCGGTAGAAACCGAAGGCTGCATTCAC
			TCACCTTGTCAAGGACCTGAGGATCTCTGC
			ACCCTTATTAAGACCCTGTGCGGTCTCAAA
			GATCTTATTCCCTTTAACTAA

90	TRZ200 E3		AGACGCCCAGGCCGAAGTTCAGATGACTAA
	region (not		CTCAGGGGCGCAGCTTGCGGGCGGCTTTCG
	full E3-this is		TCACAGGGTGCGGTCGCCCGGGCAGGGTAT
	deleted		AACTCACCTGACAATCAGAGGGCGAGGTAT
	version)		TCAGCTCAACGACGAGTCGGTGAGCTCCTC
			GCTTGGTCTCCGTCCGGACGGGACATTTCA
			GATCGGCGGCGCCGGCCGCTCTTCATTCAC
			GCCTCGTCAGGCAATCCTAACTCTGCAGAC
			CTCGTCCTCTGAGCCGCGCTCTGGAGGCAT
			TGGAACTCTGCAATTTATTGAGGAGTTTGT
			GCCATCGGTCTACTTTAACCCCTTCTCGGGA
			CCTCCCGGCCACTATCCGGATCAATTTATTC
			CTAACTTTGACGCGGTAAAGGACTCGGCGG
			ACGGCTACGACTGAATGTTAAGTGGAGAGG
			CAGAGCAACTGCGCCTGAAACACCTGGTCC
			ACTGTCGCCGCCACAAGTGCTTTGCCCGCG
			ACTCCGGTGAGTTTTGCTACTTTGAATTGCC
			CGAGGATCATATCGAGGGCCCGGCGCACGG
			CGTCCGGCTTACCGCCCAGGGAGAGCTTGC
			CCGTAGCCTGATTCGGGAGTTTACCCAGCG
			CCCCCTGCTAGTTGAGCGGGACAGGGGACC
			CTGTGTTCTCACTGTGATTTGCAACTGTCCT
			AACCCTGGATTACATCAAGATCCTCTAGTT
			AATGTCAGGTCGCCTAAGTCGATTAACTAG
			AGTACCCGGGGATCTTATTCCCTTTAACTAA

91	TRZ200		GATCTTATTCCCTTTAACTAA
	deleted E3
	14.7k partial
	ORF

92	TRZ200		ATGTTAAGTGGAGAGGCAGAGCAACTGCGC
	deleted E3		CTGAAACACCTGGTCCACTGTCGCCGCCAC
	12.5K partial		AAGTGCTTTGCCCGCGACTCCGGTGAGTTTT
	ORF		GCTACTTTGAATTGCCCGAGGATCATATCG
			AGGGCCCGGCGCACGGCGTCCGGCTTACCG
			CCCAGGGAGAGCTTGCCCGTAGCCTGATTC
			GGGAGTTTACCCAGCGCCCCCTGCTAGTTG
			AGCGGGACAGGGGACCCTGTGTTCTCACTG
			TGATTTGCAACTGTCCTAACCCTGGATTACA
			TCAAGAT

93	E3 Promoter		AGACGCCCAGGCCGAAGTTCAGATGACTA
	(E3 deleted or
	E3 intact)

94	Full TRZ200		TTTTGGATTGAAGCCAATATGATAATGAGG
	virus		GGGTGGAGTTTGTGACGTGGCGCGGGGCGT
	sequence		GGGAACGGGGCGGGTGACGTAGTAGTGTG
	beginning at		GCGGAAGTGTGATGTTGCAAGTGTGGCGGA
	5′ ITR		ACACATGTAAGCGACGGATGTGGCAAAAGT
	through 3′		GACGTTTTTGGTGTGCGCCGGTGTTTTGGGC
	ITR		GTAACCGAGTAAGATTTGGCCATTTTCGCG
			GGAAAACTGAATAAGAGGAAGTGAAATCT
			GAATAATTTTGTGTTACTCATAGCGCGTAAT
			ATTTGTCTAGGGCCGCGGGGACTTTGACCG
			TTTACGTGGAGACTCGCCCAGGTGTTTTTCT
			CAGGTGTTTTCCGCGTTCCGGGTCAAAGTT
			GGCGTTTTATTATTATAGTCAGCTGACGTGT
			AGTGTATTTATACCCGGTGAGTTCCTCAAG
			AGGCCACTCTTGAGTGCCAGCGAGTAGAGT
			TTTCTCCTCCGAGCCGCTCCGACACCGGGA
			CTGAAAATGAGACATATTATCTGCCACGGA
			GGTGTTATTACCGAAGAAATGGCCGCCAGT
			CTTTTGGACCAGCTGATCGAAGAGGTACTG
			GCTGATAATCTTCCACCTCCTAGCCATTTTG
			AACCACCTACCCTTCACGAACTGTATGATTT
			AGACGTGACGGCCCCCGAAGATCCCAACGA
			GGAGGCGGTTTCGCAGATTTTTCCCGACTCT
			GTAATGTTGGCGGTGCAGGAAGGGATTGAC
			TTACTCACTTTTCCGCCGGCGCCCGGTTCTC
			CGGAGCCGCCTCACCTTTCCCGGCAGCCCG
			AGCAGCCGGAGCAGAGAGCCTTGGGTCCG
			GTTTCTATGCCAAACCTTGTACCGGAGGTG
			ATCGATCTTACCTGCCACGAGGCTGGCTTTC
			CACCCAGTGACGACGAGGATGAAGAGGGT
			GAGGAGTTTGTGTTAGATTATGTGGAGCAC
			CCCGGGCACGGTTGCAGGTCTTGTCATTAT
			CACCGGAGGAATACGGGGGACCCAGATATT
			ATGTGTTCGCTTTGCTATATGAGGACCTGTG
			GCATGTTTGTCTACAGTAAGTGAAAATTAT
			GGGCAGTGGGTGATAGAGTGGTGGGTTTGG
			TGTGGTAATTTTTTTTTTAATTTTTACAGTTT
			TGTGGTTTAAAGAATTTTGTATTGTGATTTT
			TTTAAAAGGTCCTGTGTCTGAACCTGAGCC
			TGAGCCCGAGCCAGAACCGGAGCCTGCAA
			GACCTACCCGCCGTCCTAAAATGGCGCCTG
			CTATCCTGAGACGCCCGACATCACCTGTGT
			CTAGAGAATGCAATAGTAGTACGGATAGCT
			GTGACTCCGGTCCTTCTAACACACCTCCTGA
			GATACACCCGGTGGTCCCGCTGTGCCCCAT
			TAAACCAGTTGCCGTGAGAGTTGGTGGGCG
			TCGCCAGGCTGTGGAATGTATCGAGGACTT
			GCTTAACGAGCCTGGGCAACCTTTGGACTT
			GAGCTGTAAACGCCCCAGGCCATAAGGTGT
			AAACCTGTGATTGCGTGTGTGGTTAACGCC
			TTTGTTTGCTGAATGAGTTGATGTAAGTTTA
			ATAAAGGGTGAGATAATGTTTAACTTGCAT
			GGCGTGTTAAATGGGGCGGGGCTTAAAGGG
			TATATAATGCGCCGTGGGCTAATCTTGGTT
			ACATCTGACCTCGTCGACGCTTGGGAGTGT
			TTGGAAGATTTTTCTGCTGTGCGTAACTTGC
			TGGAACAGAGCTCTAACAGTACCTCTTGGT
			TTTGGAGGTTTCTGTGGGGCTCATCCCAGG
			CAAAGTTAGTCTGCAGAATTAAGGAGGATT
			ACAAGTGGGAATTTGAAGAGCTTTTGAAAT
			CCTGTGGTGAGCTGTTTGATTCTTTGAACTC
			GAGTCACCAGGCGCTTTTCCAAGAGAAGGT
			CATCAAGACTTTGGATTTTTCCACACCGGG
			GCGCGCTGCGGCTGCTGTTGCTTTTTTGAGT
			TTTATAAAGGATAAATGGAGCGAAGAAACC
			CATCTGAGCGGGGGGTACCTGCTGGATTTT
			CTGGCCATGCATCTGTGGAGAGCGGTTGTG
			AGACACAAGAATCGCCTGCTACTGTTGTCT
			TCCGTCCGCCCGGCGATAATACCGACGGAG
			GAGCAGCAGCAGCAGCAGGAGGAAGCCAG
			GCGGCGGCGGCAGGAGCAGAGCCCATGGA
			ACCCGAGAGCCGGCCTGGACCCTCGGGAAT
			GAATGTTGTACAGGTGGCTGAACTGTATCC
			AGAACTGAGACGCATTTTGACAATTACAGA
			GGATGGGCAGGGGCTAAAGGGGGTAAAGA
			GGGAGCGGGGGGCTTGTGAGGCTACAGAG
			GAGGCTAGGAATCTAGCTTTTAGCTTAATG
			ACCAGACACCGTCCTGAGTGTATTACTTTTC
			AACAGATCAAGGATAATTGCGCTAATGAGC
			TTGATCTGCTGGCGCAGAAGTATTCCATAG
			AGCAGCTGACCACTTACTGGCTGCAGCCAG
			GGGATGATTTTGAGGAGGCTATTAGGGTAT
			ATGCAAAGGTGGCACTTAGGCCAGATTGCA
			AGTACAAGATCAGCAAACTTGTAAATATCA
			GGAATTGTTGCTACATTTCTGGGAACGGGG
			CCGAGGTGGAGATAGATACGGAGGATAGG
			GTGGCCTTTAGATGTAGCATGATAAATATG
			TGGCCGGGGGTGCTTGGCATGGACGGGGTG
			GTTATTATGAATGTAAGGTTTACTGGCCCC
			AATTTTAGCGGTACGGTTTTCCTGGCCAATA
			CCAACCTTATCCTACACGGTGTAAGCTTCTA
			TGGGTTTAACAATACCTGTGTGGAAGCCTG
			GACCGATGTAAGGGTTCGGGGCTGTGCCTT
			TTACTGCTGCTGGAAGGGGGTGGTGTGTCG
			CCCCAAAAGCAGGGCTTCAATTAAGAAATG
			CCTCTTTGAAAGGTGTACCTTGGGTATCCTG
			TCTGAGGGTAACTCCAGGGTGCGCCACAAT
			GTGGCCTCCGACTGTGGTTGCTTCATGCTAG
			TGAAAAGCGTGGCTGTGATTAAGCATAACA
			TGGTATGTGGCAACTGCGAGGACAGGGCCT
			CTCAGATGCTGACCTGCTCGGACGGCAACT
			GTCACCTGCTGAAGACCATTCACGTAGCCA
			GCCACTCTCGCAAGGCCTGGCCAGTGTTTG
			AGCATAACATACTGACCCGCTGTTCCTTGC
			ATTTGGGTAACAGGAGGGGGGTGTTCCTAC
			CTTACCAATGCAATTTGAGTCACACTAAGA
			TATTGCTTGAGCCCGAGAGCATGTCCAAGG
			TGAACCTGAACGGGGTGTTTGACATGACCA
			TGAAGATCTGGAAGGTGCTGAGGTACGATG
			AGACCCGCACCAGGTGCAGACCCTGCGAGT
			GTGGCGGTAAACATATTAGGAACCAGCCTG
			TGATGCTGGATGTGACCGAGGAGCTGAGGC
			CCGATCACTTGGTGCTGGCCTGCACCCGCG
			CTGAGTTTGGCTCTAGCGATGAAGATACAG
			ATTGAGGTACTGAAATGTGTGGGCGTGGCT
			TAAGGGTGGGAAAGAATATATAAGGTGGG
			GGTCTTATGTAGTTTTGTATCTGTTTTGCAG
			CAGCCGCCGCCGCCATGAGCACCAACTCGT
			TTGATGGAAGCATTGTGAGCTCATATTTGA
			CAACGCGCATGCCCCCATGGGCCGGGGTGC
			GTCAGAATGTGATGGGCTCCAGCATTGATG
			GTCGCCCCGTCCTGCCCGCAAACTCTACTA
			CCTTGACCTACGAGACCGTGTCTGGAACGC
			CGTTGGAGACTGCAGCCTCCGCCGCCGCTT
			CAGCCGCTGCAGCCACCGCCCGCGGGATTG
			TGACTGACTTTGCTTTCCTGAGCCCGCTTGC
			AAGCAGTGCAGCTTCCCGTTCATCCGCCCG
			CGATGACAAGTTGACGGCTCTTTTGGCACA
			ATTGGATTCTTTGACCCGGGAACTTAATGTC
			GTTTCTCAGCAGCTGTTGGATCTGCGCCAG
			CAGGTTTCTGCCCTGAAGGCTTCCTCCCCTC
			CCAATGCGGTTTAAAACATAAATAAAAAAC
			CAGACTCTGTTTGGATTTGGATCAAGCAAG
			TGTCTTGCTGTCTTTATTTAGGGGTTTTGCG
			CGCGCGGTAGGCCCGGGACCAGCGGTCTCG
			GTCGTTGAGGGTCCTGTGTATTTTTTCCAGG
			ACGTGGTAAAGGTGACTCTGGATGTTCAGA
			TACATGGGCATAAGCCCGTCTCTGGGGTGG
			AGGTAGCACCACTGCAGAGCTTCATGCTGC
			GGGGTGGTGTTGTAGATGATCCAGTCGTAG
			CAGGAGCGCTGGGCGTGGTGCCTAAAAATG
			TCTTTCAGTAGCAAGCTGATTGCCAGGGGC
			AGGCCCTTGGTGTAAGTGTTTACAAAGCGG
			TTAAGCTGGGATGGGTGCATACGTGGGGAT
			ATGAGATGCATCTTGGACTGTATTTTTAGGT
			TGGCTATGTTCCCAGCCATATCCCTCCGGG
			GATTCATGTTGTGCAGAACCACCAGCACAG
			TGTATCCGGTGCACTTGGGAAATTTGTCAT
			GTAGCTTAGAAGGAAATGCGTGGAAGAACT
			TGGAGACGCCCTTGTGACCTCCAAGATTTT
			CCATGCATTCGTCCATAATGATGGCAATGG
			GCCCACGGGCGGCGGCCTGGGCGAAGATAT
			TTCTGGGATCACTAACGTCATAGTTGTGTTC
			CAGGATGAGATCGTCATAGGCCATTTTTAC
			AAAGCGCGGGCGGAGGGTGCCAGACTGCG
			GTATAATGGTTCCATCCGGCCCAGGGGCGT
			AGTTACCCTCACAGATTTGCATTTCCCACGC
			TTTGAGTTCAGATGGGGGGATCATGTCTAC
			CTGCGGGGCGATGAAGAAAACGGTTTCCGG
			GGTAGGGGAGATCAGCTGGGAAGAAAGCA
			GGTTCCTGAGCAGCTGCGACTTACCGCAGC
			CGGTGGGCCCGTAAATCACACCTATTACCG
			GCTGCAACTGGTAGTTAAGAGAGCTGCAGC
			TGCCGTCATCCCTGAGCAGGGGGGCCACTT
			CGTTAAGCATGTCCCTGACTCGCATGTTTTC
			CCTGACCAAATCCGCCAGAAGGCGCTCGCC
			GCCCAGCGATAGCAGTTCTTGCAAGGAAGC
			AAAGTTTTTCAACGGTTTGAGACCGTCCGC
			CGTAGGCATGCTTTTGAGCGTTTGACCAAG
			CAGTTCCAGGCGGTCCCACAGCTCGGTCAC
			CTGCTCTACGGCATCTCGATCCAGCATATCT
			CCTCGTTTCGCGGGTTGGGGCGGCTTTCGCT
			GTACGGCAGTAGTCGGTGCTCGTCCAGACG
			GGCCAGGGTCATGTCTTTCCACGGGCGCAG
			GGTCCTCGTCAGCGTAGTCTGGGTCACGGT
			GAAGGGGTGCGCTCCGGGCTGCGCGCTGGC
			CAGGGTGCGCTTGAGGCTGGTCCTGCTGGT
			GCTGAAGCGCTGCCGGTCTTCGCCCTGCGC
			GTCGGCCAGGTAGCATTTGACCATGGTGTC
			ATAGTCCAGCCCCTCCGCGGCGTGGCCCTT
			GGCGCGCAGCTTGCCCTTGGAGGAGGCGCC
			GCACGAGGGGCAGTGCAGACTTTTGAGGGC
			GTAGAGCTTGGGCGCGAGAAATACCGATTC
			CGGGGAGTAGGCATCCGCGCCGCAGGCCCC
			GCAGACGGTCTCGCATTCCACGAGCCAGGT
			GAGCTCTGGCCGTTCGGGGTCAAAAACCAG
			GTTTCCCCCATGCTTTTTGATGCGTTTCTTA
			CCTCTGGTTTCCATGAGCCGGTGTCCACGCT
			CGGTGACGAAAAGGCTGTCCGTGTCCCCGT
			ATACAGACTTGAGAGGCCTGTCCTCGAGCG
			GTGTTCCGCGGTCCTCCTCGTATAGAAACTC
			GGACCACTCTGAGACAAAGGCTCGCGTCCA
			GGCCAGCACGAAGGAGGCTAAGTGGGAGG
			GGTAGCGGTCGTTGTCCACTAGGGGGTCCA
			CTCGCTCCAGGGTGTGAAGACACATGTCGC
			CCTCTTCGGCATCAAGGAAGGTGATTGGTT
			TGTAGGTGTAGGCCACGTGACCGGGTGTTC
			CTGAAGGGGGGCTATAAAAGGGGGTGGGG
			GCGCGTTCGTCCTCACTCTCTTCCGCATCGC
			TGTCTGCGAGGGCCAGCTGTTGGGGTGAGT
			ACTCCCTCTGAAAAGCGGGCATGACTTCTG
			CGCTAAGATTGTCAGTTTCCAAAAACGAGG
			AGGATTTGATATTCACCTGGCCCGCGGTGA
			TGCCTTTGAGGGTGGCCGCATCCATCTGGT
			CAGAAAAGACAATCTTTTTGTTGTCAAGCT
			TGGTGGCAAACGACCCGTAGAGGGCGTTGG
			ACAGCAACTTGGCGATGGAGCGCAGGGTTT
			GGTTTTTGTCGCGATCGGCGCGCTCCTTGGC
			CGCGATGTTTAGCTGCACGTATTCGCGCGC
			AACGCACCGCCATTCGGGAAAGACGGTGGT
			GCGCTCGTCGGGCACCAGGTGCACGCGCCA
			ACCGCGGTTGTGCAGGGTGACAAGGTCAAC
			GCTGGTGGCTACCTCTCCGCGTAGGCGCTC
			GTTGGTCCAGCAGAGGCGGCCGCCCTTGCG
			CGAGCAGAATGGCGGTAGGGGGTCTAGCTG
			CGTCTCGTCCGGGGGGTCTGCGTCCACGGT
			AAAGACCCCGGGCAGCAGGCGCGCGTCGA
			AGTAGTCTATCTTGCATCCTTGCAAGTCTAG
			CGCCTGCTGCCATGCGCGGGCGGCAAGCGC
			GCGCTCGTATGGGTTGAGTGGGGGACCCCA
			TGGCATGGGGTGGGTGAGCGCGGAGGCGT
			ACATGCCGCAAATGTCGTAAACGTAGAGGG
			GCTCTCTGAGTATTCCAAGATATGTAGGGT
			AGCATCTTCCACCGCGGATGCTGGCGCGCA
			CGTAATCGTATAGTTCGTGCGAGGGAGCGA
			GGAGGTCGGGACCGAGGTTGCTACGGGCG
			GGCTGCTCTGCTCGGAAGACTATCTGCCTG
			AAGATGGCATGTGAGTTGGATGATATGGTT
			GGACGCTGGAAGACGTTGAAGCTGGCGTCT
			GTGAGACCTACCGCGTCACGCACGAAGGAG
			GCGTAGGAGTCGCGCAGCTTGTTGACCAGC
			TCGGCGGTGACCTGCACGTCTAGGGCGCAG
			TAGTCCAGGGTTTCCTTGATGATGTCATACT
			TATCCTGTCCCTTTTTTTTCCACAGCTCGCG
			GTTGAGGACAAACTCTTCGCGGTCTTTCCA
			GTACTCTTGGATCGGAAACCCGTCGGCCTC
			CGAACGGTAAGAGCCTAGCATGTAGAACTG
			GTTGACGGCCTGGTAGGCGCAGCATCCCTT
			TTCTACGGGTAGCGCGTATGCCTGCGCGGC
			CTTCCGGAGCGAGGTGTGGGTGAGCGCAAA
			GGTGTCCCTGACCATGACTTTGAGGTACTG
			GTATTTGAAGTCAGTGTCGTCGCATCCGCC
			CTGCTCCCAGAGCAAAAAGTCCGTGCGCTT
			TTTGGAACGCGGATTTGGCAGGGCGAAGGT
			GACATCGTTGAAGAGTATCTTTCCCGCGCG
			AGGCATAAAGTTGCGTGTGATGCGGAAGGG
			TCCCGGCACCTCGGAACGGTTGTTAATTAC
			CTGGGCGGCGAGCACGATCTCGTCAAAGCC
			GTTGATGTTGTGGCCCACAATGTAAAGTTC
			CAAGAAGCGCGGGATGCCCTTGATGGAAG
			GCAATTTTTTAAGTTCCTCGTAGGTGAGCTC
			TTCAGGGGAGCTGAGCCCGTGCTCTGAAAG
			GGCCCAGTCTGCAAGATGAGGGTTGGAAGC
			GACGAATGAGCTCCACAGGTCACGGGCCAT
			TAGCATTTGCAGGTGGTCGCGAAAGGTCCT
			AAACTGGCGACCTATGGCCATTTTTTCTGG
			GGTGATGCAGTAGAAGGTAAGCGGGTCTTG
			TTCCCAGCGGTCCCATCCAAGGTTCGCGGC
			TAGGTCTCGCGCGGCAGTCACTAGAGGCTC
			ATCTCCGCCGAACTTCATGACCAGCATGAA
			GGGCACGAGCTGCTTCCCAAAGGCCCCCAT
			CCAAGTATAGGTCTCTACATCGTAGGTGAC
			AAAGAGACGCTCGGTGCGAGGATGCGAGC
			CGATCGGGAAGAACTGGATCTCCCGCCACC
			AATTGGAGGAGTGGCTATTGATGTGGTGAA
			AGTAGAAGTCCCTGCGACGGGCCGAACACT
			CGTGCTGGCTTTTGTAAAAACGTGCGCAGT
			ACTGGCAGCGGTGCACGGGCTGTACATCCT
			GCACGAGGTTGACCTGACGACCGCGCACAA
			GGAAGCAGAGTGGGAATTTGAGCCCCTCGC
			CTGGCGGGTTTGGCTGGTGGTCTTCTACTTC
			GGCTGCTTGTCCTTGACCGTCTGGCTGCTCG
			AGGGGAGTTACGGTGGATCGGACCACCACG
			CCGCGCGAGCCCAAAGTCCAGATGTCCGCG
			CGCGGCGGTCGGAGCTTGATGACAACATCG
			CGCAGATGGGAGCTGTCCATGGTCTGGAGC
			TCCCGCGGCGTCAGGTCAGGCGGGAGCTCC
			TGCAGGTTTACCTCGCATAGACGGGTCAGG
			GCGCGGGCTAGATCCAGGTGATACCTAATT
			TCCAGGGGCTGGTTGGTGGCGGCGTCGATG
			GCTTGCAAGAGGCCGCATCCCCGCGGCGCG
			ACTACGGTACCGCGCGGCGGGCGGTGGGCC
			GCGGGGGTGTCCTTGGATGATGCATCTAAA
			AGCGGTGACGCGGGCGAGCCCCCGGAGGT
			AGGGGGGGCTCCGGACCCGCCGGGAGAGG
			GGGCAGGGGCACGTCGGCGCCGCGCGCGG
			GCAGGAGCTGGTGCTGCGCGCGTAGGTTGC
			TGGCGAACGCGACGACGCGGCGGTTGATCT
			CCTGAATCTGGCGCCTCTGCGTGAAGACGA
			CGGGCCCGGTGAGCTTGAACCTGAAAGAGA
			GTTCGACAGAATCAATTTCGGTGTCGTTGA
			CGGCGGCCTGGCGCAAAATCTCCTGCACGT
			CTCCTGAGTTGTCTTGATAGGCGATCTCGGC
			CATGAACTGCTCGATCTCTTCCTCCTGGAGA
			TCTCCGCGTCCGGCTCGCTCCACGGTGGCG
			GCGAGGTCGTTGGAAATGCGGGCCATGAGC
			TGCGAGAAGGCGTTGAGGCCTCCCTCGTTC
			CAGACGCGGCTGTAGACCACGCCCCCTTCG
			GCATCGCGGGCGCGCATGACCACCTGCGCG
			AGATTGAGCTCCACGTGCCGGGCGAAGACG
			GCGTAGTTTCGCAGGCGCTGAAAGAGGTAG
			TTGAGGGTGGTGGCGGTGTGTTCTGCCACG
			AAGAAGTACATAACCCAGCGTCGCAACGTG
			GATTCGTTGATATCCCCCAAGGCCTCAAGG
			CGCTCCATGGCCTCGTAGAAGTCCACGGCG
			AAGTTGAAAAACTGGGAGTTGCGCGCCGAC
			ACGGTTAACTCCTCCTCCAGAAGACGGATG
			AGCTCGGCGACAGTGTCGCGCACCTCGCGC
			TCAAAGGCTACAGGGGCCTCTTCTTCTTCTT
			CAATCTCCTCTTCCATAAGGGCCTCCCCTTC
			TTCTTCTTCTGGCGGCGGTGGGGGAGGGGG
			GACACGGCGGCGACGACGGCGCACCGGGA
			GGCGGTCGACAAAGCGCTCGATCATCTCCC
			CGCGGCGACGGCGCATGGTCTCGGTGACGG
			CGCGGCCGTTCTCGCGGGGGCGCAGTTGGA
			AGACGCCGCCCGTCATGTCCCGGTTATGGG
			TTGGCGGGGGGCTGCCATGCGGCAGGGATA
			CGGCGCTAACGATGCATCTCAACAATTGTT
			GTGTAGGTACTCCGCCGCCGAGGGACCTGA
			GCGAGTCCGCATCGACCGGATCGGAAAACC
			TCTCGAGAAAGGCGTCTAACCAGTCACAGT
			CGCAAGGTAGGCTGAGCACCGTGGCGGGC
			GGCAGCGGGCGGCGGTCGGGGTTGTTTCTG
			GCGGAGGTGCTGCTGATGATGTAATTAAAG
			TAGGCGGTCTTGAGACGGCGGATGGTCGAC
			AGAAGCACCATGTCCTTGGGTCCGGCCTGC
			TGAATGCGCAGGCGGTCGGCCATGCCCCAG
			GCTTCGTTTTGACATCGGCGCAGGTCTTTGT
			AGTAGTCTTGCATGAGCCTTTCTACCGGCA
			CTTCTTCTTCTCCTTCCTCTTGTCCTGCATCT
			CTTGCATCTATCGCTGCGGCGGCGGCGGAG
			TTTGGCCGTAGGTGGCGCCCTCTTCCTCCCA
			TGCGTGTGACCCCGAAGCCCCTCATCGGCT
			GAAGCAGGGCTAGGTCGGCGACAACGCGC
			TCGGCTAATATGGCCTGCTGCACCTGCGTG
			AGGGTAGACTGGAAGTCATCCATGTCCACA
			AAGCGGTGGTATGCGCCCGTGTTGATGGTG
			TAAGTGCAGTTGGCCATAACGGACCAGTTA
			ACGGTCTGGTGACCCGGCTGCGAGAGCTCG
			GTGTACCTGAGACGCGAGTAAGCCCTCGAG
			TCAAATACGTAGTCGTTGCAAGTCCGCACC
			AGGTACTGGTATCCCACCAAAAAGTGCGGC
			GGCGGCTGGCGGTAGAGGGGCCAGCGTAG
			GGTGGCCGGGGCTCCGGGGGCGAGATCTTC
			CAACATAAGGCGATGATATCCGTAGATGTA
			CCTGGACATCCAGGTGATGCCGGCGGCGGT
			GGTGGAGGCGCGCGGAAAGTCGCGGACGC
			GGTTCCAGATGTTGCGCAGCGGCAAAAAGT
			GCTCCATGGTCGGGACGCTCTGGCCGGTCA
			GGCGCGCGCAATCGTTGACGCTCTAGCGTG
			CAAAAGGAGAGCCTGTAAGCGGGCACTCTT
			CCGTGGTCTGGTGGATAAATTCGCAAGGGT
			ATCATGGCGGACGACCGGGGTTCGAGCCCC
			GTATCCGGCCGTCCGCCGTGATCCATGCGG
			TTACCGCCCGCGTGTCGAACCCAGGTGTGC
			GACGTCAGACAACGGGGGAGTGCTCCTTTT
			GGCTTCCTTCCAGGCGCGGCGGCTGCTGCG
			CTAGCTTTTTTGGCCACTGGCCGCGCGCAG
			CGTAAGCGGTTAGGCTGGAAAGCGAAAGC
			ATTAAGTGGCTCGCTCCCTGTAGCCGGAGG
			GTTATTTTCCAAGGGTTGAGTCGCGGGACC
			CCCGGTTCGAGTCTCGGACCGGCCGGACTG
			CGGCGAACGGGGGTTTGCCTCCCCGTCATG
			CAAGACCCCGCTTGCAAATTCCTCCGGAAA
			CAGGGACGAGCCCCTTTTTTGCTTTTCCCAG
			ATGCATCCGGTGCTGCGGCAGATGCGCCCC
			CCTCCTCAGCAGCGGCAAGAGCAAGAGCA
			GCGGCAGACATGCAGGGCACCCTCCCCTCC
			TCCTACCGCGTCAGGAGGGGCGACATCCGC
			GGTTGACGCGGCAGCAGATGGTGATTACGA
			ACCCCCGCGGCGCCGGGCCCGGCACTACCT
			GGACTTGGAGGAGGGCGAGGGCCTGGCGC
			GGCTAGGAGCGCCCTCTCCTGAGCGGCACC
			CAAGGGTGCAGCTGAAGCGTGATACGCGTG
			AGGCGTACGTGCCGCGGCAGAACCTGTTTC
			GCGACCGCGAGGGAGAGGAGCCCGAGGAG
			ATGCGGGATCGAAAGTTCCACGCAGGGCGC
			GAGCTGCGGCATGGCCTGAATCGCGAGCGG
			TTGCTGCGCGAGGAGGACTTTGAGCCCGAC
			GCGCGAACCGGGATTAGTCCCGCGCGCGCA
			CACGTGGCGGCCGCCGACCTGGTAACCGCA
			TACGAGCAGACGGTGAACCAGGAGATTAA
			CTTTCAAAAAAGCTTTAACAACCACGTGCG
			TACGCTTGTGGCGCGCGAGGAGGTGGCTAT
			AGGACTGATGCATCTGTGGGACTTTGTAAG
			CGCGCTGGAGCAAAACCCAAATAGCAAGC
			CGCTCATGGCGCAGCTGTTCCTTATAGTGC
			AGCACAGCAGGGACAACGAGGCATTCAGG
			GATGCGCTGCTAAACATAGTAGAGCCCGAG
			GGCCGCTGGCTGCTCGATTTGATAAACATC
			CTGCAGAGCATAGTGGTGCAGGAGCGCAGC
			TTGAGCCTGGCTGACAAGGTGGCCGCCATC
			AACTATTCCATGCTTAGCCTGGGCAAGTTTT
			ACGCCCGCAAGATATACCATACCCCTTACG
			TTCCCATAGACAAGGAGGTAAAGATCGAGG
			GGTTCTACATGCGCATGGCGCTGAAGGTGC
			TTACCTTGAGCGACGACCTGGGCGTTTATC
			GCAACGAGCGCATCCACAAGGCCGTGAGC
			GTGAGCCGGCGGCGCGAGCTCAGCGACCGC
			GAGCTGATGCACAGCCTGCAAAGGGCCCTG
			GCTGGCACGGGCAGCGGCGATAGAGAGGC
			CGAGTCCTACTTTGACGCGGGCGCTGACCT
			GCGCTGGGCCCCAAGCCGACGCGCCCTGGA
			GGCAGCTGGGGCCGGACCTGGGCTGGCGGT
			GGCACCCGCGCGCGCTGGCAACGTCGGCGG
			CGTGGAGGAATATGACGAGGACGATGAGT
			ACGAGCCAGAGGACGGCGAGTACTAAGCG
			GTGATGTTTCTGATCAGATGATGCAAGACG
			CAACGGACCCGGCGGTGCGGGCGGCGCTGC
			AGAGCCAGCCGTCCGGCCTTAACTCCACGG
			ACGACTGGCGCCAGGTCATGGACCGCATCA
			TGTCGCTGACTGCGCGCAATCCTGACGCGT
			TCCGGCAGCAGCCGCAGGCCAACCGGCTCT
			CCGCAATTCTGGAAGCGGTGGTCCCGGCGC
			GCGCAAACCCCACGCACGAGAAGGTGCTG
			GCGATCGTAAACGCGCTGGCCGAAAACAG
			GGCCATCCGGCCCGACGAGGCCGGCCTGGT
			CTACGACGCGCTGCTTCAGCGCGTGGCTCG
			TTACAACAGCGGCAACGTGCAGACCAACCT
			GGACCGGCTGGTGGGGGATGTGCGCGAGG
			CCGTGGCGCAGCGTGAGCGCGCGCAGCAGC
			AGGGCAACCTGGGCTCCATGGTTGCACTAA
			ACGCCTTCCTGAGTACACAGCCCGCCAACG
			TGCCGCGGGGACAGGAGGACTACACCAACT
			TTGTGAGCGCACTGCGGCTAATGGTGACTG
			AGACACCGCAAAGTGAGGTGTACCAGTCTG
			GGCCAGACTATTTTTTCCAGACCAGTAGAC
			AAGGCCTGCAGACCGTAAACCTGAGCCAGG
			CTTTCAAAAACTTGCAGGGGCTGTGGGGGG
			TGCGGGCTCCCACAGGCGACCGCGCGACCG
			TGTCTAGCTTGCTGACGCCCAACTCGCGCCT
			GTTGCTGCTGCTAATAGCGCCCTTCACGGA
			CAGTGGCAGCGTGTCCCGGGACACATACCT
			AGGTCACTTGCTGACACTGTACCGCGAGGC
			CATAGGTCAGGCGCATGTGGACGAGCATAC
			TTTCCAGGAGATTACAAGTGTCAGCCGCGC
			GCTGGGGCAGGAGGACACGGGCAGCCTGG
			AGGCAACCCTAAACTACCTGCTGACCAACC
			GGCGGCAGAAGATCCCCTCGTTGCACAGTT
			TAAACAGCGAGGAGGAGCGCATTTTGCGCT
			ACGTGCAGCAGAGCGTGAGCCTTAACCTGA
			TGCGCGACGGGGTAACGCCCAGCGTGGCGC
			TGGACATGACCGCGCGCAACATGGAACCGG
			GCATGTATGCCTCAAACCGGCCGTTTATCA
			ACCGCCTAATGGACTACTTGCATCGCGCGG
			CCGCCGTGAACCCCGAGTATTTCACCAATG
			CCATCTTGAACCCGCACTGGCTACCGCCCC
			CTGGTTTCTACACCGGGGGATTCGAGGTGC
			CCGAGGGTAACGATGGATTCCTCTGGGACG
			ACATAGACGACAGCGTGTTTTCCCCGCAAC
			CGCAGACCCTGCTAGAGTTGCAACAGCGCG
			AGCAGGCAGAGGCGGCGCTGCGAAAGGAA
			AGCTTCCGCAGGCCAAGCAGCTTGTCCGAT
			CTAGGCGCTGCGGCCCCGCGGTCAGATGCT
			AGTAGCCCATTTCCAAGCTTGATAGGGTCT
			CTTACCAGCACTCGCACCACCCGCCCGCGC
			CTGCTGGGCGAGGAGGAGTACCTAAACAAC
			TCGCTGCTGCAGCCGCAGCGCGAAAAAAAC
			CTGCCTCCGGCATTTCCCAACAACGGGATA
			GAGAGCCTAGTGGACAAGATGAGTAGATG
			GAAGACGTACGCGCAGGAGCACAGGGACG
			TGCCAGGCCCGCGCCCGCCCACCCGTCGTC
			AAAGGCACGACCGTCAGCGGGGTCTGGTGT
			GGGAGGACGATGACTCGGCAGACGACAGC
			AGCGTCCTGGATTTGGGAGGGAGTGGCAAC
			CCGTTTGCGCACCTTCGCCCCAGGCTGGGG
			AGAATGTTTTAAAAAAAAAAAAGCATGATG
			CAAAATAAAAAACTCACCAAGGCCATGGC
			ACCGAGCGTTGGTTTTCTTGTATTCCCCTTA
			GTATGCGGCGCGCGGCGATGTATGAGGAAG
			GTCCTCCTCCCTCCTACGAGAGTGTGGTGA
			GCGCGGCGCCAGTGGCGGCGGCGCTGGGTT
			CTCCCTTCGATGCTCCCCTGGACCCGCCGTT
			TGTGCCTCCGCGGTACCTGCGGCCTACCGG
			GGGGAGAAACAGCATCCGTTACTCTGAGTT
			GGCACCCCTATTCGACACCACCCGTGTGTA
			CCTGGTGGACAACAAGTCAACGGATGTGGC
			ATCCCTGAACTACCAGAACGACCACAGCAA
			CTTTCTGACCACGGTCATTCAAAACAATGA
			CTACAGCCCGGGGGAGGCAAGCACACAGA
			CCATCAATCTTGACGACCGGTCGCACTGGG
			GCGGCGACCTGAAAACCATCCTGCATACCA
			ACATGCCAAATGTGAACGAGTTCATGTTTA
			CCAATAAGTTTAAGGCGCGGGTGATGGTGT
			CGCGCTTGCCTACTAAGGACAATCAGGTGG
			AGCTGAAATACGAGTGGGTGGAGTTCACGC
			TGCCCGAGGGCAACTACTCCGAGACCATGA
			CCATAGACCTTATGAACAACGCGATCGTGG
			AGCACTACTTGAAAGTGGGCAGACAGAAC
			GGGGTTCTGGAAAGCGACATCGGGGTAAA
			GTTTGACACCCGCAACTTCAGACTGGGGTT
			TGACCCCGTCACTGGTCTTGTCATGCCTGGG
			GTATATACAAACGAAGCCTTCCATCCAGAC
			ATCATTTTGCTGCCAGGATGCGGGGTGGAC
			TTCACCCACAGCCGCCTGAGCAACTTGTTG
			GGCATCCGCAAGCGGCAACCCTTCCAGGAG
			GGCTTTAGGATCACCTACGATGATCTGGAG
			GGTGGTAACATTCCCGCACTGTTGGATGTG
			GACGCCTACCAGGCGAGCTTGAAAGATGAC
			ACCGAACAGGGCGGGGGTGGCGCAGGCGG
			CAGCAACAGCAGTGGCAGCGGCGCGGAAG
			AGAACTCCAACGCGGCAGCCGCGGCAATGC
			AGCCGGTGGAGGACATGAACGATCATGCCA
			TTCGCGGCGACACCTTTGCCACACGGGCTG
			AGGAGAAGCGCGCTGAGGCCGAAGCAGCG
			GCCGAAGCTGCCGCCCCCGCTGCGCAACCC
			GAGGTCGAGAAGCCTCAGAAGAAACCGGT
			GATCAAACCCCTGACAGAGGACAGCAAGA
			AACGCAGTTACAACCTAATAAGCAATGACA
			GCACCTTCACCCAGTACCGCAGCTGGTACC
			TTGCATACAACTACGGCGACCCTCAGACCG
			GAATCCGCTCATGGACCCTGCTTTGCACTCC
			TGACGTAACCTGCGGCTCGGAGCAGGTCTA
			CTGGTCGTTGCCAGACATGATGCAAGACCC
			CGTGACCTTCCGCTCCACGCGCCAGATCAG
			CAACTTTCCGGTGGTGGGCGCCGAGCTGTT
			GCCCGTGCACTCCAAGAGCTTCTACAACGA
			CCAGGCCGTCTACTCCCAACTCATCCGCCA
			GTTTACCTCTCTGACCCACGTGTTCAATCGC
			TTTCCCGAGAACCAGATTTTGGCGCGCCCG
			CCAGCCCCCACCATCACCACCGTCAGTGAA
			AACGTTCCTGCTCTCACAGATCACGGGACG
			CTACCGCTGCGCAACAGCATCGGAGGAGTC
			CAGCGAGTGACCATTACTGACGCCAGACGC
			CGCACCTGCCCCTACGTTTACAAGGCCCTG
			GGCATAGTCTCGCCGCGCGTCCTATCGAGC
			CGCACTTTTTGAGCAAGCATGTCCATCCTTA
			TATCGCCCAGCAATAACACAGGCTGGGGCC
			TGCGCTTCCCAAGCAAGATGTTTGGCGGGG
			CCAAGAAGCGCTCCGACCAACACCCAGTGC
			GCGTGCGCGGGCACTACCGCGCGCCCTGGG
			GCGCGCACAAACGCGGCCGCACTGGGCGC
			ACCACCGTCGATGACGCCATCGACGCGGTG
			GTGGAGGAGGCGCGCAACTACACGCCCAC
			GCCGCCACCAGTGTCCACAGTGGACGCGGC
			CATTCAGACCGTGGTGCGCGGAGCCCGGCG
			CTATGCTAAAATGAAGAGACGGCGGAGGC
			GCGTAGCACGTCGCCACCGCCGCCGACCCG
			GCACTGCCGCCCAACGCGCGGCGGCGGCCC
			TGCTTAACCGCGCACGTCGCACCGGCCGAC
			GGGCGGCCATGCGGGCCGCTCGAAGGCTGG
			CCGCGGGTATTGTCACTGTGCCCCCCAGGT
			CCAGGCGACGAGCGGCCGCCGCAGCAGCC
			GCGGCCATTAGTGCTATGACTCAGGGTCGC
			AGGGGCAACGTGTATTGGGTGCGCGACTCG
			GTTAGCGGCCTGCGCGTGCCCGTGCGCACC
			CGCCCCCCGCGCAACTAGATTGCAAGAAAA
			AACTACTTAGACTCGTACTGTTGTATGTATC
			CAGCGGCGGCGGCGCGCAACGAAGCTATGT
			CCAAGCGCAAAATCAAAGAAGAGATGCTC
			CAGGTCATCGCGCCGGAGATCTATGGCCCC
			CCGAAGAAGGAAGAGCAGGATTACAAGCC
			CCGAAAGCTAAAGCGGGTCAAAAAGAAAA
			AGAAAGATGATGATGATGAACTTGACGACG
			AGGTGGAACTGCTGCACGCTACCGCGCCCA
			GGCGACGGGTACAGTGGAAAGGTCGACGC
			GTAAAACGTGTTTTGCGACCCGGCACCACC
			GTAGTCTTTACGCCCGGTGAGCGCTCCACC
			CGCACCTACAAGCGCGTGTATGATGAGGTG
			TACGGCGACGAGGACCTGCTTGAGCAGGCC
			AACGAGCGCCTCGGGGAGTTTGCCTACGGA
			AAGCGGCATAAGGACATGCTGGCGTTGCCG
			CTGGACGAGGGCAACCCAACACCTAGCCTA
			AAGCCCGTAACACTGCAGCAGGTGCTGCCC
			GCGCTTGCACCGTCCGAAGAAAAGCGCGGC
			CTAAAGCGCGAGTCTGGTGACTTGGCACCC
			ACCGTGCAGCTGATGGTACCCAAGCGCCAG
			CGACTGGAAGATGTCTTGGAAAAAATGACC
			GTGGAACCTGGGCTGGAGCCCGAGGTCCGC
			GTGCGGCCAATCAAGCAGGTGGCGCCGGG
			ACTGGGCGTGCAGACCGTGGACGTTCAGAT
			ACCCACTACCAGTAGCACCAGTATTGCCAC
			CGCCACAGAGGGCATGGAGACACAAACGT
			CCCCGGTTGCCTCAGCGGTGGCGGATGCCG
			CGGTGCAGGCGGTCGCTGCGGCCGCGTCCA
			AGACCTCTACGGAGGTGCAAACGGACCCGT
			GGATGTTTCGCGTTTCAGCCCCCCGGCGCC
			CGCGCCGTTCGAGGAAGTACGGCGCCGCCA
			GCGCGCTACTGCCCGAATATGCCCTACATC
			CTTCCATTGCGCCTACCCCCGGCTATCGTGG
			CTACACCTACCGCCCCAGAAGACGAGCAAC
			TACCCGACGCCGAACCACCACTGGAACCCG
			CCGCCGCCGTCGCCGTCGCCAGCCCGTGCT
			GGCCCCGATTTCCGTGCGCAGGGTGGCTCG
			CGAAGGAGGCAGGACCCTGGTGCTGCCAAC
			AGCGCGCTACCACCCCAGCATCGTTTAAAA
			GCCGGTCTTTGTGGTTCTTGCAGATATGGCC
			CTCACCTGCCGCCTCCGTTTCCCGGTGCCGG
			GATTCCGAGGAAGAATGCACCGTAGGAGG
			GGCATGGCCGGCCACGGCCTGACGGGCGGC
			ATGCGTCGTGCGCACCACCGGCGGCGGCGC
			GCGTCGCACCGTCGCATGCGCGGCGGTATC
			CTGCCCCTCCTTATTCCACTGATCGCCGCGG
			CGATTGGCGCCGTGCCCGGAATTGCATCCG
			TGGCCTTGCAGGCGCAGAGACACTGATTAA
			AAACAAGTTGCATGTGGAAAAATCAAAATA
			AAAAGTCTGGACTCTCACGCTCGCTTGGTC
			CTGTAACTATTTTGTAGAATGGAAGACATC
			AACTTTGCGTCTCTGGCCCCGCGACACGGC
			TCGCGCCCGTTCATGGGAAACTGGCAAGAT
			ATCGGCACCAGCAATATGAGCGGTGGCGCC
			TTCAGCTGGGGCTCGCTGTGGAGCGGCATT
			AAAAATTTCGGTTCCACCGTTAAGAACTAT
			GGCAGCAAGGCCTGGAACAGCAGCACAGG
			CCAGATGCTGAGGGATAAGTTGAAAGAGC
			AAAATTTCCAACAAAAGGTGGTAGATGGCC
			TGGCCTCTGGCATTAGCGGGGTGGTGGACC
			TGGCCAACCAGGCAGTGCAAAATAAGATTA
			ACAGTAAGCTTGATCCCCGCCCTCCCGTAG
			AGGAGCCTCCACCGGCCGTGGAGACAGTGT
			CTCCAGAGGGGCGTGGCGAAAAGCGTCCGC
			GCCCCGACAGGGAAGAAACTCTGGTGACGC
			AAATAGACGAGCCTCCCTCGTACGAGGAGG
			CACTAAAGCAAGGCCTGCCCACCACCCGTC
			CCATCGCGCCCATGGCTACCGGAGTGCTGG
			GCCAGCACACACCCGTAACGCTGGACCTGC
			CTCCCCCCGCCGACACCCAGCAGAAACCTG
			TGCTGCCAGGCCCGACCGCCGTTGTTGTAA
			CCCGTCCTAGCCGCGCGTCCCTGCGCCGCG
			CCGCCAGCGGTCCGCGATCGTTGCGGCCCG
			TAGCCAGTGGCAACTGGCAAAGCACACTGA
			ACAGCATCGTGGGTCTGGGGGTGCAATCCC
			TGAAGCGCCGACGATGCTTCTGATAGCTAA
			CGTGTCGTATGTGTGTCATGTATGCGTCCAT
			GTCGCCGCCAGAGGAGCTGCTGAGCCGCCG
			CGCGCCCGCTTTCCAAGATGGCTACCCCTTC
			GATGATGCCGCAGTGGTCTTACATGCACAT
			CTCGGGCCAGGACGCCTCGGAGTACCTGAG
			CCCCGGGCTGGTGCAGTTTGCCCGCGCCAC
			CGAGACGTACTTCAGCCTGAATAACAAGTT
			TAGAAACCCCACGGTGGCGCCTACGCACGA
			CGTGACCACAGACCGGTCCCAGCGTTTGAC
			GCTGCGGTTCATCCCTGTGGACCGTGAGGA
			TACTGCGTACTCGTACAAGGCGCGGTTCAC
			CCTAGCTGTGGGTGATAACCGTGTGCTGGA
			CATGGCTTCCACGTACTTTGACATCCGCGG
			CGTGCTGGACAGGGGCCCTACTTTTAAGCC
			CTACTCTGGCACTGCCTACAACGCCCTGGC
			TCCCAAGGGTGCCCCAAATCCTTGCGAATG
			GGATGAAGCTGCTACTGCTCTTGAAATAAA
			CCTAGAAGAAGAGGACGATGACAACGAAG
			ACGAAGTAGACGAGCAAGCTGAGCAGCAA
			AAAACTCACGTATTTGGGCAGGCGCCTTAT
			TCTGGTATAAATATTACAAAGGAGGGTATT
			CAAATAGGTGTCGAAGGTCAAACACCTAAA
			TATGCCGATAAAACATTTCAACCTGAACCT
			CAAATAGGAGAATCTCAGTGGTACGAAACA
			GAAATTAATCATGCAGCTGGGAGAGTCCTA
			AAAAAGACTACCCCAATGAAACCATGTTAC
			GGTTCATATGCAAAACCCACAAATGAAAAT
			GGAGGGCAAGGCATTCTTGTAAAGCAACAA
			AATGGAAAGCTAGAAAGTCAAGTGGAAAT
			GCAATTTTTCTCAACTACTGAGGCAGCCGC
			AGGCAATGGTGATAACTTGACTCCTAAAGT
			GGTATTGTACAGTGAAGATGTAGATATAGA
			AACCCCAGACACTCATATTTCTTACATGCCC
			ACTATTAAGGAAGGTAACTCACGAGAACTA
			ATGGGCCAACAATCTATGCCCAACAGGCCT
			AATTACATTGCTTTTAGGGACAATTTTATTG
			GTCTAATGTATTACAACAGCACGGGTAATA
			TGGGTGTTCTGGCGGGCCAAGCATCGCAGT
			TGAATGCTGTTGTAGATTTGCAAGACAGAA
			ACACAGAGCTTTCATACCAGCTTTTGCTTGA
			TTCCATTGGTGATAGAACCAGGTACTTTTCT
			ATGTGGAATCAGGCTGTTGACAGCTATGAT
			CCAGATGTTAGAATTATTGAAAATCATGGA
			ACTGAAGATGAACTTCCAAATTACTGCTTT
			CCACTGGGAGGTGTGATTAATACAGAGACT
			CTTACCAAGGTAAAACCTAAAACAGGTCAG
			GAAAATGGATGGGAAAAAGATGCTACAGA
			ATTTTCAGATAAAAATGAAATAAGAGTTGG
			AAATAATTTTGCCATGGAAATCAATCTAAA
			TGCCAACCTGTGGAGAAATTTCCTGTACTC
			CAACATAGCGCTGTATTTGCCCGACAAGCT
			AAAGTACAGTCCTTCCAACGTAAAAATTTC
			TGATAACCCAAACACCTACGACTACATGAA
			CAAGCGAGTGGTGGCTCCCGGGCTAGTGGA
			CTGCTACATTAACCTTGGAGCACGCTGGTC
			CCTTGACTATATGGACAACGTCAACCCATT
			TAACCACCACCGCAATGCTGGCCTGCGCTA
			CCGCTCAATGTTGCTGGGCAATGGTCGCTA
			TGTGCCCTTCCACATCCAGGTGCCTCAGAA
			GTTCTTTGCCATTAAAAACCTCCTTCTCCTG
			CCGGGCTCATACACCTACGAGTGGAACTTC
			AGGAAGGATGTTAACATGGTTCTGCAGAGC
			TCCCTAGGAAATGACCTAAGGGTTGACGGA
			GCCAGCATTAAGTTTGATAGCATTTGCCTTT
			ACGCCACCTTCTTCCCCATGGCCCACAACA
			CCGCCTCCACGCTTGAGGCCATGCTTAGAA
			ACGACACCAACGACCAGTCCTTTAACGACT
			ATCTCTCCGCCGCCAACATGCTCTACCCTAT
			ACCCGCCAACGCTACCAACGTGCCCATATC
			CATCCCCTCCCGCAACTGGGCGGCTTTCCG
			CGGCTGGGCCTTCACGCGCCTTAAGACTAA
			GGAAACCCCATCACTGGGCTCGGGCTACGA
			CCCTTATTACACCTACTCTGGCTCTATACCC
			TACCTAGATGGAACCTTTTACCTCAACCAC
			ACCTTTAAGAAGGTGGCCATTACCTTTGAC
			TCTTCTGTCAGCTGGCCTGGCAATGACCGC
			CTGCTTACCCCCAACGAGTTTGAAATTAAG
			CGCTCAGTTGACGGGGAGGGTTACAACGTT
			GCCCAGTGTAACATGACCAAAGACTGGTTC
			CTGGTACAAATGCTAGCTAACTATAACATT
			GGCTACCAGGGCTTCTATATCCCAGAGAGC
			TACAAGGACCGCATGTACTCCTTCTTTAGA
			AACTTCCAGCCCATGAGCCGTCAGGTGGTG
			GATGATACTAAATACAAGGACTACCAACAG
			GTGGGCATCCTACACCAACACAACAACTCT
			GGATTTGTTGGCTACCTTGCCCCCACCATGC
			GCGAAGGACAGGCCTACCCTGCTAACTTCC
			CCTATCCGCTTATAGGCAAGACCGCAGTTG
			ACAGCATTACCCAGAAAAAGTTTCTTTGCG
			ATCGCACCCTTTGGCGCATCCCATTCTCCAG
			TAACTTTATGTCCATGGGCGCACTCACAGA
			CCTGGGCCAAAACCTTCTCTACGCCAACTC
			CGCCCACGCGCTAGACATGACTTTTGAGGT
			GGATCCCATGGACGAGCCCACCCTTCTTTA
			TGTTTTGTTTGAAGTCTTTGACGTGGTCCGT
			GTGCACCAGCCGCACCGCGGCGTCATCGAA
			ACCGTGTACCTGCGCACGCCCTTCTCGGCC
			GGCAACGCCACAACATAAAGAAGCAAGCA
			ACATCAACAACAGCTGCCGCCATGGGCTCC
			AGTGAGCAGGAACTGAAAGCCATTGTCAAA
			GATCTTGGTTGTGGGCCATATTTTTTGGGCA
			CCTATGACAAGCGCTTTCCAGGCTTTGTTTC
			TCCACACAAGCTCGCCTGCGCCATAGTCAA
			TACGGCCGGTCGCGAGACTGGGGGCGTACA
			CTGGATGGCCTTTGCCTGGAACCCGCACTC
			AAAAACATGCTACCTCTTTGAGCCCTTTGG
			CTTTTCTGACCAGCGACTCAAGCAGGTTTA
			CCAGTTTGAGTACGAGTCACTCCTGCGCCG
			TAGCGCCATTGCTTCTTCCCCCGACCGCTGT
			ATAACGCTGGAAAAGTCCACCCAAAGCGTA
			CAGGGGCCCAACTCGGCCGCCTGTGGACTA
			TTCTGCTGCATGTTTCTCCACGCCTTTGCCA
			ACTGGCCCCAAACTCCCATGGATCACAACC
			CCACCATGAACCTTATTACCGGGGTACCCA
			ACTCCATGCTCAACAGTCCCCAGGTACAGC
			CCACCCTGCGTCGCAACCAGGAACAGCTCT
			ACAGCTTCCTGGAGCGCCACTCGCCCTACT
			TCCGCAGCCACAGTGCGCAGATTAGGAGCG
			CCACTTCTTTTTGTCACTTGAAAAACATGTA
			AAAATAATGTACTAGAGACACTTTCAATAA
			AGGCAAATGCTTTTATTTGTACACTCTCGGG
			TGATTATTTACCCCCACCCTTGCCGTCTGCG
			CCGTTTAAAAATCAAAGGGGTTCTGCCGCG
			CATCGCTATGCGCCACTGGCAGGGACACGT
			TGCGATACTGGTGTTTAGTGCTCCACTTAAA
			CTCAGGCACAACCATCCGCGGCAGCTCGGT
			GAAGTTTTCACTCCACAGGCTGCGCACCAT
			CACCAACGCGTTTAGCAGGTCGGGCGCCGA
			TATCTTGAAGTCGCAGTTGGGGCCTCCGCC
			CTGCGCGCGCGAGTTGCGATACACAGGGTT
			GCAGCACTGGAACACTATCAGCGCCGGGTG
			GTGCACGCTGGCCAGCACGCTCTTGTCGGA
			GATCAGATCCGCGTCCAGGTCCTCCGCGTT
			GCTCAGGGCGAACGGAGTCAACTTTGGTAG
			CTGCCTTCCCAAAAAGGGCGCGTGCCCAGG
			CTTTGAGTTGCACTCGCACCGTAGTGGCAT
			CAAAAGGTGACCGTGCCCGGTCTGGGCGTT
			AGGATACAGCGCCTGCATAAAAGCCTTGAT
			CTGCTTAAAAGCCACCTGAGCCTTTGCGCC
			TTCAGAGAAGAACATGCCGCAAGACTTGCC
			GGAAAACTGATTGGCCGGACAGGCCGCGTC
			GTGCACGCAGCACCTTGCGTCGGTGTTGGA
			GATCTGCACCACATTTCGGCCCCACCGGTT
			CTTCACGATCTTGGCCTTGCTAGACTGCTCC
			TTCAGCGCGCGCTGCCCGTTTTCGCTCGTCA
			CATCCATTTCAATCACGTGCTCCTTATTTAT
			CATAATGCTTCCGTGTAGACACTTAAGCTC
			GCCTTCGATCTCAGCGCAGCGGTGCAGCCA
			CAACGCGCAGCCCGTGGGCTCGTGATGCTT
			GTAGGTCACCTCTGCAAACGACTGCAGGTA
			CGCCTGCAGGAATCGCCCCATCATCGTCAC
			AAAGGTCTTGTTGCTGGTGAAGGTCAGCTG
			CAACCCGCGGTGCTCCTCGTTCAGCCAGGT
			CTTGCATACGGCCGCCAGAGCTTCCACTTG
			GTCAGGCAGTAGTTTGAAGTTCGCCTTTAG
			ATCGTTATCCACGTGGTACTTGTCCATCAGC
			GCGCGCGCAGCCTCCATGCCCTTCTCCCAC
			GCAGACACGATCGGCACACTCAGCGGGTTC
			ATCACCGTAATTTCACTTTCCGCTTCGCTGG
			GCTCTTCCTCTTCCTCTTGCGTCCGCATACC
			ACGCGCCACTGGGTCGTCTTCATTCAGCCG
			CCGCACTGTGCGCTTACCTCCTTTGCCATGC
			TTGATTAGCACCGGTGGGTTGCTGAAACCC
			ACCATTTGTAGCGCCACATCTTCTCTTTCTT
			CCTCGCTGTCCACGATTACCTCTGGTGATGG
			CGGGCGCTCGGGCTTGGGAGAAGGGCGCTT
			CTTTTTCTTCTTGGGCGCAATGGCCAAATCC
			GCCGCCGAGGTCGATGGCCGCGGGCTGGGT
			GTGCGCGGCACCAGCGCGTCTTGTGATGAG
			TCTTCCTCGTCCTCGGACTCGATACGCCGCC
			TCATCCGCTTTTTTGGGGGCGCCCGGGGAG
			GCGGCGGCGACGGGGACGGGGACGACACG
			TCCTCCATGGTTGGGGGACGTCGCGCCGCA
			CCGCGTCCGCGCTCGGGGGTGGTTTCGCGC
			TGCTCCTCTTCCCGACTGGCCATTTCCTTCT
			CCTATAGGCAGAAAAAGATCATGGAGTCAG
			TCGAGAAGAAGGACAGCCTAACCGCCCCCT
			CTGAGTTCGCCACCACCGCCTCCACCGATG
			CCGCCAACGCGCCTACCACCTTCCCCGTCG
			AGGCACCCCCGCTTGAGGAGGAGGAAGTG
			ATTATCGAGCAGGACCCAGGTTTTGTAAGC
			GAAGACGACGAGGACCGCTCAGTACCAAC
			AGAGGATAAAAAGCAAGACCAGGACAACG
			CAGAGGCAAACGAGGAACAAGTCGGGCGG
			GGGGACGAAAGGCATGGCGACTACCTAGA
			TGTGGGAGACGACGTGCTGTTGAAGCATCT
			GCAGCGCCAGTGCGCCATTATCTGCGACGC
			GTTGCAAGAGCGCAGCGATGTGCCCCTCGC
			CATAGCGGATGTCAGCCTTGCCTACGAACG
			CCACCTATTCTCACCGCGCGTACCCCCCAA
			ACGCCAAGAAAACGGCACATGCGAGCCCA
			ACCCGCGCCTCAACTTCTACCCCGTATTTGC
			CGTGCCAGAGGTGCTTGCCACCTATCACAT
			CTTTTTCCAAAACTGCAAGATACCCCTATCC
			TGCCGTGCCAACCGCAGCCGAGCGGACAAG
			CAGCTGGCCTTGCGGCAGGGCGCTGTCATA
			CCTGATATCGCCTCGCTCAACGAAGTGCCA
			AAAATCTTTGAGGGTCTTGGACGCGACGAG
			AAGCGCGCGGCAAACGCTCTGCAACAGGA
			AAACAGCGAAAATGAAAGTCACTCTGGAGT
			GTTGGTGGAACTCGAGGGTGACAACGCGCG
			CCTAGCCGTACTAAAACGCAGCATCGAGGT
			CACCCACTTTGCCTACCCGGCACTTAACCTA
			CCCCCCAAGGTCATGAGCACAGTCATGAGT
			GAGCTGATCGTGCGCCGTGCGCAGCCCCTG
			GAGAGGGATGCAAATTTGCAAGAACAAAC
			AGAGGAGGGCCTACCCGCAGTTGGCGACG
			AGCAGCTAGCGCGCTGGCTTCAAACGCGCG
			AGCCTGCCGACTTGGAGGAGCGACGCAAAC
			TAATGATGGCCGCAGTGCTCGTTACCGTGG
			AGCTTGAGTGCATGCAGCGGTTCTTTGCTG
			ACCCGGAGATGCAGCGCAAGCTAGAGGAA
			ACATTGCACTACACCTTTCGACAGGGCTAC
			GTACGCCAGGCCTGCAAGATCTCCAACGTG
			GAGCTCTGCAACCTGGTCTCCTACCTTGGA
			ATTTTGCACGAAAACCGCCTTGGGCAAAAC
			GTGCTTCATTCCACGCTCAAGGGCGAGGCG
			CGCCGCGACTACGTCCGCGACTGCGTTTAC
			TTATTTCTATGCTACACCTGGCAGACGGCC
			ATGGGCGTTTGGCAGCAGTGCTTGGAGGAG
			TGCAACCTCAAGGAGCTGCAGAAACTGCTA
			AAGCAAAACTTGAAGGACCTATGGACGGCC
			TTCAACGAGCGCTCCGTGGCCGCGCACCTG
			GCGGACATCATTTTCCCCGAACGCCTGCTT
			AAAACCCTGCAACAGGGTCTGCCAGACTTC
			ACCAGTCAAAGCATGTTGCAGAACTTTAGG
			AACTTTATCCTAGAGCGCTCAGGAATCTTG
			CCCGCCACCTGCTGTGCACTTCCTAGCGACT
			TTGTGCCCATTAAGTACCGCGAATGCCCTC
			CGCCGCTTTGGGGCCACTGCTACCTTCTGCA
			GCTAGCCAACTACCTTGCCTACCACTCTGA
			CATAATGGAAGACGTGAGCGGTGACGGTCT
			ACTGGAGTGTCACTGTCGCTGCAACCTATG
			CACCCCGCACCGCTCCCTGGTTTGCAATTCG
			CAGCTGCTTAACGAAAGTCAAATTATCGGT
			ACCTTTGAGCTGCAGGGTCCCTCGCCTGAC
			GAAAAGTCCGCGGCTCCGGGGTTGAAACTC
			ACTCCGGGGCTGTGGACGTCGGCTTACCTT
			CGCAAATTTGTACCTGAGGACTACCACGCC
			CACGAGATTAGGTTCTACGAAGACCAATCC
			CGCCCGCCTAATGCGGAGCTTACCGCCTGC
			GTCATTACCCAGGGCCACATTCTTGGCCAA
			TTGCAAGCCATCAACAAAGCCCGCCAAGAG
			TTTCTGCTACGAAAGGGACGGGGGGTTTAC
			TTGGACCCCCAGTCCGGCGAGGAGCTCAAC
			CCAATCCCCCCGCCGCCGCAGCCCTATCAG
			CAGCAGCCGCGGGCCCTTGCTTCCCAGGAT
			GGCACCCAAAAAGAAGCTGCAGCTGCCGCC
			GCCACCCACGGACGAGGAGGAATACTGGG
			ACAGTCAGGCAGAGGAGGTTTTGGACGAG
			GAGGAGGAGGACATGATGGAAGACTGGGA
			GAGCCTAGACGAGGAAGCTTCCGAGGTCGA
			AGAGGTGTCAGACGAAACACCGTCACCCTC
			GGTCGCATTCCCCTCGCCGGCGCCCCAGAA
			ATCGGCAACCGGTTCCAGCATGGCTACAAC
			CTCCGCTCCTCAGGCGCCGCCGGCACTGCC
			CGTTCGCCGACCCAACCGTAGATGGGACAC
			CACTGGAACCAGGGCCGGTAAGTCCAAGCA
			GCCGCCGCCGTTAGCCCAAGAGCAACAACA
			GCGCCAAGGCTACCGCTCATGGCGCGGGCA
			CAAGAACGCCATAGTTGCTTGCTTGCAAGA
			CTGTGGGGGCAACATCTCCTTCGCCCGCCG
			CTTTCTTCTCTACCATCACGGCGTGGCCTTC
			CCCCGTAACATCCTGCATTACTACCGTCATC
			TCTACAGCCCATACTGCACCGGCGGCAGCG
			GCAGCAACAGCAGCGGCCACACAGAAGCA
			AAGGCGACCGGATAGCAAGACTCTGACAA
			AGCCCAAGAAATCCACAGCGGCGGCAGCA
			GCAGGAGGAGGAGCGCTGCGTCTGGCGCCC
			AACGAACCCGTATCGACCCGCGAGCTTAGA
			AACAGGATTTTTCCCACTCTGTATGCTATAT
			TTCAACAGAGCAGGGGCCAAGAACAAGAG
			CTGAAAATAAAAAACAGGTCTCTGCGATCC
			CTCACCCGCAGCTGCCTGTATCACAAAAGC
			GAAGATCAGCTTCGGCGCACGCTGGAAGAC
			GCGGAGGCTCTCTTCAGTAAATACTGCGCG
			CTGACTCTTAAGGACTAGTTTCGCGCCCTTT
			CTCAAATTTAAGCGCGAAAACTACGTCATC
			TCCAGCGGCCACACCCGGCGCCAGCACCTG
			TTGTCAGCGCCATTATGAGCAAGGAAATTC
			CCACGCCCTACATGTGGAGTTACCAGCCAC
			AAATGGGACTTGCGGCTGGAGCTGCCCAAG
			ACTACTCAACCCGAATAAACTACATGAGCG
			CGGGACCCCACATGATATCCCGGGTCAACG
			GAATACGCGCCCACCGAAACCGAATTCTCC
			TGGAACAGGCGGCTATTACCACCACACCTC
			GTAATAACCTTAATCCCCGTAGTTGGCCCG
			CTGCCCTGGTGTACCAGGAAAGTCCCGCTC
			CCACCACTGTGGTACTTCCCAGAGACGCCC
			AGGCCGAAGTTCAGATGACTAACTCAGGGG
			CGCAGCTTGCGGGCGGCTTTCGTCACAGGG
			TGCGGTCGCCCGGGCAGGGTATAACTCACC
			TGACAATCAGAGGGCGAGGTATTCAGCTCA
			ACGACGAGTCGGTGAGCTCCTCGCTTGGTC
			TCCGTCCGGACGGGACATTTCAGATCGGCG
			GCGCCGGCCGCTCTTCATTCACGCCTCGTCA
			GGCAATCCTAACTCTGCAGACCTCGTCCTCT
			GAGCCGCGCTCTGGAGGCATTGGAACTCTG
			CAATTTATTGAGGAGTTTGTGCCATCGGTCT
			ACTTTAACCCCTTCTCGGGACCTCCCGGCCA
			CTATCCGGATCAATTTATTCCTAACTTTGAC
			GCGGTAAAGGACTCGGCGGACGGCTACGA
			CTGAATGTTAAGTGGAGAGGCAGAGCAACT
			GCGCCTGAAACACCTGGTCCACTGTCGCCG
			CCACAAGTGCTTTGCCCGCGACTCCGGTGA
			GTTTTGCTACTTTGAATTGCCCGAGGATCAT
			ATCGAGGGCCCGGCGCACGGCGTCCGGCTT
			ACCGCCCAGGGAGAGCTTGCCCGTAGCCTG
			ATTCGGGAGTTTACCCAGCGCCCCCTGCTA
			GTTGAGCGGGACAGGGGACCCTGTGTTCTC
			ACTGTGATTTGCAACTGTCCTAACCCTGGAT
			TACATCAAGATCCTCTAGTTAATGTCAGGT
			CGCCTAAGTCGATTAACTAGAGTACCCGGG
			GATCTTATTCCCTTTAACTAATAAAAAAAA
			ATAATAAAGCATCACTTACTTAAAATCAGT
			TAGCAAATTTCTGTCCAGTTTATTCAGCAGC
			ACCTCCTTGCCCTCCTCCCAGCTCTGGTATT
			GCAGCTTCCTCCTGGCTGCAAACTTTCTCCA
			CAATCTAAATGGAATGTCAGTTTCCTCCTGT
			TCCTGTCCATCCGCACCCACTATCTTCATGT
			TGTTGCAGATGAAGCGCGCAAGACCGTCTG
			AAGATACCTTCAACCCCGTGTATCCATATG
			ACACGGAAACCGGTCCTCCAACTGTGCCTT
			TTCTTACTCCTCCCTTTGTATCCCCCAATGG
			GTTTCAAGAGAGTCCCCCTGGGGTACTCTC
			TTTGCGCCTATCCGAACCTCTAGTTACCTCC
			AATGGCATGCTTGCGCTCAAAATGGGCAAC
			GGCCTCTCTCTGGACGAGGCCGGCAACCTT
			ACCTCCCAAAATGTAACCACTGTGAGCCCA
			CCTCTCAAAAAAACCAAGTCAAACATAAAC
			CTGGAAATATCTGCACCCCTCACAGTTACC
			TCAGAAGCCCTAACTGTGGCTGCCGCCGCA
			CCTCTAATGGTCGCGGGCAACACACTCACC
			ATGCAATCACAGGCCCCGCTAACCGTGCAC
			GACTCCAAACTTAGCATTGCCACCCAAGGA
			CCCCTCACAGTGTCAGAAGGAAAGCTAGCC
			CTGCAAACATCAGGCCCCCTCACCACCACC
			GATAGCAGTACCCTTACTATCACTGCCTCA
			CCCCCTCTAACTACTGCCACTGGTAGCTTGG
			GCATTGACTTGAAAGAGCCCATTTATACAC
			AAAATGGAAAACTAGGACTAAAGTACGGG
			GCTCCTTTGCATGTAACAGACGACCTAAAC
			ACTTTGACCGTAGCAACTGGTCCAGGTGTG
			ACTATTAATAATACTTCCTTGCAAACTAAA
			GTTACTGGAGCCTTGGGTTTTGATTCACAA
			GGCAATATGCAACTTAATGTAGCAGGAGGA
			CTAAGGATTGATTCTCAAAACAGACGCCTT
			ATACTTGATGTTAGTTATCCGTTTGATGCTC
			AAAACCAACTAAATCTAAGACTAGGACAG
			GGCCCTCTTTTTATAAACTCAGCCCACAACT
			TGGATATTAACTACAACAAAGGCCTTTACT
			TGTTTACAGCTTCAAACAATTCCAAAAAGC
			TTGAGGTTAACCTAAGCACTGCCAAGGGGT
			TGATGTTTGACGCTACAGCCATAGCCATTA
			ATGCAGGAGATGGGCTTGAATTTGGTTCAC
			CTAATGCACCAAACACAAATCCCCTCAAAA
			CAAAAATTGGCCATGGCCTAGAATTTGATT
			CAAACAAGGCTATGGTTCCTAAACTAGGAA
			CTGGCCTTAGTTTTGACAGCACAGGTGCCA
			TTACAGTAGGAAACAAAAATAATGATAAGC
			TAACTTTGTGGACCACACCAGCTCCATCTCC
			TAACTGTAGACTAAATGCAGAGAAAGATGC
			TAAACTCACTTTGGTCTTAACAAAATGTGG
			CAGTCAAATACTTGCTACAGTTTCAGTTTTG
			GCTGTTAAAGGCAGTTTGGCTCCAATATCT
			GGAACAGTTCAAAGTGCTCATCTTATTATA
			AGATTTGACGAAAATGGAGTGCTACTAAAC
			AATTCCTTCCTGGACCCAGAATATTGGAAC
			TTTAGAAATGGAGATCTTACTGAAGGCACA
			GCCTATACAAACGCTGTTGGATTTATGCCT
			AACCTATCAGCTTATCCAAAATCTCACGGT
			AAAACTGCCAAAAGTAACATTGTCAGTCAA
			GTTTACTTAAACGGAGACAAAACTAAACCT
			GTAACACTAACCATTACACTAAACGGTACA
			CAGGAAACAGGAGACACAACTCCAAGTGC
			ATACTCTATGTCATTTTCATGGGACTGGTCT
			GGCCACAACTACATTAATGAAATATTTGCC
			ACATCCTCTTACACTTTTTCATACATTGCCC
			AAGAATAAAGAATCGTTTGTGTTATGTTTC
			AACGTGTTTATTTTTCAATTGCAGAAAATTT
			CAAGTCATTTTTCATTCAGTAGTATAGCCCC
			ACCACCACATAGCTTATACAGATCACCGTA
			CCTTAATCAAACTCACAGAACCCTAGTATT
			CAACCTGCCACCTCCCTCCCAACACACAGA
			GTACACAGTCCTTTCTCCCCGGCTGGCCTTA
			AAAAGCATCATATCATGGGTAACAGACATA
			TTCTTAGGTGTTATATTCCACACGGTTTCCT
			GTCGAGCCAAACGCTCATCAGTGATATTAA
			TAAACTCCCCGGGCAGCTCACTTAAGTTCA
			TGTCGCTGTCCAGCTGCTGAGCCACAGGCT
			GCTGTCCAACTTGCGGTTGCTTAACGGGCG
			GCGAAGGAGAAGTCCACGCCTACATGGGG
			GTAGAGTCATAATCGTGCATCAGGATAGGG
			CGGTGGTGCTGCAGCAGCGCGCGAATAAAC
			TGCTGCCGCCGCCGCTCCGTCCTGCAGGAA
			TACAACATGGCAGTGGTCTCCTCAGCGATG
			ATTCGCACCGCCCGCAGCATAAGGCGCCTT
			GTCCTCCGGGCACAGCAGCGCACCCTGATC
			TCACTTAAATCAGCACAGTAACTGCAGCAC
			AGCACCACAATATTGTTCAAAATCCCACAG
			TGCAAGGCGCTGTATCCAAAGCTCATGGCG
			GGGACCACAGAACCCACGTGGCCATCATAC
			CACAAGCGCAGGTAGATTAAGTGGCGACCC
			CTCATAAACACGCTGGACATAAACATTACC
			TCTTTTGGCATGTTGTAATTCACCACCTCCC
			GGTACCATATAAACCTCTGATTAAACATGG
			CGCCATCCACCACCATCCTAAACCAGCTGG
			CCAAAACCTGCCCGCCGGCTATACACTGCA
			GGGAACCGGGACTGGAACAATGACAGTGG
			AGAGCCCAGGACTCGTAACCATGGATCATC
			ATGCTCGTCATGATATCAATGTTGGCACAA
			CACAGGCACACGTGCATACACTTCCTCAGG
			ATTACAAGCTCCTCCCGCGTTAGAACCATA
			TCCCAGGGAACAACCCATTCCTGAATCAGC
			GTAAATCCCACACTGCAGGGAAGACCTCGC
			ACGTAACTCACGTTGTGCATTGTCAAAGTG
			TTACATTCGGGCAGCAGCGGATGATCCTCC
			AGTATGGTAGCGCGGGTTTCTGTCTCAAAA
			GGAGGTAGACGATCCCTACTGTACGGAGTG
			CGCCGAGACAACCGAGATCGTGTTGGTCGT
			AGTGTCATGCCAAATGGAACGCCGGACGTA
			GTCATATTTCCTGAAGCAAAACCAGGTGCG
			GGCGTGACAAACAGATCTGCGTCTCCGGTC
			TCGCCGCTTAGATCGCTCTGTGTAGTAGTTG
			TAGTATATCCACTCTCTCAAAGCATCCAGG
			CGCCCCCTGGCTTCGGGTTCTATGTAAACTC
			CTTCATGCGCCGCTGCCCTGATAACATCCA
			CCACCGCAGAATAAGCCACACCCAGCCAAC
			CTACACATTCGTTCTGCGAGTCACACACGG
			GAGGAGCGGGAAGAGCTGGAAGAACCATG
			TTTTTTTTTTTATTCCAAAAGATTATCCAAA
			ACCTCAAAATGAAGATCTATTAAGTGAACG
			CGCTCCCCTCCGGTGGCGTGGTCAAACTCT
			ACAGCCAAAGAACAGATAATGGCATTTGTA
			AGATGTTGCACAATGGCTTCCAAAAGGCAA
			ACGGCCCTCACGTCCAAGTGGACGTAAAGG
			CTAAACCCTTCAGGGTGAATCTCCTCTATA
			AACATTCCAGCACCTTCAACCATGCCCAAA
			TAATTCTCATCTCGCCACCTTCTCAATATAT
			CTCTAAGCAAATCCCGAATATTAAGTCCGG
			CCATTGTAAAAATCTGCTCCAGAGCGCCCT
			CCACCTTCAGCCTCAAGCAGCGAATCATGA
			TTGCAAAAATTCAGGTTCCTCACAGACCTG
			TATAAGATTCAAAAGCGGAACATTAACAAA
			AATACCGCGATCCCGTAGGTCCCTTCGCAG
			GGCCAGCTGAACATAATCGTGCAGGTCTGC
			ACGGACCAGCGCGGCCACTTCCCCGCCAGG
			AACCATGACAAAAGAACCCACACTGATTAT
			GACACGCATACTCGGAGCTATGCTAACCAG
			CGTAGCCCCGATGTAAGCTTGTTGCATGGG
			CGGCGATATAAAATGCAAGGTGCTGCTCAA
			AAAATCAGGCAAAGCCTCGCGCAAAAAAG
			AAAGCACATCGTAGTCATGCTCATGCAGAT
			AAAGGCAGGTAAGCTCCGGAACCACCACA
			GAAAAAGACACCATTTTTCTCTCAAACATG
			TCTGCGGGTTTCTGCATAAACACAAAATAA
			AATAACAAAAAAACATTTAAACATTAGAAG
			CCTGTCTTACAACAGGAAAAACAACCCTTA
			TAAGCATAAGACGGACTACGGCCATGCCGG
			CGTGACCGTAAAAAAACTGGTCACCGTGAT
			TAAAAAGCACCACCGACAGCTCCTCGGTCA
			TGTCCGGAGTCATAATGTAAGACTCGGTAA
			ACACATCAGGTTGATTCACATCGGTCAGTG
			CTAAAAAGCGACCGAAATAGCCCGGGGGA
			ATACATACCCGCAGGCGTAGAGACAACATT
			ACAGCCCCCATAGGAGGTATAACAAAATTA
			ATAGGAGAGAAAAACACATAAACACCTGA
			AAAACCCTCCTGCCTAGGCAAAATAGCACC
			CTCCCGCTCCAGAACAACATACAGCGCTTC
			CACAGCGGCAGCCATAACAGTCAGCCTTAC
			CAGTAAAAAAGAAAACCTATTAAAAAAAC
			ACCACTCGACACGGCACCAGCTCAATCAGT
			CACAGTGTAAAAAAGGGCCAAGTGCAGAG
			CGAGTATATATAGGACTAAAAAATGACGTA
			ACGGTTAAAGTCCACAAAAAACACCCAGA
			AAACCGCACGCGAACCTACGCCCAGAAAC
			GAAAGCCAAAAAACCCACAACTTCCTCAAA
			TCGTCACTTCCGTTTTCCCACGTTACGTCAC
			TTCCCATTTTAAGAAAACTACAATTCCCAA
			CACATACAAGTTACTCCGCCCTAAAACCTA
			CGTCACCCGCCCCGTTCCCACGCCCCGCGC
			CACGTCACAAACTCCACCCCCTCATTATCAT
			ATTGGCTTCAATCCAAAATAAGGTATATTA
			TTGATGATG

95	Full	(human IL-	TTTTGGATTGAAGCCAATATGATAATGAGG
	nucleotide	12 insert	GGGTGGAGTTTGTGACGTGGCGCGGGGCGT
	sequence of	sequence in	GGGAACGGGGCGGGTGACGTAGTAGTGTG
	TRZ627 hIL-	grey	GCGGAAGTGTGATGTTGCAAGTGTGGCGGA
	12 virus	highlight-	ACACATGTAAGCGACGGATGTGGCAAAAGT
	(beginning at	codon	GACGTTTTTGGTGTGCGCCGGTGTTTTGGGC
	Ad5 5′ end	optimized)	GTAACCGAGTAAGATTTGGCCATTTTCGCG
	ITR)		GGAAAACTGAATAAGAGGAAGTGAAATCT
			GAATAATTTTGTGTTACTCATAGCGCGTAAT
			ATTTGTCTAGGGCCGCGGGGACTTTGACCG
			TTTACGTGGAGACTCGCCCAGGTGTTTTTCT
			CAGGTGTTTTCCGCGTTCCGGGTCAAAGTT
			GGCGTTTTATTATTATAGTCAGCTGACGTGT
			AGTGTATTTATACCCGGTGAGTTCCTCAAG
			AGGCCACTCTTGAGTGCCAGCGAGTAGAGT
			TTTCTCCTCCGAGCCGCTCCGACACCGGGA
			CTGAAAATGAGACATATTATCTGCCACGGA
			GGTGTTATTACCGAAGAAATGGCCGCCAGT
			CTTTTGGACCAGCTGATCGAAGAGGTACTG
			GCTGATAATCTTCCACCTCCTAGCCATTTTG
			AACCACCTACCCTTCACGAACTGTATGATTT
			AGACGTGACGGCCCCCGAAGATCCCAACGA
			GGAGGCGGTTTCGCAGATTTTTCCCGACTCT
			GTAATGTTGGCGGTGCAGGAAGGGATTGAC
			TTACTCACTTTTCCGCCGGCGCCCGGTTCTC
			CGGAGCCGCCTCACCTTTCCCGGCAGCCCG
			AGCAGCCGGAGCAGAGAGCCTTGGGTCCG
			GTTTCTATGCCAAACCTTGTACCGGAGGTG
			ATCGATCTTACCTGCCACGAGGCTGGCTTTC
			CACCCAGTGACGACGAGGATGAAGAGGGT
			GAGGAGTTTGTGTTAGATTATGTGGAGCAC
			CCCGGGCACGGTTGCAGGTCTTGTCATTAT
			CACCGGAGGAATACGGGGGACCCAGATATT
			ATGTGTTCGCTTTGCTATATGAGGACCTGTG
			GCATGTTTGTCTACAGTAAGTGAAAATTAT
			GGGCAGTGGGTGATAGAGTGGTGGGTTTGG
			TGTGGTAATTTTTTTTTTAATTTTTACAGTTT
			TGTGGTTTAAAGAATTTTGTATTGTGATTTT
			TTTAAAAGGTCCTGTGTCTGAACCTGAGCC
			TGAGCCCGAGCCAGAACCGGAGCCTGCAA
			GACCTACCCGCCGTCCTAAAATGGCGCCTG
			CTATCCTGAGACGCCCGACATCACCTGTGT
			CTAGAGAATGCAATAGTAGTACGGATAGCT
			GTGACTCCGGTCCTTCTAACACACCTCCTGA
			GATACACCCGGTGGTCCCGCTGTGCCCCAT
			TAAACCAGTTGCCGTGAGAGTTGGTGGGCG
			TCGCCAGGCTGTGGAATGTATCGAGGACTT
			GCTTAACGAGCCTGGGCAACCTTTGGACTT
			GAGCTGTAAACGCCCCAGGCCATAAGGTGT
			AAACCTGTGATTGCGTGTGTGGTTAACGCC
			TTTGTTTGCTGAATGAGTTGATGTAAGTTTA
			ATAAAGGGTGAGATAATGTTTAACTTGCAT
			GGCGTGTTAAATGGGGCGGGGCTTAAAGGG
			TATATAATGCGCCGTGGGCTAATCTTGGTT



			GAAGGTCATCAAGACTTTGGATTTTTCCAC
			ACCGGGGCGCGCTGCGGCTGCTGTTGCTTT
			TTTGAGTTTTATAAAGGATAAATGGAGCGA
			AGAAACCCATCTGAGCGGGGGGTACCTGCT
			GGATTTTCTGGCCATGCATCTGTGGAGAGC
			GGTTGTGAGACACAAGAATCGCCTGCTACT
			GTTGTCTTCCGTCCGCCCGGCGATAATACC
			GACGGAGGAGCAGCAGCAGCAGCAGGAGG
			AAGCCAGGCGGCGGCGGCAGGAGCAGAGC
			CCATGGAACCCGAGAGCCGGCCTGGACCCT
			CGGGAATGAATGTTGTACAGGTGGCTGAAC
			TGTATCCAGAACTGAGACGCATTTTGACAA
			TTACAGAGGATGGGCAGGGGCTAAAGGGG
			GTAAAGAGGGAGCGGGGGGCTTGTGAGGC
			TACAGAGGAGGCTAGGAATCTAGCTTTTAG
			CTTAATGACCAGACACCGTCCTGAGTGTAT
			TACTTTTCAACAGATCAAGGATAATTGCGC
			TAATGAGCTTGATCTGCTGGCGCAGAAGTA
			TTCCATAGAGCAGCTGACCACTTACTGGCT
			GCAGCCAGGGGATGATTTTGAGGAGGCTAT
			TAGGGTATATGCAAAGGTGGCACTTAGGCC
			AGATTGCAAGTACAAGATCAGCAAACTTGT
			AAATATCAGGAATTGTTGCTACATTTCTGG
			GAACGGGGCCGAGGTGGAGATAGATACGG
			AGGATAGGGTGGCCTTTAGATGTAGCATGA
			TAAATATGTGGCCGGGGGTGCTTGGCATGG
			ACGGGGTGGTTATTATGAATGTAAGGTTTA
			CTGGCCCCAATTTTAGCGGTACGGTTTTCCT
			GGCCAATACCAACCTTATCCTACACGGTGT
			AAGCTTCTATGGGTTTAACAATACCTGTGT
			GGAAGCCTGGACCGATGTAAGGGTTCGGGG
			CTGTGCCTTTTACTGCTGCTGGAAGGGGGT
			GGTGTGTCGCCCCAAAAGCAGGGCTTCAAT
			TAAGAAATGCCTCTTTGAAAGGTGTACCTT
			GGGTATCCTGTCTGAGGGTAACTCCAGGGT
			GCGCCACAATGTGGCCTCCGACTGTGGTTG
			CTTCATGCTAGTGAAAAGCGTGGCTGTGAT
			TAAGCATAACATGGTATGTGGCAACTGCGA
			GGACAGGGCCTCTCAGATGCTGACCTGCTC
			GGACGGCAACTGTCACCTGCTGAAGACCAT
			TCACGTAGCCAGCCACTCTCGCAAGGCCTG
			GCCAGTGTTTGAGCATAACATACTGACCCG
			CTGTTCCTTGCATTTGGGTAACAGGAGGGG
			GGTGTTCCTACCTTACCAATGCAATTTGAGT
			CACACTAAGATATTGCTTGAGCCCGAGAGC
			ATGTCCAAGGTGAACCTGAACGGGGTGTTT
			GACATGACCATGAAGATCTGGAAGGTGCTG
			AGGTACGATGAGACCCGCACCAGGTGCAG
			ACCCTGCGAGTGTGGCGGTAAACATATTAG
			GAACCAGCCTGTGATGCTGGATGTGACCGA
			GGAGCTGAGGCCCGATCACTTGGTGCTGGC
			CTGCACCCGCGCTGAGTTTGGCTCTAGCGA
			TGAAGATACAGATTGAGGTACTGAAATGTG
			TGGGCGTGGCTTAAGGGTGGGAAAGAATAT
			ATAAGGTGGGGGTCTTATGTAGTTTTGTATC
			TGTTTTGCAGCAGCCGCCGCCGCCATGAGC
			ACCAACTCGTTTGATGGAAGCATTGTGAGC
			TCATATTTGACAACGCGCATGCCCCCATGG
			GCCGGGGTGCGTCAGAATGTGATGGGCTCC
			AGCATTGATGGTCGCCCCGTCCTGCCCGCA
			AACTCTACTACCTTGACCTACGAGACCGTG
			TCTGGAACGCCGTTGGAGACTGCAGCCTCC
			GCCGCCGCTTCAGCCGCTGCAGCCACCGCC
			CGCGGGATTGTGACTGACTTTGCTTTCCTGA
			GCCCGCTTGCAAGCAGTGCAGCTTCCCGTT
			CATCCGCCCGCGATGACAAGTTGACGGCTC
			TTTTGGCACAATTGGATTCTTTGACCCGGGA
			ACTTAATGTCGTTTCTCAGCAGCTGTTGGAT
			CTGCGCCAGCAGGTTTCTGCCCTGAAGGCT
			TCCTCCCCTCCCAATGCGGTTTAAAACATA
			AATAAAAAACCAGACTCTGTTTGGATTTGG
			ATCAAGCAAGTGTCTTGCTGTCTTTATTTAG
			GGGTTTTGCGCGCGCGGTAGGCCCGGGACC
			AGCGGTCTCGGTCGTTGAGGGTCCTGTGTA
			TTTTTTCCAGGACGTGGTAAAGGTGACTCT
			GGATGTTCAGATACATGGGCATAAGCCCGT
			CTCTGGGGTGGAGGTAGCACCACTGCAGAG
			CTTCATGCTGCGGGGTGGTGTTGTAGATGA
			TCCAGTCGTAGCAGGAGCGCTGGGCGTGGT
			GCCTAAAAATGTCTTTCAGTAGCAAGCTGA
			TTGCCAGGGGCAGGCCCTTGGTGTAAGTGT
			TTACAAAGCGGTTAAGCTGGGATGGGTGCA
			TACGTGGGGATATGAGATGCATCTTGGACT
			GTATTTTTAGGTTGGCTATGTTCCCAGCCAT
			ATCCCTCCGGGGATTCATGTTGTGCAGAAC
			CACCAGCACAGTGTATCCGGTGCACTTGGG
			AAATTTGTCATGTAGCTTAGAAGGAAATGC
			GTGGAAGAACTTGGAGACGCCCTTGTGACC
			TCCAAGATTTTCCATGCATTCGTCCATAATG
			ATGGCAATGGGCCCACGGGCGGCGGCCTGG
			GCGAAGATATTTCTGGGATCACTAACGTCA
			TAGTTGTGTTCCAGGATGAGATCGTCATAG
			GCCATTTTTACAAAGCGCGGGCGGAGGGTG
			CCAGACTGCGGTATAATGGTTCCATCCGGC
			CCAGGGGCGTAGTTACCCTCACAGATTTGC
			ATTTCCCACGCTTTGAGTTCAGATGGGGGG
			ATCATGTCTACCTGCGGGGCGATGAAGAAA
			ACGGTTTCCGGGGTAGGGGAGATCAGCTGG
			GAAGAAAGCAGGTTCCTGAGCAGCTGCGAC
			TTACCGCAGCCGGTGGGCCCGTAAATCACA
			CCTATTACCGGCTGCAACTGGTAGTTAAGA
			GAGCTGCAGCTGCCGTCATCCCTGAGCAGG
			GGGGCCACTTCGTTAAGCATGTCCCTGACT
			CGCATGTTTTCCCTGACCAAATCCGCCAGA
			AGGCGCTCGCCGCCCAGCGATAGCAGTTCT
			TGCAAGGAAGCAAAGTTTTTCAACGGTTTG
			AGACCGTCCGCCGTAGGCATGCTTTTGAGC
			GTTTGACCAAGCAGTTCCAGGCGGTCCCAC
			AGCTCGGTCACCTGCTCTACGGCATCTCGA
			TCCAGCATATCTCCTCGTTTCGCGGGTTGGG
			GCGGCTTTCGCTGTACGGCAGTAGTCGGTG
			CTCGTCCAGACGGGCCAGGGTCATGTCTTT
			CCACGGGCGCAGGGTCCTCGTCAGCGTAGT
			CTGGGTCACGGTGAAGGGGTGCGCTCCGGG
			CTGCGCGCTGGCCAGGGTGCGCTTGAGGCT
			GGTCCTGCTGGTGCTGAAGCGCTGCCGGTC
			TTCGCCCTGCGCGTCGGCCAGGTAGCATTT
			GACCATGGTGTCATAGTCCAGCCCCTCCGC
			GGCGTGGCCCTTGGCGCGCAGCTTGCCCTT
			GGAGGAGGCGCCGCACGAGGGGCAGTGCA
			GACTTTTGAGGGCGTAGAGCTTGGGCGCGA
			GAAATACCGATTCCGGGGAGTAGGCATCCG
			CGCCGCAGGCCCCGCAGACGGTCTCGCATT
			CCACGAGCCAGGTGAGCTCTGGCCGTTCGG
			GGTCAAAAACCAGGTTTCCCCCATGCTTTTT
			GATGCGTTTCTTACCTCTGGTTTCCATGAGC
			CGGTGTCCACGCTCGGTGACGAAAAGGCTG
			TCCGTGTCCCCGTATACAGACTTGAGAGGC
			CTGTCCTCGAGCGGTGTTCCGCGGTCCTCCT
			CGTATAGAAACTCGGACCACTCTGAGACAA
			AGGCTCGCGTCCAGGCCAGCACGAAGGAG
			GCTAAGTGGGAGGGGTAGCGGTCGTTGTCC
			ACTAGGGGGTCCACTCGCTCCAGGGTGTGA
			AGACACATGTCGCCCTCTTCGGCATCAAGG
			AAGGTGATTGGTTTGTAGGTGTAGGCCACG
			TGACCGGGTGTTCCTGAAGGGGGGCTATAA
			AAGGGGGTGGGGGCGCGTTCGTCCTCACTC
			TCTTCCGCATCGCTGTCTGCGAGGGCCAGC
			TGTTGGGGTGAGTACTCCCTCTGAAAAGCG
			GGCATGACTTCTGCGCTAAGATTGTCAGTTT
			CCAAAAACGAGGAGGATTTGATATTCACCT
			GGCCCGCGGTGATGCCTTTGAGGGTGGCCG
			CATCCATCTGGTCAGAAAAGACAATCTTTT
			TGTTGTCAAGCTTGGTGGCAAACGACCCGT
			AGAGGGCGTTGGACAGCAACTTGGCGATGG
			AGCGCAGGGTTTGGTTTTTGTCGCGATCGG
			CGCGCTCCTTGGCCGCGATGTTTAGCTGCA
			CGTATTCGCGCGCAACGCACCGCCATTCGG
			GAAAGACGGTGGTGCGCTCGTCGGGCACCA
			GGTGCACGCGCCAACCGCGGTTGTGCAGGG
			TGACAAGGTCAACGCTGGTGGCTACCTCTC
			CGCGTAGGCGCTCGTTGGTCCAGCAGAGGC
			GGCCGCCCTTGCGCGAGCAGAATGGCGGTA
			GGGGGTCTAGCTGCGTCTCGTCCGGGGGGT
			CTGCGTCCACGGTAAAGACCCCGGGCAGCA
			GGCGCGCGTCGAAGTAGTCTATCTTGCATC
			CTTGCAAGTCTAGCGCCTGCTGCCATGCGC
			GGGCGGCAAGCGCGCGCTCGTATGGGTTGA
			GTGGGGGACCCCATGGCATGGGGTGGGTGA
			GCGCGGAGGCGTACATGCCGCAAATGTCGT
			AAACGTAGAGGGGCTCTCTGAGTATTCCAA
			GATATGTAGGGTAGCATCTTCCACCGCGGA
			TGCTGGCGCGCACGTAATCGTATAGTTCGT
			GCGAGGGAGCGAGGAGGTCGGGACCGAGG
			TTGCTACGGGCGGGCTGCTCTGCTCGGAAG
			ACTATCTGCCTGAAGATGGCATGTGAGTTG
			GATGATATGGTTGGACGCTGGAAGACGTTG
			AAGCTGGCGTCTGTGAGACCTACCGCGTCA
			CGCACGAAGGAGGCGTAGGAGTCGCGCAG
			CTTGTTGACCAGCTCGGCGGTGACCTGCAC
			GTCTAGGGCGCAGTAGTCCAGGGTTTCCTT
			GATGATGTCATACTTATCCTGTCCCTTTTTT
			TTCCACAGCTCGCGGTTGAGGACAAACTCT
			TCGCGGTCTTTCCAGTACTCTTGGATCGGAA
			ACCCGTCGGCCTCCGAACGGTAAGAGCCTA
			GCATGTAGAACTGGTTGACGGCCTGGTAGG
			CGCAGCATCCCTTTTCTACGGGTAGCGCGT
			ATGCCTGCGCGGCCTTCCGGAGCGAGGTGT
			GGGTGAGCGCAAAGGTGTCCCTGACCATGA
			CTTTGAGGTACTGGTATTTGAAGTCAGTGTC
			GTCGCATCCGCCCTGCTCCCAGAGCAAAAA
			GTCCGTGCGCTTTTTGGAACGCGGATTTGG
			CAGGGCGAAGGTGACATCGTTGAAGAGTAT
			CTTTCCCGCGCGAGGCATAAAGTTGCGTGT
			GATGCGGAAGGGTCCCGGCACCTCGGAACG
			GTTGTTAATTACCTGGGCGGCGAGCACGAT
			CTCGTCAAAGCCGTTGATGTTGTGGCCCAC
			AATGTAAAGTTCCAAGAAGCGCGGGATGCC
			CTTGATGGAAGGCAATTTTTTAAGTTCCTCG
			TAGGTGAGCTCTTCAGGGGAGCTGAGCCCG
			TGCTCTGAAAGGGCCCAGTCTGCAAGATGA
			GGGTTGGAAGCGACGAATGAGCTCCACAG
			GTCACGGGCCATTAGCATTTGCAGGTGGTC
			GCGAAAGGTCCTAAACTGGCGACCTATGGC
			CATTTTTTCTGGGGTGATGCAGTAGAAGGT
			AAGCGGGTCTTGTTCCCAGCGGTCCCATCC
			AAGGTTCGCGGCTAGGTCTCGCGCGGCAGT
			CACTAGAGGCTCATCTCCGCCGAACTTCAT
			GACCAGCATGAAGGGCACGAGCTGCTTCCC
			AAAGGCCCCCATCCAAGTATAGGTCTCTAC
			ATCGTAGGTGACAAAGAGACGCTCGGTGCG
			AGGATGCGAGCCGATCGGGAAGAACTGGA
			TCTCCCGCCACCAATTGGAGGAGTGGCTAT
			TGATGTGGTGAAAGTAGAAGTCCCTGCGAC
			GGGCCGAACACTCGTGCTGGCTTTTGTAAA
			AACGTGCGCAGTACTGGCAGCGGTGCACGG
			GCTGTACATCCTGCACGAGGTTGACCTGAC
			GACCGCGCACAAGGAAGCAGAGTGGGAAT
			TTGAGCCCCTCGCCTGGCGGGTTTGGCTGG
			TGGTCTTCTACTTCGGCTGCTTGTCCTTGAC
			CGTCTGGCTGCTCGAGGGGAGTTACGGTGG
			ATCGGACCACCACGCCGCGCGAGCCCAAAG
			TCCAGATGTCCGCGCGCGGCGGTCGGAGCT
			TGATGACAACATCGCGCAGATGGGAGCTGT
			CCATGGTCTGGAGCTCCCGCGGCGTCAGGT
			CAGGCGGGAGCTCCTGCAGGTTTACCTCGC
			ATAGACGGGTCAGGGCGCGGGCTAGATCCA
			GGTGATACCTAATTTCCAGGGGCTGGTTGG
			TGGCGGCGTCGATGGCTTGCAAGAGGCCGC
			ATCCCCGCGGCGCGACTACGGTACCGCGCG
			GCGGGCGGTGGGCCGCGGGGGTGTCCTTGG
			ATGATGCATCTAAAAGCGGTGACGCGGGCG
			AGCCCCCGGAGGTAGGGGGGGCTCCGGAC
			CCGCCGGGAGAGGGGGCAGGGGCACGTCG
			GCGCCGCGCGCGGGCAGGAGCTGGTGCTGC
			GCGCGTAGGTTGCTGGCGAACGCGACGACG
			CGGCGGTTGATCTCCTGAATCTGGCGCCTCT
			GCGTGAAGACGACGGGCCCGGTGAGCTTGA
			ACCTGAAAGAGAGTTCGACAGAATCAATTT
			CGGTGTCGTTGACGGCGGCCTGGCGCAAAA
			TCTCCTGCACGTCTCCTGAGTTGTCTTGATA
			GGCGATCTCGGCCATGAACTGCTCGATCTC
			TTCCTCCTGGAGATCTCCGCGTCCGGCTCGC
			TCCACGGTGGCGGCGAGGTCGTTGGAAATG
			CGGGCCATGAGCTGCGAGAAGGCGTTGAG
			GCCTCCCTCGTTCCAGACGCGGCTGTAGAC
			CACGCCCCCTTCGGCATCGCGGGCGCGCAT
			GACCACCTGCGCGAGATTGAGCTCCACGTG
			CCGGGCGAAGACGGCGTAGTTTCGCAGGCG
			CTGAAAGAGGTAGTTGAGGGTGGTGGCGGT
			GTGTTCTGCCACGAAGAAGTACATAACCCA
			GCGTCGCAACGTGGATTCGTTGATATCCCC
			CAAGGCCTCAAGGCGCTCCATGGCCTCGTA
			GAAGTCCACGGCGAAGTTGAAAAACTGGG
			AGTTGCGCGCCGACACGGTTAACTCCTCCT
			CCAGAAGACGGATGAGCTCGGCGACAGTGT
			CGCGCACCTCGCGCTCAAAGGCTACAGGGG
			CCTCTTCTTCTTCTTCAATCTCCTCTTCCATA
			AGGGCCTCCCCTTCTTCTTCTTCTGGCGGCG
			GTGGGGGAGGGGGGACACGGCGGCGACGA
			CGGCGCACCGGGAGGCGGTCGACAAAGCG
			CTCGATCATCTCCCCGCGGCGACGGCGCAT
			GGTCTCGGTGACGGCGCGGCCGTTCTCGCG
			GGGGCGCAGTTGGAAGACGCCGCCCGTCAT
			GTCCCGGTTATGGGTTGGCGGGGGGCTGCC
			ATGCGGCAGGGATACGGCGCTAACGATGCA
			TCTCAACAATTGTTGTGTAGGTACTCCGCCG
			CCGAGGGACCTGAGCGAGTCCGCATCGACC
			GGATCGGAAAACCTCTCGAGAAAGGCGTCT
			AACCAGTCACAGTCGCAAGGTAGGCTGAGC
			ACCGTGGCGGGCGGCAGCGGGCGGCGGTC
			GGGGTTGTTTCTGGCGGAGGTGCTGCTGAT
			GATGTAATTAAAGTAGGCGGTCTTGAGACG
			GCGGATGGTCGACAGAAGCACCATGTCCTT
			GGGTCCGGCCTGCTGAATGCGCAGGCGGTC
			GGCCATGCCCCAGGCTTCGTTTTGACATCG
			GCGCAGGTCTTTGTAGTAGTCTTGCATGAG
			CCTTTCTACCGGCACTTCTTCTTCTCCTTCCT
			CTTGTCCTGCATCTCTTGCATCTATCGCTGC
			GGCGGCGGCGGAGTTTGGCCGTAGGTGGCG
			CCCTCTTCCTCCCATGCGTGTGACCCCGAAG
			CCCCTCATCGGCTGAAGCAGGGCTAGGTCG
			GCGACAACGCGCTCGGCTAATATGGCCTGC
			TGCACCTGCGTGAGGGTAGACTGGAAGTCA
			TCCATGTCCACAAAGCGGTGGTATGCGCCC
			GTGTTGATGGTGTAAGTGCAGTTGGCCATA
			ACGGACCAGTTAACGGTCTGGTGACCCGGC
			TGCGAGAGCTCGGTGTACCTGAGACGCGAG
			TAAGCCCTCGAGTCAAATACGTAGTCGTTG
			CAAGTCCGCACCAGGTACTGGTATCCCACC
			AAAAAGTGCGGCGGCGGCTGGCGGTAGAG
			GGGCCAGCGTAGGGTGGCCGGGGCTCCGG
			GGGCGAGATCTTCCAACATAAGGCGATGAT
			ATCCGTAGATGTACCTGGACATCCAGGTGA
			TGCCGGCGGCGGTGGTGGAGGCGCGCGGA
			AAGTCGCGGACGCGGTTCCAGATGTTGCGC
			AGCGGCAAAAAGTGCTCCATGGTCGGGACG
			CTCTGGCCGGTCAGGCGCGCGCAATCGTTG
			ACGCTCTAGCGTGCAAAAGGAGAGCCTGTA
			AGCGGGCACTCTTCCGTGGTCTGGTGGATA
			AATTCGCAAGGGTATCATGGCGGACGACCG
			GGGTTCGAGCCCCGTATCCGGCCGTCCGCC
			GTGATCCATGCGGTTACCGCCCGCGTGTCG
			AACCCAGGTGTGCGACGTCAGACAACGGG
			GGAGTGCTCCTTTTGGCTTCCTTCCAGGCGC
			GGCGGCTGCTGCGCTAGCTTTTTTGGCCACT
			GGCCGCGCGCAGCGTAAGCGGTTAGGCTGG
			AAAGCGAAAGCATTAAGTGGCTCGCTCCCT
			GTAGCCGGAGGGTTATTTTCCAAGGGTTGA
			GTCGCGGGACCCCCGGTTCGAGTCTCGGAC
			CGGCCGGACTGCGGCGAACGGGGGTTTGCC
			TCCCCGTCATGCAAGACCCCGCTTGCAAAT
			TCCTCCGGAAACAGGGACGAGCCCCTTTTT
			TGCTTTTCCCAGATGCATCCGGTGCTGCGGC
			AGATGCGCCCCCCTCCTCAGCAGCGGCAAG
			AGCAAGAGCAGCGGCAGACATGCAGGGCA
			CCCTCCCCTCCTCCTACCGCGTCAGGAGGG
			GCGACATCCGCGGTTGACGCGGCAGCAGAT
			GGTGATTACGAACCCCCGCGGCGCCGGGCC
			CGGCACTACCTGGACTTGGAGGAGGGCGAG
			GGCCTGGCGCGGCTAGGAGCGCCCTCTCCT
			GAGCGGCACCCAAGGGTGCAGCTGAAGCG
			TGATACGCGTGAGGCGTACGTGCCGCGGCA
			GAACCTGTTTCGCGACCGCGAGGGAGAGGA
			GCCCGAGGAGATGCGGGATCGAAAGTTCCA
			CGCAGGGCGCGAGCTGCGGCATGGCCTGAA
			TCGCGAGCGGTTGCTGCGCGAGGAGGACTT
			TGAGCCCGACGCGCGAACCGGGATTAGTCC
			CGCGCGCGCACACGTGGCGGCCGCCGACCT
			GGTAACCGCATACGAGCAGACGGTGAACC
			AGGAGATTAACTTTCAAAAAAGCTTTAACA
			ACCACGTGCGTACGCTTGTGGCGCGCGAGG
			AGGTGGCTATAGGACTGATGCATCTGTGGG
			ACTTTGTAAGCGCGCTGGAGCAAAACCCAA
			ATAGCAAGCCGCTCATGGCGCAGCTGTTCC
			TTATAGTGCAGCACAGCAGGGACAACGAG
			GCATTCAGGGATGCGCTGCTAAACATAGTA
			GAGCCCGAGGGCCGCTGGCTGCTCGATTTG
			ATAAACATCCTGCAGAGCATAGTGGTGCAG
			GAGCGCAGCTTGAGCCTGGCTGACAAGGTG
			GCCGCCATCAACTATTCCATGCTTAGCCTG
			GGCAAGTTTTACGCCCGCAAGATATACCAT
			ACCCCTTACGTTCCCATAGACAAGGAGGTA
			AAGATCGAGGGGTTCTACATGCGCATGGCG
			CTGAAGGTGCTTACCTTGAGCGACGACCTG
			GGCGTTTATCGCAACGAGCGCATCCACAAG
			GCCGTGAGCGTGAGCCGGCGGCGCGAGCTC
			AGCGACCGCGAGCTGATGCACAGCCTGCAA
			AGGGCCCTGGCTGGCACGGGCAGCGGCGAT
			AGAGAGGCCGAGTCCTACTTTGACGCGGGC
			GCTGACCTGCGCTGGGCCCCAAGCCGACGC
			GCCCTGGAGGCAGCTGGGGCCGGACCTGGG
			CTGGCGGTGGCACCCGCGCGCGCTGGCAAC
			GTCGGCGGCGTGGAGGAATATGACGAGGA
			CGATGAGTACGAGCCAGAGGACGGCGAGT
			ACTAAGCGGTGATGTTTCTGATCAGATGAT
			GCAAGACGCAACGGACCCGGCGGTGCGGG
			CGGCGCTGCAGAGCCAGCCGTCCGGCCTTA
			ACTCCACGGACGACTGGCGCCAGGTCATGG
			ACCGCATCATGTCGCTGACTGCGCGCAATC
			CTGACGCGTTCCGGCAGCAGCCGCAGGCCA
			ACCGGCTCTCCGCAATTCTGGAAGCGGTGG
			TCCCGGCGCGCGCAAACCCCACGCACGAGA
			AGGTGCTGGCGATCGTAAACGCGCTGGCCG
			AAAACAGGGCCATCCGGCCCGACGAGGCC
			GGCCTGGTCTACGACGCGCTGCTTCAGCGC
			GTGGCTCGTTACAACAGCGGCAACGTGCAG
			ACCAACCTGGACCGGCTGGTGGGGGATGTG
			CGCGAGGCCGTGGCGCAGCGTGAGCGCGC
			GCAGCAGCAGGGCAACCTGGGCTCCATGGT
			TGCACTAAACGCCTTCCTGAGTACACAGCC
			CGCCAACGTGCCGCGGGGACAGGAGGACT
			ACACCAACTTTGTGAGCGCACTGCGGCTAA
			TGGTGACTGAGACACCGCAAAGTGAGGTGT
			ACCAGTCTGGGCCAGACTATTTTTTCCAGA
			CCAGTAGACAAGGCCTGCAGACCGTAAACC
			TGAGCCAGGCTTTCAAAAACTTGCAGGGGC
			TGTGGGGGGTGCGGGCTCCCACAGGCGACC
			GCGCGACCGTGTCTAGCTTGCTGACGCCCA
			ACTCGCGCCTGTTGCTGCTGCTAATAGCGC
			CCTTCACGGACAGTGGCAGCGTGTCCCGGG
			ACACATACCTAGGTCACTTGCTGACACTGT
			ACCGCGAGGCCATAGGTCAGGCGCATGTGG
			ACGAGCATACTTTCCAGGAGATTACAAGTG
			TCAGCCGCGCGCTGGGGCAGGAGGACACG
			GGCAGCCTGGAGGCAACCCTAAACTACCTG
			CTGACCAACCGGCGGCAGAAGATCCCCTCG
			TTGCACAGTTTAAACAGCGAGGAGGAGCGC
			ATTTTGCGCTACGTGCAGCAGAGCGTGAGC
			CTTAACCTGATGCGCGACGGGGTAACGCCC
			AGCGTGGCGCTGGACATGACCGCGCGCAAC
			ATGGAACCGGGCATGTATGCCTCAAACCGG
			CCGTTTATCAACCGCCTAATGGACTACTTGC
			ATCGCGCGGCCGCCGTGAACCCCGAGTATT
			TCACCAATGCCATCTTGAACCCGCACTGGC
			TACCGCCCCCTGGTTTCTACACCGGGGGAT
			TCGAGGTGCCCGAGGGTAACGATGGATTCC
			TCTGGGACGACATAGACGACAGCGTGTTTT
			CCCCGCAACCGCAGACCCTGCTAGAGTTGC
			AACAGCGCGAGCAGGCAGAGGCGGCGCTG
			CGAAAGGAAAGCTTCCGCAGGCCAAGCAG
			CTTGTCCGATCTAGGCGCTGCGGCCCCGCG
			GTCAGATGCTAGTAGCCCATTTCCAAGCTT
			GATAGGGTCTCTTACCAGCACTCGCACCAC
			CCGCCCGCGCCTGCTGGGCGAGGAGGAGTA
			CCTAAACAACTCGCTGCTGCAGCCGCAGCG
			CGAAAAAAACCTGCCTCCGGCATTTCCCAA
			CAACGGGATAGAGAGCCTAGTGGACAAGA
			TGAGTAGATGGAAGACGTACGCGCAGGAG
			CACAGGGACGTGCCAGGCCCGCGCCCGCCC
			ACCCGTCGTCAAAGGCACGACCGTCAGCGG
			GGTCTGGTGTGGGAGGACGATGACTCGGCA
			GACGACAGCAGCGTCCTGGATTTGGGAGGG
			AGTGGCAACCCGTTTGCGCACCTTCGCCCC
			AGGCTGGGGAGAATGTTTTAAAAAAAAAA
			AAGCATGATGCAAAATAAAAAACTCACCA
			AGGCCATGGCACCGAGCGTTGGTTTTCTTG
			TATTCCCCTTAGTATGCGGCGCGCGGCGAT
			GTATGAGGAAGGTCCTCCTCCCTCCTACGA
			GAGTGTGGTGAGCGCGGCGCCAGTGGCGGC
			GGCGCTGGGTTCTCCCTTCGATGCTCCCCTG
			GACCCGCCGTTTGTGCCTCCGCGGTACCTG
			CGGCCTACCGGGGGGAGAAACAGCATCCGT
			TACTCTGAGTTGGCACCCCTATTCGACACC
			ACCCGTGTGTACCTGGTGGACAACAAGTCA
			ACGGATGTGGCATCCCTGAACTACCAGAAC
			GACCACAGCAACTTTCTGACCACGGTCATT
			CAAAACAATGACTACAGCCCGGGGGAGGC
			AAGCACACAGACCATCAATCTTGACGACCG
			GTCGCACTGGGGCGGCGACCTGAAAACCAT
			CCTGCATACCAACATGCCAAATGTGAACGA
			GTTCATGTTTACCAATAAGTTTAAGGCGCG
			GGTGATGGTGTCGCGCTTGCCTACTAAGGA
			CAATCAGGTGGAGCTGAAATACGAGTGGGT
			GGAGTTCACGCTGCCCGAGGGCAACTACTC
			CGAGACCATGACCATAGACCTTATGAACAA
			CGCGATCGTGGAGCACTACTTGAAAGTGGG
			CAGACAGAACGGGGTTCTGGAAAGCGACA
			TCGGGGTAAAGTTTGACACCCGCAACTTCA
			GACTGGGGTTTGACCCCGTCACTGGTCTTGT
			CATGCCTGGGGTATATACAAACGAAGCCTT
			CCATCCAGACATCATTTTGCTGCCAGGATG
			CGGGGTGGACTTCACCCACAGCCGCCTGAG
			CAACTTGTTGGGCATCCGCAAGCGGCAACC
			CTTCCAGGAGGGCTTTAGGATCACCTACGA
			TGATCTGGAGGGTGGTAACATTCCCGCACT
			GTTGGATGTGGACGCCTACCAGGCGAGCTT
			GAAAGATGACACCGAACAGGGCGGGGGTG
			GCGCAGGCGGCAGCAACAGCAGTGGCAGC
			GGCGCGGAAGAGAACTCCAACGCGGCAGC
			CGCGGCAATGCAGCCGGTGGAGGACATGA
			ACGATCATGCCATTCGCGGCGACACCTTTG
			CCACACGGGCTGAGGAGAAGCGCGCTGAG
			GCCGAAGCAGCGGCCGAAGCTGCCGCCCCC
			GCTGCGCAACCCGAGGTCGAGAAGCCTCAG
			AAGAAACCGGTGATCAAACCCCTGACAGA
			GGACAGCAAGAAACGCAGTTACAACCTAAT
			AAGCAATGACAGCACCTTCACCCAGTACCG
			CAGCTGGTACCTTGCATACAACTACGGCGA
			CCCTCAGACCGGAATCCGCTCATGGACCCT
			GCTTTGCACTCCTGACGTAACCTGCGGCTC
			GGAGCAGGTCTACTGGTCGTTGCCAGACAT
			GATGCAAGACCCCGTGACCTTCCGCTCCAC
			GCGCCAGATCAGCAACTTTCCGGTGGTGGG
			CGCCGAGCTGTTGCCCGTGCACTCCAAGAG
			CTTCTACAACGACCAGGCCGTCTACTCCCA
			ACTCATCCGCCAGTTTACCTCTCTGACCCAC
			GTGTTCAATCGCTTTCCCGAGAACCAGATTT
			TGGCGCGCCCGCCAGCCCCCACCATCACCA
			CCGTCAGTGAAAACGTTCCTGCTCTCACAG
			ATCACGGGACGCTACCGCTGCGCAACAGCA
			TCGGAGGAGTCCAGCGAGTGACCATTACTG
			ACGCCAGACGCCGCACCTGCCCCTACGTTT
			ACAAGGCCCTGGGCATAGTCTCGCCGCGCG
			TCCTATCGAGCCGCACTTTTTGAGCAAGCA
			TGTCCATCCTTATATCGCCCAGCAATAACA
			CAGGCTGGGGCCTGCGCTTCCCAAGCAAGA
			TGTTTGGCGGGGCCAAGAAGCGCTCCGACC
			AACACCCAGTGCGCGTGCGCGGGCACTACC
			GCGCGCCCTGGGGCGCGCACAAACGCGGCC
			GCACTGGGCGCACCACCGTCGATGACGCCA
			TCGACGCGGTGGTGGAGGAGGCGCGCAACT
			ACACGCCCACGCCGCCACCAGTGTCCACAG
			TGGACGCGGCCATTCAGACCGTGGTGCGCG
			GAGCCCGGCGCTATGCTAAAATGAAGAGAC
			GGCGGAGGCGCGTAGCACGTCGCCACCGCC
			GCCGACCCGGCACTGCCGCCCAACGCGCGG
			CGGCGGCCCTGCTTAACCGCGCACGTCGCA
			CCGGCCGACGGGCGGCCATGCGGGCCGCTC
			GAAGGCTGGCCGCGGGTATTGTCACTGTGC
			CCCCCAGGTCCAGGCGACGAGCGGCCGCCG
			CAGCAGCCGCGGCCATTAGTGCTATGACTC
			AGGGTCGCAGGGGCAACGTGTATTGGGTGC
			GCGACTCGGTTAGCGGCCTGCGCGTGCCCG
			TGCGCACCCGCCCCCCGCGCAACTAGATTG
			CAAGAAAAAACTACTTAGACTCGTACTGTT
			GTATGTATCCAGCGGCGGCGGCGCGCAACG
			AAGCTATGTCCAAGCGCAAAATCAAAGAA
			GAGATGCTCCAGGTCATCGCGCCGGAGATC
			TATGGCCCCCCGAAGAAGGAAGAGCAGGA
			TTACAAGCCCCGAAAGCTAAAGCGGGTCAA
			AAAGAAAAAGAAAGATGATGATGATGAAC
			TTGACGACGAGGTGGAACTGCTGCACGCTA
			CCGCGCCCAGGCGACGGGTACAGTGGAAA
			GGTCGACGCGTAAAACGTGTTTTGCGACCC
			GGCACCACCGTAGTCTTTACGCCCGGTGAG
			CGCTCCACCCGCACCTACAAGCGCGTGTAT
			GATGAGGTGTACGGCGACGAGGACCTGCTT
			GAGCAGGCCAACGAGCGCCTCGGGGAGTTT
			GCCTACGGAAAGCGGCATAAGGACATGCTG
			GCGTTGCCGCTGGACGAGGGCAACCCAACA
			CCTAGCCTAAAGCCCGTAACACTGCAGCAG
			GTGCTGCCCGCGCTTGCACCGTCCGAAGAA
			AAGCGCGGCCTAAAGCGCGAGTCTGGTGAC
			TTGGCACCCACCGTGCAGCTGATGGTACCC
			AAGCGCCAGCGACTGGAAGATGTCTTGGAA
			AAAATGACCGTGGAACCTGGGCTGGAGCCC
			GAGGTCCGCGTGCGGCCAATCAAGCAGGTG
			GCGCCGGGACTGGGCGTGCAGACCGTGGAC
			GTTCAGATACCCACTACCAGTAGCACCAGT
			ATTGCCACCGCCACAGAGGGCATGGAGACA
			CAAACGTCCCCGGTTGCCTCAGCGGTGGCG
			GATGCCGCGGTGCAGGCGGTCGCTGCGGCC
			GCGTCCAAGACCTCTACGGAGGTGCAAACG
			GACCCGTGGATGTTTCGCGTTTCAGCCCCCC
			GGCGCCCGCGCCGTTCGAGGAAGTACGGCG
			CCGCCAGCGCGCTACTGCCCGAATATGCCC
			TACATCCTTCCATTGCGCCTACCCCCGGCTA
			TCGTGGCTACACCTACCGCCCCAGAAGACG
			AGCAACTACCCGACGCCGAACCACCACTGG
			AACCCGCCGCCGCCGTCGCCGTCGCCAGCC
			CGTGCTGGCCCCGATTTCCGTGCGCAGGGT
			GGCTCGCGAAGGAGGCAGGACCCTGGTGCT
			GCCAACAGCGCGCTACCACCCCAGCATCGT
			TTAAAAGCCGGTCTTTGTGGTTCTTGCAGAT
			ATGGCCCTCACCTGCCGCCTCCGTTTCCCGG
			TGCCGGGATTCCGAGGAAGAATGCACCGTA
			GGAGGGGCATGGCCGGCCACGGCCTGACG
			GGCGGCATGCGTCGTGCGCACCACCGGCGG
			CGGCGCGCGTCGCACCGTCGCATGCGCGGC
			GGTATCCTGCCCCTCCTTATTCCACTGATCG
			CCGCGGCGATTGGCGCCGTGCCCGGAATTG
			CATCCGTGGCCTTGCAGGCGCAGAGACACT
			GATTAAAAACAAGTTGCATGTGGAAAAATC
			AAAATAAAAAGTCTGGACTCTCACGCTCGC
			TTGGTCCTGTAACTATTTTGTAGAATGGAA
			GACATCAACTTTGCGTCTCTGGCCCCGCGA
			CACGGCTCGCGCCCGTTCATGGGAAACTGG
			CAAGATATCGGCACCAGCAATATGAGCGGT
			GGCGCCTTCAGCTGGGGCTCGCTGTGGAGC
			GGCATTAAAAATTTCGGTTCCACCGTTAAG
			AACTATGGCAGCAAGGCCTGGAACAGCAG
			CACAGGCCAGATGCTGAGGGATAAGTTGAA
			AGAGCAAAATTTCCAACAAAAGGTGGTAG
			ATGGCCTGGCCTCTGGCATTAGCGGGGTGG
			TGGACCTGGCCAACCAGGCAGTGCAAAATA
			AGATTAACAGTAAGCTTGATCCCCGCCCTC
			CCGTAGAGGAGCCTCCACCGGCCGTGGAGA
			CAGTGTCTCCAGAGGGGCGTGGCGAAAAGC
			GTCCGCGCCCCGACAGGGAAGAAACTCTGG
			TGACGCAAATAGACGAGCCTCCCTCGTACG
			AGGAGGCACTAAAGCAAGGCCTGCCCACC
			ACCCGTCCCATCGCGCCCATGGCTACCGGA
			GTGCTGGGCCAGCACACACCCGTAACGCTG
			GACCTGCCTCCCCCCGCCGACACCCAGCAG
			AAACCTGTGCTGCCAGGCCCGACCGCCGTT
			GTTGTAACCCGTCCTAGCCGCGCGTCCCTG
			CGCCGCGCCGCCAGCGGTCCGCGATCGTTG
			CGGCCCGTAGCCAGTGGCAACTGGCAAAGC
			ACACTGAACAGCATCGTGGGTCTGGGGGTG
			CAATCCCTGAAGCGCCGACGATGCTTCTGA
			TAGCTAACGTGTCGTATGTGTGTCATGTATG
			CGTCCATGTCGCCGCCAGAGGAGCTGCTGA
			GCCGCCGCGCGCCCGCTTTCCAAGATGGCT
			ACCCCTTCGATGATGCCGCAGTGGTCTTAC
			ATGCACATCTCGGGCCAGGACGCCTCGGAG
			TACCTGAGCCCCGGGCTGGTGCAGTTTGCC
			CGCGCCACCGAGACGTACTTCAGCCTGAAT
			AACAAGTTTAGAAACCCCACGGTGGCGCCT
			ACGCACGACGTGACCACAGACCGGTCCCAG
			CGTTTGACGCTGCGGTTCATCCCTGTGGACC
			GTGAGGATACTGCGTACTCGTACAAGGCGC
			GGTTCACCCTAGCTGTGGGTGATAACCGTG
			TGCTGGACATGGCTTCCACGTACTTTGACAT
			CCGCGGCGTGCTGGACAGGGGCCCTACTTT
			TAAGCCCTACTCTGGCACTGCCTACAACGC
			CCTGGCTCCCAAGGGTGCCCCAAATCCTTG
			CGAATGGGATGAAGCTGCTACTGCTCTTGA
			AATAAACCTAGAAGAAGAGGACGATGACA
			ACGAAGACGAAGTAGACGAGCAAGCTGAG
			CAGCAAAAAACTCACGTATTTGGGCAGGCG
			CCTTATTCTGGTATAAATATTACAAAGGAG
			GGTATTCAAATAGGTGTCGAAGGTCAAACA
			CCTAAATATGCCGATAAAACATTTCAACCT
			GAACCTCAAATAGGAGAATCTCAGTGGTAC
			GAAACAGAAATTAATCATGCAGCTGGGAG
			AGTCCTAAAAAAGACTACCCCAATGAAACC
			ATGTTACGGTTCATATGCAAAACCCACAAA
			TGAAAATGGAGGGCAAGGCATTCTTGTAAA
			GCAACAAAATGGAAAGCTAGAAAGTCAAG
			TGGAAATGCAATTTTTCTCAACTACTGAGG
			CAGCCGCAGGCAATGGTGATAACTTGACTC
			CTAAAGTGGTATTGTACAGTGAAGATGTAG
			ATATAGAAACCCCAGACACTCATATTTCTT
			ACATGCCCACTATTAAGGAAGGTAACTCAC
			GAGAACTAATGGGCCAACAATCTATGCCCA
			ACAGGCCTAATTACATTGCTTTTAGGGACA
			ATTTTATTGGTCTAATGTATTACAACAGCAC
			GGGTAATATGGGTGTTCTGGCGGGCCAAGC
			ATCGCAGTTGAATGCTGTTGTAGATTTGCA
			AGACAGAAACACAGAGCTTTCATACCAGCT
			TTTGCTTGATTCCATTGGTGATAGAACCAG
			GTACTTTTCTATGTGGAATCAGGCTGTTGAC
			AGCTATGATCCAGATGTTAGAATTATTGAA
			AATCATGGAACTGAAGATGAACTTCCAAAT
			TACTGCTTTCCACTGGGAGGTGTGATTAAT
			ACAGAGACTCTTACCAAGGTAAAACCTAAA
			ACAGGTCAGGAAAATGGATGGGAAAAAGA
			TGCTACAGAATTTTCAGATAAAAATGAAAT
			AAGAGTTGGAAATAATTTTGCCATGGAAAT
			CAATCTAAATGCCAACCTGTGGAGAAATTT
			CCTGTACTCCAACATAGCGCTGTATTTGCCC
			GACAAGCTAAAGTACAGTCCTTCCAACGTA
			AAAATTTCTGATAACCCAAACACCTACGAC
			TACATGAACAAGCGAGTGGTGGCTCCCGGG
			CTAGTGGACTGCTACATTAACCTTGGAGCA
			CGCTGGTCCCTTGACTATATGGACAACGTC
			AACCCATTTAACCACCACCGCAATGCTGGC
			CTGCGCTACCGCTCAATGTTGCTGGGCAAT
			GGTCGCTATGTGCCCTTCCACATCCAGGTG
			CCTCAGAAGTTCTTTGCCATTAAAAACCTCC
			TTCTCCTGCCGGGCTCATACACCTACGAGT
			GGAACTTCAGGAAGGATGTTAACATGGTTC
			TGCAGAGCTCCCTAGGAAATGACCTAAGGG
			TTGACGGAGCCAGCATTAAGTTTGATAGCA
			TTTGCCTTTACGCCACCTTCTTCCCCATGGC
			CCACAACACCGCCTCCACGCTTGAGGCCAT
			GCTTAGAAACGACACCAACGACCAGTCCTT
			TAACGACTATCTCTCCGCCGCCAACATGCT
			CTACCCTATACCCGCCAACGCTACCAACGT
			GCCCATATCCATCCCCTCCCGCAACTGGGC
			GGCTTTCCGCGGCTGGGCCTTCACGCGCCTT
			AAGACTAAGGAAACCCCATCACTGGGCTCG
			GGCTACGACCCTTATTACACCTACTCTGGCT
			CTATACCCTACCTAGATGGAACCTTTTACCT
			CAACCACACCTTTAAGAAGGTGGCCATTAC
			CTTTGACTCTTCTGTCAGCTGGCCTGGCAAT
			GACCGCCTGCTTACCCCCAACGAGTTTGAA
			ATTAAGCGCTCAGTTGACGGGGAGGGTTAC
			AACGTTGCCCAGTGTAACATGACCAAAGAC
			TGGTTCCTGGTACAAATGCTAGCTAACTAT
			AACATTGGCTACCAGGGCTTCTATATCCCA
			GAGAGCTACAAGGACCGCATGTACTCCTTC
			TTTAGAAACTTCCAGCCCATGAGCCGTCAG
			GTGGTGGATGATACTAAATACAAGGACTAC
			CAACAGGTGGGCATCCTACACCAACACAAC
			AACTCTGGATTTGTTGGCTACCTTGCCCCCA
			CCATGCGCGAAGGACAGGCCTACCCTGCTA
			ACTTCCCCTATCCGCTTATAGGCAAGACCG
			CAGTTGACAGCATTACCCAGAAAAAGTTTC
			TTTGCGATCGCACCCTTTGGCGCATCCCATT
			CTCCAGTAACTTTATGTCCATGGGCGCACTC
			ACAGACCTGGGCCAAAACCTTCTCTACGCC
			AACTCCGCCCACGCGCTAGACATGACTTTT
			GAGGTGGATCCCATGGACGAGCCCACCCTT
			CTTTATGTTTTGTTTGAAGTCTTTGACGTGG
			TCCGTGTGCACCAGCCGCACCGCGGCGTCA
			TCGAAACCGTGTACCTGCGCACGCCCTTCT
			CGGCCGGCAACGCCACAACATAAAGAAGC
			AAGCAACATCAACAACAGCTGCCGCCATGG
			GCTCCAGTGAGCAGGAACTGAAAGCCATTG
			TCAAAGATCTTGGTTGTGGGCCATATTTTTT
			GGGCACCTATGACAAGCGCTTTCCAGGCTT
			TGTTTCTCCACACAAGCTCGCCTGCGCCATA
			GTCAATACGGCCGGTCGCGAGACTGGGGGC
			GTACACTGGATGGCCTTTGCCTGGAACCCG
			CACTCAAAAACATGCTACCTCTTTGAGCCC
			TTTGGCTTTTCTGACCAGCGACTCAAGCAG
			GTTTACCAGTTTGAGTACGAGTCACTCCTGC
			GCCGTAGCGCCATTGCTTCTTCCCCCGACCG
			CTGTATAACGCTGGAAAAGTCCACCCAAAG
			CGTACAGGGGCCCAACTCGGCCGCCTGTGG
			ACTATTCTGCTGCATGTTTCTCCACGCCTTT
			GCCAACTGGCCCCAAACTCCCATGGATCAC
			AACCCCACCATGAACCTTATTACCGGGGTA
			CCCAACTCCATGCTCAACAGTCCCCAGGTA
			CAGCCCACCCTGCGTCGCAACCAGGAACAG
			CTCTACAGCTTCCTGGAGCGCCACTCGCCCT
			ACTTCCGCAGCCACAGTGCGCAGATTAGGA
			GCGCCACTTCTTTTTGTCACTTGAAAAACAT
			GTAAAAATAATGTACTAGAGACACTTTCAA
			TAAAGGCAAATGCTTTTATTTGTACACTCTC
			GGGTGATTATTTACCCCCACCCTTGCCGTCT
			GCGCCGTTTAAAAATCAAAGGGGTTCTGCC
			GCGCATCGCTATGCGCCACTGGCAGGGACA
			CGTTGCGATACTGGTGTTTAGTGCTCCACTT
			AAACTCAGGCACAACCATCCGCGGCAGCTC
			GGTGAAGTTTTCACTCCACAGGCTGCGCAC
			CATCACCAACGCGTTTAGCAGGTCGGGCGC
			CGATATCTTGAAGTCGCAGTTGGGGCCTCC
			GCCCTGCGCGCGCGAGTTGCGATACACAGG
			GTTGCAGCACTGGAACACTATCAGCGCCGG
			GTGGTGCACGCTGGCCAGCACGCTCTTGTC
			GGAGATCAGATCCGCGTCCAGGTCCTCCGC
			GTTGCTCAGGGCGAACGGAGTCAACTTTGG
			TAGCTGCCTTCCCAAAAAGGGCGCGTGCCC
			AGGCTTTGAGTTGCACTCGCACCGTAGTGG
			CATCAAAAGGTGACCGTGCCCGGTCTGGGC
			GTTAGGATACAGCGCCTGCATAAAAGCCTT
			GATCTGCTTAAAAGCCACCTGAGCCTTTGC
			GCCTTCAGAGAAGAACATGCCGCAAGACTT
			GCCGGAAAACTGATTGGCCGGACAGGCCGC
			GTCGTGCACGCAGCACCTTGCGTCGGTGTT
			GGAGATCTGCACCACATTTCGGCCCCACCG
			GTTCTTCACGATCTTGGCCTTGCTAGACTGC
			TCCTTCAGCGCGCGCTGCCCGTTTTCGCTCG
			TCACATCCATTTCAATCACGTGCTCCTTATT
			TATCATAATGCTTCCGTGTAGACACTTAAG
			CTCGCCTTCGATCTCAGCGCAGCGGTGCAG
			CCACAACGCGCAGCCCGTGGGCTCGTGATG
			CTTGTAGGTCACCTCTGCAAACGACTGCAG
			GTACGCCTGCAGGAATCGCCCCATCATCGT
			CACAAAGGTCTTGTTGCTGGTGAAGGTCAG
			CTGCAACCCGCGGTGCTCCTCGTTCAGCCA
			GGTCTTGCATACGGCCGCCAGAGCTTCCAC
			TTGGTCAGGCAGTAGTTTGAAGTTCGCCTTT
			AGATCGTTATCCACGTGGTACTTGTCCATCA
			GCGCGCGCGCAGCCTCCATGCCCTTCTCCC
			ACGCAGACACGATCGGCACACTCAGCGGGT
			TCATCACCGTAATTTCACTTTCCGCTTCGCT
			GGGCTCTTCCTCTTCCTCTTGCGTCCGCATA
			CCACGCGCCACTGGGTCGTCTTCATTCAGC
			CGCCGCACTGTGCGCTTACCTCCTTTGCCAT
			GCTTGATTAGCACCGGTGGGTTGCTGAAAC
			CCACCATTTGTAGCGCCACATCTTCTCTTTC
			TTCCTCGCTGTCCACGATTACCTCTGGTGAT
			GGCGGGCGCTCGGGCTTGGGAGAAGGGCG
			CTTCTTTTTCTTCTTGGGCGCAATGGCCAAA
			TCCGCCGCCGAGGTCGATGGCCGCGGGCTG
			GGTGTGCGCGGCACCAGCGCGTCTTGTGAT
			GAGTCTTCCTCGTCCTCGGACTCGATACGCC
			GCCTCATCCGCTTTTTTGGGGGCGCCCGGG
			GAGGCGGCGGCGACGGGGACGGGGACGAC
			ACGTCCTCCATGGTTGGGGGACGTCGCGCC
			GCACCGCGTCCGCGCTCGGGGGTGGTTTCG
			CGCTGCTCCTCTTCCCGACTGGCCATTTCCT
			TCTCCTATAGGCAGAAAAAGATCATGGAGT
			CAGTCGAGAAGAAGGACAGCCTAACCGCC
			CCCTCTGAGTTCGCCACCACCGCCTCCACC
			GATGCCGCCAACGCGCCTACCACCTTCCCC
			GTCGAGGCACCCCCGCTTGAGGAGGAGGA
			AGTGATTATCGAGCAGGACCCAGGTTTTGT
			AAGCGAAGACGACGAGGACCGCTCAGTAC
			CAACAGAGGATAAAAAGCAAGACCAGGAC
			AACGCAGAGGCAAACGAGGAACAAGTCGG
			GCGGGGGGACGAAAGGCATGGCGACTACC
			TAGATGTGGGAGACGACGTGCTGTTGAAGC
			ATCTGCAGCGCCAGTGCGCCATTATCTGCG
			ACGCGTTGCAAGAGCGCAGCGATGTGCCCC
			TCGCCATAGCGGATGTCAGCCTTGCCTACG
			AACGCCACCTATTCTCACCGCGCGTACCCC
			CCAAACGCCAAGAAAACGGCACATGCGAG
			CCCAACCCGCGCCTCAACTTCTACCCCGTAT
			TTGCCGTGCCAGAGGTGCTTGCCACCTATC
			ACATCTTTTTCCAAAACTGCAAGATACCCCT
			ATCCTGCCGTGCCAACCGCAGCCGAGCGGA
			CAAGCAGCTGGCCTTGCGGCAGGGCGCTGT
			CATACCTGATATCGCCTCGCTCAACGAAGT
			GCCAAAAATCTTTGAGGGTCTTGGACGCGA
			CGAGAAGCGCGCGGCAAACGCTCTGCAAC
			AGGAAAACAGCGAAAATGAAAGTCACTCT
			GGAGTGTTGGTGGAACTCGAGGGTGACAAC
			GCGCGCCTAGCCGTACTAAAACGCAGCATC
			GAGGTCACCCACTTTGCCTACCCGGCACTT
			AACCTACCCCCCAAGGTCATGAGCACAGTC
			ATGAGTGAGCTGATCGTGCGCCGTGCGCAG
			CCCCTGGAGAGGGATGCAAATTTGCAAGAA
			CAAACAGAGGAGGGCCTACCCGCAGTTGGC
			GACGAGCAGCTAGCGCGCTGGCTTCAAACG
			CGCGAGCCTGCCGACTTGGAGGAGCGACGC
			AAACTAATGATGGCCGCAGTGCTCGTTACC
			GTGGAGCTTGAGTGCATGCAGCGGTTCTTT
			GCTGACCCGGAGATGCAGCGCAAGCTAGA
			GGAAACATTGCACTACACCTTTCGACAGGG
			CTACGTACGCCAGGCCTGCAAGATCTCCAA
			CGTGGAGCTCTGCAACCTGGTCTCCTACCTT
			GGAATTTTGCACGAAAACCGCCTTGGGCAA
			AACGTGCTTCATTCCACGCTCAAGGGCGAG
			GCGCGCCGCGACTACGTCCGCGACTGCGTT
			TACTTATTTCTATGCTACACCTGGCAGACGG
			CCATGGGCGTTTGGCAGCAGTGCTTGGAGG
			AGTGCAACCTCAAGGAGCTGCAGAAACTGC
			TAAAGCAAAACTTGAAGGACCTATGGACGG
			CCTTCAACGAGCGCTCCGTGGCCGCGCACC
			TGGCGGACATCATTTTCCCCGAACGCCTGC
			TTAAAACCCTGCAACAGGGTCTGCCAGACT
			TCACCAGTCAAAGCATGTTGCAGAACTTTA
			GGAACTTTATCCTAGAGCGCTCAGGAATCT
			TGCCCGCCACCTGCTGTGCACTTCCTAGCG
			ACTTTGTGCCCATTAAGTACCGCGAATGCC
			CTCCGCCGCTTTGGGGCCACTGCTACCTTCT
			GCAGCTAGCCAACTACCTTGCCTACCACTC
			TGACATAATGGAAGACGTGAGCGGTGACG
			GTCTACTGGAGTGTCACTGTCGCTGCAACC
			TATGCACCCCGCACCGCTCCCTGGTTTGCA
			ATTCGCAGCTGCTTAACGAAAGTCAAATTA
			TCGGTACCTTTGAGCTGCAGGGTCCCTCGC
			CTGACGAAAAGTCCGCGGCTCCGGGGTTGA
			AACTCACTCCGGGGCTGTGGACGTCGGCTT
			ACCTTCGCAAATTTGTACCTGAGGACTACC
			ACGCCCACGAGATTAGGTTCTACGAAGACC
			AATCCCGCCCGCCTAATGCGGAGCTTACCG
			CCTGCGTCATTACCCAGGGCCACATTCTTG
			GCCAATTGCAAGCCATCAACAAAGCCCGCC
			AAGAGTTTCTGCTACGAAAGGGACGGGGG
			GTTTACTTGGACCCCCAGTCCGGCGAGGAG
			CTCAACCCAATCCCCCCGCCGCCGCAGCCC
			TATCAGCAGCAGCCGCGGGCCCTTGCTTCC
			CAGGATGGCACCCAAAAAGAAGCTGCAGC
			TGCCGCCGCCACCCACGGACGAGGAGGAAT
			ACTGGGACAGTCAGGCAGAGGAGGTTTTGG
			ACGAGGAGGAGGAGGACATGATGGAAGAC
			TGGGAGAGCCTAGACGAGGAAGCTTCCGA
			GGTCGAAGAGGTGTCAGACGAAACACCGTC
			ACCCTCGGTCGCATTCCCCTCGCCGGCGCC
			CCAGAAATCGGCAACCGGTTCCAGCATGGC
			TACAACCTCCGCTCCTCAGGCGCCGCCGGC
			ACTGCCCGTTCGCCGACCCAACCGTAGATG
			GGACACCACTGGAACCAGGGCCGGTAAGTC
			CAAGCAGCCGCCGCCGTTAGCCCAAGAGCA
			ACAACAGCGCCAAGGCTACCGCTCATGGCG
			CGGGCACAAGAACGCCATAGTTGCTTGCTT
			GCAAGACTGTGGGGGCAACATCTCCTTCGC
			CCGCCGCTTTCTTCTCTACCATCACGGCGTG
			GCCTTCCCCCGTAACATCCTGCATTACTACC
			GTCATCTCTACAGCCCATACTGCACCGGCG
			GCAGCGGCAGCAACAGCAGCGGCCACACA
			GAAGCAAAGGCGACCGGATAGCAAGACTC
			TGACAAAGCCCAAGAAATCCACAGCGGCG
			GCAGCAGCAGGAGGAGGAGCGCTGCGTCT
			GGCGCCCAACGAACCCGTATCGACCCGCGA
			GCTTAGAAACAGGATTTTTCCCACTCTGTAT
			GCTATATTTCAACAGAGCAGGGGCCAAGAA
			CAAGAGCTGAAAATAAAAAACAGGTCTCTG
			CGATCCCTCACCCGCAGCTGCCTGTATCAC
			AAAAGCGAAGATCAGCTTCGGCGCACGCTG
			GAAGACGCGGAGGCTCTCTTCAGTAAATAC
			TGCGCGCTGACTCTTAAGGACTAGTTTCGC
			GCCCTTTCTCAAATTTAAGCGCGAAAACTA
			CGTCATCTCCAGCGGCCACACCCGGCGCCA
			GCACCTGTTGTCAGCGCCATTATGAGCAAG
			GAAATTCCCACGCCCTACATGTGGAGTTAC
			CAGCCACAAATGGGACTTGCGGCTGGAGCT
			GCCCAAGACTACTCAACCCGAATAAACTAC
			ATGAGCGCGGGACCCCACATGATATCCCGG
			GTCAACGGAATACGCGCCCACCGAAACCGA
			ATTCTCCTGGAACAGGCGGCTATTACCACC
			ACACCTCGTAATAACCTTAATCCCCGTAGTT
			GGCCCGCTGCCCTGGTGTACCAGGAAAGTC
			CCGCTCCCACCACTGTGGTACTTCCCAGAG
			ACGCCCAGGCCGAAGTTCAGATGACTAACT
			CAGGGGCGCAGCTTGCGGGCGGCTTTCGTC
			ACAGGGTGCGGTCGCCCGGGCAGGGTATAA
			CTCACCTGACAATCAGAGGGCGAGGTATTC
			AGCTCAACGACGAGTCGGTGAGCTCCTCGC
			TTGGTCTCCGTCCGGACGGGACATTTCAGA
			TCGGCGGCGCCGGCCGCTCTTCATTCACGC
			CTCGTCAGGCAATCCTAACTCTGCAGACCT
			CGTCCTCTGAGCCGCGCTCTGGAGGCATTG
			GAACTCTGCAATTTATTGAGGAGTTTGTGC
			CATCGGTCTACTTTAACCCCTTCTCGGGACC
			TCCCGGCCACTATCCGGATCAATTTATTCCT
			AACTTTGACGCGGTAAAGGACTCGGCGGAC
			GGCTACGACTGAATGTTAAGTGGAGAGGCA
			GAGCAACTGCGCCTGAAACACCTGGTCCAC
			TGTCGCCGCCACAAGTGCTTTGCCCGCGAC
			TCCGGTGAGTTTTGCTACTTTGAATTGCCCG
			AGGATCATATCGAGGGCCCGGCGCACGGCG
			TCCGGCTTACCGCCCAGGGAGAGCTTGCCC
			GTAGCCTGATTCGGGAGTTTACCCAGCGCC
			CCCTGCTAGTTGAGCGGGACAGGGGACCCT
			GTGTTCTCACTGTGATTTGCAACTGTCCTAA
			CCCTGGATTACATCAAGATCCTCTAGTTAAT
			GTCAGGTCGCCTAAGTCGATTAACTAGAGT
			ACCCGGGGATCTTATTCCCTTTAACTAATAA
			AAAAAAATAATAAAGCATCACTTACTTAAA
			ATCAGTTAGCAAATTTCTGTCCAGTTTATTC
			AGCAGCACCTCCTTGCCCTCCTCCCAGCTCT
			GGTATTGCAGCTTCCTCCTGGCTGCAAACTT
			TCTCCACAATCTAAATGGAATGTCAGTTTCC
			TCCTGTTCCTGTCCATCCGCACCCACTATCT
			TCATGTTGTTGCAGATGAAGCGCGCAAGAC
			CGTCTGAAGATACCTTCAACCCCGTGTATC
			CATATGACACGGAAACCGGTCCTCCAACTG
			TGCCTTTTCTTACTCCTCCCTTTGTATCCCCC
			AATGGGTTTCAAGAGAGTCCCCCTGGGGTA
			CTCTCTTTGCGCCTATCCGAACCTCTAGTTA
			CCTCCAATGGCATGCTTGCGCTCAAAATGG
			GCAACGGCCTCTCTCTGGACGAGGCCGGCA
			ACCTTACCTCCCAAAATGTAACCACTGTGA
			GCCCACCTCTCAAAAAAACCAAGTCAAACA
			TAAACCTGGAAATATCTGCACCCCTCACAG
			TTACCTCAGAAGCCCTAACTGTGGCTGCCG
			CCGCACCTCTAATGGTCGCGGGCAACACAC
			TCACCATGCAATCACAGGCCCCGCTAACCG
			TGCACGACTCCAAACTTAGCATTGCCACCC
			AAGGACCCCTCACAGTGTCAGAAGGAAAG
			CTAGCCCTGCAAACATCAGGCCCCCTCACC
			ACCACCGATAGCAGTACCCTTACTATCACT
			GCCTCACCCCCTCTAACTACTGCCACTGGTA
			GCTTGGGCATTGACTTGAAAGAGCCCATTT
			ATACACAAAATGGAAAACTAGGACTAAAG
			TACGGGGCTCCTTTGCATGTAACAGACGAC
			CTAAACACTTTGACCGTAGCAACTGGTCCA
			GGTGTGACTATTAATAATACTTCCTTGCAA
			ACTAAAGTTACTGGAGCCTTGGGTTTTGATT
			CACAAGGCAATATGCAACTTAATGTAGCAG
			GAGGACTAAGGATTGATTCTCAAAACAGAC
			GCCTTATACTTGATGTTAGTTATCCGTTTGA
			TGCTCAAAACCAACTAAATCTAAGACTAGG
			ACAGGGCCCTCTTTTTATAAACTCAGCCCA
			CAACTTGGATATTAACTACAACAAAGGCCT
			TTACTTGTTTACAGCTTCAAACAATTCCAAA
			AAGCTTGAGGTTAACCTAAGCACTGCCAAG
			GGGTTGATGTTTGACGCTACAGCCATAGCC
			ATTAATGCAGGAGATGGGCTTGAATTTGGT
			TCACCTAATGCACCAAACACAAATCCCCTC
			AAAACAAAAATTGGCCATGGCCTAGAATTT
			GATTCAAACAAGGCTATGGTTCCTAAACTA
			GGAACTGGCCTTAGTTTTGACAGCACAGGT
			GCCATTACAGTAGGAAACAAAAATAATGAT
			AAGCTAACTTTGTGGACCACACCAGCTCCA
			TCTCCTAACTGTAGACTAAATGCAGAGAAA
			GATGCTAAACTCACTTTGGTCTTAACAAAA
			TGTGGCAGTCAAATACTTGCTACAGTTTCA
			GTTTTGGCTGTTAAAGGCAGTTTGGCTCCA
			ATATCTGGAACAGTTCAAAGTGCTCATCTT
			ATTATAAGATTTGACGAAAATGGAGTGCTA
			CTAAACAATTCCTTCCTGGACCCAGAATAT
			TGGAACTTTAGAAATGGAGATCTTACTGAA
			GGCACAGCCTATACAAACGCTGTTGGATTT
			ATGCCTAACCTATCAGCTTATCCAAAATCTC
			ACGGTAAAACTGCCAAAAGTAACATTGTCA
			GTCAAGTTTACTTAAACGGAGACAAAACTA
			AACCTGTAACACTAACCATTACACTAAACG
			GTACACAGGAAACAGGAGACACAACTCCA
			AGTGCATACTCTATGTCATTTTCATGGGACT
			GGTCTGGCCACAACTACATTAATGAAATAT
			TTGCCACATCCTCTTACACTTTTTCATACAT
			TGCCCAAGAATAAAGAATCGTTTGTGTTAT
			GTTTCAACGTGTTTATTTTTCAATTGCAGAA
			AATTTCAAGTCATTTTTCATTCAGTAGTATA
			GCCCCACCACCACATAGCTTATACAGATCA
			CCGTACCTTAATCAAACTCACAGAACCCTA
			GTATTCAACCTGCCACCTCCCTCCCAACAC
			ACAGAGTACACAGTCCTTTCTCCCCGGCTG
			GCCTTAAAAAGCATCATATCATGGGTAACA
			GACATATTCTTAGGTGTTATATTCCACACGG
			TTTCCTGTCGAGCCAAACGCTCATCAGTGA
			TATTAATAAACTCCCCGGGCAGCTCACTTA
			AGTTCATGTCGCTGTCCAGCTGCTGAGCCA
			CAGGCTGCTGTCCAACTTGCGGTTGCTTAA
			CGGGCGGCGAAGGAGAAGTCCACGCCTAC
			ATGGGGGTAGAGTCATAATCGTGCATCAGG
			ATAGGGCGGTGGTGCTGCAGCAGCGCGCGA
			ATAAACTGCTGCCGCCGCCGCTCCGTCCTG
			CAGGAATACAACATGGCAGTGGTCTCCTCA
			GCGATGATTCGCACCGCCCGCAGCATAAGG
			CGCCTTGTCCTCCGGGCACAGCAGCGCACC
			CTGATCTCACTTAAATCAGCACAGTAACTG
			CAGCACAGCACCACAATATTGTTCAAAATC
			CCACAGTGCAAGGCGCTGTATCCAAAGCTC
			ATGGCGGGGACCACAGAACCCACGTGGCC
			ATCATACCACAAGCGCAGGTAGATTAAGTG
			GCGACCCCTCATAAACACGCTGGACATAAA
			CATTACCTCTTTTGGCATGTTGTAATTCACC
			ACCTCCCGGTACCATATAAACCTCTGATTA
			AACATGGCGCCATCCACCACCATCCTAAAC
			CAGCTGGCCAAAACCTGCCCGCCGGCTATA
			CACTGCAGGGAACCGGGACTGGAACAATG
			ACAGTGGAGAGCCCAGGACTCGTAACCATG
			GATCATCATGCTCGTCATGATATCAATGTTG
			GCACAACACAGGCACACGTGCATACACTTC
			CTCAGGATTACAAGCTCCTCCCGCGTTAGA
			ACCATATCCCAGGGAACAACCCATTCCTGA
			ATCAGCGTAAATCCCACACTGCAGGGAAGA
			CCTCGCACGTAACTCACGTTGTGCATTGTCA
			AAGTGTTACATTCGGGCAGCAGCGGATGAT
			CCTCCAGTATGGTAGCGCGGGTTTCTGTCTC
			AAAAGGAGGTAGACGATCCCTACTGTACGG
			AGTGCGCCGAGACAACCGAGATCGTGTTGG
			TCGTAGTGTCATGCCAAATGGAACGCCGGA
			CGTAGTCATATTTCCTGAAGCAAAACCAGG
			TGCGGGCGTGACAAACAGATCTGCGTCTCC
			GGTCTCGCCGCTTAGATCGCTCTGTGTAGTA
			GTTGTAGTATATCCACTCTCTCAAAGCATCC
			AGGCGCCCCCTGGCTTCGGGTTCTATGTAA
			ACTCCTTCATGCGCCGCTGCCCTGATAACAT
			CCACCACCGCAGAATAAGCCACACCCAGCC
			AACCTACACATTCGTTCTGCGAGTCACACA
			CGGGAGGAGCGGGAAGAGCTGGAAGAACC
			ATGTTTTTTTTTTTATTCCAAAAGATTATCC
			AAAACCTCAAAATGAAGATCTATTAAGTGA
			ACGCGCTCCCCTCCGGTGGCGTGGTCAAAC
			TCTACAGCCAAAGAACAGATAATGGCATTT
			GTAAGATGTTGCACAATGGCTTCCAAAAGG
			CAAACGGCCCTCACGTCCAAGTGGACGTAA
			AGGCTAAACCCTTCAGGGTGAATCTCCTCT
			ATAAACATTCCAGCACCTTCAACCATGCCC
			AAATAATTCTCATCTCGCCACCTTCTCAATA
			TATCTCTAAGCAAATCCCGAATATTAAGTC
			CGGCCATTGTAAAAATCTGCTCCAGAGCGC
			CCTCCACCTTCAGCCTCAAGCAGCGAATCA
			TGATTGCAAAAATTCAGGTTCCTCACAGAC
			CTGTATAAGATTCAAAAGCGGAACATTAAC
			AAAAATACCGCGATCCCGTAGGTCCCTTCG
			CAGGGCCAGCTGAACATAATCGTGCAGGTC
			TGCACGGACCAGCGCGGCCACTTCCCCGCC
			AGGAACCATGACAAAAGAACCCACACTGA
			TTATGACACGCATACTCGGAGCTATGCTAA
			CCAGCGTAGCCCCGATGTAAGCTTGTTGCA
			TGGGCGGCGATATAAAATGCAAGGTGCTGC
			TCAAAAAATCAGGCAAAGCCTCGCGCAAA
			AAAGAAAGCACATCGTAGTCATGCTCATGC
			AGATAAAGGCAGGTAAGCTCCGGAACCAC
			CACAGAAAAAGACACCATTTTTCTCTCAAA
			CATGTCTGCGGGTTTCTGCATAAACACAAA
			ATAAAATAACAAAAAAACATTTAAACATTA
			GAAGCCTGTCTTACAACAGGAAAAACAACC
			CTTATAAGCATAAGACGGACTACGGCCATG
			CCGGCGTGACCGTAAAAAAACTGGTCACCG
			TGATTAAAAAGCACCACCGACAGCTCCTCG
			GTCATGTCCGGAGTCATAATGTAAGACTCG
			GTAAACACATCAGGTTGATTCACATCGGTC
			AGTGCTAAAAAGCGACCGAAATAGCCCGG
			GGGAATACATACCCGCAGGCGTAGAGACA
			ACATTACAGCCCCCATAGGAGGTATAACAA
			AATTAATAGGAGAGAAAAACACATAAACA
			CCTGAAAAACCCTCCTGCCTAGGCAAAATA
			GCACCCTCCCGCTCCAGAACAACATACAGC
			GCTTCCACAGCGGCAGCCATAACAGTCAGC
			CTTACCAGTAAAAAAGAAAACCTATTAAAA
			AAACACCACTCGACACGGCACCAGCTCAAT
			CAGTCACAGTGTAAAAAAGGGCCAAGTGC
			AGAGCGAGTATATATAGGACTAAAAAATG
			ACGTAACGGTTAAAGTCCACAAAAAACACC
			CAGAAAACCGCACGCGAACCTACGCCCAG
			AAACGAAAGCCAAAAAACCCACAACTTCCT
			CAAATCGTCACTTCCGTTTTCCCACGTTACG
			TCACTTCCCATTTTAAGAAAACTACAATTCC
			CAACACATACAAGTTACTCCGCCCTAAAAC
			CTACGTCACCCGCCCCGTTCCCACGCCCCG
			CGCCACGTCACAAACTCCACCCCCTCATTA
			TCATATTGGCTTCAATCCAAAATAAGGTAT
			ATTATTGATGAT

96	human IL-12	DNA	GTCGACGCCACCATGTGTCACCAGCAGCT
	insert	Sequence	CGTGATTAGCTGGTTCAGCCTGGTGTTTCTG
	(including		GCTAGCCCTCTGGTGGCCATCTGGGAGCTG
	restriction		AAGAAGGACGTGTACGTGGTGGAGCTCGAC
	sites and		TGGTACCCTGACGCTCCCGGCGAGATGGTC
	Kozak		GTGCTGACCTGCGACACCCCTGAGGAAGAT
	sequence)		GGCATCACCTGGACCCTGGATCAAAGCTCC
			GAAGTGCTCGGCAGCGGCAAGACACTCACC
			ATCCAGGTGAAAGAGTTCGGAGACGCCGGC
			CAGTACACCTGCCACAAAGGCGGCGAGGTG
			CTGTCCCATTCCCTGCTGCTGCTGCACAAGA
			AAGAGGATGGCATCTGGTCCACCGACATCC
			TGAAGGACCAGAAGGAACCCAAGAACAAG
			ACCTTTCTGAGATGTGAGGCCAAGAACTAC
			AGCGGCAGGTTCACCTGCTGGTGGCTGACA
			ACAATCTCCACCGACCTGACCTTCAGCGTC
			AAGAGCAGCAGGGGCAGCAGCGACCCTCA
			AGGCGTGACATGTGGAGCCGCTACCCTGAG
			CGCTGAGAGAGTCAGGGGCGACAATAAGG
			AGTACGAGTACTCCGTGGAATGCCAGGAGG
			ACTCCGCCTGCCCTGCCGCCGAAGAGTCCC
			TCCCTATCGAAGTGATGGTTGATGCCGTGC
			ACAAGCTCAAGTATGAGAATTACACCAGCA
			GCTTTTTCATCAGGGACATCATCAAGCCCG
			ACCCCCCCAAAAACCTCCAGCTGAAACCCC
			TCAAGAATAGCAGGCAGGTGGAGGTCTCCT
			GGGAGTATCCTGACACCTGGAGCACCCCCC
			ACAGCTACTTCTCCCTGACCTTCTGTGTGCA
			GGTGCAGGGCAAGAGCAAAAGGGAGAAGA
			AGGATAGGGTCTTTACCGACAAGACCAGCG
			CCACAGTGATCTGCAGGAAGAACGCCAGCA
			TTTCCGTCAGGGCCCAGGACAGGTACTACA
			GCAGCAGCTGGTCCGAGTGGGCTAGCGTGC
			CTTGTTCCGGCGGCGGAGGATCTGGCGGAG
			GCGGAAGTGGCGGAGGGGGCTCTAGAAAC
			CTCCCCGTGGCCACACCCGACCCTGGCATG
			TTCCCCTGCCTCCACCACAGCCAGAACCTG
			CTGAGAGCCGTGAGCAATATGCTGCAGAAG
			GCCAGGCAAACCCTGGAGTTCTACCCCTGT
			ACCTCCGAGGAGATTGACCATGAGGACATC
			ACAAAGGACAAAACCAGCACCGTGGAGGC
			CTGTCTCCCCCTCGAACTGACCAAGAACGA
			GTCCTGCCTGAACTCCAGGGAGACATCCTT
			CATCACCAACGGCTCCTGCCTGGCCTCCAG
			AAAGACCAGCTTCATGATGGCCCTCTGCCT
			GAGCAGCATCTACGAGGACCTCAAGATGTA
			CCAGGTGGAGTTTAAAACAATGAACGCCAA
			GCTCCTCATGGACCCTAAGAGGCAGATTTT
			CCTCGACCAGAATATGCTGGCTGTCATTGA
			CGAGCTGATGCAGGCCCTCAATTTCAACTC
			CGAGACCGTCCCCCAGAAGTCCTCCCTGGA
			AGAGCCCGACTTTTACAAGACCAAGATCAA
			GCTCTGCATCCTGCTGCACGCCTTCAGAATT
			AGAGCCGTGACCATTGACAGGGTGATGAGC
			TACCTCAACGCCTCCTGATGACTCGAG

97	human IL-		GTCGACGCCACCACATCCGCGGCAACGCC
	insert		TCCTTGGTGTCGTCCGCTTCCAATAACCCAG
			CTTGCGTCCTGC
			ACACTTGTGGCTTCCGTGCACACATTAACA
			ACTCATGGTTCTAGCTCCCAGTCGCCAAGC
			GTTGCCAAGGCGTTGAGAGATCATCTGGGA
			AGTCTTTTACCCAGAATTGCTTTGATTCAG
			GCCAGCTGGTTTTTCCTGCGGTGATTCGGA
			AATTCGCGAATTCCTCTGGTCCTCATCCAG
			GTGCGCGGGAAGCAGGTGCCCAGGAGAGA
			GGGGATAATGAAGATTCCATGCTGATGATC
			C
			CAAAGATTGAACCTGCAGACCAAGCGCAA
			AGTAGAAACTGAAAGTACACTGCTGGCGGA
			T
			CCTACGGAAGTTATGGAAAAGGCAAAGCG
			CAGAGCCACGCCGTAGTGTGTGCCGCCCCC
			C
			TTGGGATGGATGAAACTGCAGTCGCGGCGT
			GGGTAAGAGGAACCAGCTGCAGAGATCAC
			C
			CTGCCCAACACAGACTCGGCAACTCCGCGG
			AAGACCAGGGTCCTGGGAGTGACTATGGGC
			GGTGAGAGCTTGCTCCTGCTCCAGTTGCGG
			TCATCATGACTACGCCCGCCTCCCGCAGAC
			CATGTTCCATGTTTCTTTTAGGTATATCTTT
			GGACTTCCTCCCCTGATCCTTGTTCTGTT
			GCCAGTAGCATCATCTGATTGTGATATTGA
			AGGTAAAGATGGCAAACAATATGAGAGTG
			T
			TCTAATGGTCAGCATCGATCAATTATTGGA
			CAGCATGAAAGAAATTGGTAGCAATTGCCT
			GAATAATGAATTTAACTTTTTTAAAAGACA
			TATCTGTGATGCTAATAAGGAAGGTATGTT
			TTTATTCCGTGCTGCTCGCAAGTTGAGGCA
			ATTTCTTAAAATGAATAGCACTGGTGATTT
			TGATCTCCACTTATTAAAAGTTTCAGAAGG
			CACAACAATACTGTTGAACTGCACTGGCCA
			GGTTAAAGGAAGAAAACCAGCTGCCCTGG
			GTGAAGCCCAACCAACAAAGAGTTTGGAA
			GA
			AAATAAATCTTTAAAGGAACAGAAAAAACT
			GAATGACTTGTGTTTCCTAAAGAGACTATT
			ACAAGAGATAAAAACTTGTTGGAATAAAAT
			TTTGATGGGCACTAAAGAACACTGAAAAAT
			ATGGAGTGGCAATATAGAAACACGAACTTT
			AGCTGCATCCTCCAAGAATCTATCTGCTTA
			TGCAGTTTTTCAGAGTGGAATGCTTCCTAG
			AAGTTACTGAATGCACCATGGTCAAAACGG
			ATTAGGGCATTTGAGAAATGCATATTGTAT
			TACTAGAAGATGAATACAAACAATGGAAA
			C
			TGAATGCTCCAGTCAACAAACTATTTCTTAT
			ATATGTGAACATTTATCAATCAGTATAAT
			TCTGTACTGATTTTTGTAAGACAATCCATGT
			AAGGTATCAGTTGCAATAATACTTCTCAA
			ACCTGTTTAAATATTTCAAGACATTAAATCT
			ATGAAGTATATAATGGTTTCAAAGATTCA
			AAATTGACATTGCTTTACTGTCAAAATAATT
			TTATGGCTCACTATGAATCTATTATACTG
			TATTAAGAGTGAAAATTGTCTTCTTCTGTGC
			TGGAGATGTTTTAGAGTTAACAATGATAT
			ATGGATAATGCCGGTGAGAATAAGAGAGTC
			ATAAACCTTAAGTAAGCAACAGCATAACAA
			GGTCCAAGATACCTAAAAGAGATTTCAAGA
			GATTTAATTAATCATGAATGTGTAACACAG
			TGCCTTCAATAAATGGTATAGCAAATGTTTT
			GACATGAAAAAAGGACAATTTCAAAAAAA
			TAAAATAAAATAAAAATAAATTCACCTAGT
			CTAAGGATGCTAAACCTTAGTACTGAGTTA
			CATTGTCATTTATATAGATTATAACTTGTCT
			AAATAAGTTTGCAATTTGGGAGATATATT
			TTTAAGATAATAATATATGTTTACCTTTTAA
			TTAATGAAATATCTGTATTTAATTTTGAC
			ACTATATCTGTATATAAAATATTTTCATACA
			GCATTACAAATTGCTTACTTTGGAATACA
			TTTCTCCTTTGATAAAATAAATGAGCTATGT
			ATTAAAAAAAAAAAAAAA

98	human CD70	NCBI	GTCGACGCCACCCCAGAGAGGGGCAGGCT
	insert	Reference	GGTCCCCTGACAGGTTGAAGCAAGTAGACG
		Sequence:	CCCAGGAGCCCCG
		NM_001252.4	GGAGGGGGCTGCAGTTTCCTTCCTTCCTTCT
			CGGCAGCGCTCCGCGCCCCCATCGCCCCT
			CCTGCGCTAGCGGAGGTGATCGCCGCGGCG
			ATGCCGGAGGAGGGTTCGGGCTGCTCGGTG
			CGGCGCAGGCCCTATGGGTGCGTCCTGCGG
			GCTGCTTTGGTCCCATTGGTCGCGGGCTTG
			GTGATCTGCCTCGTGGTGTGCATCCAGCGC
			TTCGCACAGGCTCAGCAGCAGCTGCCGCTC
			GAGTCACTTGGGTGGGACGTAGCTGAGCTG
			CAGCTGAATCACACAGGACCTCAGCAGGAC
			CCCAGGCTATACTGGCAGGGGGGCCCAGCA
			CTGGGCCGCTCCTTCCTGCATGGACCAGAG
			CTGGACAAGGGGCAGCTACGTATCCATCGT
			GATGGCATCTACATGGTACACATCCAGGTG
			ACGCTGGCCATCTGCTCCTCCACGACGGCC
			TCCAGGCACCACCCCACCACCCTGGCCGTG
			GGAATCTGCTCTCCCGCCTCCCGTAGCATC
			AGCCTGCTGCGTCTCAGCTTCCACCAAGGT
			TGTACCATTGCCTCCCAGCGCCTGACGCCC
			CTGGCCCGAGGGGACACACTCTGCACCAAC
			CTCACTGGGACACTTTTGCCTTCCCGAAAC
			ACTGATGAGACCTTCTTTGGAGTGCAGTGG
			GTGCGCCCCTGACCACTGCTGCTGATTAGG
			GTTTTTTAAATTTTATTTTATTTTATTTAA
			GTTCAAGAGAAAAAGTGTACACACAGGGG
			CCACCCGGGGTTGGGGTGGGAGTGTGGTGG
			G
			GGGTAGTGGTGGCAGGACAAGAGAAGGCA
			TTGAGCTTTTTCTTTCATTTTCCTATTAAAA
			AATACAAAAATCA

99	NV1	Adenovirus	GGCGGAAGTGTGATGTTGCAAGTGTGGCGG
		E1a	AACACATGTAAGCGACGGATGTGGCGCAA
		Enhancer	GTCTATGTTGTAGTAAATTTGGGCGTAACC
		Region	GAGTAAGATTTGGCCATTTTCGCGGGAAAA
		Mutant 1	CTGAATAAGAGGAAGTGAAATC

100	NV2	Adenovirus	ACAGGAAGTGACATGTTGCAAGTGTGGCGG
		E1a	AACACATGTAAGCGACGGATGTGGCGCAA
		Enhancer	GTCTATGTTGTAGTAAATTTGGGCGTAACC
		Region	GAGTAAGATTTGGCCATTTTCGCGGGAAAA
		Mutant 2	CTGAATAAGAGGAAGTGAAATCT

101	NV3	Adenovirus	ACAGGAAGTGACAATTTTCGCGCGGTTTTA
		E1a	GGCGGATGTGGCGCAAGTCTATGTTGTAGT
		Enhancer	AAATTTGGGCGTAACCGAGTAAGATTTGGC
		Region	CATTTTCGCGGGAAAACTGAATAAGAGGAA
		Mutant 3	GTGAAATCT

102	NV4	Adenovirus	GGCGGAAGTGTGATGTTGCAAGTGTGGCGG
		E1a	AACACATGTAAGCGACGGATGTGGCAAAA
		Enhancer	GTGACGTTTTTGGTGTGCGCCGGTGTACGG
		Region	CGGAAGTGTGAATTTTCGCGCGGTTTTAGA
		Mutant 4	CGGATGTGGCAGTAAATTTGGGCGTAACCG
			AGTAAGATTTGGCCATTTTCGCGGGAAAAC
			TGAATAAGAGGAAGTGAAATCT

103	NV5	Adenovirus	GGCGGAAGTGTGATGTTGCAAGTGTGGCGG
		E1a	AACACATGTAAGCGACGGATGTGGCAAAA
		Enhancer	GTGACGTTTTTGGTGTGCGCCGGTGTACGG
		Region	CGGAAGTGTGAATTTTCGCGCGGTTTTAGA
		Mutant 5	CGGATGTGGCAGTAAATTTGGGCGTAACCG
			AGTAAGATTTGGCCATTTTCGCGGGAAAAC
			TGAATAAGAGGATGTGAAATCT

104	NV6	Adenovirus	GGCGGAAGTGTGATGTTGCAAGTGTGGCGG
		E1a	AACACATGTAAGCGACGGATGTGGCAAAA
		Enhancer	GTGACGTTTTTGGTGTGCGCCGGTGTACGG
		Region	CGGAAGTGTGAATTTTCGCGCGGTTTTAGA
		Mutant 6	CGGATGTGGCAGTAAATTTGGGCGTAACCG
			AGTAAGATTTGGCCATTTTCGCGGGAAAAC
			TGAATAGGCGGAAGTGTGATCT

105	NV7	Adenovirus	GGCGGAAGTGTGATGTTGCAAGTGTGGCGG
		E1a	AACACATGTAAGCGACGGATGTGGCAAAA
		Enhancer	GTGACGTTTTTGGTGTGCGCCGGTGTACAC
		Region	AGGAAGTGACAATTTTCGCGCGGTTTTAGA
		Mutant 7	CGGATGTGGCAGTAAATTTGGGCGTAACCG
			AGTAAGATTTGGCCATTTTCGCGGGAAAAC
			TGAATAGGCGGAAGTGTGATCT

Claims

What is claimed is:

1. A pharmaceutical composition comprising an effective amount of a recombinant adenoviral vector comprising:

(a) a first transgene insertion site located between the start site of adenoviral E1b-19K and the start site of adenoviral E1b-55K, the transgene insertion site comprising:

a first DNA sequence encoding a first polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, wherein the first DNA sequence is present in the first transgene insertion site and is operably linked to a first endogenous promoter, and

a second DNA sequence encoding a second polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, wherein the second DNA sequence is present in the second transgene insertion site and is operably linked to a second endogenous promoter,

wherein the first DNA sequence and the second DNA sequence are operably linked or the first polypeptide and the second polypeptide are operably linked, and

(b) a modified adenoviral E1a regulatory sequence, wherein at least one Pea3 binding site of the adenoviral E1a regulatory sequence is modified or deleted.

2. The pharmaceutical composition of claim 1, wherein the first DNA sequence and the second DNA sequence are operably linked by an IRES element.

3. The pharmaceutical composition of claim 1, wherein the first polypeptide and the second polypeptide are operably linked by a self-cleaving 2A peptide.

4. The pharmaceutical composition of claim 1, wherein a sequence between two Pea3 sites of the adenoviral E1a regulatory sequence is deleted.

5. The pharmaceutical composition of claim 1, wherein the first DNA sequence or the second DNA sequence encodes the chimeric human IL-12, wherein the chimeric human IL-12 comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide.

6. The pharmaceutical composition of claim 1, wherein the first DNA sequence or the second DNA sequence encodes human IL-2.

7. The pharmaceutical composition of claim 1, wherein the first DNA sequence or the second DNA sequence encodes human IL-7.

8. The pharmaceutical composition of claim 1, wherein the first polypeptide comprises human IL-7 and the second polypeptide comprises chimeric human IL-12.

9. A pharmaceutical composition comprising an effective amount of a recombinant adenoviral vector comprising:

(a) a first transgene insertion site located in the adenoviral E3 region, the transgene insertion site comprising a first DNA sequence encoding a first polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, wherein the first DNA sequence is present in the first transgene insertion site and is operably linked to a first endogenous promoter, and

10. The pharmaceutical composition of claim 9, further comprising in the transgene insertion site a second DNA sequence encoding a second polypeptide selected from the group consisting of: a chimeric human IL-12, a human IL-7, an anti-CTLA-4 antibody, an IL-10Rtrap, a human CD70, a human IL-2 polypeptide, a human CD40 ligand, and a human OX40 ligand, wherein the second DNA sequence is present in the second transgene insertion site and is operably linked to a second endogenous promoter.

11. The pharmaceutical composition of claim 9, wherein a sequence between two Pea3 sites of the adenoviral E1a regulatory sequence is deleted.

12. The pharmaceutical composition of claim 10, wherein the first DNA sequence or the second DNA sequence encodes the chimeric human IL-12, wherein the chimeric human IL-12 comprises a p40 polypeptide, a p35 polypeptide, and a linker polypeptide.

13. The pharmaceutical composition of claim 10, wherein the first DNA sequence or the second DNA sequence encodes human IL-2.

14. The pharmaceutical composition of claim 10, wherein the first DNA sequence or the second DNA sequence encodes human IL-7.

15. The pharmaceutical composition of claim 10, comprising a truncation in the E3 12.5K coding region, the E3 7.1K coding region, the E3 gp19K, the E3 10.5, or a combination thereof.

16. A method for treating a tumor in a human subject in need thereof, comprising administering to the human with a tumor a therapeutic amount of the pharmaceutical composition of claim 1 by systemic or intratumor administration.

17. The method of claim 16, further comprising treating the human subject with an anti-PD-1 antibody or an anti-PD-L1 antibody.

18. The method of claim 16, further comprising treating the human subject with:

(a) an agonist of a co-stimulatory signal selected from glucocorticoid-induced tumor necrosis factor receptor (GITR), Inducible T-cell co-stimulator (ICOS or CD278), OX40 (CD134), CD27, CD28, 4-IBB (CD137), CD40, lymphotoxin alpha (LT alpha), LIGHT (lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes), CD226, cytotoxic and regulatory T cell molecule (CRT AM), death receptor 3 (DR3), lymphotoxin-beta receptor (LTBR), transmembrane activator and CAML interactor (TACI), B cell-activating factor receptor (BAFFR), and B cell maturation protein (BCMA), or

(b) an antagonist of an inhibitory molecule selected from cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD1 or CD279), B and T-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), T-cell membrane protein 3 (TIM3), CD 160, adenosine A2a receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and CD 160.

19. A method for treating a tumor in a human subject in need thereof, comprising administering to the human with a tumor a therapeutic amount of the pharmaceutical composition of claim 9 by systemic or intratumor administration.

20. The method of claim 19, further comprising treating the human subject with:

(b) an antagonist of an inhibitory molecule selected from cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 or CD52), programmed cell death protein 1 (PD1 or CD279), B and I-lymphocyte attenuator (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), T-cell membrane protein 3 (TIM3), CD 160, adenosine A2a receptor (A2aR), T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and CD 160.