US20240011098A1

US20240011098A1 - Diagnostic and therapeutic methods for cancer

Info

Publication number: US20240011098A1
Application number: US17/901,481
Authority: US
Inventors: Yinghui Guan; Yasin SENBABAOGLU; Shannon TURLEY; Yulei Wang
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2017-11-06
Filing date: 2022-09-01
Publication date: 2024-01-11
Also published as: CA3077664A1; AU2018359967A1; IL274246A; CN111213059B; KR20200075860A; TW201923089A; MX2020004567A; JP2021502066A; US11473151B2; WO2019090263A1; EP3707510A1; CN111213059A; US20200263261A1

Abstract

The present invention provides diagnostic methods, therapeutic methods, and compositions for the treatment of cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer). The invention is based, at least in part, on the discovery that expression levels of one or more biomarkers described herein in a sample from an individual having cancer can be used in methods of identifying an individual having a cancer who may benefit with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist such as an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist), methods for selecting a therapy for an individual having cancer, methods of treating an individual having cancer, methods for assessing a response or monitoring the response of an individual to treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist such as an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist), and related kits, anti-cancer therapies, and uses.

Description

FIELD OF THE INVENTION

The present invention is directed to diagnostic and therapeutic methods for the treatment of cancer. Also provided are related kits and assays.

BACKGROUND OF THE INVENTION

Cancer remains one of the most deadly threats to human health. In the U.S., cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for approximately 1 in 4 deaths. It is also predicted that cancer may surpass cardiovascular diseases as the number one cause of death within 5 years. Solid tumors are responsible for most of those deaths. Although there have been significant advances in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved only by about 10% in the past 20 years. Malignant solid tumors, in particular, metastasize and grow rapidly in an uncontrolled manner, making their timely detection and treatment extremely difficult.
Studies in humans with immune checkpoint inhibitors have demonstrated the promise of harnessing the immune system to control and eradicate tumor growth. The programmed death 1 (PD-1) receptor and its ligand programmed death-ligand 1 (PD-L1) are immune checkpoint proteins that have been implicated in the suppression of immune system responses during chronic infections, pregnancy, tissue allografts, autoimmune diseases, and cancer. PD-L1 regulates the immune response by binding to the inhibitory receptor PD-1, which is expressed on the surface of T-cells, B-cells, and monocytes. PD-L1 negatively regulates T-cell function also through interaction with another receptor, B7-1. Formation of the PD-L1/PD-1 and PD-L1/B7-1 complexes negatively regulates T-cell receptor signaling, resulting in the subsequent downregulation of T-cell activation and suppression of anti-tumor immune activity.
Despite the significant advancement in the treatment of cancer, improved diagnostic methods and cancer therapies and are still being sought.

SUMMARY OF THE INVENTION

The present invention provides diagnostic and therapeutic methods and compositions for treating an individual having a cancer, including, but not limited to, a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer.
In one aspect, the invention features a method of identifying an individual having a cancer who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the method comprising determining the expression level of three or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein an expression level of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample that is at or above a reference expression level of the three or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In another aspect, the invention features a method for selecting a therapy for an individual having a cancer, the method comprising determining the expression level of three or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein an expression level of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample that is at or above a reference expression level of the three or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In some embodiments of any of the preceding aspects, the expression level of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample is at or above a reference expression level of the three or more genes, and the method further comprises administering to the individual an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist. In some embodiments, the expression level of at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 is at or above a reference expression level of the three or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 is at or above a reference level of TGFB1 and/or TGFBR1. In some embodiments, the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 is at or above a reference expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In another aspect, the invention features a method of treating an individual having a cancer, the method comprising: (a) determining the expression level of three or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein the expression level of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample is determined to be at or above a reference expression level of the three or more genes; and (b) administering an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual based on the expression level of the three or more genes determined in step (a). In some embodiments, the expression level of at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 is determined to be at or above a reference expression level of the three or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 is determined to be at or above a reference level of TGFB1 and/or TGFBR1. In some embodiments, the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 is determined to be at or above a reference expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In another aspect, the invention features a method of treating an individual having a cancer, the method comprising administering to the individual an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, wherein prior to treatment the expression level of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample has been determined to be at or above a reference expression level of the three or more genes. In some embodiments, the expression level of at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been determined to be at or above a reference expression level of the three or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 has been determined to be at or above a reference level of TGFB1 and/or TGFBR1. In some embodiments, the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 in the sample has been determined to be at or above a reference expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In another aspect, the invention provides a method of treating cancer in an individual having been identified as having an expression level in a sample from the individual of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 that is at or above a reference expression level of the three or more genes, the method comprising administering to the individual an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist. In some embodiments, the expression level of at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been identified to be at or above a reference expression level of the three or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 has been identified to be at or above a reference level of TGFB1 and/or TGFBR1. In some embodiments, the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 in the sample has been identified to be at or above a reference expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In another aspect, the invention features a method of identifying an individual having a cancer who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the method comprising determining the expression level of one or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein the one or more genes includes at least ADAM19 or COMP, and wherein an expression level of the one or more genes in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In another aspect, the invention features a method for selecting a therapy for an individual having a cancer, the method comprising determining the expression level of one or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein the one or more genes includes at least ADAM19 or COMP, and wherein an expression level of the one or more genes in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In some embodiments of any of the preceding aspects, the expression level of the one or more genes in the sample is at or above a reference expression level of the one or more genes, and the method further comprises administering to the individual an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist. In some embodiments, the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 is at or above a reference expression level of the one or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 is at or above a reference level of TGFB1 and/or TGFBR1.
In another aspect, the invention features a method of treating an individual having a cancer, the method comprising: (a) determining the expression level of one or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein the one or more genes includes at least ADAM19 or COMP, and wherein the expression level of the one or more genes in the sample is determined to be at or above a reference expression level of the one or more genes; and (b) administering an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual based on the expression level of the one or more genes determined in step (a). In some embodiments, the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 is determined to be at or above a reference expression level of the one or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 is at or above a reference level of TGFB1 and/or TGFBR1.
In another aspect, the invention features a method of treating an individual having a cancer, the method comprising administering to the individual an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, wherein prior to treatment the expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample has been determined to be at or above a reference expression level of the one or more genes, wherein the one or more genes includes at least ADAM19 or COMP. In some embodiments, the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been determined to be at or above a reference expression level of the one or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 has been determined to be at or above a reference level of TGFB1 and/or TGFBR1.
In another aspect, the invention provides a method of treating cancer in an individual having been identified as having an expression level in a sample from the individual of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 that is at or above a reference expression level of the one or more genes, the method comprising administering to the individual an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, wherein the one or more genes includes at least ADAM19 or COMP. In some embodiments, the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been identified to be at or above a reference expression level of the one or more genes. In some embodiments, the expression level of TGFB1 and/or TGFBR1 has been identified to be at or above a reference level of TGFB1 and/or TGFBR1.
In another aspect, the invention features a method for assessing a response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the method comprising: (a) determining the expression level of five or more of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and (b) maintaining, adjusting, or stopping the treatment based on a comparison of the expression level of the five or more genes in the sample with a reference expression level of the five or more genes, wherein a change in the expression level of the five or more genes in the sample from the individual compared to the reference expression level of the five or more genes is indicative of a response to treatment with the anti-cancer therapy. In some embodiments, the method comprises determining the expression level of at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1. In some embodiments, the method comprises determining the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the expression level of at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 is changed relative to a reference expression level of the five or more genes. In some embodiments, the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 is changed relative to a reference expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the change is an increase in the expression level of the five or more genes and the treatment is adjusted or stopped. In some embodiments, the change is a decrease in the expression level of the five or more genes and the treatment is maintained.
In another aspect, the invention features a method for monitoring the response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the method comprising: (a) determining the expression level of five or more of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and (b) comparing the expression level of the five or more genes in the sample from the individual with a reference expression level of the five or more genes, thereby monitoring the response of the individual undergoing treatment with the anti-cancer therapy. In some embodiments, the method comprises determining the expression level of at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1. In some embodiments, the method comprises determining the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the expression level of at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 is changed relative to a reference expression level of the five or more genes. In some embodiments, the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 is changed relative to a reference expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, an increase in the expression level of the five or more genes is indicative of a lack of response of the individual to the treatment. In some embodiments, a decrease in the expression level of the five or more genes is indicative of a response of the individual to the treatment.
In another aspect, the invention features a method for assessing a response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the method comprising: (a) determining the expression level of one or more of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1, wherein the one or more genes includes at least ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and (b) maintaining, adjusting, or stopping the treatment based on a comparison of the expression level of the one or more genes in the sample with a reference expression level of the one or more genes, wherein a change in the expression level of the one or more genes in the sample from the individual compared to the reference expression level of the one or more genes is indicative of a response to treatment with the anti-cancer therapy. In some embodiments, the method comprises determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1. In some embodiments, the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 is changed relative to a reference expression level of the one or more genes. In some embodiments, the change is an increase in the expression level of the one or more genes and the treatment is adjusted or stopped. In some embodiments, the change is a decrease in the expression level of the one or more genes and the treatment is maintained.
In another aspect, the invention features a method for monitoring the response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the method comprising: (a) determining the expression level of one or more of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1, wherein the one or more genes includes at least ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and (b) comparing the expression level of the one or more genes in the sample from the individual with a reference expression level of the one or more genes, thereby monitoring the response of the individual undergoing treatment with the anti-cancer therapy. In some embodiments, the method comprises determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1. In some embodiments, the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 is changed relative to a reference expression level of the five or more genes. In some embodiments, an increase in the expression level of the one or more genes is indicative of a lack of response of the individual to the treatment. In some embodiments, a decrease in the expression level of the one or more genes is indicative of a response of the individual to the treatment.
In some embodiments of any of the preceding aspects, the method further comprises determining the expression level in the sample of one or more additional genes selected from the group consisting of PD-L1, CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the one or more additional genes is PD-L1. In some embodiments, the one or more additional genes is selected from the group consisting of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the method further comprises determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the method further comprises determining the expression level of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21.
In some embodiments of any of the preceding aspects, the method further comprises determining a tumor mutational burden (TMB) in a tumor sample from the individual.
In another aspect, the invention features a method of selecting a therapy for an individual having a cancer, the method comprising: determining (i) the expression level of one or more of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and (ii) the expression level of one or more additional genes selected from the group consisting of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21 in the sample from the individual, or a TMB score in a tumor sample from the individual, wherein: an expression level of the one or more genes of (i) that is at or above a reference expression level of the one or more genes of (i) identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, and an expression level of the one or more genes of (i) that is below a reference expression level of the one or more genes and an expression level of the one or more additional genes of (ii) that is at or above a reference expression level of the one or more additional genes of (ii), or a TMB score in the tumor sample that is at or above a reference TMB score, identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy alone. In some embodiments, the method further comprises administering to the individual the anti-cancer therapy for which the individual may benefit from treatment.
In some embodiments of any of the preceding aspects, the cancer is selected from the group consisting of a bladder cancer, a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer. In some embodiments, the cancer is a bladder cancer. In some embodiments, the bladder cancer is a urothelial carcinoma (UC). In some embodiments, the UC is a metastatic UC. In some embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).
In some embodiments of any of the preceding aspects, a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment. In some embodiments, the CD8+ T-cells localize at or near collagen fibers.
In some embodiments of any of the preceding aspects, the reference expression level is determined from a population of individuals having a cancer. In some embodiments, the reference expression level is a median expression level and/or is determined by principle component analysis of Z-score-transformed expression levels.
In some embodiments of any of the preceding aspects, the expression level is a nucleic acid expression level. In some embodiments, the nucleic acid expression level is an mRNA expression level. In some embodiments, the mRNA expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof.
In other embodiments of any of the preceding aspects, the expression level is a protein expression level. In some embodiments, the protein expression level is determined by immunohistochemistry (IHC), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, radioimmunoassay, or mass spectrometry.
In some embodiments of any of the preceding aspects, the sample is a tissue sample, a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the tissue sample is a tumor tissue sample. In some embodiments, the tumor tissue sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some embodiments, the tumor tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archival sample, a fresh sample, or a frozen sample.
In some embodiments of any of the preceding aspects, the suppressive stromal antagonist is a transforming growth factor beta (TGF-β), podoplanin (PDPN), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), SMAD, anaplastic lymphoma kinase (ALK), connective tissue growth factor (CTGF/CCN2), endothelial-1 (ET-1), AP-1, interleukin (IL)-13, lysyl oxidase homolog 2 (LOXL2), endoglin (CD105), fibroblast activation protein (FAP), vascular cell adhesion protein 1 (CD106), thymocyte antigen 1 (THY1), beta 1 integrin (CD29), platelet-derived growth factor (PDGF), PDGF receptor A (PDGFRα), PDGF receptor B (PDGFRβ), vimentin, smooth muscle actin alpha (ACTA2), desmin, endosialin (CD248), or S100 calcium-binding protein A4 (S100A4) antagonist. In some embodiments, the suppressive stromal antagonist is pirfenidone, galunisertib, or nintedanib. In some embodiments, the suppressive stromal antagonist is a TGF-β antagonist. In some embodiments, the TGF-β antagonist is a TGF-β binding antagonist. In some embodiments, the TGF-β binding antagonist inhibits the binding of TGF-β to its ligand binding partners. In some embodiments, the TGF-β binding antagonist inhibits the binding of TGF-β to a cellular receptor for TGF-β. In some embodiments, the TGF-β binding antagonist inhibits activation of TGF-β. In some embodiments, the TGF-β antagonist inhibits TGF-β1, TGF-β2, and/or TGF-β3. In some embodiments, the TGF-β antagonist inhibits TGF-β1, TGF-β2, and TGF-β3. In some embodiments, the TGF-β antagonist inhibits TGF-β receptor-1 (TGFBR1), TGF-β receptor-2 (TGFBR2), and/or TGF-β receptor-3 (TGFBR3). In some embodiments, the TGF-β antagonist is a polypeptide, a small molecule, or a nucleic acid. In some embodiments, the TGF-β antagonist is a polypeptide. In some embodiments, the polypeptide is an anti-TGF-β antibody, a soluble TGF-β receptor, or a peptide. In some embodiments, the polypeptide is an anti-TGF-β antibody. In some embodiments, the anti-TGF-β antibody is a pan-specific anti-TGF-β antibody. In some embodiments, the anti-TGF-β antibody is fresolimumab, metelimumab, lerdelimumab, 1 D11, 2G7, or derivatives thereof. In some embodiments, the peptide is disitertide (P144). In some embodiments, the TGF-β antagonist is a small molecule. In some embodiments, the small molecule is selected from the group consisting of galunisertib (LY2157299), LY2382770, LY3022859, SB-431542, SD208, SM16, tranilast, pirfenidone, TEW-7197, PF-03446962, and pyrrole-imidazole polyamide. In some embodiments, the TGF-β antagonist is a nucleic acid. In some embodiments, the nucleic acid is trabedersen (AP12009) or belagenpumatucel-L.
In some embodiments of any of the preceding aspects, the immunotherapy comprises a CD28, OX40, GITR, CD137, CD27, CD40, ICOS, HVEM, NKG2D, MICA, 2B4, IL-2, IL-12, IFNγ, IFNα, TNFα, IL-1, CDN, HMGB1, or TLR agonist.
In other embodiments of any of the preceding aspects, the immunotherapy comprises a PD-L1 axis, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, B7H4, CD96, TIGIT, CD226, prostaglandin, VEGF, endothelin B, IDO, arginase, MICA/MICB, TIM-3, IL-10, IL-4, or IL-13 antagonist. In some embodiments, the immunotherapy is a PD-L1 axis antagonist. In some embodiments, the PD-L1 axis antagonist is a PD-L1 axis binding antagonist. In some embodiments, the PD-L1 axis binding antagonist is selected from the group consisting of a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist. In some embodiments, the PD-L1 axis binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is a monoclonal anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is a human, humanized, or chimeric anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of: MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MED14736 (durvalumab), and MSB0010718C (avelumab). In some embodiments, the anti-PD-L1 antibody comprises the following hypervariable regions (HVRs): (a) an HVR-H1 sequence of GFTFSDSWIH (SEQ ID NO: 63); (b) an HVR-H2 sequence of AWISPYGGSTYYADSVKG (SEQ ID NO: 64); (c) an HVR-H3 sequence of RHWPGGFDY (SEQ ID NO: 65); (d) an HVR-L1 sequence of RASQDVSTAVA (SEQ ID NO: 66); (e) an HVR-L2 sequence of SASFLYS (SEQ ID NO: 67); and (f) an HVR-L3 sequence of QQYLYHPAT (SEQ ID NO: 68). In some embodiments, the anti-PD-L1 antibody comprises: (a) a heavy chain variable (VH) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of
(SEQ ID NO: 69)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA

WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR

RHWPGGFDYWGQGTLVTVSS;

(b) a light chain variable (VL) domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR (SEQ ID NO: 70); or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the anti-PD-L1 antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 69; (b) a VL domain comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 70; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the anti-PD-L1 antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 69; (b) a VL domain comprising an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 70; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the anti-PD-L1 antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 69; (b) a VL domain comprising an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 70; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the anti-PD-L1 antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 69; (b) a VL domain comprising an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 70; or
(c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the anti-PD-L1 antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 69; (b) a VL domain comprising an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 70; or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the anti-PD-L1 antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 69; (b) a VL domain comprising the amino acid sequence of SEQ ID NO: or (c) a VH domain as in (a) and a VL domain as in (b). In some embodiments, the anti-PD-L1 antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 69; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a monoclonal anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a human, humanized, or chimeric anti-PD-1 antibody.
In some embodiments, the method further comprises administering an additional therapeutic agent to the individual. In some embodiments, the additional therapeutic agent is selected from the group consisting of an immunotherapy agent, a cytotoxic agent, a growth inhibitory agent, a radiation therapy agent, an anti-angiogenic agent, and combinations thereof. In some embodiments, the individual is a human.
In another aspect, the invention features a kit for identifying an individual having a cancer who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the kit comprising: (a) reagents for determining the expression level of three or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2; and, optionally, (b) instructions for using the reagents to identify an individual having a cancer who may benefit from a treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In another aspect, the invention features a kit for identifying an individual having a cancer who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist, the kit comprising: (a) reagents for determining the expression level of one or more of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein the one or more genes includes at least ADAM19 or COMP; and, optionally, (b) instructions for using the reagents to identify an individual having a cancer who may benefit from a treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In another aspect, the invention features an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist for use in a method of treating an individual suffering from a cancer, wherein prior to treatment the expression level of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been determined to be at or above a reference expression level of the one or more genes.
In another aspect, the invention features an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist for use in a method of treating an individual suffering from a cancer, wherein prior to treatment the expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been determined to be at or above a reference expression level of the one or more genes, wherein the one or more genes includes at least ADAM19 or COMP.
In another aspect, the invention provides for the use of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist in the manufacture of a medicament for treating an individual suffering from a cancer, wherein prior to treatment the expression level of three or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been determined to be at or above a reference expression level of the one or more genes.
In another aspect, the invention provides for the use of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist in the manufacture of a medicament for treating an individual suffering from a cancer, wherein prior to treatment the expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been determined to be at or above a reference expression level of the one or more genes, wherein the one or more genes includes at least ADAM19 or COMP.
In another aspect, the invention features a kit for monitoring a response of an individual having a cancer, the kit comprising: (a) reagents for determining the expression level of five or more of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and, optionally, (b) instructions for using the reagents to monitor the response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In another aspect, the invention features a kit for monitoring a response of an individual having a cancer, the kit comprising: (a) reagents for determining the expression level of one or more of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1, wherein the one or more genes includes at least ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and, optionally, (b) instructions for using the reagents to monitor the response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIG. 1A is a graph showing that programmed death-ligand 1 (PD-L1) protein expression on tumor-infiltrating immune cells (IC) is associated with response to atezolizumab (Fisher exact test; p=0.0038). IC2+ tumors had a significantly higher complete response (CR) rate (p=0.0006), but the proportion of the partial responders (PR) was similar across all IC levels (p=0.53).

FIG. 1B is a graph showing genes associated with PD-L1 immunohistochemistry (IHC) positivity on IC. Normalized log₂-transformed gene expression was compared with PD-L1 IC protein expression, and adjusted p values (−log₁₀transformed, y-axis) and effect size of the association (x-axis) are plotted. Interferon gamma-stimulated (“IFNg inducible”) genes and previously-reported CD8 T-effector and immune checkpoint molecule gene sets were among the most dramatically up-regulated.

FIGS. 1C and 1D are a series of graphs showing an association between CD8 T-effector signature score and PD-L1 IC or response. CD8+T-effector signature score (y-axis) is plotted against PD-L1 IC score (FIG. 1C) and response category (FIG. 1D). There was a significant positive relationship between the signature score and both PD-L1 IC staining (p=4.2×10⁻³⁵) and response to atezolizumab (p=0.0087). The association between CD8 T-effector signature and response was driven by the CR group, which had a significantly higher CD8 T-effector signature (t test; p=0.002); differences among the other three response groups were not significant (analysis of variance (ANOVA) p=0.08).

FIGS. 1E and 1F are a series of graphs showing the relationship between response status and tumor mutation burden (TMB) or tumor neoantigen burden (TNB). Shown are mutations per megabase (y-axis) as determined by Foundation Medicine's FOUNDATIONONE® (also referred to herein as “FMOne”) panel (FIG. 1E) or whole-exome filtering for those mutations that are predicted to be expressed neoantigens (FIG. 1F). Both metrics are positively associated with response to atezolizumab (Wilcoxon p for FMOne-based mutations=1.4×10⁻⁷, for predicted neoantigens=5.3×10⁻⁹).

FIG. 1G is a graph showing KEGG pathways enriched in genes whose expression is correlated with TMB. Shown are adjusted −log₁₀p-values for enrichment of KEGG gene sets significantly (FDR<0.05) enriched in genes that are correlated with TMB. Sets inferred to reflect key underlying biological processes are colored as follows: proliferation (turquoise), DNA damage response (DDR) (magenta), TGF-β signaling (orange). Only the top seven genes per set (ranked by single-gene p-value) are shown.

FIG. 1H is a graph showing the relationship between different gene expression signatures as well as the single-gene expression values for MKI67, a marker for proliferation. In the left corner, correlation between signature scores/gene expression is visualized; in the right corner, Pearson correlation coefficients are given. Gene set membership is given in Table 10. See Example 1 for information regarding computation of signature scores. Pan-F-TBRS: pan-fibroblast TGF-β response signature.

FIGS. 1I and 1J are a series of graphs showing apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) 3A (FIG. 1I) and APOBEC3B (FIG. 1J) gene expression and its association with response and TMB. Normalized log₂-transformed counts for gene expression are shown on the y-axes; response categories and TMB are shown on the x-axes. Both APOBEC3A (p=0.015) and APOBEC3B (p=exhibited higher mean expression in responders. For TMB, Pearson correlation coefficients and p-values are given. Trend lines for the relationship between gene expression and TMB are plotted. For FIG. 1J, the two extreme expression outliers were excluded when calculating correlation between gene expression and TMB.

FIG. 1K is a graph showing that mutations in DDR genes are associated with TMB. Genes are in rows and patients are in columns, with a mutation marked with a black rectangle. The upper bar graph depicts TMB, with patients sorted from high to low TMB. The lower rows represent the mutation status of the entire pathway, with or without inclusion of TP53. The percentage of patients carrying a mutation in a gene or pathway is given to the left.

FIG. 1L is a graph showing set-wise mutation status for DDR genes versus response. Patients were labelled DDR mutant if they harbored a mutation in one or more DDR genes, excluding TP53. The fraction of responders (CR/PR) and non-responders (stable disease (SD)/progressive disease (PD)) is plotted for these two patient subgroups. There was a significant association between DDR mutation status and response (p=0.0117).

FIG. 1M is a graph showing that mutations in cell cycle regulator genes are associated with TMB. Genes are plotted in rows and patients in columns, marking patients with a mutation with a black rectangle. The upper bar plot depicts TMB in each patient, with patients sorted from TMB high to TMB low. The final rows represent the mutation status of the pathway as a whole, with or without TP53. Percentages to the left of the plot indicate prevalence. Genes with significant single gene associations with TMB are marked by an asterisk.

FIG. 1N is a graph showing mutation status in cell cycle regulation (CCR) pathway by response. For each patient, it was determined whether they harbor any mutation in a gene belonging to the CCR pathway, except for TP53. If patients harbored mutation(s) in CCR pathway gene(s), they were called mutant for the pathway. Otherwise, they were considered non-mutant. The fraction of responders (CR/PR) and non-responders (SD/PD) is plotted for these two patient subgroups. Excluding TP53, there was no association between mutation status for the CCR pathway and response (p=0.31104; Table 6).

FIG. 1O shows KEGG pathways that are significantly associated with response to atezolizumab. Shown are adjusted −log₁₀p-values for KEGG pathways significantly (adj. p<0.1) enriched in genes associated with response. Sets inferred to reflect key underlying biological processes are colored: proliferation (turquoise), DDR (magenta), TGF-β signaling (orange). Only the top seven genes per set (ranked by single-gene p-value) are shown. Complete lists of differentially expressed genes are given in Table 3.

FIG. 1P is a graph showing that the TGF-β 2-gene signature (y-axis) comprised of TGFB1 and TGFBR2 was significantly associated with lack of response to atezolizumab (p=9.6×10⁻⁶).

FIG. 1Q is a schematic representation of the relationship between three core axes, assays that interrogate those axes, and patient response.

FIG. 1R is a graph showing explained variance in patient response. Generalized linear models were fit using binary response (CR/PR versus SD/PD) as the dependent variable and scores from single input or input combinations (x-axis) as independent variables. Percent explained variance of response is plotted on the y-axis. Comparisons between different models were made via likelihood ratio test. A model that includes both DNA (TMB) and RNA markers (CD8+ T-effector (Teff) and TGF-β 2-gene sets) explained 42% of the variance observed in response, and it significantly improved on all singleton models.

FIG. 1S is a graph showing explained variation in patient response. Generalized linear models were fit using binary response (CR/PR versus SD/PD) as the dependent variable and scores from single input or input combinations (x-axis) as independent variables. Percent explained variance of response is plotted on the y-axis. Comparisons between different models were made via likelihood ratio test; a significant p-value means that the additional variable contributed independent information to the model. The triple model including TMB as well as Teff and TGF-β 2-gene signatures explains 42% of the variance observed in response and significantly improves on all singleton and two-biology models. This shows that all three biologies have non-redundant explanatory value.

FIG. 1T is a graph showing explained variation in patient response. Generalized linear models were fit using binary response (CR/PR vs. SD/PD) as the dependent variable and scores from single input or input combinations (x-axis) as independent variables. Percent explained variance of response is plotted on the y-axis. Comparisons between different models were made via likelihood ratio test. The association between TMB with response was significantly stronger than that of its proxy measurements (APOBEC3B expression, MKI67 expression or mutation in members of the DDR set). APOBEC3B and DDR gene set mutation provided no additional explanatory information that was independent of direct measurement of TMB. Combining TMB with MKI67 expression did marginally improve on TMB alone, possibly through MKI67's negative association with TFG-β (see FIG. 1H).

FIG. 1U is a graph showing that Teff signature score quartiles (“quart”) were associated with overall survival (OS) (p=0.0092).

FIG. 1V is a graph showing that TMB was associated with overall survival (OS) (likelihood ratio test p=2×10⁻⁵).

FIG. 1W is a graph showing that TNB was associated with OS (likelihood ratio test p=0.00015).

FIG. 1X is a graph showing that TGFB1 expression was associated with poor objective response (p=0.0001).

FIG. 1Y is a graph showing that TGFB1 expression quartiles were associated with reduced OS.

FIG. 1Z is a series of graphs showing that MKI67 expression (left panel) and DNA replication score (right panel) are associated with response to atezolizumab.

FIG. 2A is a heatmap representing all patients evaluated, sorted by molecular subtype and then response. For comparison, immune cell (IC) and tumor cell (TC) PD-L1 status are given. In addition, TMB and mutation status (gray: patients without mutation data) for key genes of interest are shown. The rows of the heat map show expression (Z-scores) of genes of interest, grouped into the following pathways: A: FGFR3 gene signature, B: Teff signature, C: antigen processing machinery, D: immune checkpoint signature, E: MKI67 and cell cycle genes, F: DNA replication-dependent histones, G: DNA damage repair genes, H: extracellular matrix (ECM) organization gene set, I: TGF-β 2-gene signature, J: angiogenesis signature, K: epithelial-mesenchymal transition (EMT) markers, and L: cancer-associated fibroblast genes.

FIG. 2B is a graph showing TMB (y-axis) plotted against Lund (left panel) and TOGA subtypes (right panel) (x-axis). The Lund genomically-unstable (Wilcoxon test; p=0.0002) and TOGA luminal II subtypes (p=5.94×10⁻⁵) had a higher median TMB. UroA: Urothelial-like A, GU: Genomically-unstable, Inf: Infiltrated, UroB: Urothelial-like B, SCOL: SCC-like.

FIG. 2C is a graph showing patients split into TMB low (grey) and TMB high (black) subgroups, based on median TMB, and the fraction of patients in these two subgroups is shown for each of the Lund (left panel) and TOGA (right panel) molecular subtypes, respectively.

FIG. 2D is a graph showing the fraction of patients in each of the indicated response categories, divided by Lund molecular subtype. The GU subtype had a significantly higher response rate compared with all other subtypes (Fisher exact test; p=1.6×10⁻⁵).

FIG. 2E is a graph showing the fraction of patients in each of the indicated response categories, divided by TOGA subtype. Although the luminal II subgroup achieved the highest response rate, consistent with previously reported results for the platinum-resistant cohort alone, the difference between luminal II versus other subtypes was not significant (Fisher exact test; p=0.12).

FIG. 2F is a graph showing that TGF-β is a likely driver of differential response in Lund GU and TOGA luminal II subtypes. The Venn diagram represents the three subgroups dissected further: (i) Lund subtyped as GU but not TOGA luminal II (GU only), (ii) both GU Lund luminal II (GU and II), or (iii) luminal II, but not GU (II only). The heat map shows the assessment of CD8 Teff and TGF-β 2-gene signatures as well as TMB in these subgroups (columns). Biologies of interest (rows) were scaled before medians were calculated across groups. Red means high, blue means low. Response differed significantly between the three subgroups (p=0.00062), as shown in the bar graph: while “GU only” and “GU and II” have a high fraction of partial and/or complete responders, “II only” shows limited response. Teff−TGF-β indicates CD8 Teff signature score minus TGF-β 2-gene signature score.

FIG. 2G is a heatmap showing assessment of MKI67 expression and signatures of interest as well as TMB relative to molecular subtypes. Biologies of interest were scaled before medians were calculated across the Lund (left) and TOGA (right) molecular subtypes (columns). Red means high, blue means low. DNA rep.: DNA replication.

FIG. 2H is a heatmap showing a comparison between Lund and TOGA subtyping schemes. The heatmap represents all patients evaluated, except for patients without defined response, arranged in columns and sorted first by molecular subtype, then by response to atezolizumab. For the left hand panel, patients were sorted based on a TOGA-based subtyping scheme; for the right hand panel, patients were sorted by a Lund-based subtyping scheme (like FIG. 2A). IC and TC PD-L1 status are given. In addition, TMB and mutation status (either mutated, black, or not-mutated, white; grey indicates patients without mutation data) for a few genes of interest are shown. The rows of the heatmap show expression (z-scores) of genes of interest, grouped into the following biologies/pathways: TOGA: TOGA subtyping genes, A: FGFR3 gene signature, B: Teff signature, C: antigen processing machinery, D: immune checkpoint signature, E: MKI67 and cell cycle genes, F: DNA replication-dependent histones, G: DNA damage repair genes, H: ECM gene set, I: TGFB two-gene signature, J: angiogenesis signature, K: EMT markers, and L: Pan-F-TBRS genes (for details on these signatures see Example 1).

FIG. 3A is a series of images showing histology of the three tumor-immune phenotypes: immune “desert,” immune “excluded,” and immune “inflamed.” Categorization of tumors into one of three immunophenotypic entities was performed on formalin-fixed, paraffin-embedded (FFPE) sections stained immunohistochemically for the presence of CD8+ T-cells. The categorization was based on prevalence of CD8+ cells as well as pattern of infiltration with respect to malignant epithelial cells. Tumors were categorized as “desert” when the prevalence of CD8+ cells was low (<10 CD8+ cells in an area of tumor and tumor-associated stroma at a magnification of 200×; in larger specimens, this was calculated as the average of 10 representative fields of view). Tumors were categorized as “excluded” if CD8+ cells were exclusively seen in stroma immediately adjacent to or within the main tumor mass. Tumors were categorized as “inflamed” if CD8+ cells were seen in direct contact with malignant epithelial cells either in the form of spillover of stromal infiltrates into tumor cell aggregates or of diffuse infiltration of CD8+ cells in aggregates or sheets of tumor cells. As these features were frequently observed in a focal manner, any such observation in a tumor lesion led to a categorization as “inflamed.” H&E, hematoxylin and eosin stain.

FIG. 3B is an image showing combined CD8 IHC-trichrome stain performed on FFPE sections to visualize the spatial distribution of CD8+ T-cells and collagenous stroma. CD8+ T-cells outlined by 3,3′-diaminobenzidine (DAB) stain (brown) are primarily localized within collagenous (blue) stroma (white arrows). Rare CD8+ T-cells are interspersed between tumor cells (green arrows).

FIGS. 3C and 3D are a series of graphs showing the relationship between TGF-β 2-gene signature and pan-fibroblast TGF-β response signature (Pan-F-TBRS) scores and response status within each tumor-immune phenotype group. TGF-β 2-gene signature (FIG. 3C) or Pan-F-TBRS (FIG. 3D) scores (y-axis) are plotted by immune phenotype and response group. FIG. 3C shows that the TGF-β 2-gene signature was comparable across immune phenotypes but was significantly associated with response only in the excluded phenotype (adj. p=5.7×10⁻⁵; t test p-values Bonferroni corrected for three tests). A likelihood ratio test for interaction confirmed a phenotype-specific relationship between the TGF-β signature and response (p=0.02251). FIG. 3D shows that the Pan-F-TBRS was significantly associated with response only in the excluded phenotype (adj. p=0.0066; t test p-values Bonferroni corrected for three tests).

FIG. 3E is a graph showing TMB (y-axis) plotted against tumor-immune phenotype. There was no significant difference in TMB between cancer-immune phenotypes (Kruskal Wallis p=0.091).

FIG. 4A is a series of images showing collagen (green) and T-cells (CD3, red) in EMT6 tumors stained by immunofluorescence.

FIG. 4B is a graph showing quantification of TGF-β and PD-L1 RNA in whole EMT6 tumors by RNA sequencing (RNAseq). The tumors were inoculated orthotopically and collected when volume reached 300 mm³(N=5; data from one experiment).

FIG. 4C is a graph showing quantification of TGF-β protein within whole EMT6 tumors by enzyme-linked immunosorbent assay (ELISA). Tumors were collected 14 days after inoculation, flash frozen, and lysed for protein quantification (N=4; data from one experiment).

FIGS. 4D-4F is a series of graphs showing the results of studies in which BALB/c mice were inoculated with EMT6 tumor cells orthotopically in the mammary fat pad. When tumor volumes reached ≈160 mm³(mean±SD, 161.8±20.2 mm³) approximately eight days after inoculation, mice were treated with isotype control, an anti-PD-L1 antibody, an anti-TGF-β antibody, or a combination of an anti-PD-L1 antibody with an anti-TGF-β antibody. Tumors were measured two times per week for approximately eight weeks by caliper. When tumor volumes fell below 32 mm³(lowest limit of detection), they were considered CR (100% tumor growth inhibition). FIG. 4D shows the percentage of CR across 2-6 independent studies. ****p<0.0001 by one-way ANOVA, Sidak multiple comparisons test. FIG. 4E shows tumor growth curves for each individual mouse. The data are from one representative of six independent experiments with 10 mice/group. FIG. 4F shows the change in tumor volume compared with baseline (onset of treatment). Data are from one representative of 6 independent experiments with 10 mice/group.

FIG. 4G is a series of images showing the distribution of tumor-infiltrating lymphocytes (TILs) assessed by IHC and digital imaging seven days after the initiation of treatment as described in FIGS. 4D-4F. Brown indicates representative CD3 staining. Scale bar, 100 microns.

FIGS. 4H-4J are a series of graphs showing cytofluorimetric enumeration of TILs seven days after the initiation of treatment as described with respect to FIGS. 4D-4F. Abundance of total T-cells (FIG. 4H), CD8+ T-cells (FIG. 4I), and the mean fluorescence intensity (MFI) of granzyme B in CD8+ T-cells (FIG. 4J) are shown (N=15 for all groups, except anti-TGF-β antibody alone in which N=10; data from three combined experiments expressed as fold change relative to the isotype cell/mg average). *p<0.05; **p<0.01; ****p<0.0001 by one-way ANOVA, Sidak multiple comparisons test.

FIG. 4K is a graph showing TIL localization quantification at seven days after initiation of treatment as described in FIGS. 4D-4F. T-cells were stained by IHC (as in FIG. 4G) and their localization was digitally analyzed. Normalized mean distances of CD3 T-cells from the tumor periphery are shown as percentages (N=19-20; data from three combined experiments). ****p<0.0001 by Tukey HSD multiple comparison test.

FIG. 4L is a graph showing phosphoflow analysis of SMAD2/3 in tumors seven days after the initiation of treatment as described in FIGS. 4D-4F. MFI of phospho-SMAD2/3 among total cells, CD45−, and CD45+ cells is shown. Data are expressed as fold change (FC) relative to the isotype MFI average. 10 mice/group from two combined experiments. *, p<0.05; **, p<0.01, one-way ANOVA, Dunnett's multiple comparisons test, compared to isotype.

FIG. 4M is a graph showing quantification of VEGF-A protein in the plasma by ELISA seven days after the initiation of treatment as described in FIGS. 4D-4F. N=8 from one experiment. p=0.0194, one-way ANOVA, Dunnett's multiple comparisons test, compared to isotype.

FIG. 4N is a graph showing cytofluorimetric analysis of T-cells seven days after initiation of the treatment as described in FIGS. 4D-4F. The percentage of GzmB+ CD8 T-cells is shown. N=15 for all groups except for anti-TGF-β alone, in which N=10. These data are from three combined experiments expressed as fold change relative to the isotype cell/mg average. **, p<0.01 one-way ANOVA, Sidak's multiple comparisons test.

FIG. 4O is a graph showing distribution of TILs as assessed by immunohistochemistry and digital imaging seven days after the initiation of treatment as described in FIGS. 4D-4F. Brown indicates representative CD3 staining. Scale bar, 500 microns.

FIGS. 4P-4S are a series of graphs showing RNAseq analysis on whole tumors collected seven days after the initiation of treatment as described in FIGS. 4D-4F. Scores of Teff (FIG. 4P), Pan-F-TBRS (FIG. 4Q), T-cell and macrophage signatures (FIG. 4R), and the ratio of Teff to Pan-F-TBRS (FIG. 4S) in different treatment arms are shown. *p<0.05; **p<0.01; ***p<0.001.

FIG. 4T is a heatmap showing expression of Teff and cancer-associated fibroblast remodeling (CAF) genes after the initiation of treatment as described with respect to FIGS. 4D-4F. Therapeutic administration of anti-TGF-β in combination with anti-PD-L1 promoted T-cell infiltration and CAF remodeling, resulting in complete responses.

FIG. 4U is a series of graphs showing the expression of the indicated genes after the initiation of treatment as described with respect to FIGS. 4D-4F. Therapeutic administration of anti-TGF-β in combination with anti-PD-L1 resulted in increased expression of Teff genes including IFNG, GZMB, and ZAP70 (top) and decreased expression of CAF genes including LOXL2, TNC, and POSTN (bottom). RPKM, reads per kilobase per million mapped reads.

FIG. 5 is a Venn diagram showing overlap of the efficacy-evaluable patient populations with whole transcriptome (RNAseq), FMOne, cancer-immune phenotyping, and whole-exome sequencing (WES) data (n=326 for one or more of these assays). For gene expression analyses with respect to response, the complete RNAseq data set was used (n=298). For gene expression analyses in the context of TMB or immune phenotype, the intersect between RNAseq and FMOne (n=237) or immune phenotype (n=244) was used, respectively. For mutation analysis around immune phenotypes, the intersect between FMOne and immune phenotype was used (n=220). For associations between response or genes mutation status with TMB, the complete FMOne data set was used (n=251).

FIGS. 6A-6C are a series of graphs showing that high expression of the 6-gene signature (“6TBRS”) is associated with poor prognosis (FIG. 6A) and with lack of benefit from atezolizumab monotherapy (FIGS. 6B and 6C). FIG. 6A shows data from the TOGA colorectal cancer dataset for the 6-gene signature, TGFB1, TGFB2, and TGFB3. FIG. 6B shows data from the IMvigor210 UC dataset for the 6-gene signature, TGFB1, TGFB2, and TGFB3. FIG. 6C shows data from the POPLAR NSCLC dataset for TGFB1+TGFBR2 and the 6-gene signature.

FIG. 7A is a graph showing that expression of the 6-gene signature (also referred to herein as “curTBRS”) is associated with lack of response to atezolizumab.

FIG. 7B is a graph showing that high expression of the 6-gene signature is enriched in the CMS4 molecular subtype, a poor survival subgroup of CRC patients.

FIGS. 8A-8C are a series of graphs showing that high TMB and high PD-L1 IC scores are associated with improved OS benefit from atezolizumab therapy. FIG. 8A shows OS by PD-L1 status; FIG. 8B shows OS by TMB status; and FIG. 8C shows OS by combined PD-L1 IC and TMB status.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides diagnostic methods and uses, therapeutic methods and uses, and compositions for the treatment of cancer, including, but not limited to, a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer. The invention is based, at least in part, on the discovery that the expression level of one or more genes (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a sample obtained from an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) can be used as a biomarker (including, but not limited to, a predictive biomarker and/or a pharmacodynamic biomarker) in methods of identifying whether the individual may benefit or is likely to respond to treatment with an anti-cancer therapy that includes an immunotherapy (including, but not limited to, a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (including, but not limited to, a TGF-β antagonist, e.g., an anti-TGF-β antibody); selecting a therapy for treating the individual; optimizing therapeutic efficacy of a treatment that includes with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody); and/or monitoring the response of the individual to a treatment that includes with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).

I. Definitions

It is to be understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments. As used herein, the singular form “a,” “an,” and “the” includes plural references unless indicated otherwise.
The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
The term “biomarker” as used herein refers to an indicator, e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample. The biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and/or clinical features. In some embodiments, a biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA, and/or RNA), polynucleotide copy number alterations (e.g., DNA copy numbers), polypeptides, polypeptide and polynucleotide modifications (e.g. posttranslational modifications), carbohydrates, and/or glycolipid-based molecular markers.
Such biomarkers include, but are not limited to, TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1. In some embodiments, the biomarker is one or more biomarkers selected from the group consisting of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2. In other embodiments, the biomarker is one or more biomarkers selected from the group consisting of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. Expression of such a biomarker may be determined to be higher or lower in a sample obtained from a patient sensitive or responsive to a treatment (e.g., treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) than a reference level (including, e.g., the median expression level of the biomarker in a sample from a group/population of patients, e.g., patients having cancer, and being tested for responsiveness to a treatment; the median expression level of the biomarker in a sample from a group/population of patients, e.g., patients having cancer, and identified as not responding to a treatment; the level in a sample previously obtained from the individual at a prior time; or the level in a sample from a patient who received prior treatment (e.g., treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) in a primary tumor setting, and who now may be experiencing metastasis).
The terms “biomarker signature,” “signature,” “biomarker expression signature,” or “expression signature” are used interchangeably herein and refer to one or a combination of biomarkers whose expression is an indicator, e.g., predictive, diagnostic, and/or prognostic. The biomarker signature may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker signature is a “gene signature.” The term “gene signature” is used interchangeably with “gene expression signature” and refers to one or a combination of polynucleotides whose expression is an indicator, e.g., predictive, diagnostic, and/or prognostic. In some embodiments, the biomarker signature is a “protein signature.” The term “protein signature” is used interchangeably with “protein expression signature” and refers to one or a combination of polypeptides whose expression is an indicator, e.g., predictive, diagnostic, and/or prognostic. In some instances, the biomarker signature includes one or more biomarkers selected from the group consisting of TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1, which is also referred to herein as the “22-gene signature.” In some instances, the biomarker signature includes one or more biomarkers selected from the group consisting of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, which is also referred to herein as the “6-gene signature.” In other instances, the biomarker signature includes one or more biomarkers selected from the group consisting of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 which is also referred to herein as is the “Pan-F-TBRS.”
The term “TGFB1” as used herein, refers to any native TGFB1 (transforming growth factor beta 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TGFB1 as well as any form of TGFB1 that results from processing in the cell. The term also encompasses naturally occurring variants of TGFB1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human TGFB1 is shown under NCBI Reference Sequence: NM_000660.6 or in SEQ ID NO: 1. The amino acid sequence of an exemplary protein encoded by human TGFB1 is shown under UniProt Accession No. P01137 or in SEQ ID NO: 2.
The term “TGFBR2” as used herein, refers to any native TGFBR2 (transforming growth factor beta receptor 2; also known as TGFR-2 or TGFbeta-RII) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TGFBR2 as well as any form of TGFBR2 that results from processing in the cell. The term also encompasses naturally occurring variants of TGFBR2, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human TGFBR2 is shown under NCBI Reference Sequence: NM_001024847 or in SEQ ID NO: 3. The amino acid sequence of an exemplary protein encoded by human TGFBR2 is shown under UniProt Accession No. P37173 or in SEQ ID NO: 4.
The term “ACTA2” as used herein, refers to any native ACTA2 (alpha-actin-2, also known as aortic smooth muscle actin or alpha smooth muscle actin) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed ACTA2 as well as any form of ACTA2 that results from processing in the cell. The term also encompasses naturally occurring variants of ACTA2, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human ACTA2 is shown under NCBI Reference Sequence: NM_001141945 or in SEQ ID NO: 5. The amino acid sequence of an exemplary protein encoded by human ACTA2 is shown under UniProt Accession No. P62736 or in SEQ ID NO: 6.
The term “ACTG2” as used herein, refers to any native ACTG2 (actin, gamma-enteric smooth muscle, also known as actin, gamma 2) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed ACTG2 as well as any form of ACTG2 that results from processing in the cell. The term also encompasses naturally occurring variants of ACTG2, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human ACTG2 is shown under NCBI Reference Sequence: NM_001615 or in SEQ ID NO: 7. The amino acid sequence of an exemplary protein encoded by human ACTG2 is shown under UniProt Accession No. P63267 or in SEQ ID NO: 8.
The term “ADAM12” as used herein, refers to any native ADAM12 (a disintegrin and metalloproteinase domain-containing protein 12, also known as ADAM metallopeptidase domain 12, MCMP, CAR10, and MLTN) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed ADAM12 as well as any form of ADAM12 that results from processing in the cell. The term also encompasses naturally occurring variants of ADAM12, e.g., splice variants or allelic variants. Splice variants include ADAM12-L, which has a transmembrane region and ADAM12-S, a shorter variant, which is soluble and lacks the transmembrane and cytoplasmic domains. The nucleic acid sequence of an exemplary human ADAM12 is shown under NCBI Reference Sequence: NM_003474 or in SEQ ID NO: 9. The amino acid sequence of an exemplary protein encoded by human ADAM12 is shown under UniProt Accession No. O43184 or in SEQ ID NO: 10.
The term “ADAM19” as used herein, refers to any native ADAM19 (a disintegrin and metalloproteinase domain-containing protein 19; also known as MADDAM or meltrin beta) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed ADAM19 as well as any form of ADAM19 that results from processing in the cell. The term also encompasses naturally occurring variants of ADAM19, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human ADAM19 is shown under NCBI Reference Sequence: NM_033274 or in SEQ ID NO: 11. The amino acid sequence of an exemplary protein encoded by human ADAM19 is shown under UniProt Accession No. Q9H013 or in SEQ ID NO: 12.
The term “COMP” as used herein, refers to any native COMP (cartilage oligomeric matrix protein; also known as thrombospondin-5) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed COMP as well as any form of COMP that results from processing in the cell. The term also encompasses naturally occurring variants of COMP, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human COMP is shown under NCBI Reference Sequence: NM_000095 or in SEQ ID NO: 81. The amino acid sequence of an exemplary protein encoded by human COMP is shown under UniProt Accession No. P49747 or in SEQ ID NO: 82.
The term “CNN1” as used herein, refers to any native CNN1 (calponin 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CNN1 as well as any form of CNN1 that results from processing in the cell. The term also encompasses naturally occurring variants of CNN1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human CNN1 is shown under NCBI Reference Sequence: NM_001299 or in SEQ ID NO: 13. The amino acid sequence of an exemplary protein encoded by human CNN1 is shown under UniProt Accession No. P51911 or in SEQ ID NO: 14.
The term “COL4A1” as used herein, refers to any native COL4A1 (collagen, type IV, alpha I, also known as collagen alpha-1(IV) chain) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed COL4A1 as well as any form of COL4A1 that results from processing in the cell. The term also encompasses naturally occurring variants of COL4A1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human COL4A1 is shown under NCBI Reference Sequence: NM_001845 or in SEQ ID NO: 15. The amino acid sequence of an exemplary protein encoded by human COL4A1 is shown under UniProt Accession No. P02462 or in SEQ ID NO: 16.
The term “CTGF” as used herein, refers to any native CTGF (connective tissue growth factor, also known as CCN2 or IGFBP8) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CTGF as well as any form of CTGF that results from processing in the cell. The term also encompasses naturally occurring variants of CTGF, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human CTGF is shown under NCBI Reference Sequence: NM_001901 or in SEQ ID NO: 17. The amino acid sequence of an exemplary protein encoded by human CTGF is shown under UniProt Accession No. P29279 or in SEQ ID NO: 18.
The term “CTPS1” as used herein, refers to any native CTPS1 (CTP synthase 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CTPS1 as well as any form of CTPS1 that results from processing in the cell. The term also encompasses naturally occurring variants of CTPS1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human CTPS1 is shown under NCBI Reference Sequence: NM_001905 or in SEQ ID NO: 19. The amino acid sequence of an exemplary protein encoded by human CTPS1 is shown under UniProt Accession No. P17812 or in SEQ ID NO: 20.
The term “FAM101B” as used herein, refers to any native FAM101B (family with sequence similarity 101, member B, also known as filamin-interacting protein FAM101B, refilin B, or RFLNB) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed FAM101 B as well as any form of FAM101B that results from processing in the cell. The term also encompasses naturally occurring variants of FAM101B, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human FAM101B is shown under NCBI Reference Sequence: NM_182705 or in SEQ ID NO: 21. The amino acid sequence of an exemplary protein encoded by human FAM101B is shown under UniProt Accession No. Q8N5W9 or in SEQ ID NO: 22.
The term “FSTL3” as used herein, refers to any native FSTL3 (follistatin-related protein 3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed FSTL3 as well as any form of FSTL3 that results from processing in the cell. The term also encompasses naturally occurring variants of FSTL3, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human FSTL3 is shown under NCBI Reference Sequence: NM_005860 or in SEQ ID NO: 23. The amino acid sequence of an exemplary protein encoded by human FSTL3 is shown under UniProt Accession No. 095633 or in SEQ ID NO: 24.
The term “HSPB1” as used herein, refers to any native HSPB1 (heat shock protein beta-1, also known as heat shock protein 27 or HSP27) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed HSPB1 as well as any form of HSPB1 that results from processing in the cell. The term also encompasses naturally occurring variants of HSPB1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human HSPB1 is shown under NCBI Reference Sequence: NM_001540 or in SEQ ID NO: 25. The amino acid sequence of an exemplary protein encoded by human HSPB1 is shown under UniProt Accession No. P04792 or in SEQ ID NO: 26.
The term “IGFBP3” as used herein, refers to any native IGFBP3 (insulin-like growth factor-binding protein 3, also known as BP-53 or IBP3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed IGFBP3 as well as any form of IGFBP3 that results from processing in the cell. The term also encompasses naturally occurring variants of IGFBP3, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human IGFBP3 is shown under NCBI Reference Sequence: NM_001013398 or in SEQ ID NO: 27. The amino acid sequence of an exemplary protein encoded by human IGFBP3 is shown under UniProt Accession No. P17936 or in SEQ ID NO: 28.
The term “PXDC1” as used herein, refers to any native PXDC1 (PX domain-containing protein 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PXDC1 as well as any form of PXDC1 that results from processing in the cell. The term also encompasses naturally occurring variants of PXDC1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human PXDC1 is shown under NCBI Reference Sequence: NM_183373 or in SEQ ID NO: 29. The amino acid sequence of an exemplary protein encoded by human PXDC1 is shown under UniProt Accession No. Q5TGL8 or in SEQ ID NO: 30.
The term “SEMA7A” as used herein, refers to any native SEMA7A (semaphorin 7A, also known as CD108 or John-Milton-Hagen blood group antigen) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed SEMA7A as well as any form of SEMA7A that results from processing in the cell. The term also encompasses naturally occurring variants of SEMA7A, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human SEMA7A is shown under NCBI Reference Sequence: NM_003612 or in SEQ ID NO: 31. The amino acid sequence of an exemplary protein encoded by human SEMA7A is shown under UniProt Accession No. 075326 or in SEQ ID NO: 32.
The term “SH3PXD2A” as used herein, refers to any native SH3PXD2A (SH3 and PX domain-containing protein 2A) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed SH3PXD2A as well as any form of SH3PXD2A that results from processing in the cell. The term also encompasses naturally occurring variants of SH3PXD2A, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human SH3PXD2A is shown under NCBI Reference Sequence: NM_014631 or in SEQ ID NO: 33. The amino acid sequence of an exemplary protein encoded by human SH3PXD2A is shown under UniProt Accession No. Q5TCZ1 or in SEQ ID NO: 34.
The term “TAGLN” as used herein, refers to any native TAGLN (transgelin) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TAGLN as well as any form of TAGLN that results from processing in the cell. The term also encompasses naturally occurring variants of TAGLN, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human TAGLN is shown under NCBI Reference Sequence: NM_001001522 or in SEQ ID NO: 35. The amino acid sequence of an exemplary protein encoded by human TAGLN is shown under UniProt Accession No. Q01995 or in SEQ ID NO: 36.
The term “TGFBI” as used herein, refers to any native TGFBI (transforming growth factor, beta-induced) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TGFBI as well as any form of TGFBI that results from processing in the cell. The term also encompasses naturally occurring variants of TGFBI, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human TGFBI is shown under NCBI Reference Sequence: NM_000358 or in SEQ ID NO: 37. The amino acid sequence of an exemplary protein encoded by human TGFBI is shown under UniProt Accession No. Q15582 or in SEQ ID NO: 38.
The term “TNS1” as used herein, refers to any native TNS1 (tensin-1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TNS1 as well as any form of TNS1 that results from processing in the cell. The term also encompasses naturally occurring variants of TNS1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human TNS1 is shown under NCBI Reference Sequence: NM_022648 or in SEQ ID NO: 39. The amino acid sequence of an exemplary protein encoded by human TNS1 is shown under UniProt Accession No. Q9HBL0 or in SEQ ID NO: 40.
The term “TPM1” as used herein, refers to any native TPM1 (tropomyosin alpha-1 chain) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TPM1 as well as any form of TPM1 that results from processing in the cell. The term also encompasses naturally occurring variants of TPM1, e.g., splice variants or allelic variants. The human TPM1 gene encodes at least 10 variants via alternative splicing and/or the use of two promoters. The nucleic acid sequence of an exemplary human TPM1 is shown under NCBI Reference Sequence: NM_001018005.1 or in SEQ ID NO: 41. The amino acid sequence of an exemplary protein encoded by human TPM1 is shown under UniProt Accession No. P09493 or in SEQ ID NO: 42. Sequences of human TPM1 protein isoforms are shown under UniProt Accession Nos. P09493-1, P09493-2, P09493-3, P09493-4, P09493-5, P09493-6, P09493-7, P09493-8, P09493-9, and P09493-10. In some embodiments, the human TPM1 protein has the amino acid sequence shown under UniProt Accession No. P09493-1.
The term “CD8A” as used herein, refers to any native CD8A from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CD8A as well as any form of CD8A that results from processing in the cell. The term also encompasses naturally occurring variants of CD8A, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human CD8A is shown under EMBL Accession No. M12824 or in SEQ ID NO: 43. The amino acid sequence of an exemplary protein encoded by human CD8A is shown under UniProt Accession No. P01732-1 or in SEQ ID NO: 44.
The term “CXCL9” as used herein, refers to any native CXCL9 (chemokine (C-X-C motif) ligand 9) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CXCL9 as well as any form of CXCL9 that results from processing in the cell. The term also encompasses naturally occurring variants of CXCL9, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human CXCL9 is set forth in SEQ ID NO: 45. The amino acid sequence of an exemplary protein encoded by human CXCL9 is shown in SEQ ID NO: 46.
The term “CXCL10” as used herein, refers to any native CXCL10 (chemokine (C-X-C motif) ligand from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CXCL10 as well as any form of CXCL10 that results from processing in the cell. The term also encompasses naturally occurring variants of CXCL10, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human CXCL10 is set forth in SEQ ID NO: 47. The amino acid sequence of an exemplary protein encoded by human CXCL10 is shown in SEQ ID NO: 48.
The term “GZMA” as used herein, refers to any native GZMA (granzyme A) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed GZMA as well as any form of GZMA that results from processing in the cell. The term also encompasses naturally occurring variants of GZMA, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human GZMA is set forth in SEQ ID NO: 49. The amino acid sequence of an exemplary protein encoded by human GZMA is shown in SEQ ID NO: 50.
The term “GZMB” as used herein, refers to any native GZMB (granzyme B) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed GZMB as well as any form of GZMB that results from processing in the cell. The term also encompasses naturally occurring variants of GZMB, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human GZMB is set forth in SEQ ID NO: 51. The amino acid sequence of an exemplary protein encoded by human GZMB is shown in SEQ ID NO: 52.
The term “PRF1” as used herein, refers to any native PRF1 (perforin 1; also known as pore forming protein) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PRF1 as well as any form of PRF1 that results from processing in the cell. The term also encompasses naturally occurring variants of PRF1, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human RF1 is set forth in SEQ ID NO: 53. The amino acid sequence of an exemplary protein encoded by human PRF1 is shown in SEQ ID NO: 54.
The term “IFNG” as used herein, refers to any native IFNG (interferon, gamma; also referred to herein as IFN-γ) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed IFNG as well as any form of IFNG that results from processing in the cell. The term also encompasses naturally occurring variants of IFNG, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human IFNG is set forth in SEQ ID NO: 55. The amino acid sequence of an exemplary protein encoded by human IFNG is shown in SEQ ID NO: 56.
The term “TBX21” as used herein, refers to any native TBX21 (T-box transcription factor 21) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed TBX21 as well as any form of TBX21 that results from processing in the cell. The term also encompasses naturally occurring variants of TBX21, e.g., splice variants or allelic variants. The nucleic acid sequence of an exemplary human TBX21 is shown under NCBI Reference Sequence: NM_013351.1 or in SEQ ID NO: 57. The amino acid sequence of an exemplary protein encoded by human TBX21 is shown under UniProt Accession No. Q9UL17 or in SEQ ID NO: 58.
The terms “Programmed Death Ligand 1” and “PD-L1” refer herein to a native sequence PD-L1 polypeptide, polypeptide variants, and fragments of a native sequence polypeptide and polypeptide variants (which are further defined herein). The PD-L1 polypeptide described herein may be that which is isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
A “native sequence PD-L1 polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PD-L1 polypeptide derived from nature. The term encompasses “full-length,” unprocessed PD-L1 as well as any form of PD-L1 that results from processing in the cell. The term also encompasses naturally occurring variants of PD-L1, e.g., splice variants or allelic variants. For example, a native sequence PD-L1 polypeptide may be PD-L1 isoform 1 (also known as PD-L1I; see, e.g., UniProt Accession No. Q9NZQ7-1), PD-L1 isoform 2 (also known as PD-L1II; see, e.g., UniProt Accession No. Q9NZQ7-2), or PD-L1 isoform 3 (see, e.g., UniProt Accession No. Q9NZQ7-3). The nucleic acid sequence of an exemplary human PD-L1 is shown under NCBI Reference Sequence: NM_014143 or in SEQ ID NO: 59. The amino acid sequence of an exemplary protein encoded by human PD-L1 is shown under UniProt Accession No. Q9NZQ7 or in SEQ ID NO: 60.
A “PD-L1 polypeptide variant,” or variations thereof, means a PD-L1 polypeptide, generally an active PD-L1 polypeptide, as defined herein having at least about 80% amino acid sequence identity with any of the native sequence PD-L1 polypeptide sequences as disclosed herein. Such PD-L1 polypeptide variants include, for instance, PD-L1 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of a native amino acid sequence. Ordinarily, a PD-L1 polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a native sequence PD-L1 polypeptide sequence as disclosed herein. Ordinarily, PD-L1 variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, or 289 amino acids in length, or more. Optionally, PD-L1 variant polypeptides will have no more than one conservative amino acid substitution as compared to a native PD-L1 polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to the native PD-L1 polypeptide sequence.
The term “detecting” is used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels. Detecting may be direct or indirect.
The terms “level of expression” or “expression level” in general are used interchangeably and generally refer to the amount of a biomarker in a biological sample. “Expression” generally refers to the process by which information (e.g., gene-encoded and/or epigenetic information) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). An expression level for more than one gene of interest may be determined by aggregation methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the median or mean of all the expression levels of the genes of interest. Before aggregation, the expression level of each gene of interest may be normalized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, normalized to the expression level of one or more housekeeping genes, or normalized to a total library size, or normalized to the median or mean expression level value across all genes measured. In some instances, before aggregation across multiple genes of interest, the normalized expression level of each gene of interest may be standardized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the Z-score of the normalized expression level of each gene of interest.
The term “sample,” as used herein, refers to a composition that is obtained or derived from a patient and/or individual of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example, based on physical, biochemical, chemical, and/or physiological characteristics. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof.
A sample or cell that “expresses” a protein of interest is one in which mRNA encoding the protein, or the protein, including fragments thereof, is determined to be present in the sample or cell.
As used herein, the terms “reference expression level” and “reference level” are used interchangeably to refer to an expression level against which another expression level, e.g., the expression level of one or more genes described herein (e.g., any gene set forth in Table 1 or any combination thereof (e.g., any combination set forth in any one of Tables 2-5) in a sample from an individual is compared, e.g., to make a predictive, diagnostic, prognostic, and/or therapeutic determination. For example, the reference expression level may be derived from expression levels in a reference population (e.g., the median expression level in a reference population, e.g., a population of patients having a cancer), a reference sample, and/or a pre-assigned value (e.g., a cut-off value which was previously determined to significantly (e.g., statistically significantly) separate a first subset of individuals who have been treated with an anti-cancer therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) in a reference population and a second subset of individuals who have been treated with a different anti-cancer therapy (or who have not been treated with the anti-cancer therapy) in the same reference population based on a significant difference between an individual's responsiveness to treatment with the anti-cancer therapy and an individual's responsiveness to treatment with the different anti-cancer therapy above the cut-off value and/or below the cut-off value). In some embodiments, the cut-off value may be the median or mean expression level in the reference population. In other embodiments, the reference level may be the top 40%, the top 30%, the top 20%, the top 10%, the top 5%, or the top 1% of the expression level in the reference population. In particular embodiments, the cut-off value may be the median expression level in the reference population. It will be appreciated by one skilled in the art that the numerical value for the reference expression level may vary depending on the indication or disorder (e.g., a cancer (e.g., a bladder cancer, a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer)), the methodology used to detect expression levels (e.g., RNAseq or RT-qPCR), and/or the specific combinations of genes examined (e.g., any combination of the genes set forth in Table 1; or any one of the combinations of genes listed in Tables 2-6).
Expression “above” a level (e.g., above a reference level), “increased expression,” “increased expression level,” “increased levels,” “elevated expression,” “elevated expression levels,” or “elevated levels” refers to an increased expression or increased levels of a biomarker in an individual relative to the expression level of the biomarker in a control (e.g., an individual or individuals who are not suffering from the disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or the level of a biomarker in a sample obtained prior to administration of a therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)), or relative to a reference level (e.g., the median expression level of the biomarker in samples from a group/population of patients, e.g., patients having cancer who are being tested for responsiveness to an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody); the median expression level of the biomarker in samples from a group/population of patients, e.g., patients having cancer who have been identified as not responding to an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody); or the level in a sample previously obtained from the individual at a prior time).
Expression “below” a level (e.g., below a reference level), “decreased expression,” “decreased expression level,” “decreased levels,” “reduced expression,” “reduced expression levels,” or “reduced levels” refers to a decrease expression or decreased levels of a biomarker in an individual relative to the expression level of the biomarker in a control (e.g., an individual or individuals who are not suffering from the disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or the level of a biomarker in a sample obtained prior to administration of a therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)), or relative to a reference level (e.g., the median expression level of the biomarker in samples from a group/population of patients, e.g., patients having cancer who are being tested for responsiveness to an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody); the median expression level of the biomarker in samples from a group/population of patients, e.g., patients having cancer who have been identified as not responding to an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody); or the level in a sample previously obtained from the individual at a prior time). In some embodiments, reduced expression is little or no expression.
A “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue,” as used herein, refers to a sample, cell, tissue, or standard that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same patient or individual. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and/or cell of the body of the same patient or individual. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the patient or individual. In even another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the patient or individual. In another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a patient prior to administration of a therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)).
The phrase “based on” when used herein means that the information about one or more biomarkers is used to inform a treatment decision, information provided on a package insert, or marketing/promotional guidance, and the like.
The term “housekeeping biomarker” refers to a biomarker or group of biomarkers (e.g., polynucleotides and/or polypeptides) which are typically similarly present in all cell types. In some embodiments, the housekeeping biomarker is a “housekeeping gene.” A “housekeeping gene” refers herein to a gene or group of genes which encode proteins whose activities are essential for the maintenance of cell function and which are typically similarly present in all cell types.
By “correlate” or “correlating” is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed. With respect to the embodiment of polypeptide analysis or protocol, one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. With respect to the embodiment of polynucleotide analysis or protocol, one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.
“Amplification,” as used herein generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” mean at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and/or sequence errors that occur during amplification.
The term “multiplex-PCR” refers to a single PCR reaction carried out on nucleic acid obtained from a single source (e.g., an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.
The technique of “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5′ terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263 (1987) and Erlich, ed., PCR Technology, (Stockton Press, NY, 1989). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid.
“Quantitative real-time polymerase chain reaction” or “qRT-PCR” refers to a form of PCR wherein the amount of PCR product is measured at each step in a PCR reaction. This technique has been described in various publications including, for example, Cronin et al., Am. J. Pathol. 164(1):35-42 (2004) and Ma et al., Cancer Cell 5:607-616 (2004).
The term “microarray” refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.
The term “RNAseq,” also called “RNA-seq,” and “Whole Transcriptome Shotgun Sequencing (WTSS),” refers to the use of high-throughput sequencing technologies to sequence and/or quantify cDNA to obtain information about a sample's RNA content. Publications describing RNAseq include: Wang et al. Nature Reviews Genetics 10(1):57-63, 2009; Ryan et al. Bio Techniques 45(1):81-94, 2008; and Maher et al. Nature 458(7234):97-101, 2009.
The term “diagnosis” is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer)). In some embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC). For example, “diagnosis” may refer to identification of a particular type of cancer. “Diagnosis” may also refer to the classification of a particular subtype of cancer, for instance, by histopathological criteria, or by molecular features (e.g., a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).
A “tumor-infiltrating immune cell,” as used herein, refers to any immune cell present in a tumor or a sample thereof. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, other tumor stroma cells (e.g., fibroblasts), or any combination thereof. Such tumor-infiltrating immune cells can be, for example, T lymphocytes (such as CD8⁺T lymphocytes and/or CD4⁺T lymphocytes), B lymphocytes, or other bone marrow-lineage cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages (e.g., CD68⁺/CD163⁺ macrophages), dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer (NK) cells.
A “tumor cell” as used herein, refers to any tumor cell present in a tumor or a sample thereof. Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and/or described herein.
As used herein, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, more preferably a mammal (including such non-human animals as, for example, cats, dogs, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates) for which treatment is desired. In particular embodiments, the patient herein is a human. The patient may be a “cancer patient,” i.e., one who is suffering from cancer (e.g., bladder cancer, kidney cancer, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, colorectal cancer, or breast cancer), or at risk for suffering from cancer, or suffering from one or more symptoms of cancer.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma (UC), including metastatic UC (mUC); muscle-invasive bladder cancer (MIBC), and non-muscle-invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); lung cancer, including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung; cancer of the urinary tract; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC), which are estrogen receptors (ER−), progesterone receptors (PR−), and HER2 (HER2−) negative); prostate cancer, such as castration-resistant prostate cancer (CRPC); cancer of the peritoneum; hepatocellular cancer; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., hepatocellular carcinoma (HCC)); hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine carcinoma; salivary gland carcinoma; prostate cancer; vulval cancer; thyroid cancer; hepatic carcinoma; anal carcinoma; penile carcinoma; melanoma, including superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, and nodular melanomas; multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myelogenous leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain cancer, head and neck cancer, and associated metastases. In some embodiments, the cancer is bladder cancer (e.g., UC, e.g., mUC).
By “early stage cancer” or “early stage tumor” is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, I, or II cancer.
An “advanced” cancer is one which has spread outside the site or organ of origin, either by local invasion or metastasis.
A “refractory” cancer is one which progresses even though an anti-tumor agent, such as a chemotherapeutic agent, is being administered to the cancer patient. An example of a refractory cancer is one which is platinum refractory.
A “recurrent” cancer is one which has regrown, either at the initial site or at a distant site, after a response to initial therapy.
The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.
The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as referred to herein.
The term “immune inflamed tumor,” as used herein, refers to a tumor (e.g., a solid tumor) characterized by CD8+ T-cell infiltration and PD-L1 expression. See, e.g., Herbst et al. Nature 515:563-567, 2014 and Hegde et al. Clin. Canc. Res. 22: 1865-1874, 2016. In some embodiments, a tumor is categorized as an immune inflamed tumor if CD8+ cells are observed in direct contact with malignant epithelial cells, either in the form of spillover of stromal infiltrates into tumor cell aggregates or of diffuse infiltration of CD8+ cells in aggregates or sheets of tumor cells.
The term “immune excluded tumor,” as used herein, refers to a tumor (e.g., a solid tumor) characterized by accumulation of T-cells in the extracellular matrix-rich stroma. See, e.g., Herbst et al. Nature 515:563-567, 2014 and Hegde et al. Clin. Canc. Res. 22: 1865-1874, 2016. In immune excluded tumors, the majority of T-cells migrate along aligned collagen and fibronectin fibers that run circumferentially around the tumor. See, e.g., Salmon et al. J. Clin. Invest. 122:899-910, 2012. In some embodiments, a tumor is categorized as an immune excluded tumor if CD8+ cells are observed substantially or exclusively in stroma immediately adjacent to or within a main tumor mass.
The term “immune desert tumor,” as used herein, refers to a tumor (e.g., a solid tumor) with a paucity of infiltrating lymphocytes within the tumor or surrounding stroma. See, e.g., Herbst et al. Nature 515:563-567, 2014 and Hegde et al. Clin. Canc. Res. 22: 1865-1874, 2016. In some embodiments, a tumor is categorized as an immune desert tumor if the prevalence of CD8+ cells is low (e.g., less than about 10 CD8+ cells (e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 CD8+ cells) in an area of tumor and tumor-associated stroma at a magnification of about 200×, for example, as calculated as the average of 10 representative fields of view).
A “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, treatment is with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody). In some embodiments, antibodies (e.g., anti-PD-L1 antibodies, anti-PD-1 antibodies, and/or anti-TGF-β antibodies) are used to delay development of a disease or to slow the progression of a disease or disorder (e.g., a cancer (e.g., a bladder cancer, a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer)).
As used herein, “administering” is meant a method of giving a dosage of a compound (e.g., an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) to a patient. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, intravitreally (e.g., by intravitreal injection), by eye drop, orally, topically, transdermally, parenterally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).
A “therapeutically effective amount” refers to an amount of a therapeutic agent to treat or prevent a disease or disorder (e.g., a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer)) in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival (e.g., overall survival or progression-free survival), time to disease progression (TTP), response rates (e.g., complete response (CR) and partial response (PR)), duration of response, and/or quality of life.
The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time. Accordingly, concurrent administration includes a dosing regimen when the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s). For example, in some embodiments, a VEGF antagonist and a PD-L1 axis binding antagonist may be administered concurrently.
By “reduce or inhibit” is meant the ability to cause an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer, for example, to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.
A “loading” dose herein generally comprises an initial dose of a therapeutic agent administered to a patient, and is followed by one or more maintenance dose(s) thereof. Generally, a single loading dose is administered, but multiple loading doses are contemplated herein. Usually, the amount of loading dose(s) administered exceeds the amount of the maintenance dose(s) administered and/or the loading dose(s) are administered more frequently than the maintenance dose(s), so as to achieve the desired steady-state concentration of the therapeutic agent earlier than can be achieved with the maintenance dose(s).
A “maintenance” dose or “extended” dose herein refers to one or more doses of a therapeutic agent administered to the patient over a treatment period. Usually, the maintenance doses are administered at spaced treatment intervals, such as approximately every week, approximately every 2 weeks, approximately every 3 weeks, or approximately every 4 weeks.
“Response to a treatment,” “responsiveness to treatment,” or “benefit from a treatment” can be assessed using any endpoint indicating a benefit to the individual, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., cancer progression), including slowing down and complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down or complete stopping) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e. reduction, slowing down or complete stopping) of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer); (6) increase or extend in the length of survival, including overall survival (OS HR<1) and progression free survival (PFS HR<1); and/or (9) decreased mortality at a given point of time following treatment (e.g., treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)).
An “objective response” refers to a measurable response, including complete response (CR) or partial response (PR). In some embodiments, the “objective response rate (ORR)” refers to the sum of complete response (CR) rate and partial response (PR) rate.
By “complete response” or “CR” is intended the disappearance of all signs of cancer (e.g., disappearance of all target lesions) in response to treatment. This does not always mean the cancer has been cured.
As used herein, “partial response” or “PR” refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment. For example, in some embodiments, PR refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD.
“Sustained response” refers to the sustained effect on reducing tumor growth after cessation of a treatment. For example, the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase. In some embodiments, the sustained response has a duration at least the same as the treatment duration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatment duration, or longer.
As used herein, “stable disease” or “SD” refers to neither sufficient shrinkage of target lesions to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started.
As used herein, “progressive disease” or “PD” refers to at least a 20% increase in the SLD of target lesions, taking as reference the smallest SLD recorded since the treatment started or the presence of one or more new lesions.
The term “survival” refers to the patient remaining alive, and includes overall survival as well as progression-free survival.
As used herein, “progression-free survival” or “PFS” refers to the length of time during and after treatment during which the disease being treated (e.g., cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer)) does not progress or get worse. Progression-free survival may include the amount of time individuals have experienced a complete response or a partial response, as well as the amount of time individuals have experienced stable disease.
As used herein, “overall survival” or “OS” refers to the percentage of subjects in a group who are likely to be alive after a particular duration of time (e.g., 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, years, 15 years, 20 years, or more than 20 years from the time of diagnosis or treatment).
By “extending survival” is meant increasing overall or progression-free survival in a treated patient relative to an untreated patient (i.e. relative to a patient not treated with the medicament), or relative to a patient who does not express a biomarker at the designated level, and/or relative to a patient treated with an approved anti-tumor agent (e.g., an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)).
As used herein, “hazard ratio” or “HR” is a statistical definition for rates of events. For the purpose of the invention, hazard ratio is defined as representing the probability of an event (e.g., PFS or OS) in the experimental (e.g., treatment) group/arm divided by the probability of an event in the control group/arm at any specific point in time. An HR with a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the “treatment” and “control” groups; a value greater than 1 indicates that the risk is greater in the treatment group relative to the control group; and a value less than 1 indicates that the risk is greater in the control group relative to the treatment group. “Hazard ratio” in progression-free survival analysis (i.e., PFS HR) is a summary of the difference between two progression-free survival curves, representing the reduction in the risk of death on treatment compared to control, over a period of follow-up. “Hazard ratio” in overall survival analysis (i.e., OS HR) is a summary of the difference between two overall survival curves, representing the reduction in the risk of death on treatment compared to control, over a period of follow-up.
The term “anti-cancer therapy” refers to a therapy useful in treating cancer. Examples of anti-cancer therapeutic agents include, but are limited to, cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, for example, anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g., GLEEVEC™ (imatinib mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: PDGFR-6, BlγS, APRIL, BCMA receptor(s), TRAIL/Apo2, other bioactive and organic chemical agents, and the like. Combinations thereof are also included in the invention.
“Active” or “activity” for the purposes herein refer to form(s) of a polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring polypeptide, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by a native or naturally-occurring polypeptide other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring polypeptide and an “immunological” activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring polypeptide.
The term “antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide. In a similar manner, the term “agonist” is used in the broadest sense and includes any molecule that mimics a biological activity of a native polypeptide. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, and the like. Methods for identifying agonists or antagonists of a polypeptide may comprise contacting a polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide.
The term “immunotherapy” refers the use of a therapeutic agent that modulates an immune response. An immunotherapy may be an activating immunotherapy or a suppressing immunotherapy. The term “activating immunotherapy” refers to the use of a therapeutic agent that induces, enhances, or promotes an immune response, including, e.g., a T cell response. The term “suppressing immunotherapy” refers to the use of a therapeutic agent that interferes with, suppresses, or inhibits an immune response, including, e.g., a T cell response.
The term “PD-L1 axis binding antagonist” refers to a molecule that inhibits the interaction of a PD-L1 axis binding partner with one or more of its binding partners, so as to remove T cell dysfunction resulting from signaling on the PD-1 signaling axis, with a result being restored or enhanced T cell function. As used herein, a PD-L1 axis binding antagonist includes a PD-L1 binding antagonist and a PD-1 binding antagonist as well as molecules that interfere with the interaction between PD-L1 and PD-1 (e.g., a PD-L2-Fc fusion).
The term “PD-L1 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1 or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners. In a specific aspect, the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, the PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 or B7-1. In one embodiment, a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L1 so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition). In some embodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific embodiment, the anti-PD-L1 antibody is atezolizumab (CAS Registry Number: 1422185-06-5), also known as MPDL3280A, and described herein. In another specific embodiment, the anti-PD-L1 antibody is YW243.55.S70, described herein. In another specific embodiment, the anti-PD-L1 antibody is MDX-1105, described herein. In still another specific aspect, the anti-PD-L1 antibody is MED14736 (durvalumab), described herein. In still another specific aspect, the anti-PD-L1 antibody is MSB0010718C (avelumab), described herein.
As used herein, a “PD-1 binding antagonist” is a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and/or PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include anti PD-1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces the negative signal mediated by or through cell surface proteins expressed on T lymphocytes, and other cells, mediated signaling through PD-1 or PD-L1 so as render a dysfunctional T cell less dysfunctional. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, a PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific aspect, a PD-1 binding antagonist is CT-011 (pidilizumab). In another specific aspect, a PD-1 binding antagonist is MEDI-0680 (AMP-514). In another specific aspect, a PD-1 binding antagonist is PDR001. In another specific aspect, a PD-1 binding antagonist is REGN2810. In another specific aspect, a PD-1 binding antagonist is BGB-108. In another specific aspect, a PD-1 binding antagonist is AMP-224.
The terms “anti-PD-L1 antibody” and “an antibody that binds to PD-L1” refer to an antibody that is capable of binding PD-L1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-L1. In one embodiment, the extent of binding of an anti-PD-L1 antibody to an unrelated, non-PD-L1 protein is less than about 10% of the binding of the antibody to PD-L1 as measured, for example, by a RIA. In certain embodiments, an anti-PD-L1 antibody binds to an epitope of PD-L1 that is conserved among PD-L1 from different species.
The terms “anti-PD-1 antibody” and “an antibody that binds to PD-1” refer to an antibody that is capable of binding PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-1. In one embodiment, the extent of binding of an anti-PD-1 antibody to an unrelated, non-PD-1 protein is less than about 10% of the binding of the antibody to PD-1 as measured, for example, by a RIA. In certain embodiments, an anti-PD-1 antibody binds to an epitope of PD-1 that is conserved among PD-1 from different species.
A “suppressive stromal antagonist” as defined herein is any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity and/or function of a gene or gene product associated with stroma (e.g, tumor-associated stroma). In some embodiments, the suppressive stromal antagonist partially or fully blocks, inhibits, or neutralizes a biological activity and/or function of a gene or gene product associated with fibrotic tumors. In some embodiments, treatment with a suppressive stromal antagonist results in the reduction of stroma thereby resulting in an increase activity of an immunotherapy; for example, by increasing the ability of activating immune cells (e.g., proinflammatory cells) to infiltrate a fibrotic tissue (e.g., a fibrotic tumor). Targets for stromal gene antagonists are known in the art; for example, see Turley et al., Nature Reviews Immunology 15:669-682, 2015 and Rosenbloom et al., Biochimica et Biophysica Acta 1832:1088-1103, 2013. In some embodiments, the suppressive stromal antagonist is a transforming growth factor beta (TGF-β), podoplanin (PDPN), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), SMAD, anaplastic lymphoma kinase (ALK), connective tissue growth factor (CTGF/CCN2), endothelial-1 (ET-1), AP-1, interleukin (IL)-13, lysyl oxidase homolog 2 (LOXL2), endoglin (CD105), fibroblast activation protein (FAP), vascular cell adhesion protein 1 (CD106), thymocyte antigen 1 (THY1), beta 1 integrin (CD29), platelet-derived growth factor (PDGF), PDGF receptor A (PDGFRα), PDGF receptor B (PDGFRβ), vimentin, smooth muscle actin alpha (ACTA2), desmin, endosialin (CD248), or S100 calcium-binding protein A4 (S100A4) antagonist.
A “TGF-β antagonist” as defined herein is any molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of TGF-β with one or more of its interaction partners, such as a TGF-β cellular receptor. In some embodiments, a “TGF-β binding antagonist” is a molecule that inhibits the binding of TGF-β to its binding partners. In some embodiments, the TGF-β antagonist inhibits the activation of TGF-β. In some embodiments, the TGF-β antagonist includes an anti-TGF-β antibody, antigen binding fragments thereof, an immunoadhesin, a fusion protein, an oligopeptide, and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of TGF-β with one or more of its interaction partners. In some embodiments, the TGF-β antagonist is a polypeptide, a small molecule, or a nucleic acid. In some embodiments, the TGF-β antagonist (e.g., the TGF-β binding antagonist) inhibits TGF-β1, TGF-β2, and/or TGF-β3. In some embodiments, the TGF-β antagonist (e.g., the TGF-β binding antagonist) inhibits TGF-β receptor-1 (TGFBR1), TGF-β receptor-2 (TGFBR2), and/or TGF-β receptor-3 (TGFBR3).
The terms “anti-TGF-β antibody” and “an antibody that binds to TGF-β” refer to an antibody that is capable of binding TGF-β with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting TGF-β. In one embodiment, the extent of binding of an anti-TGF-β antibody to an unrelated, non-TGF-β protein is less than about 10% of the binding of the antibody to TGF-β as measured, for example, by a RIA. In certain embodiments, an anti-TGF-β antibody binds to an epitope of TGF-β that is conserved among TGF-β from different species. In some embodiments, the anti-TGF-β antibody inhibits TGF-β1, TGF-β2, and/or TGF-β3. In some embodiments, the anti-TGF-β antibody inhibits TGF-β1, TGF-β2, and TGF-β3. In some embodiments, the anti-TGF-β antibody is a pan-specific anti-TGF-β antibody. In some embodiments, the anti-TGF-β antibody may be any anti-TGF-β antibody disclosed in, for example, U.S. Pat. No. 5,571,714 or in International Patent Application Nos. WO 92/00330, WO 92/08480, WO 95/26203, WO 97/13844, WO 00/066631, WO 05/097832, WO 06/086469, WO 05/010049, WO 06/116002, WO 07/076391, WO 12/167143, WO 13/134365, WO 14/164709, or WO 16/201282, each of which is incorporated herein by reference in its entirety. In particular embodiments, the anti-TGF-β antibody is fresolimumab, metelimumab, lerdelimumab, 1 D11, 2G7, or a derivative thereof.
An “angiogenesis inhibitor” or “anti-angiogenesis agent” refers to a small molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly. It should be understood that the anti-angiogenesis agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor. For example, an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined above, e.g., antibodies to VEGF-A or the VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as GLEEVEC™ (imatinib mesylate). Anti-angiogenesis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, for example, Klagsbrun and D'Amore, Annu. Rev. Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003) and, Sato Int. J. Clin. Oncol, 8:200-206 (2003).
The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², and radioactive isotopes of Lu), chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof (e.g., nucleolytic enzymes), antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof, and the various antitumor or anticancer agents disclosed below. A tumoricidal agent causes destruction of tumor cells.
A “chemotherapeutic agent” includes chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Pfizer), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredepa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductases including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (Angew. Chem. Intl. Ed. Engl. 33:183-186, 1994); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, idoxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifene citrate); aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all transretinoic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); protein kinase inhibitors; lipid kinase inhibitors; antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories), which is a recombinant, exclusively human-sequence, full-length IgG1λ antibody genetically modified to recognize interleukin-12 p40 protein.
Chemotherapeutic agents also include “EGFR inhibitors,” which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an “EGFR antagonist.” Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in U.S. Pat. No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF or panitumumab (see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH). EGFR antagonists include small molecules such as compounds described in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 6,002,008, and 5,747,498, as well as the following PCT publications: WO98/14451, WO98/50038, WO99/09016, and WO99/24037. Particular small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSI Pharmaceuticals); PD 183805 (011033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3′-Chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3 fluorophenyl) methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).
Chemotherapeutic agents also include “tyrosine kinase inhibitors” including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or as described in any of the following patent publications: U.S. Pat. No. 5,804,396, WO 1999/09016, WO 1998/43960, WO 1997/38983, WO 1999/06378, WO 1999/06396, WO 1996/30347, WO 1996/33978, WO 1996/3397, and WO 1996/33980.
Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, all-trans retinoic acid (ATRA), valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.
The term “prodrug” as used herein refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. See, for example, Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, 6-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in this invention include, but are not limited to, those chemotherapeutic agents described above.
A “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth and/or proliferation of a cell (e.g., a cell whose growth is dependent on PD-L1 expression) either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as the anthracycline antibiotic doxorubicin ((8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione), epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in “The Molecular Basis of Cancer,” Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
By “radiation therapy” is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one-time administration and typical dosages range from 10 to 200 units (Grays) per day.
The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a patient to which the formulation would be administered.
A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a patient. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings concerning the use of such therapeutic products.
A “sterile” formulation is aseptic or free from all living microorganisms and their spores.
An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.
The term “small molecule” refers to any molecule with a molecular weight of about 2000 daltons or less, preferably of about 500 daltons or less.
The word “label” when used herein refers to a compound or composition that is conjugated or fused directly or indirectly to a reagent such as a polynucleotide probe or an antibody and facilitates detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, half-antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term “immunoglobulin” (Ig) is used interchangeably with antibody herein.
“Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
An “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic, and/or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.
A “blocking” antibody or an antibody “antagonist” is one which inhibits or reduces biological activity of the antigen it binds. For example, a PD-L1-specific antagonist antibody binds PD-L1 and decreases, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2. Preferred blocking antibodies or antagonist antibodies completely inhibit the biological activity of the antigen.
Unless indicated otherwise, the expression “multivalent antibody” is used throughout this specification to denote an antibody comprising three or more antigen binding sites. The multivalent antibody is preferably engineered to have the three or more antigen binding sites and is generally not a native sequence IgM or IgA antibody.
The “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“κ”) and lambda (“λ”), based on the amino acid sequences of their constant domains.
The term “constant domain” refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CH1, CH2, and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH.” The variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The variable or “V” domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The term “hypervariable region” or “HVR” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from, for example, around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about residues 26-35 (H1), 49-65 (H2) and 95-102 (H3) in the VH (in one embodiment, H1 is around about residues 31-35); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a “hypervariable loop” (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the VL, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the VH; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987). The variable domains of native heavy and light chains each comprise four FRs, largely adopting a beta-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).
An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.
The term “hypervariable region,” “HVR,” or “HV,” as used herein, refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
A number of HVR delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The “contact” HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.


Loop	Kabat	AbM	Chothia	Contact

L1	L24-L34	L24-L34	L26-L32	L30-L36
L2	L50-L56	L50-L56	L50-L52	L46-L55
L3	L89-L97	L89-L97	L91-L96	L89-L96
H1	H31-H35b	H26-H35b	H26-H32	H30-H35b (Kabat
				Numbering)
H1	H31-H35	H26-H35	H26-H32	H30-H35 (Chothia
				Numbering)
H2	H50-H65	H50-H58	H53-H55	H47-H58
H3	H95-H102	H95-H102	H96-H101	H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.
“Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.
A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one embodiment, for the VH, the subgroup is subgroup III as in Kabat et al., supra.
The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system. Unless stated otherwise herein, references to residue numbers in the constant domain of antibodies means residue numbering by the EU numbering system (e.g., see U.S. Provisional Application No. 60/640,323, Figures for EU numbering).
Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
“Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen-binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; inear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).
“Effector functions” refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.
“Fv” is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.
The term “multispecific antibody” is used in the broadest sense and specifically covers an antibody comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VH-VL unit has polyepitopic specificity (i.e., is capable of binding to two different epitopes on one biological molecule or each epitope on a different biological molecule). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies having two or more VL and VH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies and triabodies, antibody fragments that have been linked covalently or non-covalently. “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). “Dual specificity” or “bispecificity” refers to the ability to specifically bind to two different epitopes on the same or different target(s). However, in contrast to bispecific antibodies, dual-specific antibodies have two antigen-binding arms that are identical in amino acid sequence and each Fab arm is capable of recognizing two antigens. Dual-specificity allows the antibodies to interact with high affinity with two different antigens as a single Fab or IgG molecule. According to one embodiment, the multispecific antibody in an IgG1 form binds to each epitope with an affinity of 5 μM to 0.001 μM, 3 μM to 0.001 μM, 1 μM to 0.001 μM, 0.5 μM to 0.001 μM or 0.1 μM to 0.001 μM. “Monospecific” refers to the ability to bind only one epitope.
The term “diabodies” refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).
The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628, 1991; Marks et al., J. Mol. Biol. 222: 581-597, 1992; Sidhu et al., J. Mol. Biol. 338(2): 299-310, 2004; Lee et al., J. Mol. Biol. 340(5): 1073-1093, 2004; Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472, 2004; and Lee et al., J. Immunol. Methods 284(1-2): 119-132, 2004; and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551, 1993; Jakobovits et al., Nature 362: 255-258, 1993; Bruggemann et al., Year in Immunol. 7:33, 1993; U.S. Pat. Nos. 5,545,807; 5,545,806; 5,625,126; 5,633,425; and U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859, 1994; Morrison, Nature 368: 812-813, 1994; Fishwild et al., Nature Biotechnol. 14: 845-851, 1996; Neuberger, Nature Biotechnol. 14: 826, 1996; and Lonberg et al., Intern. Rev. Immunol. 13: 65-93, 1995.
The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-329, 1988; and Presta, Curr. Op. Struct. Biol. 2:593-596, 1992.
A “wild-type (WT)” or “reference” sequence or the sequence of a “wild-type” or “reference” protein/polypeptide, such as an HVR or a variable domain of a reference antibody, may be the reference sequence from which variant polypeptides are derived through the introduction of mutations. In general, the “wild-type” sequence for a given protein is the sequence that is most common in nature. Similarly, a “wild-type” gene sequence is the sequence for that gene which is most commonly found in nature. Mutations may be introduced into a “wild-type” gene (and thus the protein it encodes) either through natural processes or through man-induced means. The products of such processes are “variant” or “mutant” forms of the original “wild-type” protein or gene.
A “variant” or “mutant” of a starting or reference polypeptide (e.g., a reference antibody or its variable domain(s)/HVR(s)), is a polypeptide that (1) has an amino acid sequence different from that of the starting or reference polypeptide and (2) was derived from the starting or reference polypeptide through either natural or artificial (man-made) mutagenesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the polypeptide of interest, referred to herein as “amino acid residue alterations.” Thus, a variant HVR refers to a HVR comprising a variant sequence with respect to a starting or reference polypeptide sequence (such as that of a source antibody or antigen binding fragment). An amino acid residue alteration, in this context, refers to an amino acid different from the amino acid at the corresponding position in a starting or reference polypeptide sequence (such as that of a reference antibody or fragment thereof). Any combination of deletion, insertion, and substitution may be made to arrive at the final variant or mutant construct, provided that the final construct possesses the desired functional characteristics. The amino acid changes also may alter post-translational processes of the polypeptide, such as changing the number or position of glycosylation sites.
“Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.
With regard to the binding of an antibody to a target molecule, the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of 10⁻⁴M or lower, alternatively 10⁻⁵M or lower, alternatively 10⁻⁶M or lower, alternatively 10⁻⁷M or lower, alternatively 10⁻⁸M or lower, alternatively 10⁻⁹M or lower, alternatively 10⁻¹⁰M or lower, alternatively 10⁻¹¹M or lower, alternatively 10⁻¹²M or lower or a Kd in the range of 10⁻⁴M to 10⁻⁶M or 10⁻⁶M to 10⁻¹⁰M or 10⁻⁷M to 10⁻⁹M. As will be appreciated by the skilled artisan, affinity and Kd values are inversely related. A high affinity for an antigen is measured by a low Kd value. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
An “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
An “antibody that binds to the same epitope” as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
An “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
A “fusion protein” and a “fusion polypeptide” refer to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, and the like. The two portions may be linked directly by a single peptide bond or through a peptide linker but are in reading frame with each other.
As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the binding specificity of a heterologous protein (an “adhesin”) with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is “heterologous”), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG1, IgG2 (including IgG2A and IgG2B), IgG3, or IgG4 subtypes, IgA (including IgA1 and IgA2), IgE, IgD or IgM. The Ig fusions preferably include the substitution of a domain of a polypeptide or antibody described herein in the place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Pat. No. 5,428,130. For example, useful immunoadhesins as medicaments useful for therapy herein include polypeptides that comprise the extracellular domain (ECD) or PD-1-binding portions of PD-L1 or PD-L2, or the extracellular or PD-L1- or PD-L2-binding portions of PD-1, fused to a constant domain of an immunoglobulin sequence, such as a PD-L1 ECD-Fc, a PD-L2 ECD-Fc, and a PD-1 ECD-Fc, respectively. Immunoadhesin combinations of Ig Fc and ECD of cell surface receptors are sometimes termed soluble receptors.
“Percent (%) amino acid sequence identity” with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
“Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The term “polynucleotide” specifically includes cDNAs.
A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl-, 2′-fluoro-, or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂(“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
“Oligonucleotide,” as used herein, generally refers to short, single stranded, polynucleotides that are, but not necessarily, less than about 250 nucleotides in length. Oligonucleotides may be synthetic. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides.
The term “primer” refers to a single-stranded polynucleotide that is capable of hybridizing to a nucleic acid and allowing polymerization of a complementary nucleic acid, generally by providing a free 3′-OH group.
The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
An “isolated” nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
As used herein, the terms “mutational load,” “mutation load,” “mutational burden,” “tumor mutational burden score,” “TMB score,” “tissue tumor mutational burden score,” and “tTMB score” each of which may be used interchangeably, refer to the level (e.g., number) of an alteration (e.g., one or more alterations, e.g., one or more somatic alterations) per a pre-selected unit (e.g., per megabase) in a pre-determined set of genes (e.g., in the coding regions of the pre-determined set of genes) detected in a tumor tissue sample (e.g., a FFPE tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample). The TMB score can be measured, for example, on a whole genome or exome basis, or on the basis of a subset of the genome or exome. In certain embodiments, the TMB score measured on the basis of a subset of the genome or exome can be extrapolated to determine a whole genome or exome mutation load. In some embodiments, a TMB score refers to the level of accumulated somatic mutations within an individual (e.g., an animal (e.g., a human)). The TMB score may refer to accumulated somatic mutations in a patient with cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer). In some embodiments, a TMB score refers to the accumulated mutations in the whole genome of an individual. In some embodiments, a TMB score refers to the accumulated mutations within a particular tissue sample (e.g., tumor tissue sample biopsy, e.g., a bladder cancer tumor sample, e.g., an UC tumor sample) collected from an individual.
The term “somatic mutation” or “somatic alteration” refers to a genetic alteration occurring in the somatic tissues (e.g., cells outside the germline). Examples of genetic alterations include, but are not limited to, point mutations (e.g., the exchange of a single nucleotide for another (e.g., silent mutations, missense mutations, and nonsense mutations)), insertions and deletions (e.g., the addition and/or removal of one or more nucleotides (e.g., indels)), amplifications, gene duplications, copy number alterations (CNAs), rearrangements, and splice variants. The presence of particular mutations can be associated with disease states (e.g., cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer)).
In certain embodiments, the somatic alteration is a silent mutation (e.g., a synonymous alteration). In other embodiments, the somatic alteration is a non-synonymous single nucleotide variant (SNV). In other embodiments, the somatic alteration is a passenger mutation (e.g., an alteration that has no detectable effect on the fitness of a clone). In certain embodiments, the somatic alteration is a variant of unknown significance (VUS), for example, an alteration, the pathogenicity of which can neither be confirmed nor ruled out. In certain embodiments, the somatic alteration has not been identified as being associated with a cancer phenotype.
In certain embodiments, the somatic alteration is not associated with, or is not known to be associated with, an effect on cell division, growth, or survival. In other embodiments, the somatic alteration is associated with an effect on cell division, growth, or survival.
In certain embodiments, the number of somatic alterations excludes a functional alteration in a sub-genomic interval.
In some embodiments, the functional alteration is an alteration that, compared with a reference sequence (e.g., a wild-type or unmutated sequence) has an effect on cell division, growth, or survival (e.g., promotes cell division, growth, or survival). In certain embodiments, the functional alteration is identified as such by inclusion in a database of functional alterations, e.g., the COSMIC database (see Forbes et al. Nucl. Acids Res. 43 (D1): D805-D811, 2015, which is herein incorporated by reference in its entirety). In other embodiments, the functional alteration is an alteration with known functional status (e.g., occurring as a known somatic alteration in the COSMIC database). In certain embodiments, the functional alteration is an alteration with a likely functional status (e.g., a truncation in a tumor suppressor gene). In certain embodiments, the functional alteration is a driver mutation (e.g., an alteration that gives a selective advantage to a clone in its microenvironment, e.g., by increasing cell survival or reproduction). In other embodiments, the functional alteration is an alteration capable of causing clonal expansions. In certain embodiments, the functional alteration is an alteration capable of causing one, two, three, four, five, or all six of the following: (a) self-sufficiency in a growth signal; (b) decreased, e.g., insensitivity, to an antigrowth signal; (c) decreased apoptosis; (d) increased replicative potential; (e) sustained angiogenesis; or (f) tissue invasion or metastasis.
In certain embodiments, the functional alteration is not a passenger mutation (e.g., is not an alteration that has no detectable effect on the fitness of a clone of cells). In certain embodiments, the functional alteration is not a variant of unknown significance (VUS) (e.g., is not an alteration, the pathogenicity of which can neither be confirmed nor ruled out).
In certain embodiments, a plurality (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) of functional alterations in a pre-selected tumor gene in the pre-determined set of genes are excluded. In certain embodiments, all functional alterations in a pre-selected gene (e.g., tumor gene) in the pre-determined set of genes are excluded. In certain embodiments, a plurality of functional alterations in a plurality of pre-selected genes (e.g., tumor genes) in the pre-determined set of genes are excluded. In certain embodiments, all functional alterations in all genes (e.g., tumor genes) in the pre-determined set of genes are excluded.
In certain embodiments, the number of somatic alterations excludes a germline mutation in a sub-genomic interval.
In certain embodiments, the germline alteration is an SNP, a base substitution, an insertion, a deletion, an indel, or a silent mutation (e.g., synonymous mutation).
In certain embodiments, the germline alteration is excluded by use of a method that does not use a comparison with a matched normal sequence. In other embodiments, the germline alteration is excluded by a method comprising the use of an algorithm. In certain embodiments, the germline alteration is identified as such by inclusion in a database of germline alterations, for example, the dbSNP database (see Sherry et al. Nucleic Acids Res. 29(1): 308-311, 2001, which is herein incorporated by reference in its entirety). In other embodiments, the germline alteration is identified as such by inclusion in two or more counts of the ExAC database (see Exome Aggregation Consortium et al. bioRxiv preprint, Oct. 30, 2015, which is herein incorporated by reference in its entirety). In some embodiments, the germline alteration is identified as such by inclusion in the 1000 Genome Project database (McVean et al. Nature 491, 56-65, 2012, which is herein incorporated by reference in its entirety). In some embodiments, the germline alteration is identified as such by inclusion in the ESP database (Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA).
As used herein, the term “reference TMB score” refers to a TMB score against which another TMB score is compared, e.g., to make a diagnostic, predictive, prognostic, and/or therapeutic determination. For example, the reference TMB score may be a TMB score in a reference sample, a reference population, and/or a pre-determined value. In some instances, the reference TMB score is a cutoff value that significantly separates a first subset of individuals (e.g., patients) who have been treated with an immunotherapy, for example, a PD-L1 axis binding antagonist therapy, in a reference population and a second subset of individuals (e.g., patients) who have been treated with a non-PD-L1 axis binding antagonist therapy that does not comprise an immunotherapy, for example, a PD-L1 axis binding antagonist, in the same reference population based on a significant difference between an individual's responsiveness to treatment with the immunotherapy, for example, a PD-L1 axis binding antagonist therapy, and an individual's responsiveness to treatment with the non-PD-L1 axis binding antagonist therapy at or above the cutoff value and/or below the cutoff value. In some instances, the individual's responsiveness to treatment with the immunotherapy, for example, a PD-L1 axis binding antagonist therapy, is significantly improved relative to the individual's responsiveness to treatment with the non-PD-L1 axis binding antagonist therapy at or above the cutoff value. In some instances, the individual's responsiveness to treatment with the non-PD-L1 axis binding antagonist therapy is significantly improved relative to the individual's responsiveness to treatment with the immunotherapy, for example, a PD-L1 axis binding antagonist therapy, below the cutoff value.
It will be appreciated by one skilled in the art that the numerical value for the reference TMB score may vary depending on the type of, the methodology used to measure a TMB score, and/or the statistical methods used to generate a TMB score.
The term “equivalent TMB value” refers to a numerical value that corresponds to a TMB score that can be calculated by dividing the count of somatic variants (e.g., somatic mutations) by the number of bases sequenced (e.g., about 1.1 Mb (e.g., about 1.125 Mb), e.g., as assessed by the FOUNDATIONONE® panel). It is to be understood that, in general, the TMB score is linearly related to the size of the genomic region sequenced. Such equivalent TMB values indicate an equivalent degree of tumor mutational burden as compared to a TMB score and can be used interchangeably in the methods described herein, for example, to predict response of a cancer patient to an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody). As an example, in some embodiments, an equivalent TMB value is a normalized TMB value that can be calculated by dividing the count of somatic variants (e.g., somatic mutations) by the number of bases sequenced. For example, an equivalent TMB value can be represented as the number of somatic mutations counted over a defined number of sequenced bases (e.g., about 1.1 Mb (e.g., about 1.125 Mb), e.g., as assessed by the FOUNDATIONONE® panel). For example, a TMB score of about 25 (as determined as the number of somatic mutations counted over about 1.1 Mb) corresponds to an equivalent TMB value of about 23 mutations/Mb. It is to be understood that TMB scores as described herein (e.g., TMB scores represented as the number of somatic mutations counted over a defined number of sequenced bases (e.g., about 1.1 Mb (e.g., about 1.125 Mb), e.g., as assessed by the FOUNDATIONONE® panel)) encompass equivalent TMB values obtained using different methodologies (e.g., whole-exome sequencing or whole-genome sequencing). As an example, for a whole-exome panel, the target region may be approximately 50 Mb, and a sample with about 500 somatic mutations detected is an equivalent TMB value to a TMB score of about 10 mutations/Mb. In some embodiments, a TMB score determined as the number of somatic mutations counted over a defined number of sequenced bases (e.g., about 1.1 Mb (e.g., about 1.125 Mb), e.g., as assessed by the FOUNDATIONONE® panel) in a subset of the genome or exome (e.g., a predetermined set of genes) deviates by less than about 30% (e.g., less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, about 1%, or less) from a TMB score determined by whole-exome sequencing. See, e.g., Chalmers et al. Genome Medicine 9:34, 2017.

II. Diagnostic Methods and Assays

Provided herein are methods and uses for identifying an individual having a cancer (including, but not limited to, a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from a treatment with an anti-cancer therapy that includes an immunotherapy (including, but not limited to, a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (including, but not limited to, a TGF-β antagonist, e.g., an anti-TGF-β antibody).
The methods and uses described herein are based, in part, on the finding that the expression level of one or more genes (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a sample from the individual may be used to predict the therapeutic efficacy of an an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).
Further provided herein are methods and assays for selecting a therapy for an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer); methods for determining whether an individual having a cancer is likely to respond to treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody); methods for predicting the responsiveness of an individual having a cancer to treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody); and methods for monitoring the response of an individual having a cancer to treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody. Any of the methods provided herein may further include administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) (e.g., as described below in Section IV) to the individual.
In any embodiment described herein in which the expression level of more than one biomarker is determined in a patient sample and compared to a reference expression level, it is to be understood that, in some embodiments, the expression level of each individual biomarker in the patient sample is compared to a reference expression level for each individual biomarker. For example, if the expression level of TGFB1 and TGFBR1 are determined in a sample from an individual and compared to reference expression levels for TGFB1 and TGFBR1, in some embodiments, the expression level of TGFB1 in the sample from the individual is compared to the reference expression level for TGFB1, and the expression level of TGFBR1 in the sample from the individual is compared to the reference expression level for TGFBR1. In other embodiments, an expression level for more than one gene of interest may be determined by aggregation methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the median or mean of all the expression levels of the genes of interest. Before aggregation, the expression level of each gene of interest may be normalized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, normalized to the expression level of one or more housekeeping genes, or normalized to a total library size, or normalized to the median or mean expression level value across all genes measured. In some instances, before aggregation across multiple genes of interest, the normalized expression level of each gene of interest may be standardized by using statistical methods known to one skilled in the art and also disclosed herein, including, for example, by calculating the Z-score of the normalized expression level of each gene of interest.
A. Exemplary 22-Gene Signature
In some embodiments, the methods and uses herein involve determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) genes selected from a 22-gene signature, which includes the genes set forth in Table 1.
For example, provided herein is a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) that involves determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes set forth in Table 1 in a sample from the individual, wherein a change in the expression level of one or more of the genes set forth in Table 1 identifies the individual as one who may benefit from treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody). In some instances, the change is an increase. In other instances, the change is a decrease.

TABLE 1

Exemplary Biomarkers
Biomarkers

	TGFB1
	TGFBR2
	ACTA2
	ACTG2
	ADAM12
	ADAM19
	COMP
	CNN1
	COL4A1
	CTGF
	CTPS1
	FAM101B
	FSTL3
	HSPB1
	IGFBP3
	PXDC1
	SEMA7A
	SH3PXD2A
	TAGLN
	TGFBI
	TNS1
	TPM1

The invention also provides for selecting a therapy for an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes set forth in Table 1 in a sample from the individual, wherein a change in the expression level of one or more of the genes set forth in Table 1 identifies the individual as one who may benefit from treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody). In some instances, the change is an increase. In other instances, the change is a decrease.
In another embodiment, provided herein is a method of diagnosing or prognosing a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) biomarkers in a sample from an individual and comparing the expression level of the one or more biomarkers in the sample with a reference expression level, thereby diagnosing or prognosing the cancer. In some embodiments, a change in the expression level (e.g., an increase or a decrease) of the one or more biomarkers in the sample relative to the reference expression level diagnoses or prognoses the individual. In some embodiments, the biomarker is set forth in Table 1. In particular embodiments, the change is an increase.
In yet another embodiment, provided herein is a method of determining whether an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) is likely to respond to treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) biomarkers in a sample from the individual and comparing the expression level of the one or more biomarkers in the sample with a reference expression level, thereby identifying the individual as one who is likely to respond to the anti-cancer therapy. In some embodiments, a change in the expression level (e.g., an increase or a decrease) of the one or more biomarkers in the biological sample relative to the reference expression level identifies the individual as likely to respond to treatment with the anti-cancer therapy. In some embodiments, the biomarker is set forth in Table 1. In particular embodiments, the change is an increase.
In other embodiments, provided herein is a method of optimizing therapeutic efficacy of an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) biomarkers in a biological sample obtained from an individual and comparing the expression level of the one or more biomarkers in the sample with a reference expression level, wherein a change (e.g., an increase or decrease) in the expression level of the one or more biomarkers in the biological sample relative to the reference expression level identifies the individual as one who is likely to respond to the anti-cancer therapy. In some embodiments, the biomarker is set forth in Table 1. In particular embodiments, the change is an increase.
In some embodiments of any of the preceding methods involving the 22-gene signature, a reference expression level is the expression level of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) genes (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a reference population, for example, a population of individuals having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer). In particular embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC). In certain embodiments, a reference expression level is the median expression level of the one or more genes in a reference population, for example, a population of individuals having a cancer. In other embodiments, the reference expression level may be the top 40%, the top 30%, the top 20%, the top 10%, the top 5%, or the top 1% of the expression level in the reference population. In some embodiments, the reference expression level is determined by principle component analysis of Z-score-transformed expression levels. In certain embodiments, the reference expression level is a pre-assigned expression level for the one or more genes. In some embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a previous time point, wherein the previous time point is following administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, a reference expression level is the expression level of the one or more genes (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a biological sample from the patient obtained prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In other embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a subsequent time point (e.g., minutes, hours, days, weeks, months, or years after administration of an anti-cancer therapy).
In some embodiments of any of the preceding methods involving the 22-gene signature, an expression level above a reference expression level, or an elevated or increased expression or number, refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level or number of a biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by methods such as those described herein and/or known in the art, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, the elevated expression or number refers to the increase in expression level/amount of a biomarker (e.g., one or more of TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the increase is at least about any of 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×, 500×, or 1000× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression or number refers to an overall increase in expression level/amount of a biomarker (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 22-gene signature, an expression level below a reference expression level, or a reduced (decreased) expression or number, refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by standard art known methods such as those described herein, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression or number refers to the decrease in expression level/amount of a biomarker (e.g., one or more of TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the decrease is at least about any of 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or 0.01× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, reduced (decreased) expression or number refers to an overall decrease in expression level/amount of a biomarker (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 22-gene signature, a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment. In some embodiments, the CD8+ T-cells localize at or near collagen fibers.
B. Exemplary 6-Gene Signature
In some embodiments, the methods and uses herein involve determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) genes selected from a 6-gene signature which includes ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
For example, provided herein is a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein an expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In yet another embodiment, provided herein is a method for selecting a therapy for an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein an expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).
In another embodiment, provided herein is a method of diagnosing or prognosing a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2; and comparing the expression level of the one or more genes in the sample with a reference expression level, thereby diagnosing or prognosing the cancer. In some embodiments, a change in the expression level (e.g., an increase or a decrease) of the one or more biomarkers in the sample relative to the reference expression level diagnoses or prognoses the individual. In particular embodiments, the change is an increase.
In yet another embodiment, provided herein is a method of determining whether an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) is likely to respond to treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein an expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who is likely to respond to treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).
In other embodiments, provided herein is a method of optimizing therapeutic efficacy of an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2; and comparing the expression level of the one or more genes in the sample with a reference expression level, wherein an expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who is likely to respond to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist. In any of the preceding methods involving the 6-gene signature, the method may include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2. In some embodiments, the method includes determining the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2. In some embodiments, the method includes determining the expression level of two of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 2. In some embodiments, the method includes determining the expression level of three of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 3. In some embodiments, the method includes determining the expression level of four of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 4. In some embodiments, the method includes determining the expression level of five of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 5. In some embodiments, the method involves determining the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In any of the preceding methods involving the 6-gene signature, the method may include determining the expression level of TGFB1 and/or TGFBR2. In any of the preceding methods, the method may include determining the expression level of TGFB1 and TGFBR2.
In some embodiments of any of the preceding methods involving the 6-gene signature, the one or more genes includes at least ADAM19 or COMP. In some embodiments, the one or more genes includes ADAM19. In other embodiments, the one or more genes includes COMP. In still further embodiments, the one or more genes includes ADAM19 and COMP.

TABLE 2

Two-Gene Combinations of ACTA2, ADAM19,
COMP, CTGF, TGFB1, and TGFBR2

	ACTA2 and ADAM19
	ACTA2 and COMP
	ACTA2 and CTGF
	ACTA2 and TGFB1
	ACTA2 and TGFBR2
	ADAM19 and COMP
	ADAM19 and CTGF
	ADAM19 and TGFB1
	ADAM19 and TGFBR2
	COMP and CTGF
	COMP and TGFB1
	COMP and TGFBR2
	CTGF and TGFB1
	CTGF and TGFBR2
	TGFB1 and TGFBR2

TABLE 3

Three-Gene Combinations of ACTA2, ADAM19,
COMP, CTGF, TGFB1, and TGFBR2

	ACTA2, ADAM19, and COMP
	ACTA2, ADAM19, and CTGF
	ACTA2, ADAM19, and TGFB1
	ACTA2, ADAM19, and TGFBR2
	ACTA2, COMP, and CTGF
	ACTA2, COMP, and TGFB1
	ACTA2, COMP, and TGFBR2
	ACTA2, CTGF, and TGFB1
	ACTA2, CTGF, and TGFBR2
	ACTA2, TGFB1, and TGFBR2
	ADAM19, COMP, and CTGF
	ADAM19, COMP, and TGFB1
	ADAM19, COMP, and TGFBR2
	ADAM19, CTGF, and TGFB1
	ADAM19, CTGF, and TGFBR2
	ADAM19, TGFB1, and TGFBR2
	COMP, CTGF, and TGFB1
	COMP, CTGF, and TGFBR2
	COMP, TGFB1, and TGFBR2
	CTGF, TGFB1, and TGFBR2

TABLE 4

Four-Gene Combinations of ACTA2, ADAM19,
COMP, CTGF, TGFB1, and TGFBR2

	ACTA2, ADAM19, COMP, and CTGF
	ACTA2, ADAM19, COMP, and TGFB1
	ACTA2, ADAM19, COMP, and TGFBR2
	ACTA2, ADAM19, CTGF, and TGFB1
	ACTA2, ADAM19, CTGF, and TGFBR2
	ACTA2, ADAM19, TGFB1, and TGFBR2
	ACTA2, COMP, CTGF, and TGFB1
	ACTA2, COMP, CTGF, and TGFBR2
	ACTA2, COMP, TGFB1, and TGFBR2
	ACTA2, CTGF, TGFB1, and TGFBR2
	ADAM19, COMP, CTGF, and TGFB1
	ADAM19, COMP, CTGF, and TGFBR2
	ADAM19, COMP, TGFB1, and TGFBR2
	ADAM19, CTGF, TGFB1, and TGFBR2
	COMP, CTGF, TGFB1, and TGFBR2

TABLE 5

Five-Gene Combinations of ACTA2, ADAM19,
COMP, CTGF, TGFB1, and TGFBR2

	ACTA2, ADAM19, COMP, CTGF, and TGFB1
	ACTA2, ADAM19, COMP, CTGF, and TGFBR2
	ACTA2, ADAM19, COMP, TGFB1, and TGFBR2
	ACTA2, ADAM19, CTGF, TGFB1, and TGFBR2
	ACTA2, COMP, CTGF, TGFB1, and TGFBR2
	ADAM19, COMP, CTGF, TGFB1, and TGFBR2

In some embodiments of any of the preceding methods involving the 6-gene signature, the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 in the sample is at or above a reference expression level of the one or more genes, and the method further includes administering to the individual an effective amount of the anti-cancer therapy. For example, in some embodiments, the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 is at or above a reference expression level of the one or more genes. In some embodiments, the expression level of one or more of the exemplary combinations set forth in Tables 2-5 in the sample is at or above a reference expression level of the one or more genes. In some embodiments, the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 is at or above a reference expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In some embodiments of any of the preceding methods involving the 6-gene signature, the expression level of TGFB1 and/or TGFBR1 is at or above a reference expression level of TGFB1 and/or TGFBR1. For example, in some embodiments, the expression level of TGFB1 is at or above a reference expression level of TGFB1. In some embodiments, the expression level of TGFBR1 is at or above a reference expression level of TGFBR1. In some embodiments, the expression level of TGFB1 and TGFBR1 is at or above a reference expression level of TGFB1 and TGFBR1.
In some embodiments of any of the preceding methods involving the 6-gene signature, the one or more genes includes at least ADAM19 or COMP. In some embodiments, the one or more genes includes ADAM19. In other embodiments, the one or more genes includes COMP. In still further embodiments, the one or more genes includes ADAM19 and COMP.
In other embodiments of any of the preceding methods involving the 6-gene signature, the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 in the sample is below a reference expression level of the one or more genes, and the method further includes administering to the individual an effective amount of an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) alone. For example, in some embodiments, the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 is below a reference expression level of the one or more genes. In some embodiments, the expression level of one or more of the exemplary combinations set forth in Tables 2-5 in the sample is below a reference expression level of the one or more genes. In some embodiments, the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 is below a reference expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2. In some embodiments, the one or more genes includes at least ADAM19 or COMP. In some embodiments, the one or more genes includes ADAM19. In other embodiments, the one or more genes includes COMP. In still further embodiments, the one or more genes includes ADAM19 and COMP.
For example, in certain embodiments of any of the preceding methods involving the 6-gene signature, a reference expression level is the expression level of the one or more (e.g., 1, 2, 3, 4, 5, or 6) genes (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in a reference population, for example, a population of individuals having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer). In particular embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC). In certain embodiments, a reference expression level is the median expression level of the one or more genes in a reference population, for example, a population of individuals having a cancer. In other embodiments, the reference expression level may be the top 40%, the top 30%, the top 20%, the top 10%, the top 5%, or the top 1% of the expression level in the reference population. In some embodiments, the reference expression level is determined by principle component analysis of Z-score-transformed expression levels. In certain embodiments, the reference expression level is a pre-assigned expression level for the one or more genes. In some embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a previous time point, wherein the previous time point is following administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, a reference expression level is the expression level of the one or more genes (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in a biological sample from the patient obtained prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In other embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a subsequent time point (e.g., minutes, hours, days, weeks, months, or years after administration of an anti-cancer therapy).
In some embodiments of any of the preceding methods involving the 6-gene signature, an expression level above a reference expression level, or an elevated or increased expression or number, refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level or number of a biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by methods such as those described herein and/or known in the art, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, the elevated expression or number refers to the increase in expression level/amount of a biomarker (e.g., one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in the sample wherein the increase is at least about any of 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×, 500×, or 1000× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression or number refers to an overall increase in expression level/amount of a biomarker (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 6-gene signature, an expression level below a reference expression level, or a reduced (decreased) expression or number, refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by standard art known methods such as those described herein, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression or number refers to the decrease in expression level/amount of a biomarker (e.g., one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in the sample wherein the decrease is at least about any of 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, reduced (decreased) expression or number refers to an overall decrease in expression level/amount of a biomarker (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 6-gene signature, a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment. In some embodiments, the CD8+ T-cells localize at or near collagen fibers.
C. Exemplary 19-Gene Signature (Pan-F-TBRS)
In some embodiments, the methods and uses herein involve determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) genes selected from a 19-gene signature which includes ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1, which is also referred to herein as Pan-F-TBRS.
For example, provided herein is a method of identifying an individual having a cancer who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1, wherein an expression level of one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In yet another embodiment, provided herein is a method for selecting a therapy for an individual having a cancer, the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1, wherein an expression level of one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).
In another embodiment, provided herein is a method of diagnosing or prognosing a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and comparing the expression level of the one or more genes in the sample with a reference expression level, thereby diagnosing or prognosing the cancer. In some embodiments, a change in the expression level (e.g., an increase or a decrease) of the one or more biomarkers in the sample relative to the reference expression level diagnoses or prognoses the individual. In particular embodiments, the change is an increase.
In yet another embodiment, provided herein is a method of determining whether an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) is likely to respond to treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1, wherein an expression level of one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who is likely to respond to treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).
In other embodiments, provided herein is a method of optimizing therapeutic efficacy of an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) that includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and comparing the expression level of the one or more genes in the sample with a reference expression level, wherein an expression level of one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 in the sample that is at or above a reference expression level of the one or more genes identifies the individual as one who is likely to respond to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In any of the preceding methods involving the 19-gene signature, the method may include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the method includes determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the method involves determining the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In some embodiments of any of the preceding methods involving the 19-gene signature, the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 in the sample is at or above a reference expression level of the one or more genes, and the method further includes administering to the individual an effective amount of the anti-cancer therapy. For example, in some embodiments, the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 is at or above a reference expression level of the one or more genes. In some embodiments, the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 is at or above a reference expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In some embodiments of any of the preceding methods involving the 19-gene signature, the one or more genes includes at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1.
In some embodiments of any of the preceding methods involving the 19-gene signature, a reference expression level is the expression level of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) genes (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a reference population, for example, a population of individuals having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer). In particular embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC). In certain embodiments, a reference expression level is the median expression level of the one or more genes in a reference population, for example, a population of individuals having a cancer. In other embodiments, the reference level may be the top 40%, the top 30%, the top 20%, the top 10%, the top 5%, or the top 1% of the expression level in the reference population. In some embodiments, the reference expression level is determined by principle component analysis of Z-score-transformed expression levels. In certain embodiments, the reference expression level is a pre-assigned expression level for the one or more genes. In some embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a previous time point, wherein the previous time point is following administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, a reference expression level is the expression level of the one or more genes (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a biological sample from the patient obtained prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In other embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a subsequent time point (e.g., minutes, hours, days, weeks, months, or years after administration of an anti-cancer therapy).
In some embodiments of any of the preceding methods involving the 19-gene signature, an expression level above a reference expression level, or an elevated or increased expression or number, refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level or number of a biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by methods such as those described herein and/or known in the art, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, the elevated expression or number refers to the increase in expression level/amount of a biomarker (e.g., one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the increase is at least about any of 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×, 500×, or 1000× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression or number refers to an overall increase in expression level/amount of a biomarker (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 19-gene signature, an expression level below a reference expression level, or a reduced (decreased) expression or number, refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by standard art known methods such as those described herein, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression or number refers to the decrease in expression level/amount of a biomarker (e.g., one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the decrease is at least about any of 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or 0.01× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, reduced (decreased) expression or number refers to an overall decrease in expression level/amount of a biomarker (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 19-gene signature, a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment. In some embodiments, the CD8+ T-cells localize at or near collagen fibers.
D. Exemplary Additional Biomarkers (e.g., CD8+ T-Effector Signature)
Any of the preceding methods (e.g., as described in Section II, Subsections A-C above, relating to the 22-, 6-, and 19-gene (Pan-F-TBRS) signatures, respectively) can further include determining the expression level in the sample of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) additional genes selected from the group consisting of PD-L1, CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the one or more additional gene is PD-L1. In other embodiments, the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) additional genes is selected from the group consisting of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21, which is also referred to herein as a CD8+T-effector (Teff) signature. In some embodiments, the method further includes determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the one or more additional genes are CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21.
For example, provided herein is a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following 22-gene signature genes in the sample: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1, wherein an expression level of one or more of the Teff genes in the sample that is at or above a reference expression level of the one or more Teff genes and an expression level of one or more of the 22-gene signature genes that is below a reference expression level of the one or more 22-gene signature genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
In another embodiment, provided herein is a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, or 6) of the following 6-gene signature genes in the sample: ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, wherein an expression level of one or more of the Teff genes in the sample that is at or above a reference expression level of the one or more Teff genes and an expression level of one or more of the 6-gene signature genes that is below a reference expression level of the one or more 6-gene signature genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
In another embodiment, provided herein is a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following Pan-F-TBRS genes in the sample: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1, wherein an expression level of one or more of the Teff genes in the sample that is at or above a reference expression level of the one or more Teff genes and an expression level of one or more of the Pan-F-TBRS genes that is below a reference expression level of the one or more Pan-F-TBRS genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
For example, provided herein is a method for selecting a therapy for an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following 22-gene signature genes in the sample: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1, wherein an expression level of one or more of the Teff genes in the sample that is at or above a reference expression level of the one or more Teff genes and an expression level of one or more of the 22-gene signature genes that is below a reference expression level of the one or more 22-gene signature genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
In another embodiment, provided herein is a method for selecting a therapy for an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following 6-gene signature genes in the sample: ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, wherein an expression level of one or more of the Teff genes in the sample that is at or above a reference expression level of the one or more Teff genes and an expression level of one or more of the 6-gene signature genes that is below a reference expression level of the one or more 6-gene signature genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
In yet another embodiment, provided herein is a method for selecting a therapy for an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method comprising determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following Pan-F-TBRS genes in the sample: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1, wherein an expression level of one or more of the Teff genes in the sample that is at or above a reference expression level of the one or more Teff genes and an expression level of one or more of the Pan-F-TBRS genes that is below a reference level of the one or more Pan-F-TBRS genes identifies the individual as one who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)). In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
E. Tumor Mutational Burden (TMB)
Any of the preceding embodiments (e.g., as described in Section II, Subsections A-C above, relating to the 22-, 6-, and 19-gene (Pan-F-TBRS) signatures, respectively, or in Section II, Subsection D) may further include determining a TMB in a tumor sample from the individual. TMB may be determined using any suitable approach, for example, as described in Example 1 below or in International Patent Application No.: PCT/US2017/055669, which is incorporated herein by reference in its entirety. In some embodiments, the individual may have a TMB in a tumor sample that is at or above a reference TMB. In other embodiments, the individual may have a TMB that is below a reference TMB.
For example, the invention provides a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from a treatment with an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising determining (i) the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following 22-gene signature genes: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 in a sample from the patient; and (ii) a TMB score from a tumor sample from the individual, wherein an expression level of the one or more 22-gene signature genes in the sample that is below a reference level and a TMB score from the tumor sample that is at or above a reference TMB score identifies the individual as one who may benefit from a treatment comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody)). In some embodiments, the immunotherapy is a monotherapy.
The invention provides a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from a treatment with an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising determining (i) the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following 6-gene signature genes: ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 in a sample from the patient; and (ii) a TMB score from a tumor sample from the individual, wherein an expression level of the one or more 6-gene signature genes in the sample that is below a reference expression level and a TMB score from the tumor sample that is at or above a reference TMB score identifies the individual as one who may benefit from a treatment comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody)). In some embodiments, the immunotherapy is a monotherapy.
The invention provides a method of identifying an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from a treatment with an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), the method comprising determining (i) the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following Pan-F-TBRS genes: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 in a sample from the patient; and (ii) a TMB score from a tumor sample from the individual, wherein an expression level of the one or more Pan-F-TBRS genes in the sample that is below a reference expression level and a TMB score from the tumor sample that is at or above a reference TMB score identifies the individual as one who may benefit from a treatment comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab))). In some embodiments, the immunotherapy is a monotherapy.
Any of the preceding methods involving TMB can further include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21.
F. Exemplary Approaches for Determination of Biomarker Expression Levels
The presence and/or expression level of any of the biomarkers described above (e.g., as described in Section II, Subsections A-C above, relating to the 22-, 6-, and 19-gene (Pan-F-TBRS) signatures, respectively, or in Section II, Subsections D and E) may be assessed qualitatively and/or quantitatively based on any suitable criterion known in the art, including but not limited to DNA, mRNA, cDNA, proteins, protein fragments, and/or gene copy number. Methodologies for measuring such biomarkers are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood based assays (e.g., Serum ELISA), biochemical enzymatic activity assays, in situ hybridization (ISH), fluorescence in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (PCR) including quantitative real time PCR (qRT-PCR) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like, RNASeq, microarray analysis, gene expression profiling, whole-genome sequencing (WGS), and/or serial analysis of gene expression (“SAGE”), as well as any one of the wide variety of assays that can be performed by protein, gene, and/or tissue array analysis. Typical protocols for evaluating the status of genes and gene products are found, for example, in Ausubel et al. eds. (Current Protocols In Molecular Biology, 1995), Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.
In some embodiments of any of the preceding methods, the expression level of a biomarker may be a nucleic acid expression level (e.g., a DNA expression level or an RNA expression level (e.g., an mRNA expression level)). Any suitable method of determining a nucleic acid expression level may be used. In some embodiments, the nucleic acid expression level is determined using RNAseq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof.
Methods for the evaluation of mRNAs in cells are well known and include, for example, serial analysis of gene expression (SAGE), whole genome sequencing (WGS), hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR (e.g., q RT-PCR) using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). In addition, such methods can include one or more steps that allow one to determine the levels of target mRNA in a biological sample (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a “housekeeping” gene such as an actin family member). Optionally, the sequence of the amplified target cDNA can be determined. Optional methods include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes whose expression correlates with increased or reduced clinical benefit of treatment comprising an immunotherapy and a suppressive stromal antagonist may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
In other embodiments of any of the preceding methods, the expression level of a biomarker may be a protein expression level. In certain embodiments, the method comprises contacting the sample with antibodies that specifically bind to a biomarker described herein under conditions permissive for binding of the biomarker, and detecting whether a complex is formed between the antibodies and biomarker. Such method may be an in vitro or in vivo method. In some instances, an antibody is used to select patients eligible for treatment with an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), e.g., a biomarker for selection of individuals. Any method of measuring protein expression levels known in the art or provided herein may be used.
For example, in some embodiments, a protein expression level of a biomarker is determined using a method selected from the group consisting of flow cytometry (e.g., fluorescence-activated cell sorting (FACS™)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC. In some embodiments, the protein expression level of the biomarker is determined in tumor-infiltrating immune cells. In some embodiments, the protein expression level of the biomarker is determined in tumor cells. In some embodiments, the protein expression level of the biomarker is determined in tumor-infiltrating immune cells and/or in tumor cells. In some embodiments, the protein expression level of the biomarker is determined in peripheral blood mononuclear cells (PBMCs).
In certain embodiments, the presence and/or expression level/amount of a biomarker protein in a sample is examined using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of proteins in a sample. In some embodiments of any of the methods, assays and/or kits, the biomarker is one or more of the protein expression products of the following genes: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1. In one embodiment, an expression level of biomarker is determined using a method comprising: (a) performing IHC analysis of a sample (such as a tumor sample obtained from a patient) with an antibody; and (b) determining expression level of a biomarker in the sample. In some embodiments, IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line staining sample, a tissue sample from non-cancerous patient, or a tumor sample that is determined to be negative for the biomarker of interest).
IHC may be performed in combination with additional techniques such as morphological staining and/or in situ hybridization (e.g., ISH). Two general methods of IHC are available; direct and indirect assays. According to the first assay, binding of antibody to the target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
The primary and/or secondary antibody used for IHC typically will be labeled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories: (a) radioisotopes, such as ³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I; (b) colloidal gold particles; (c) fluorescent labels including, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerytherin, phycocyanin, or commercially-available fluorophores such as SPECTRUM ORANGE7 and SPECTRUM GREEN7 and/or derivatives of any one or more of the above; (d) various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; see, e.g., U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
Examples of enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate; alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase). For a general review of these, see, for example, U.S. Pat. Nos. 4,275,149 and 4,318,980.
Specimens may be prepared, for example, manually, or using an automated staining instrument (e.g., a Ventana BenchMark XT or Benchmark ULTRA instrument). Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, for example, using a microscope, and staining intensity criteria, routinely used in the art, may be employed. In one embodiment, it is to be understood that when cells and/or tissue from a tumor is examined using IHC, staining is generally determined or assessed in tumor cell(s) and/or tissue (as opposed to stromal or surrounding tissue that may be present in the sample). In some embodiments, it is understood that when cells and/or tissue from a tumor is examined using IHC, staining includes determining or assessing in tumor-infiltrating immune cells, including intratumoral or peritumoral immune cells. In some embodiments, the presence of a biomarker is detected by IHC in >0% of the sample, in at least 1% of the sample, in at least 5% of the sample, in at least 10% of the sample, in at least 15% of the sample, in at least 15% of the sample, in at least 20% of the sample, in at least 25% of the sample, in at least 30% of the sample, in at least 35% of the sample, in at least 40% of the sample, in at least 45% of the sample, in at least 50% of the sample, in at least 55% of the sample, in at least 60% of the sample, in at least 65% of the sample, in at least 70% of the sample, in at least 75% of the sample, in at least 80% of the sample, in at least 85% of the sample, in at least 90% of the sample, in at least 95% of the sample, or more. Samples may be scored using any method known in the art, for example, by a pathologist or automated image analysis.
In some embodiments of any of the methods, the biomarker is detected by immunohistochemistry using a diagnostic antibody (i.e., primary antibody). In some embodiments, the diagnostic antibody specifically binds human antigen. In some embodiments, the diagnostic antibody is a non-human antibody. In some embodiments, the diagnostic antibody is a rat, mouse, or rabbit antibody. In some embodiments, the diagnostic antibody is a rabbit antibody. In some embodiments, the diagnostic antibody is a monoclonal antibody. In some embodiments, the diagnostic antibody is directly labeled. In other embodiments, the diagnostic antibody is indirectly labeled (e.g., by a secondary antibody).
In some embodiments of any of the preceding embodiments, the sample is obtained from the individual prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, the sample from the individual is obtained about 2 to about 10 weeks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks) following administration of the anti-cancer therapy. In some embodiments, the sample from the individual is obtained about 4 to about 6 weeks following administration of the anti-cancer therapy.
In some embodiments of any of the preceding methods, the expression level or number of a biomarker is detected in a tissue sample, a primary or cultured cells or cell line, a cell supernatant, a cell lysate, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, or any combination thereof. In some embodiments, the sample is a tissue sample (e.g., a tumor tissue sample), a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the tumor tissue sample wherein the tumor tissue sample includes tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some embodiments, the tumor tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archival sample, a fresh sample, or a frozen sample.
For example, in some embodiments of any of the preceding methods, the expression level of a biomarker is detected in tumor-infiltrating immune cells, tumor cells, PBMCs, or combinations thereof using known techniques (e.g., flow cytometry or IHC). Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells or any combinations thereof, and other tumor stroma cells (e.g., fibroblasts). Such tumor infiltrating immune cells may be T lymphocytes (such as CD8⁺ T lymphocytes (e.g., CD8⁺ T effector (Teff) cells) and/or CD4⁺ T lymphocytes (e.g., CD4⁺ Teff cells), B lymphocytes, or other bone marrow-lineage cells including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer (NK) cells. In some embodiments, the staining for a biomarker is detected as membrane staining, cytoplasmic staining, or combinations thereof. In other embodiments, the absence of a biomarker is detected as absent or no staining in the sample, relative to a reference sample.
In particular embodiments of any of the preceding methods, the expression level of a biomarker is assessed in a sample that contains or is suspected to contain cancer cells. The sample may be, for example, a tissue biopsy or a metastatic lesion obtained from a patient suffering from, suspected to suffer from, or diagnosed with cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer). In some embodiments, the sample is a sample of bladder tissue, a biopsy of a bladder tumor, a known or suspected metastatic bladder cancer lesion or section, or a blood sample, e.g., a peripheral blood sample, known or suspected to comprise circulating cancer cells, e.g., bladder cancer cells. The sample may comprise both cancer cells, i.e., tumor cells, and non-cancerous cells (e.g., lymphocytes, such as T cells or NK cells), and, in certain embodiments, comprises both cancerous and non-cancerous cells. Methods of obtaining biological samples including tissue resections, biopsies, and body fluids, e.g., blood samples comprising cancer/tumor cells, are well known in the art.
In some embodiments of any of the preceding methods, the patient has carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. In some embodiments, the cancer is bladder cancer (e.g., UC, e.g., mUC), kidney cancer (e.g., renal cell carcinoma (RCC), e.g., advanced RCC or metastatic RCC (mRCC)), squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., PDAC), glioblastoma, cervical cancer, ovarian cancer, liver cancer (e.g., HCC), hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, Merkel cell cancer, mycoses fungoids, testicular cancer, esophageal cancer, tumors of the biliary tract, head and neck cancer, B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs' syndrome. In some embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer (e.g., HCC), an ovarian cancer, or a breast cancer (e.g., TNBC). In preferred embodiments, the patient has a bladder cancer (e.g., UC, e.g., mUC). The patient may optionally have an advanced, refractory, recurrent, chemotherapy-resistant, and/or platinum-resistant form of the cancer.
In certain embodiments, the presence and/or expression levels/amount of a biomarker in a first sample is increased or elevated as compared to presence/absence and/or expression levels/amount in a second sample. In certain embodiments, the presence/absence and/or expression levels/amount of a biomarker in a first sample is decreased or reduced as compared to presence and/or expression levels/amount in a second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or combined multiple samples from the same patient or individual that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained at an earlier time point from the same patient or individual than when the test sample is obtained. Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.
In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more healthy individuals who are not the patient. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the patient or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the patient. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the patient.

III. Therapeutic Methods and Uses

Provided herein are methods and uses for treating an individual having a cancer (including, but not limited to, a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer). In particular embodiments, the cancer is a bladder cancer, such as UC, e.g., metastatic UC. In some instances, the methods of the invention include administering to the individual an anti-cancer therapy that includes an immunotherapy (including, but not limited to, a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (including, but not limited to, a TGF-β antagonist, e.g., an anti-TGF-β antibody) based on the expression level of a biomarker of the invention. Any of the immunotherapies (e.g., PD-L1 axis binding antagonists (e.g., anti-PD-L1 antibodies)), suppressive stromal antagonists (e.g., TGF-β antagonists (e.g., anti-TGF-β antibodies)), or other anti-cancer agents described herein (e.g., as described below in Section IV and/or the Examples) or known in the art may be used in the methods and uses. The invention further relates to methods for improving PFS and/or OS of a patient suffering from a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) by administration of an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody). The invention further relates to methods for improving response (e.g., ORR) of a patient suffering from a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) by administration of an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody). The expression level or number of any of the biomarkers described herein may be determined using any method known in the art and/or described herein, for example, in Section II above and/or in the working Examples.
In some embodiments, therapy with an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in combination with a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) preferably extends and/or improves survival, including PFS and/or OS. In one embodiment, therapy with an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in combination with a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) extends survival by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, relative to the survival achieved by administering an approved anti-tumor agent, or standard of care, for the cancer being treated. In another embodiment, therapy with an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) in combination with a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) improves a response rate (e.g., an ORR, a CR rate, or a PR rate) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more, relative to the response rate achieved by administering an approved anti-tumor agent, or standard of care, for the cancer being treated.
A. Exemplary 22-Gene Signature
In some embodiments, the methods and uses herein involve determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) genes selected from a 22-gene signature, which includes the genes set forth in Table 1. For example, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) that includes (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes set forth in Table 1 in a sample from the individual, wherein the expression level of one or more of the genes set forth in Table 1 is determined to be changed relative to a reference expression level; and (b) administering an effective amount of an anti-cancer therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) to the individual based on the expression level of the one or more genes determined in step (a). In some instances, the change is an increase. In other instances, the change is a decrease.
Also provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) that includes administering an effective amount of an anti-cancer therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) to the individual, wherein the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes set forth in Table 1 in a sample from the individual has been determined to be changed relative to a reference level, wherein a change in the expression level of one or more of the genes set forth in Table 1 identifies the individual as one who may benefit from treatment with an anti-cancer therapy. In some instances, the change is an increase. In other instances, the change is a decrease.
In another aspect, the invention provides a method of treating cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) in an individual having been identified as having an expression level in a sample from the individual of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes set forth in Table 1 that is at or above a reference expression level of the one or more genes, the method including administering to the individual an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)).
In another aspect, provided herein is a method for assessing a response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes listed in Table 1 in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual; and (b) maintaining, adjusting, or stopping the treatment based on a comparison of the expression level of the one or more genes in the sample with a reference expression level of the one or more genes, wherein a change in the expression level of the one or more genes in the sample from the individual compared to the reference expression level of the one or more genes is indicative of a response to treatment with the anti-cancer therapy.
In another aspect, provided herein is a method for monitoring the response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes listed in Table 1 in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual; and (b) comparing the expression level of the one or more genes in the sample from the individual with a reference expression level of the one or more genes, thereby monitoring the response of the individual undergoing treatment with the anti-cancer therapy.
In some embodiments of any of the preceding methods involving the 22-gene signature, the change is an increase in the expression level of the one or more genes and the treatment is adjusted or stopped. In other embodiments of any of the preceding methods, the change is a decrease in the expression level of the one or more genes and the treatment is maintained.
In some embodiments of any of the preceding methods involving the 22-gene signature, an increase in the expression level of the five or more genes is indicative of a lack of response of the individual to the treatment. In other embodiments of any of the preceding methods, a decrease in the expression level of the five or more genes is indicative of a response of the individual to the treatment.
In some embodiments of any of the preceding methods involving the 22-gene signature, a reference expression level is the expression level of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) genes (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a reference population, for example, a population of individuals having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer). In particular embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC). In certain embodiments, a reference expression level is the median expression level of the one or more genes in a reference population, for example, a population of individuals having a cancer. In other embodiments, the reference expression level may be the top 40%, the top 30%, the top 20%, the top 10%, the top 5%, or the top 1% of the expression level in the reference population. In some embodiments, the reference expression level is determined by principle component analysis of Z-score-transformed expression levels. In certain embodiments, the reference expression level is a pre-assigned expression level for the one or more genes. In some embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a previous time point, wherein the previous time point is following administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, a reference expression level is the expression level of the one or more genes (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a biological sample from the patient obtained prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In other embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a subsequent time point (e.g., minutes, hours, days, weeks, months, or years after administration of an anti-cancer therapy).
In some embodiments of any of the preceding methods involving the 22-gene signature, an expression level above a reference expression level, or an elevated or increased expression or number, refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level or number of a biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by methods such as those described herein and/or known in the art, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, the elevated expression or number refers to the increase in expression level/amount of a biomarker (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the increase is at least about any of 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×, 500×, or 1000× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression or number refers to an overall increase in expression level/amount of a biomarker (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 22-gene signature, an expression level below a reference expression level, or reduced (decreased) expression or number, refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by standard art known methods such as those described herein, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression or number refers to the decrease in expression level/amount of a biomarker (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the decrease is at least about any of 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or 0.01× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, reduced (decreased) expression or number refers to an overall decrease in expression level/amount of a biomarker (e.g., TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 22-gene signature, a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment. In some embodiments, the CD8+ T-cells localize at or near collagen fibers.
B. Exemplary 6-Gene Signature
In some embodiments, the methods and uses herein involve determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) genes selected from a 6-gene signature which includes ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
For example, in one embodiment, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2, wherein the expression level of one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample is determined to be at or above a reference expression level of the one or more genes; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more genes determined in step (a)).
The method may include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2. In some embodiments, the method includes determining the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2. In some embodiments, the method includes determining the expression level of two of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 2. In some embodiments, the method includes determining the expression level of three of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 3. In some embodiments, the method includes determining the expression level of four of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 4. In some embodiments, the method includes determining the expression level of five of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 5. In some embodiments, the method involves determining the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In any of the preceding methods involving the 6-gene signature, the method may include determining the expression level of TGFB1 and/or TGFBR2. In any of the preceding methods, the method may include determining the expression level of TGFB1 and TGFBR2.
In some embodiments of any of the preceding methods involving the 6-gene signature, the one or more genes includes at least ADAM19 or COMP. In some embodiments, the one or more genes includes ADAM19. In other embodiments, the one or more genes includes COMP. In still further embodiments, the one or more genes includes ADAM19 and COMP.
In another embodiment, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 in the sample has been determined to be at or above a reference expression level of the one or more genes.
In another aspect, the invention provides a method of treating cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) in an individual having been identified as having an expression level in a sample from the individual of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 that is at or above a reference expression level of the one or more genes, the method including administering to the individual an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)).
In any of the preceding methods involving the 6-gene signature, the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2 has been determined to be at or above a reference level of the one or more genes. In some embodiments, the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 has been determined to be at or above a reference level of the one or more genes. In some embodiments, the expression level of two of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 2, has been determined to be at or above a reference level of the one or more genes. In some embodiments, the expression level of three of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 3, has been determined to be at or above a reference level of the one or more genes. In some embodiments, the expression level of four of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 4, has been determined to be at or above a reference level of the one or more genes. In some embodiments, the expression level of five of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 5, has been determined to be at or above a reference level of the one or more genes. In some embodiments, the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 has been determined to be at or above a reference level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In any of the preceding methods involving the 6-gene signature, the expression level of TGFB1 and/or TGFBR2 has been determined to be at or above a reference expression level of TGFB1 and/or TGFBR2. For example, in some embodiments, the expression level of TGFB1 is at or above a reference expression level of TGFB1. In some embodiments, the expression level of TGFBR1 is at or above a reference expression level of TGFBR1. In any of the preceding methods, the expression level of TGFB1 and TGFBR2 has been determined to be at or above a reference expression level of TGFB1 and TGFBR2.
In some embodiments of any of the preceding methods involving the 6-gene signature, the expression level of ADAM19 or COMP has been determined to be at or above a reference level of ADAM19 or COMP. In some embodiments, the expression level of ADAM19 has been determined to be at or above a reference level of ADAM19. In some embodiments, the expression level of COMP has been determined to be at or above a reference level of COMP. In still further embodiments, the one or more genes includes ADAM19 and COMP. In some embodiments, the expression level of ADAM19 and COMP has been determined to be at or above a reference level of ADAM19 and COMP.
In another aspect, provided herein is a method for assessing a response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2; and (b) maintaining, adjusting, or stopping the treatment based on a comparison of the expression level of the one or more genes in the sample with a reference expression level of the one or more genes, wherein a change in the expression level of the one or more genes in the sample from the individual compared to the reference expression level of the one or more genes is indicative of a response to treatment with the anti-cancer therapy.
In another aspect, provided herein is a method for monitoring the response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2; and (b) comparing the expression level of the one or more genes in the sample from the individual with a reference expression level of the one or more genes, thereby monitoring the response of the individual undergoing treatment with the anti-cancer therapy.
In any of the preceding methods involving the 6-gene signature, the method may include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2. In some embodiments, the method includes determining the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2. In some embodiments, the method includes determining the expression level of two of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 2. In some embodiments, the method includes determining the expression level of three of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 3. In some embodiments, the method includes determining the expression level of four of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 4. In some embodiments, the method includes determining the expression level of five of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 5. In some embodiments, the method involves determining the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In any of the preceding methods involving the 6-gene signature, the method may include determining the expression level of TGFB1 and/or TGFBR2. In any of the preceding methods, the method may include determining the expression level of TGFB1 and TGFBR2.
In some embodiments of any of the preceding methods involving the 6-gene signature, the one or more genes includes at least ADAM19 or COMP. In some embodiments, the one or more genes includes ADAM19. In other embodiments, the one or more genes includes COMP. In still further embodiments, the one or more genes includes ADAM19 and COMP.
In some embodiments of any of the preceding methods involving the 6-gene signature, the change is an increase in the expression level of the one or more genes and the treatment is adjusted or stopped. In other embodiments of any of the preceding methods, the change is a decrease in the expression level of the one or more genes and the treatment is maintained.
In some embodiments of any of the preceding methods involving the 6-gene signature, an increase in the expression level of the five or more genes is indicative of a lack of response of the individual to the treatment. In other embodiments of any of the preceding methods, a decrease in the expression level of the five or more genes is indicative of a response of the individual to the treatment.
In some embodiments of any of the preceding methods involving the 6-gene signature, a reference expression level is the expression level of the one or more (e.g., 1, 2, 3, 4, 5, or 6) genes (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in a reference population, for example, a population of individuals having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer). In particular embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC). In certain embodiments, a reference expression level is the median expression level of the one or more genes in a reference population, for example, a population of individuals having a cancer. In other embodiments, the reference expression level may be the top 40%, the top 30%, the top 20%, the top 10%, the top 5%, or the top 1% of the expression level in the reference population. In some embodiments, the reference expression level is determined by principle component analysis of Z-score-transformed expression levels. In certain embodiments, the reference expression level is a pre-assigned expression level for the one or more genes. In some embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a previous time point, wherein the previous time point is following administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, a reference expression level is the expression level of the one or more genes (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in a biological sample from the patient obtained prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In other embodiments, the reference expression level is the expression level of the one or more genes in a biological sample obtained from the patient at a subsequent time point (e.g., minutes, hours, days, weeks, months, or years after administration of an anti-cancer therapy).
In some embodiments of any of the preceding methods involving the 6-gene signature, an expression level above a reference expression level, or an elevated or increased expression or number, refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level or number of a biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by methods such as those described herein and/or known in the art, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, the elevated expression or number refers to the increase in expression level/amount of a biomarker (e.g., one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in the sample wherein the increase is at least about any of 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×, 500×, or 1000× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression or number refers to an overall increase in expression level/amount of a biomarker (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 6-gene signature, an expression level below a reference expression level, or a reduced (decreased) expression or number, refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by standard art known methods such as those described herein, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression or number refers to the decrease in expression level/amount of a biomarker (e.g., one or more of ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) in the sample wherein the decrease is at least about any of 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or 0.01× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, reduced (decreased) expression or number refers to an overall decrease in expression level/amount of a biomarker (e.g., ACTA2, ADAM19, COMP, CTGF, TGFB1, and/or TGFBR2) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 6-gene signature, a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment. In some embodiments, the CD8+ T-cells localize at or near collagen fibers.
C. Exemplary 19-Gene Signature (Pan-F-TBRS)
In some embodiments, the methods and uses herein involve determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) genes selected from a 19-gene signature which includes ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1, which is also referred to herein as Pan-F-TBRS.
For example, in one embodiment, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1, wherein the expression level of one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 in the sample is determined to be at or above a reference expression level of the one or more genes; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more genes determined in step (a)).
The method may include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the method includes determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the method involves determining the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In some embodiments of any of the preceding methods involving the 19-gene signature, the method includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) genes selected from ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In another embodiment, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 in the sample has been determined to be at or above a reference expression level of the one or more genes.
In another aspect, the invention provides a method of treating cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) in an individual having been identified as having an expression level in a sample from the individual of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1 that is at or above a reference expression level of the one or more genes, the method including administering to the individual an anti-cancer therapy comprising an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)).
In some embodiments of any of the preceding methods involving the 19-gene signature, the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be at or above a reference expression level of the one or more genes. For example, in some embodiments, the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be at or above a reference expression level of the one or more genes. In some embodiments, the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be at or above a reference expression level ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In some embodiments of any of the preceding methods involving the 19-gene signature, the the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be at or above a reference level of the one or more genes.
In another aspect, provided herein is a method for assessing a response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and (b) maintaining, adjusting, or stopping the treatment based on a comparison of the expression level of the one or more genes in the sample with a reference expression level of the one or more genes, wherein a change in the expression level of the one or more genes in the sample from the individual compared to the reference expression level of the one or more genes is indicative of a response to treatment with the anti-cancer therapy.
In another aspect, provided herein is a method for monitoring the response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1; and (b) comparing the expression level of the one or more genes in the sample from the individual with a reference expression level of the one or more genes, thereby monitoring the response of the individual undergoing treatment with the anti-cancer therapy.
In any of the preceding methods involving the 19-gene signature, the method may include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the method includes determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the method involves determining the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In some embodiments of any of the preceding methods involving the 19-gene signature, the method includes determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) genes selected from ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In some embodiments of any of the preceding methods involving the 19-gene signature, the change is an increase in the expression level of the one or more genes and the treatment is adjusted or stopped. In other embodiments of any of the preceding methods, the change is a decrease in the expression level of the one or more genes and the treatment is maintained.
In some embodiments of any of the preceding methods involving the 19-gene signature, an increase in the expression level of the five or more genes is indicative of a lack of response of the individual to the treatment. In other embodiments of any of the preceding methods, a decrease in the expression level of the five or more genes is indicative of a response of the individual to the treatment.
In some embodiments of any of the preceding methods involving the 19-gene signature, a reference level is the expression level of the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) genes (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a reference population, for example, a population of individuals having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer, a lung cancer, a liver cancer, an ovarian cancer, a pancreatic cancer, a colorectal cancer, or a breast cancer). In particular embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC). In certain embodiments, a reference level is the median expression level of the one or more genes in a reference population, for example, a population of individuals having a cancer. In other embodiments, the reference level may be the top 40%, the top 30%, the top 20%, the top 10%, the top 5%, or the top 1% of the expression level in the reference population. In some embodiments, the reference expression level is determined by principle component analysis of Z-score-transformed expression levels. In certain embodiments, the reference level is a pre-assigned expression level for the one or more genes. In some embodiments, the reference level is the expression level of the one or more genes in a biological sample obtained from the patient at a previous time point, wherein the previous time point is following administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, a reference level is the expression level of the one or more genes (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in a biological sample from the patient obtained prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In other embodiments, the reference level is the expression level of the one or more genes in a biological sample obtained from the patient at a subsequent time point (e.g., minutes, hours, days, weeks, months, or years after administration of an anti-cancer therapy).
In some embodiments of any of the preceding methods involving the 19-gene signature, an expression level above a reference expression level, or an elevated or increased expression or number, refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level or number of a biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by methods such as those described herein and/or known in the art, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, the elevated expression or number refers to the increase in expression level/amount of a biomarker (e.g., one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the increase is at least about any of 1.1×, 1.2×, 1.3×, 1.4×, 1.5×, 1.6×, 1.7×, 1.8×, 1.9×, 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 30×, 40×, 50×, 100×, 500×, or 1000× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression or number refers to an overall increase in expression level/amount of a biomarker (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 19-gene signature, an expression level below a reference expression level, or a reduced (decreased) expression or number, refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein, nucleic acid (e.g., gene or mRNA), or cell), detected by standard art known methods such as those described herein, as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression or number refers to the decrease in expression level/amount of a biomarker (e.g., one or more of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) in the sample wherein the decrease is at least about any of 0.9×, 0.8×, 0.7×, 0.6×, 0.5×, 0.4×, 0.3×, 0.2×, 0.1×, 0.05×, or 0.01× the expression level/amount of the respective biomarker in a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, reduced (decreased) expression or number refers to an overall decrease in expression level/amount of a biomarker (e.g., ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and/or TPM1) of greater than about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1-fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 500-fold, about 1,000-fold or greater as compared to a reference expression level, reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
In some embodiments of any of the preceding methods involving the 19-gene signature, a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment. In some embodiments, the CD8+ T-cells localize at or near collagen fibers.
D. Exemplary Additional Biomarkers (e.g., Teff Genes and CAF Genes)
Any of the preceding methods (e.g., as described in Section III, Subsections A-C above, relating to the 22-, 6-, and 19-gene (Pan-F-TBRS) signatures, respectively) can further include determining the expression level in the sample of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9) additional genes selected from the group consisting of PD-L1, CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the one or more additional gene is PD-L1. In other embodiments, the one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) additional genes is selected from the group consisting of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the method further includes determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21. In some embodiments, the one or more additional genes are CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21.
For example, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following 22-gene signature genes in the sample: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more Teff genes and the one or more Pan-F-TBRS genes determined in step (a)). In some embodiments, the expression level of the one or more Pan-F-TBRS genes is at or above a reference expression level, and the expression level of the one or more Teff genes is at or above or below a reference expression level, and the method comprises administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual. In other embodiments, the expression level of the one or more Pan-F-TBRS genes is below a reference expression level, and the expression level of the one or more Teff genes is at or above a reference expression level, and the method comprises administering an anti-cancer therapy that includes an immunotherapy to the individual. In some embodiments, the immunotherapy is a monotherapy.
In yet another embodiment, provided herein is a method for treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21 has been determined to be at or above a reference expression level of the one or more Teff genes, and the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following 22-gene signature genes in the sample: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be below a reference expression level of the one or more 22-gene signature genes. In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
In another embodiment, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following 6-gene signature genes in the sample: of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more Teff genes and the one or more 6-gene signature genes determined in step (a)). In some embodiments, the expression level of the one or more 6-gene signature genes is at or above a reference expression level, and the expression level of the one or more Teff genes is at or above or below a reference expression level, and the method comprises administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual. In other embodiments, the expression level of the one or more 6-gene signature genes is below a reference expression level, and the expression level of the one or more Teff genes is at or above a reference expression level, and the method comprises administering an anti-cancer therapy that includes an immunotherapy to the individual. In some embodiments, the immunotherapy is a monotherapy.
For example, provided herein is a method for treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21 has been determined to be at or above a reference expression level of the one or more Teff genes, and the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following 6-gene signature genes in the sample: of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 has been determined to be below a reference expression level of the one or more 6-gene signature genes. In some embodiments, the immunotherapy is a monotherapy.
In another embodiment, provided herein is a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including (a) determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following Pan-F-TBRS genes in the sample: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more Teff genes and the one or more Pan-F-TBRS genes determined in step (a)). In some embodiments, the expression level of the one or more Pan-F-TBRS genes is at or above a reference expression level, and the expression level of the one or more Teff genes is at or above or below a reference expression level, and the method comprises administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual. In other embodiments, the expression level of the one or more Pan-F-TBRS genes is below a reference expression level, and the expression level of the one or more Teff genes is at or above a reference expression level, and the method comprises administering an anti-cancer therapy that includes an immunotherapy to the individual. In some embodiments, the immunotherapy is a monotherapy.
In yet another embodiment, provided herein is a method for treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21 has been determined to be at or above a reference expression level of the one or more Teff genes, and the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following Pan-F-TBRS genes in the sample: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be below a reference expression level of the one or more Pan-F-TBRS genes. In some embodiments, the immunotherapy is a monotherapy. In some embodiments, the method further includes administering the anti-cancer therapy to the patient.
In another aspect, provided herein is a method for assessing a response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more Teff genes (e.g., 1, 2, or 3 Teff genes selected from IFNG, GZMB, and ZAP70) and/or one or more CAF genes (e.g., 1, 2, or 3 CAF genes selected from LOXL2, TNC, and POSTN) in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual; and (b) maintaining, adjusting, or stopping the treatment based on a comparison of the expression level of the one or more Teff genes and/or the one or more CAF genes in the sample with a reference expression level of the one or more Teff genes and/or the one or more CAF genes, wherein a change in the expression level of the one or more Teff genes and/or the one or more CAF genes in the sample from the individual compared to the reference expression level of the one or more Teff genes and/or the one or more CAF genes is indicative of a response to treatment with the anti-cancer therapy. In some embodiments, the change is an increase in the expression level of one or more Teff genes (e.g., IFNG, GZMB, and/or ZAP70). In some embodiments, the change is a decrease in the expression level of one or more CAF genes (e.g., LOXL2, TNC, and POSTN).
In another aspect, provided herein is a method for monitoring the response of an individual having a cancer to treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the method including: (a) determining the expression level of one or more Teff genes (e.g., 1, 2, or 3 Teff genes selected from IFNG, GZMB, and ZAP70) and/or one or more CAF genes (e.g., 1, 2, or 3 CAF genes selected from LOXL2, TNC, and POSTN) in a sample from the individual at a time point during or after administration of an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist to the individual; and (b) comparing the expression level of the one or more Teff genes and/or the one or more CAF genes in the sample from the individual with a reference expression level of the one or more Teff genes and/or the one or more CAF genes, thereby monitoring the response of the individual undergoing treatment with the anti-cancer therapy.
E. Tumor Mutational Burden (TMB)
Any of the preceding embodiments (e.g., as described in Section III, Subsections A-C above, relating to the 22-, 6-, and 19-gene (Pan-F-TBRS) signatures, respectively, or in Section III, Subsection D) may include determining a TMB in a tumor sample from the individual. TMB may be determined using any suitable approach, for example, as described in Example 1 or in International Patent Application No.: PCT/US2017/055669, which is incorporated herein by reference in its entirety. In some embodiments, the individual may have a TMB in a tumor sample that is at or above a reference TMB. In other embodiments, the individual may have a TMB that is below a reference TMB.
The invention provides a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including: (a) determining (i) the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following 22-gene signature genes in a sample from the individual: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1; and (ii) a TMB score from a tumor sample from the individual; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more 22-gene signature genes and TMB score determined in step (a)). In some embodiments, the expression level of the one or more 22-gene signature genes is at or above a reference expression level, and the TMB score from the tumor sample is at or above or below a reference TMB score, and the method comprises administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual. In other embodiments, the expression level of the one or more 22-gene signature genes is below a reference expression level, and the TMB score from the tumor sample is at or above a reference TMB score, and the method comprises administering an anti-cancer therapy that includes an immunotherapy to the individual. In some embodiments, the immunotherapy is a monotherapy.
The invention further provides a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including: administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the following 22-gene signature genes in a sample from the individual: TGFB1, TGFBR2, ACTA2, ACTG2, ADAM12, ADAM19, COMP, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be below a reference expression level of the one or more 22-gene signature genes, and a TMB score in a tumor sample from the patient has been determined to be at or above a reference TMB score. In some embodiments, the immunotherapy is a monotherapy.
The invention provides a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including: (a) determining (i) the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following 6-gene signature genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2; and (ii) a TMB score from a tumor sample from the individual; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more 6-gene signature genes and TMB score determined in step (a)). In some embodiments, the expression level of the one or more 6-gene signature genes is at or above a reference expression level, and the TMB score from the tumor sample is at or above or below a reference TMB score, and the method comprises administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) to the individual. In other embodiments, the expression level of the one or more 6-gene signature genes is below a reference expression level, and the TMB score from the tumor sample is at or above a reference TMB score, and the method comprises administering an anti-cancer therapy that includes an immunotherapy to the individual. In some embodiments, the immunotherapy is a monotherapy.
The invention further provides a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including: administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following 6-gene signature genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2 has been determined to be below a reference expression level of the one or more 6-gene signature genes, and a TMB score in a tumor sample from the patient has been determined to be at or above a reference TMB score. In some embodiments, the immunotherapy is a monotherapy.
The invention provides a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including: (a) determining (i) the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following Pan-F-TBRS genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1; and (ii) a TMB score from a tumor sample from the individual; and (b) administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody) to the individual based on the expression level of the one or more Pan-F-TBRS genes and TMB score determined in step (a)). In some embodiments, the expression level of the one or more Pan-F-TBRS genes is at or above a reference expression level, and the TMB score from the tumor sample is at or above or below a reference TMB score, and the method comprises administering an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody) to the individual. In other embodiments, the expression level of the one or more Pan-F-TBRS genes is below a reference expression level, and the TMB score from the tumor sample is at or above a reference TMB score, and the method comprises administering an anti-cancer therapy that includes an immunotherapy to the individual. In some embodiments, the immunotherapy is a monotherapy.
The invention further provides a method of treating an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer), the method including: administering to the individual an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), wherein prior to treatment the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following Pan-F-TBRS genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1 has been determined to be below a reference expression level of the one or more Teff genes, and a TMB score in a tumor sample from the patient has been determined to be at or above a reference TMB score. In some embodiments, the immunotherapy is a monotherapy. Any of the preceding methods involving TMB can further include determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following Teff genes in a sample from the individual: CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, or TBX21.
F. Exemplary Approaches for Determination of Biomarker Expression Levels
In some embodiments of any of the preceding embodiments (e.g., as described in Section III, Subsections A-C above, relating to the 22-, 6-, and 19-gene (Pan-F-TBRS) signatures, respectively, or in Section III, Subsections D and E), the sample is obtained from the individual prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the anti-cancer therapy. In some embodiments of any of the preceding methods, the sample from the individual is obtained about 2 to about 10 weeks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks) following administration of the anti-cancer therapy. In some embodiments, the sample from the individual is obtained about 4 to about 6 weeks following administration of the anti-cancer therapy.
In some embodiments of any of the preceding methods, the expression level or number of a biomarker is detected in a tissue sample, a primary or cultured cells or cell line, a cell supernatant, a cell lysate, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, or any combination thereof. In some embodiments, the sample is a tissue sample (e.g., a tumor tissue sample), a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof. In some embodiments, the tumor tissue sample wherein the tumor tissue sample includes tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof. In some embodiments, the tumor tissue sample is a FFPE sample, an archival sample, a fresh sample, or a frozen sample.
For example, in some embodiments of any of the preceding methods, the expression level of a biomarker is detected in tumor-infiltrating immune cells, tumor cells, PBMCs, or combinations thereof using known techniques (e.g., flow cytometry or IHC). Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells or any combinations thereof, and other tumor stroma cells (e.g., fibroblasts). Such tumor infiltrating immune cells may be T lymphocytes (such as CD8⁺ T lymphocytes (e.g., CD8⁺ Teff cells) and/or CD4⁺ T lymphocytes (e.g., CD4⁺ Teff cells), B lymphocytes, or other bone marrow-lineage cells including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer (NK) cells. In some embodiments, the staining for a biomarker is detected as membrane staining, cytoplasmic staining, or combinations thereof. In other embodiments, the absence of a biomarker is detected as absent or no staining in the sample, relative to a reference sample.
In particular embodiments of any of the preceding methods, the expression level of a biomarker is assessed in a sample that contains or is suspected to contain cancer cells. The sample may be, for example, a tissue biopsy or a metastatic lesion obtained from a patient suffering from, suspected to suffer from, or diagnosed with cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC)). In some embodiments, the sample is a sample of bladder tissue, a biopsy of an bladder tumor, a known or suspected metastatic bladder cancer lesion or section, or a blood sample, e.g., a peripheral blood sample, known or suspected to comprise circulating cancer cells, e.g., bladder cancer cells. The sample may comprise both cancer cells, i.e., tumor cells, and non-cancerous cells (e.g., lymphocytes, such as T cells or NK cells), and, in certain embodiments, comprises both cancerous and non-cancerous cells. Methods of obtaining biological samples including tissue resections, biopsies, and body fluids, e.g., blood samples comprising cancer/tumor cells, are well known in the art.
In some embodiments of any of the preceding methods, the patient has carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. In some embodiments, the cancer is bladder cancer (e.g., UC, e.g., mUC), kidney cancer (e.g., renal cell carcinoma (RCC), e.g., advanced RCC or metastatic RCC (mRCC)), squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., PDAC), glioblastoma, cervical cancer, ovarian cancer, liver cancer (e.g., HCC), hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, Merkel cell cancer, mycoses fungoids, testicular cancer, esophageal cancer, tumors of the biliary tract, head and neck cancer, B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), or Meigs' syndrome. In some embodiments, the cancer is a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer (e.g., HCC), an ovarian cancer, or a breast cancer (e.g., TNBC). In preferred embodiments, the patient has a bladder cancer (e.g., UC, e.g., mUC). The patient may optionally have an advanced, refractory, recurrent, chemotherapy-resistant, and/or platinum-resistant form of the cancer.
In certain embodiments, the presence and/or expression levels/amount of a biomarker in a first sample is increased or elevated as compared to presence/absence and/or expression levels/amount in a second sample. In certain embodiments, the presence/absence and/or expression levels/amount of a biomarker in a first sample is decreased or reduced as compared to presence and/or expression levels/amount in a second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or combined multiple samples from the same patient or individual that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained at an earlier time point from the same patient or individual than when the test sample is obtained. Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.
In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more healthy individuals who are not the patient. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the patient or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the patient. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the patient.
G. Exemplary Therapeutic Approaches
In embodiments of any of the preceding methods and uses (e.g., as described in Section III, Subsections A-F), for the prevention or treatment of cancer, the dose of an anti-cancer therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) will depend on the type of cancer to be treated, as defined above, the severity and course of the cancer, whether the anti-cancer therapy is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attending physician.
In some embodiments, the anti-cancer therapy (e.g., an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody)) may be suitably administered to the patient at one time or over a series of treatments. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives, for example, from about two to about twenty, or e.g., about six doses of the anti-cancer therapy). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
For example, as a general proposition, the therapeutically effective amount of an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) administered to human will be in the range of about 0.01 to about 50 mg/kg of patient body weight, whether by one or more administrations. In some embodiments, the therapeutic agent (e.g, antibody) used is about 0.01 mg/kg to about 45 mg/kg, about 0.01 mg/kg to about 40 mg/kg, about 0.01 mg/kg to about 35 mg/kg, about 0.01 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 25 mg/kg, about 0.01 mg/kg to about 20 mg/kg, about 0.01 0.01 mg/kg to about 15 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 1 mg/kg administered daily, weekly, every two weeks, every three weeks, or monthly, for example. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosage regimens may be useful. In one embodiment, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) is administered to a human at a dose of about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 420 mg, about 500 mg, about 525 mg, about 600 mg, about 700 mg, about 800 mg, about 840 mg, about 900 mg, about 1000 mg, about 1050 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, or about 1800 mg, for example, on day 1 of 21-day cycles (every three weeks, q3w). For example, the fixed dose may be approximately 420 mg, approximately 525 mg, approximately 840 mg, or approximately 1050 mg. In some embodiments, atezolizumab is administered at 1200 mg intravenously every three weeks (q3w). Where a fixed dose is administered, preferably it is in the range from about 5 mg to about 2000 mg. The dose of an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered as a single dose or as multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more doses). Where a series of doses are administered, these may, for example, be administered approximately every week, approximately every 2 weeks, approximately every 3 weeks, or approximately every 4 weeks. The dose of the antagonist administered in a combination treatment may be reduced as compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.
Immunotherapies (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) described herein (and any additional therapeutic agent) may be formulated, dosed, and administered in a fashion consistent with good medical practice. Likewise, suppressive stromal antagonists (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or the suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) need not be, but is optionally formulated with and/or administered concurrently with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of the immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or the suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.
In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered concurrently with a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody). In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) are administered as part of the same formulation. In other embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) is administered separately from a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).
In some embodiments, any of the preceding methods may further include administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from the group consisting of an immunotherapy agent, a cytotoxic agent, a growth inhibitory agent, a radiation therapy agent, an anti-angiogenic agent, and combinations thereof.
In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) is administered concurrently with an agonist directed against an activating co-stimulatory molecule. In some embodiments, an activating co-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist directed against an activating co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an antagonist directed against an inhibitory co-stimulatory molecule. In some embodiments, an inhibitory co-stimulatory molecule may include CTLA-4 (also known as CD152), TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonist directed against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an antagonist directed against CTLA-4 (also known as CD152), e.g., a blocking antibody. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with ipilimumab (also known as MDX-010, MDX-101, or YERVOY®). In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an antagonist directed against B7-H3 (also known as CD276), e.g., a blocking antibody. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with MGA271.
In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an agonist directed against CD137 (also known as TNFRSF9, 4-1 BB, or ILA), e.g., an activating antibody. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with urelumab (also known as BMS-663513). In some embodiments an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an agonist directed against CD40, e.g., an activating antibody. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with CP-870893. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an agonist directed against OX40 (also known as CD134), e.g., an activating antibody. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an anti-OX40 antibody (e.g., AgonOX). In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an agonist directed against CD27, e.g., an activating antibody. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with CDX-1127. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an antagonist directed against TIGIT, for example, an anti-TIGIT antibody. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an antagonist directed against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).
In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci. 104:14-21, 2013). In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an adjuvant. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with a treatment comprising a TLR agonist, e.g., Poly-ICLC (also known as HILTONOL®), LPS, MPL, or CpG ODN. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with tumor necrosis factor (TNF) alpha. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with IL-1. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with HMGB1. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an IL-10 antagonist. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an IL-4 antagonist. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an IL-13 antagonist. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an HVEM antagonist. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with a treatment targeting CX3CL1. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with a treatment targeting CXCL9. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with a treatment targeting CXCL10. In some embodiments an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with a treatment targeting CCLS. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with an LFA-1 or ICAM1 agonist. In some embodiments, an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) may be administered in conjunction with a Selectin agonist.
A chemotherapeutic agent, if administered, is usually administered at dosages known therefore, or optionally lowered due to combined action of the drugs or negative side effects attributable to administration of the chemotherapeutic agent. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Where the chemotherapeutic agent is paclitaxel, preferably, it is administered at a dose between about 130 mg/m²to 200 mg/m²(e.g., approximately 175 mg/m²), for instance, over 3 hours, once every 3 weeks. Where the chemotherapeutic agent is carboplatin, preferably it is administered by calculating the dose of carboplatin using the Calvert formula which is based on a patients preexisting renal function or renal function and desired platelet nadir. Renal excretion is the major route of elimination for carboplatin. The use of this dosing formula, as compared to empirical dose calculation based on body surface area, allows compensation for patient variations in pretreatment renal function that might otherwise result in either underdosing (in patients with above average renal function) or overdosing (in patients with impaired renal function). The target AUC of 4-6 mg/mL/min using single agent carboplatin appears to provide the most appropriate dose range in previously treated patients.
In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of tumors and/or cancer cells.
Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent or agents. In one embodiment, administration of an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other.
In embodiments where either the immunotherapy or the suppressive stromal antagonist is an antibody (e.g., an anti-PD-L1 antibody or an anti-TGF-β antibody), the administered antibody may be a naked antibody. The immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or the suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody) administered may be conjugated with a cytotoxic agent. Preferably, the conjugated and/or antigen to which it is bound is/are internalized by the cell, resulting in increased therapeutic efficacy of the conjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with nucleic acid in the cancer cell. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonucleases, and DNA endonucleases.
The compositions utilized in the methods described herein can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, intravitreally (e.g., by intravitreal injection), parenterally, by eye drop, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In some embodiments, the immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or the suppressive stromal antagonist (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody) is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the multi-targeted tyrosine kinase inhibitor is administered orally. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

IV. Compositions and Pharmaceutical Formulations

In one aspect, the invention is based, in part, on the discovery that biomarkers of the invention can be used to identify individuals having a cancer (including, but not limited to, a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from anti-cancer therapies that include immunotherapies (including, but not limited to, a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonists (including, but not limited to, a TGF-8 antagonist, e.g., an anti-TGF-β antibody). In some embodiments, the individual is less likely to respond to the immunotherapy alone. In another aspect, the invention is based, in part, on the discovery that biomarkers of the invention can be used to monitor and/or assess treatment response for individuals having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who are treated with anti-cancer therapies that include immunotherapies (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonists (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody). These agents, and combinations thereof, are useful for the treatment of cancer, e.g., as part of any of the methods and uses described herein, for example, in Sections II and III above. Any suitable immunotherapy and/or suppressive stromal antagonist can be used in the methods and uses described herein. Non-limiting examples suitable for use in the methods and uses of the invention are described further below.
A. Exemplary Immunotherapies
Any suitable immunotherapy may be used in the context of the invention. Immunotherapies are described in the art (see, e.g., Chen et al. Immunity 39:1-10, 2013). The immunotherapy may be an activating immunotherapy or a suppressing immunotherapy. In some embodiments, the activating immunotherapy includes a CD28, OX40, GITR, CD137, CD27, ICOS, HVEM, NKG2D, MICA, 2B4, IL-2, IL-12, IFNγ, IFNα, TNFα, IL-1, CDN, HMBG1, or TLR agonist. In particular embodiments, the agonist (e.g., a CD28, OX40, GITR, CD137, CD27, ICOS, HVEM, NKG2D, MICA, 2B4, IL-2, IL-12, IFNγ, IFNα, TNFα, IL-1, CDN, HMBG1, or TLR agonist) increases, enhances, or stimulates an immune response or function in a patient having cancer. In some embodiments, the agonist modulates the expression and/or activity of a ligand (e.g., a T cell receptor ligand), and/or increases or stimulates the interaction of the ligand with its immune receptor, and/or increases or stimulates the intracellular signaling mediated by ligand binding to the immune receptor. In other embodiments, the suppressing immunotherapy includes a PD-L1 axis, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, B7H4, CD96, TIGIT, CD226, prostaglandin, VEGF, endothelin B, IDO, arginase, MICA/MICB, TIM-3, IL-10, IL-4, or IL-13 antagonist. In particular embodiments, the antagonist (e.g., a PD-L1 axis, CTLA-4, TIM-3, BTLA, VISTA, LAG-3, B7H4, CD96, TIGIT, CD226, prostaglandin, VEGF, endothelin B, IDO, arginase, MICA/MICB, TIM-3, IL-10, IL-4, or IL-13 antagonist) is an agent that inhibits and/or blocks the interaction of a ligand (e.g., a T cell receptor ligand) with its immune receptor or is an antagonist of ligand and/or receptor expression and/or activity, or is an agent that blocks the intracellular signaling mediated by a ligand (e.g., a T cell receptor ligand) with its immune receptor. In some embodiments, the immunotherapy is an immune checkpoint inhibitor. Immunotherapy antibodies may have any of the features, singly or in combination, described in Sections i-vii of Subsection D below.
B. Exemplary PD-L1 Axis Binding Antagonists
PD-L1 axis binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. PD-1 (programmed death 1) is also referred to in the art as “programmed cell death 1,” “PDCD1,” “CD279,” and “SLEB2.” An exemplary human PD-1 is shown in UniProtKB/Swiss-Prot Accession No. 015116. PD-L1 (programmed death ligand 1) is also referred to in the art as “programmed cell death 1 ligand 1,” “PDCD1LG1,” “CD274,” “B7-H,” and “PDL1.” An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1. PD-L2 (programmed death ligand 2) is also referred to in the art as “programmed cell death 1 ligand 2,” “PDCD1LG2,” “CD273,” “B7-DC,” “Btdc,” and “PDL2.” An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2. The PD-L1 axis binding antagonist may, in some instances, be a PD-1 binding antagonist, a PD-L1 binding antagonist, or a PD-L2 binding antagonist. It is expressly contemplated that such PD-L1 axis binding antagonist antibodies (e.g., anti-PD-L1 antibodies, anti-PD-1 antibodies, and anti-PD-L2 antibodies), or other antibodies described herein (e.g., anti-PD-L1 antibodies for detection of PD-L1 expression levels) for use in any of the embodiments enumerated herein may have any of the features, singly or in combination, described in Sections i-vii of Subsection D below.
(i) PD-L1 Binding Antagonists
In some instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to one or more of its ligand binding partners. In other instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1. In yet other instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1. In some instances, the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. The PD-L1 binding antagonist may be, without limitation, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1. In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PD-L1 and VISTA. In some embodiments, the PD-L1 binding antagonist is CA-170 (also known as AUPM-170). In some embodiments, the PD-L1 binding antagonist is a small molecule that inhibits PDL1 and TIM3. In some embodiments, the small molecule is a compound described in WO2015/033301 and WO2015/033299.
In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. A variety of anti-PD-L1 antibodies are contemplated and described herein. In any of the embodiments herein, the isolated anti-PD-L1 antibody can bind to a human PD-L1, for example a human PD-L1 as shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof. In some embodiments, the anti-PD-L1 antibody is capable of inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1. In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab′)2 fragments. In some embodiments, the anti-PD-L1 antibody is a chimeric or humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody. Examples of anti-PD-L1 antibodies useful in the methods of this invention and methods of making them are described in International Patent Application Publication No. WO 2010/077634 and U.S. Pat. No. 8,217,149, each of which is incorporated herein by reference in its entirety.
In some embodiments, the anti-PD-L1 antibody is atezolizumab (CAS Registry Number: 1422185-06-5). Atezolizumab (Genentech), also known as MPDL3280A, is an anti-PD-L1 antibody.
Atezolizumab comprises:

- (a) an HVR-H1, HVR-H2, and HVR-H3 sequence of GFTFSDSWIH (SEQ ID NO: 63), AWISPYGGSTYYADSVKG (SEQ ID NO: 64) and RHWPGGFDY (SEQ ID NO: 65), respectively, and
- (b) an HVR-L1, HVR-L2, and HVR-L3 sequence of RASQDVSTAVA (SEQ ID NO: 66), SASFLYS (SEQ ID NO:67) and QQYLYHPAT (SEQ ID NO: 68), respectively.

Atezolizumab comprises a heavy chain and a light chain sequence, wherein:

(a) the heavy chain variable region sequence

comprises the amino acid sequence:

(SEQ ID NO: 69)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA

WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR

RHWPGGFDYWGQGTLVTVSS,

and

(b) the light chain variable region sequence

comprises the amino acid sequence:

(SEQ ID NO: 70)

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY

SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF

GQGTKVEIKR.

In some instances, the anti-PD-L1 antibody comprises (a) a VH domain comprising an amino acid sequence comprising having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of (SEQ ID NO: 69); (b) a VL domain comprising an amino acid sequence comprising having at least 95% sequence identity (e.g., at least 95%, 96%, 97%, 98%, or 99% sequence identity) to, or the sequence of (SEQ ID NO: 70); or (c) a VH domain as in (a) and a VL domain as in (b).
Atezolizumab comprises a heavy chain and a light chain sequence, wherein:

(a) the heavy chain comprises the amino acid

sequence:

(SEQ ID NO: 71)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA

WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAR

RHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV

KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT

QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY

KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

LSLSPG,

and

(b) the light chain comprises the amino acid

sequence:

(SEQ ID NO: 72)

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY

SASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATF

GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

In some embodiments, the anti-PD-L1 antibody is avelumab (CAS Registry Number: 1537032-82-8). Avelumab, also known as MSB0010718C, is a human monoclonal IgG1 anti-PD-L1 antibody (Merck KGaA, Pfizer). Avelumab comprises a heavy chain and a light chain sequence, wherein:

(a) the heavy chain comprises the amino acid sequence:
(SEQ ID NO: 73)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISR

DNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK

KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE

LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

HEALHNHYTQKSLSLSPG,
and

(b) the light chain comprises the amino acid sequence:
(SEQ ID NO: 74)
QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGN

TASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYP

GAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

S

In some embodiments, the anti-PD-L1 antibody comprises the six HVR sequences from SEQ ID NO: 73 and SEQ ID NO: 74 (e.g., the three heavy chain HVRs from SEQ ID NO: 73 and the three light chain HVRs from SEQ ID NO: 74). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain from SEQ ID NO: 73 and the light chain variable domain from SEQ ID NO: 74.
In some embodiments, the anti-PD-L1 antibody is durvalumab (CAS Registry Number: 1428935-60-7). Durvalumab, also known as MED14736, is an Fc-optimized human monoclonal IgG1 kappa anti-PD-L1 antibody (MedImmune, AstraZeneca) described in WO2011/066389 and US2013/034559. Durvalumab comprises a heavy chain and a light chain sequence, wherein:

(a) the heavy chain comprises the amino acid sequence:
(SEQ ID NO: 75)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTI

SRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN

TKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLP

PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

SCSVMHEALHNHYTQKSLSLSPG,
and

(b) the light chain comprises the amino acid sequence:
(SEQ ID NO: 76)
EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDF

TLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In some embodiments, the anti-PD-L1 antibody comprises the six HVR sequences from SEQ ID NO: 75 and SEQ ID NO: 76 (e.g., the three heavy chain HVRs from SEQ ID NO: 75 and the three light chain HVRs from SEQ ID NO: 76). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain from SEQ ID NO: 75 and the light chain variable domain from SEQ ID NO: 76.
In some embodiments, the anti-PD-L1 antibody is MDX-1105 (Bristol Myers Squibb). MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874.
In some embodiments, the anti-PD-L1 antibody is LY3300054 (Eli Lilly).
In some embodiments, the anti-PD-L1 antibody is STI-A1014 (Sorrento). STI-A1014 is a human anti-PD-L1 antibody.
In some embodiments, the anti-PD-L1 antibody is KN035 (Suzhou Alphamab). KN035 is single-domain antibody (dAB) generated from a camel phage display library.
In some embodiments, the anti-PD-L1 antibody comprises a cleavable moiety or linker that, when cleaved (e.g., by a protease in the tumor microenvironment), activates an antibody antigen binding domain to allow it to bind its antigen, e.g., by removing a non-binding steric moiety. In some embodiments, the anti-PD-L1 antibody is CX-072 (CytomX Therapeutics).
In some embodiments, the anti-PD-L1 antibody comprises the six HVR sequences (e.g., the three heavy chain HVRs and the three light chain HVRs) and/or the heavy chain variable domain and light chain variable domain from an anti-PD-L1 antibody described in US20160108123 (Assigned to Novartis), WO2016/000619 (Applicant: Beigene), WO2012/145493 (Applicant: Amplimmune), U.S. Pat. No. 9,205,148 (Assigned to MedImmune), WO2013/181634 (Applicant: Sorrento), and WO2016/061142 (Applicant: Novartis).
In some instances, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, YW243.55.570, MDX-1105, MED14736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.570 is an anti-PD-L1 described in PCT Pub. No. WO 2010/077634. Further examples of anti-PD-L1 antibodies useful for the methods of this invention, and methods for making thereof are described in PCT Pub. Nos. WO 2010/077634, WO 2007/005874, and WO 2011/066389, and also in U.S. Pat. No. 8,217,149, and U.S. Pub. No. 2013/034559, which are incorporated herein by reference.
In a still further specific aspect, the anti-PD-L1 antibody has reduced or minimal effector function. In a still further specific aspect the minimal effector function results from an “effector-less Fc mutation” or aglycosylation mutation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites from an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).
(ii) PD-1 Binding Antagonists
In some instances, the PD-L1 axis binding antagonist is a PD-1 binding antagonist. For example, in some instances, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners. In some instances, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In other instances, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In yet other instances, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. The PD-1 binding antagonist may be, without limitation, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). For example, in some instances, the PD-1 binding antagonist is an Fc-fusion protein. In some embodiments, the PD-1 binding antagonist is AMP-224. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO 2010/027827 and WO 2011/066342. In some embodiments, the PD-1 binding antagonist is a peptide or small molecule compound. In some embodiments, the PD-1 binding antagonist is AUNP-12 (PierreFabre/Aurigene). See, e.g., WO2012/168944, WO2015/036927, WO2015/044900, WO2015/033303, WO2013/144704, WO2013/132317, and WO2011/161699. In some embodiments, the PD-1 binding antagonist is a small molecule that inhibits PD-1.
In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. A variety of anti-PD-1 antibodies can be utilized in the methods and uses disclosed herein. In any of the embodiments herein, the PD-1 antibody can bind to a human PD-1 or a variant thereof. In some embodiments the anti-PD-1 antibody is a monoclonal antibody. In some embodiments, the anti-PD-1 antibody is an antibody fragment selected from the group consisting of Fab, Fab′, Fab′-SH, Fv, scFv, and (Fab′)₂fragments. In some embodiments, the anti-PD-1 antibody is a chimeric or humanized antibody. In other embodiments, the anti-PD-1 antibody is a human antibody.
In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Nivolumab (Bristol-Myers Squibb/Ono), also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in WO2006/121168. Nivolumab comprises a heavy chain and a light chain sequence, wherein:

(a) the heavy chain comprises the amino acid sequence:
(SEQ ID NO: 77)
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTI

SRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP

REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN

HYTQKSLSLSLGK,
and

(b) the light chain comprises the amino acid sequence:
(SEQ ID NO: 78)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTL

TISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In some embodiments, the anti-PD-1 antibody comprises the six HVR sequences from SEQ ID NO: 77 and SEQ ID NO: 78 (e.g., the three heavy chain HVRs from SEQ ID NO: 77 and the three light chain HVRs from SEQ ID NO: 78). In some embodiments, the anti-PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 77 and the light chain variable domain from SEQ ID NO: 78.
In some embodiments, the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4). Pembrolizumab (Merck), also known as MK-3475, Merck 3475, lambrolizumab, SCH-900475, and KEYTRUDA®, is an anti-PD-1 antibody described in WO2009/114335. Pembrolizumab comprises a heavy chain and a light chain sequence, wherein:

(a) the heavy chain comprises the amino acid sequence:
(SEQ ID NO: 79)
QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVT

LTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSE

STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT

KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE

VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ

EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS

VMHEALHNHYTQKSLSLSLGK,
and

(b) the light chain comprises the amino acid sequence:
(SEQ ID NO: 80)
EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSG

TDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR

EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

C.

In some embodiments, the anti-PD-1 antibody comprises the six HVR sequences from SEQ ID NO: 79 and SEQ ID NO: 80 (e.g., the three heavy chain HVRs from SEQ ID NO: 79 and the three light chain HVRs from SEQ ID NO: 80). In some embodiments, the anti-PD-1 antibody comprises the heavy chain variable domain from SEQ ID NO: 79 and the light chain variable domain from SEQ ID NO: 80.
In some embodiments, the anti-PD-1 antibody is MEDI-0680 (AMP-514; AstraZeneca). MEDI-0680 is a humanized IgG4 anti-PD-1 antibody.
In some embodiments, the anti-PD-1 antibody is PDR001 (CAS Registry No. 1859072-53-9; Novartis). PDR001 is a humanized IgG4 anti-PD-1 antibody that blocks the binding of PD-L1 and PD-L2 to PD-1.
In some embodiments, the anti-PD-1 antibody is REGN2810 (Regeneron). REGN2810 is a human anti-PD-1 antibody.
In some embodiments, the anti-PD-1 antibody is BGB-108 (BeiGene). In some embodiments, the anti-PD-1 antibody is BGB-A317 (BeiGene).
In some embodiments, the anti-PD-1 antibody is JS-001 (Shanghai Junshi). JS-001 is a humanized anti-PD-1 antibody.
In some embodiments, the anti-PD-1 antibody is STI-A1110 (Sorrento). STI-A1110 is a human anti-PD-1 antibody.
In some embodiments, the anti-PD-1 antibody is INCSHR-1210 (Incyte). INCSHR-1210 is a human IgG4 anti-PD-1 antibody.
In some embodiments, the anti-PD-1 antibody is PF-06801591 (Pfizer).
In some embodiments, the anti-PD-1 antibody is TSR-042 (also known as ANB011; Tesaro/AnaptysBio).
In some embodiments, the anti-PD-1 antibody is AM0001 (ARMO Biosciences).
In some embodiments, the anti-PD-1 antibody is ENUM 244C8 (Enumeral Biomedical Holdings). ENUM 244C8 is an anti-PD-1 antibody that inhibits PD-1 function without blocking binding of PD-L1 to PD-1.
In some embodiments, the anti-PD-1 antibody is ENUM 388D4 (Enumeral Biomedical Holdings). ENUM 388D4 is an anti-PD-1 antibody that competitively inhibits binding of PD-L1 to PD-1.
In some embodiments, the anti-PD-1 antibody comprises the six HVR sequences (e.g., the three heavy chain HVRs and the three light chain HVRs) and/or the heavy chain variable domain and light chain variable domain from an anti-PD-1 antibody described in WO2015/112800 (Applicant: Regeneron), WO2015/112805 (Applicant: Regeneron), WO2015/112900 (Applicant: Novartis), US20150210769 (Assigned to Novartis), WO2016/089873 (Applicant: Celgene), WO2015/035606 (Applicant: Beigene), WO2015/085847 (Applicants: Shanghai Hengrui Pharmaceutical/Jiangsu Hengrui Medicine), WO2014/206107 (Applicants: Shanghai Junshi Biosciences/Junmeng Biosciences), WO2012/145493 (Applicant: Amplimmune), U.S. Pat. No. 9,205,148 (Assigned to MedImmune), WO2015/119930 (Applicants: Pfizer/Merck), WO2015/119923 (Applicants: Pfizer/Merck), WO2016/032927 (Applicants: Pfizer/Merck), WO2014/179664 (Applicant: AnaptysBio), WO2016/106160 (Applicant: Enumeral), and WO2014/194302 (Applicant: Sorrento).
In a still further embodiment, provided is an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 61 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 62.
In a still further embodiment, provided is an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:
(SEQ ID NO: 61)
QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTI

SRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL

VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP

REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV

SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN

HYTQKSLSLSLGK,
and

(b) the light chain sequences has at least 85%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% sequence identity to the light chain sequence:
(SEQ ID NO: 62)
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTL

TISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In a still further specific aspect, the anti-PD-1 antibody has reduced or minimal effector function. In a still further specific aspect the minimal effector function results from an “effector-less Fc mutation” or aglycosylation mutation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some embodiments, the isolated anti-PD-1 antibody is aglycosylated. Removal of glycosylation sites from an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed. The alteration may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).
(iii) PD-L2 Binding Antagonists
In some embodiments, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its ligand binding partners. In a specific aspect, the PD-L2 binding ligand partner is PD-1. The PD-L2 binding antagonist may be, without limitation, an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, an oligopeptide, or a small molecule.
In some embodiments, the PD-L2 binding antagonist is an anti-PD-L2 antibody. In any of the embodiments herein, the anti-PD-L2 antibody can bind to a human PD-L2 or a variant thereof. In some embodiments the anti-PD-L2 antibody is a monoclonal antibody. In some embodiments, the anti-PD-L2 antibody is an antibody fragment selected from the group consisting of Fab, Fab′, Fab′-SH, Fv, scFv, and (Fab′)₂fragments. In some embodiments, the anti-PD-L2 antibody is a chimeric or humanized antibody. In other embodiments, the anti-PD-L2 antibody is a human antibody. In a still further specific aspect, the anti-PD-L2 antibody has reduced or minimal effector function. In a still further specific aspect the minimal effector function results from an “effector-less Fc mutation” or aglycosylation mutation. In still a further embodiment, the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region. In some embodiments, the isolated anti-PD-L2 antibody is aglycosylated.
C. Exemplary Suppressive Stromal Antagonists
Any suitable suppressive stromal antagonist can be used in the context of the invention. Targets for stromal gene antagonists are known in the art; for example, see Turley et al., Nature Reviews Immunology 15:669-682, 2015 and Rosenbloom et al., Biochimica et Biophysica Acta 1832:1088-1103, 2013. In some embodiments, the suppressive stromal antagonist targets TGF-β (e.g., TGF-β1 and TGF-β2). In some embodiments, the suppressive stromal antagonist targets a surface glycoprotein, including, without limitation, endoglin (CD105), 3G5 antigen, FAP, CSPG4 (NG2), and/or podoplanin (GP38). In some embodiments, the suppressive stromal antagonist targets an adhesion molecule, including, without limitation, PECAM (CD31), VCAM (CD106), ICAM1 (CD54), THY1 (CD90) and/or β1 integrin (CD29). In some embodiments, the suppressive stromal antagonist targets a growth factor receptor, including, without limitation, VEGFR1 (FLT1), VEGFR2 (KDR), VEGFR3 (FLT4), PDGFRα (CD140α), and/or PDGFRβ (CD140β). In some embodiments, the suppressive stromal antagonist targets an intracellular structural protein, including, without limitation, vimentin, αSMA (ACTA2) and/or desmin. Other targets for suppressive stromal antagonist include, but are not limited to, 5NT (CD73 or NT5E) RGS3, endosialin (CD248), and FSP1 (S100A4). In some embodiments, the suppressive stromal antagonist targets a cancer-associated fibroblast associated polypeptide. Examples of cancer-associated fibroblast associated polypeptides include, but are not limited to, endoglin (CD105), FAP, podoplanin, VCAM1, THY1, β1 integrin, PDGFRα, PDGFRβ, vimentin, αSMA, desmin, endosialin, and/or FSP1.
In some embodiments, the suppressive stromal antagonist targets TGF-β expression and activation. Examples include pirfenidone, αv66 antibodies, ATI and ATII receptor blockers, ACE inhibitors, CAT-192 (anti-TGF-β1 monoclonal AB), and caveolin scaffolding domain (CSD). In some embodiments, the suppressive stromal antagonist targets TGF-β signaling pathways including canonical signaling pathways TBR1 (exemplary inhibitor is SM305) and SMAD3 (exemplary inhibitor is SIS3). In some embodiments, the suppressive stromal antagonist targets TGF-β signaling pathways including noncanonical signaling pathways including c-Abl (exemplary inhibitor is imatinib mesylate), PDGFR (exemplary inhibitor is dasatinib), c-kit (exemplary inhibitor is nilotinib), and PKC-δ (exemplary inhibitors include small specific polypeptides and rottlerin derivatives). In some embodiments, the suppressive stromal antagonist targets CTGF (exemplary inhibitors include monoclonal CTGF antibodies). In some embodiments, the suppressive stromal antagonist targets inhibition of fibrocyte homing, including CXCL12 (exemplary inhibitor includes CXCL12 antibodies), CXCR4 (exemplary inhibitors include AMD3100), and CCR2 (exemplary inhibitors include PF-04136309). In some embodiments, the suppressive stromal antagonist targets Src (exemplary inhibitors includes dasatinib and SU6656). In some embodiments, the suppressive stromal antagonist targets VEGFR (exemplary inhibitors include nintedanib and BIBF1120). In some embodiments, the suppressive stromal antagonist targets FGFR (exemplary inhibitor includes sorafenib). In some embodiments, the suppressive stromal antagonist targets IL-13 (exemplary inhibitor includes humanized monoclonal antibodies to IL-13). In some embodiments, the suppressive stromal antagonist targets IL-6 receptor (exemplary inhibitor includes tocilizumab). In some embodiments, the suppressive stromal antagonist targets TLR (exemplary inhibitors include TLR inhibitors E5564, TAK-242). In some embodiments, the suppressive stromal antagonist targets Nox4 (ROS) (exemplary inhibitor includes GKT136901). In some embodiments, the suppressive stromal antagonist targets ET-1 (exemplary inhibitors include Bosentan and other ET receptor blockers). In some embodiments, the suppressive stromal antagonist is a TGF-β, PDPN, LAIR-1, SMAD, ALK, connective tissue growth factor (CTGF/CCN2), endothelial-1 (ET-1), AP-1, IL-13, PDGF, LOXL2, endoglin (CD105), FAP, podoplanin (GP38), VCAM1 (CD106), THY1, β1 integrin (CD29), PDGFRα (CD140α), PDGFRβ (CD140β), vimentin, αSMA (ACTA2), desmin, endosialin (CD248), or FSP1 (S100A4) antagonist.
In some embodiments, the suppressive stromal antagonist is pirfenidone, galunisertib, dasetininb, nintedanib, nilotinib, rottlerin and derivatives, or sorafenib.
In some embodiments, the suppressive stromal antagonist is a TGF-β antagonist. In some embodiments, the TGF-β antagonist inhibits the binding of TGF-β to its ligand binding partners. In some embodiments, the TGF-β antagonist inhibits the binding of TGF-β to a cellular receptor of TGF-β. In some embodiments, the TGF-β antagonist inhibits activation of TGF-β. In some embodiments, the TGF-β antagonist targets TGF-β1. In some embodiments, the TGF-β antagonist targets TGF-β2. In some embodiments, the TGF-β antagonist targets TGF-β3. In some embodiments, the TGF-β antagonist targets TGF-β receptor 1. In some embodiments, the TGF-β antagonist targets one or more of TGF-β1, TGF-β2, or TGF-β3. In some embodiments, the TGF-β antagonist targets TGF-β receptor 2. In some embodiments, the TGF-β antagonist targets TGF-β receptor 3. In some embodiments, the TGF-β antagonist targets one or more of TGF-β receptor 1, TGF-β receptor 2, or TGF-β receptor 3.
In some embodiments, the TGF-β antagonist is an anti-TGF-β antibody. In some embodiments, the anti-TGF-β antibody is capable of inhibiting binding between anti-TGF-β and one or more of its ligands. In some embodiments, the anti-TGF-β antibody is capable of inhibiting activation of TGF-β. In some embodiments, the anti-TGF-β antibody is a monoclonal antibody. In some embodiments, the anti-TGF-β antibody is an antibody fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab′)₂fragments. In some embodiments, the anti-TGF-β antibody is a humanized antibody. In some embodiments, the anti-TGF-β antibody is a human antibody. In some embodiments, the anti-TGF-β antibody inhibits TGF-β1, TGF-β2, and/or TGF-β3. In some embodiments, the anti-TGF-β antibody inhibits TGF-β1, TGF-β2, and TGF-β3. In some embodiments, the anti-TGF-β antibody is a pan-specific anti-TGF-β antibody. In some embodiments, the anti-TGF-β antibody may be any anti-TGF-β antibody disclosed in, for example, U.S. Pat. No. 5,571,714 or in International Patent Application Nos. WO 92/00330, WO 92/08480, WO 95/26203, WO 97/13844, WO 00/066631, WO 05/097832, WO 06/086469, WO 05/010049, WO 06/116002, WO 07/076391, WO 12/167143, WO 13/134365, WO 14/164709, or WO 16/201282, each of which is incorporated herein by reference in its entirety. In particular embodiments, the anti-TGF-β antibody is fresolimumab, metelimumab, lerdelimumab, 1 D11, 2G7, or a derivative thereof. It is expressly contemplated that such suppressive stromal antagonist antibodies (e.g., anti-TGF-β antibodies) for use in any of the embodiments enumerated herein may have any of the features, singly or in combination, described in Sections i-vii of Subsection D below.
In some embodiments, treatment with the suppressive stromal antagonist allows increased immune cell infiltration in a tissue. Without being bound by theory, the suppressive stromal antagonist modulates the stromal in the target tissue to facilitate infiltration of immune cells to the target tissue. For example, treatment of a fibrotic tumor expressing a biomarker described herein with a suppressive stromal antagonist modulates the stroma in and around the tumor to allow infiltration of immune cells (e.g., modulated by the immunotherapy) to the tumor. In some embodiments, the increased immune cell infiltration is an increased infiltration of one or more of T-cells, B cells, macrophages, or dendritic cells. In some embodiments, the T-cells are CD8+ T-cells and/or Teff cells. In some embodiments, the individual is resistant to immunotherapy prior to treatment with the suppressive stromal antagonist. In some embodiments, the individual has already been administered monotherapy immunotherapy.
D. Antibodies
i. Antibody Affinity
In certain embodiments, an antibody provided herein (e.g., an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-TGF-β antibody) has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, e.g., from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³M).
In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA). In one embodiment, an RIA is performed with the Fab version of an antibody of interest and its antigen. For example, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881, 1999). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599, 1997). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 μl/well of scintillant (MICROSCINT-Packard) is added, and the plates are counted on a TOPCOUNT™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.
According to another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25° C. with immobilized antigen CM5 chips at ˜10 response units (RU). In one embodiment, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TH) surfactant (PBST) at 25° C. at a flow rate of approximately 25 μl/min. Association rates (k_on) and dissociation rates (k_off) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k_off/k_on. See, for example, Chen et al., (J. Mol. Biol. 293:865-881, 1999). If the on-rate exceeds 10⁶M⁻¹s⁻¹by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.
ii. Antibody Fragments
In certain embodiments, an antibody (e.g., an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-TGF-β antibody) provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. (Nat. Med. 9:129-134, 2003). For a review of scFv fragments, see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab′)₂fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.
Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097, WO 1993/01161, Hudson et al. Nat. Med. 9:129-134, 2003, and Hollinger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993. Triabodies and tetrabodies are also described in Hudson et al. (Nat. Med. 9:129-134, 2003).
Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, M A; see, e.g., U.S. Pat. No. 6,248,516 B1).
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), according to known methods.
iii. Chimeric and Humanized Antibodies
In certain embodiments, an antibody (e.g., an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-TGF-β antibody) provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al. (Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, (Front. Biosci. 13:1619-1633, 2008), and are further described, e.g., in Riechmann et al. (Nature 332:323-329, 1988); Queen et al. (Proc. Natl. Acad. Sci. USA 86:10029-10033, 1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. (Methods 36:25-34, 2005) (describing specificity determining region (SDR) grafting); Padlan, (Mol. Immunol. 28:489-498, 1991) (describing “resurfacing”); Dall'Acqua et al. (Methods 36:43-60, 2005) (describing “FR shuffling”); Osbourn et al. (Methods 36:61-68, 2005), and Klimka et al. (Br. J. Cancer, 83:252-260, 2000) (describing the “guided selection” approach to FR shuffling).
Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151:2296, 1993); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285, 1992; and Presta et al. J. Immunol., 151:2623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633, 2008); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684, 1997; and Rosok et al. J. Biol. Chem. 271:22611-22618, 1996).
iv. Human Antibodies
In certain embodiments, an antibody (e.g., an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-TGF-β antibody) provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, (Curr. Opin. Pharmacol. 5: 368-74, 2001) and Lonberg (Curr. Opin. Immuno. 20:450-459, 2008).
Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, (Nat. Biotech. 23:1117-1125, 2005). See also, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. See, e.g., Kozbor, (J. Immunol. 133: 3001, 1984); Brodeur et al. (Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987); and Boerner et al. (J. Immunol., 147: 86, 1991). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562, 2006. Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, (Xiandai Mianyixue, 26(4):265-268, 2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, (Histology and Histopathology, 20(3):927-937, 2005) and Vollmers and Brandlein, (Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91, 2005).
Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.
v. Library-Derived Antibodies
Antibodies (e.g., anti-PD-L1 antibodies, anti-PD-1 antibodies, or anti-TGF-β antibodies) may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. (Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in McCafferty et al. (Nature 348:552-554, 1990); Clackson et al. (Nature 352: 624-628, 1991); Marks et al. (J. Mol. Biol. 222: 581-597, 1992); Marks and Bradbury, (Methods in Molecular Biology 248:161-175, Lo, ed., Human Press, Totowa, N J, 2003); Sidhu et al. (J. Mol. Biol. 338(2): 299-310, 2004); Lee et al. (J. Mol. Biol. 340(5): 1073-1093, 2004); Fellouse, (Proc. Natl. Acad. Sci. USA 101(34): 12467-12472, 2004); and Lee et al. (J. Immunol. Methods 284(1-2): 119-132, 2004).
In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. (Ann. Rev. Immunol., 12: 433-455, 1994). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and self antigens without any immunization as described by Griffiths et al. (EMBO J, 12: 725-734, 1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, (J. Mol. Biol., 227: 381-388, 1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.
vi. Multispecific Antibodies
In any one of the above aspects, an antibody (e.g., an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-TGF-β antibody) provided herein may be a multispecific antibody, for example, a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, an antibody provided herein is a multispecific antibody, e.g., a bispecific antibody. In certain embodiments, one of the binding specificities is for PD-L1 and the other is for any other antigen. In certain embodiments, one of the binding specificities is for TGF-β and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of PD-L1. In certain embodiments, bispecific antibodies may bind to two different epitopes of TGF-β. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express PD-L1 or TGF-β. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537, 1983), WO 93/08829 and Traunecker et al. EMBO J. 10: 3655, 1991) and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al. Science 229: 81, 1985); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al. J. Immunol. 148(5): 1547-1553, 1992); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al. J. Immunol. 152:5368, 1994); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60, 1991).
Engineered antibodies with three or more functional antigen binding sites, including “Octopus antibodies,” are also included herein (see, e.g., US 2006/0025576A1).
The antibody or fragment herein includes a “Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to PD-L1 and another, different antigen. The antibody or fragment herein also includes a DAF comprising an antigen binding site that binds to VEGF and another, different antigen.
vii. Antibody Variants
In certain embodiments, amino acid sequence variants of the antibodies (e.g., anti-PD-L1 antibodies, anti-PD-1 antibodies, and anti-TGF-β antibodies) are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, for example, antigen-binding.
a. Substitution, Insertion, and Deletion Variants
In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 6 under the heading of “preferred substitutions.” More substantial changes are provided in Table 6 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 6

Exemplary and Preferred Amino Acid Substitutions

Original	Exemplary	Preferred
Residue	Substitutions	Substitutions

Ala (A)	Val; Leu; Ile	Val
Arg (R)	Lys; Gln; Asn	Lys
Asn (N)	Gln; His; Asp, Lys; Arg	Gln
Asp (D)	Glu; Asn	Glu
Cys (C)	Ser; Ala	Ser
Gln (Q)	Asn; Glu	Asn
Glu (E)	Asp; Gln	Asp
Gly (G)	Ala	Ala
His (H)	Asn; Gln; Lys; Arg	Arg
Ile (I)	Leu; Val; Met; Ala; Phe; Norleucine	Leu
Leu (L)	Norleucine; Ile; Val; Met; Ala; Phe	Ile
Lys (K)	Arg; Gln; Asn	Arg
Met (M)	Leu; Phe; Ile	Leu
Phe (F)	Trp; Leu; Val; Ile; Ala; Tyr	Tyr
Pro (P)	Ala	Ala
Ser (S)	Thr	Thr
Thr (T)	Val; Ser	Ser
Trp (W)	Tyr; Phe	Tyr
Tyr (Y)	Trp; Phe; Thr; Ser	Phe
Val (V)	Ile; Leu; Met; Phe; Ala; Norleucine	Leu

Amino acids may be grouped according to common side-chain properties:

- (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
- (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
- (3) acidic: Asp, Glu;
- (4) basic: His, Lys, Arg;
- (5) residues that influence chain orientation: Gly, Pro;
- (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity and/or reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g., binding affinity).
Alterations (e.g., substitutions) may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196, 2008), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. (Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, NJ, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs. Such alterations may, for example, be outside of antigen-contacting residues in the HVRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (Science, 244:1081-1085, 1989). In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
b. Glycosylation Variants
In certain embodiments, antibodies of the invention can be altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody of the invention may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32, 1997. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003/0157108; US 2004/0093621. Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. (J. Mol. Biol. 336:1239-1249, 2004); and Yamane-Ohnuki et al. (Biotech. Bioeng. 87: 614, 2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545, 1986); U.S. Pat. Appl. No. US 2003/0157108 A1; and WO 2004/056312 A1, especially at Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614, 2004; Kanda, Y. et al. Biotechnol. Bioeng. 94(4):680-688, 2006; and WO 2003/085107).
Antibody variants are further provided with bisected oligosaccharides, for example, in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878; U.S. Pat. No. 6,602,684; and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.
c. Fc Region Variants
In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, (Annu. Rev. Immunol. 9:457-492, 1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Natl. Acad. Sci. USA 83:7059-7063, 1986) and Hellstrom, I et al. Proc. Natl. Acad. Sci. USA 82:1499-1502, 1985; U.S. Pat. No. 5,821,337; Bruggemann et al.J. Exp. Med. 166:1351-1361, 1987). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CYTOTOX 96® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. (Proc. Natl. Acad. Sci. USA 95:652-656, 1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods 202:163, 1996; Cragg et al. Blood. 101:1045-1052, 2003; and Cragg et al. Blood. 103:2738-2743, 2004). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova et al. Int'l. Immunol. 18(12):1759-1769, 2006).
Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. Nos. 6,737,056 and 8,219,149). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. Nos. 7,332,581 and 8,219,149).
Certain antibody variants with improved or diminished binding to FcRs are described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604, 2001).
In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. (J. Immunol. 164: 4178-4184, 2000).
Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587, 1976; and Kim et al. J. Immunol. 24:249, 1994), are described in U.S. Pub. No. 2005/0014934A1. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
See also Duncan and Winter, (Nature 322:738-40, 1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351, concerning other examples of Fc region variants.
d. Cysteine Engineered Antibody Variants
In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., “thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine engineered antibodies may be generated as described, e.g., in U.S. Pat. No. 7,521,541.
e. Antibody Derivatives
In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al. Proc. Natl. Acad. Sci. USA 102: 11600-11605, 2005). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.
f. Immunoconjugates
The invention also provides immunoconjugates comprising an antibody herein (e.g., an anti-PD-L1 antibody, an anti-PD-1 antibody, or an anti-TGF-β antibody) conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020 and 5,416,064 and European Patent EP 0 425 235 B1); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al. Cancer Res. 53:3336-3342, 1993; and Lode et al. Cancer Res. 58:2925-2928, 1998); an anthracycline such as daunomycin or doxorubicin (see Kratz et al. Current Med. Chem. 13:477-523, 2006; Jeffrey et al. Bioorganic & Med. Chem. Letters 16:358-362, 2006; Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532, 2002; King et al., J. Med. Chem. 45:4336-4343, 2002; and U.S. Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.
In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, R¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹²and radioactive isotopes of Lu. When the radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al. (Science 238:1098, 1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO94/11026. The linker may be a “cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al. Cancer Res. 52:127-131, 1992; and U.S. Pat. No. 5,208,020) may be used.
The immunoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, STAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).
D. Pharmaceutical Formulations
Therapeutic formulations of the therapeutic agents, e.g., immunotherapies (e.g., PD-L1 axis binding antagonists such as anti-PD-L1 antibodies (e.g., atezolizumab)) and/or the suppressive stromal antagonists (e.g., TGF-β antagonists such as an anti-TGF-β antibody) used in accordance with the present invention are prepared for storage by mixing the therapeutic agent having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. For general information concerning formulations, see, e.g., Gilman et al. (eds.) The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press, 1990; A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Pennsylvania, 1990; Avis et al. (eds.) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York, 1993; Lieberman et al. (eds.) Pharmaceutical Dosage Forms: Tablets Dekker, New York, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York, 1990; and Walters (ed.) Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol 119, Marcel Dekker, 2002.
Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).
The formulation herein may also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other. The type and effective amounts of such medicaments depend, for example, on the amount and type of therapeutic agent present in the formulation, and clinical parameters of the patients.
The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the therapeutic agent, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.
The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

V. Articles of Manufacture and Kits

In another aspect of the invention, a kit or an article of manufacture containing materials useful for the treatment, prevention, diagnosis, and/or monitoring of individuals is provided.
In some instances, such kits or articles of manufacture can be used to identify an individual having a cancer (including, but not limited to, a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from an anti-cancer therapy that includes an immunotherapy (including, but not limited to, a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (including, but not limited to, a TGF-β antagonist, e.g., an anti-TGF-β antibody). Such articles of manufacture or kits may include (a) reagents for determining the expression level of one or more genes set forth in Table 1, or any combination thereof (e.g., any combination set forth in any one of Tables 2-5) in a sample from the individual and (b) instructions for using the reagents to identify an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) who may benefit from treatment with a an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody). In additional aspects, the articles of manufacture or kits may include (a) reagents for determining the expression level of one or more genes set forth in Table 1, or any combination thereof (e.g., any combination set forth in any one of Tables 2-5) in a sample from the individual and (b) instructions for using the reagents to monitor and/or assess the response of an individual having a cancer (e.g., a bladder cancer (e.g., UC, e.g., mUC), a kidney cancer (e.g., RCC), a lung cancer (e.g., NSCLC), a liver cancer, an ovarian cancer, a pancreatic cancer (e.g., PDAC), a colorectal cancer, or a breast cancer) to treatment with a an anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and/or a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody).
Any of the kits or articles of manufacture described may include a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. Where the article of manufacture or kit utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as an enzymatic, florescent, or radioisotope label.
In some instances, the article of manufacture or kit includes the container described above and one or more other containers including materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use, such as those described above. For example, the article of manufacture or kit may further include a container including a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution.
The kits or articles of manufacture described herein may have a number of embodiments. In one instance, the kits or articles of manufacture includes a container, a label on said container, and a composition contained within said container, wherein the composition includes one or more polynucleotides that hybridize to a complement of a gene listed herein (e.g., a gene set forth in Table 1, or any combination of genes set forth in Tables 2-5) under stringent conditions, and the label on said container indicates that the composition can be used to evaluate the presence of a gene listed herein (e.g., a gene set forth in Table 1, or any combination of genes set forth in Tables 2-5) in a sample, and wherein the kit includes instructions for using the polynucleotide(s) for evaluating the presence of the gene RNA or DNA in a particular sample type.
For oligonucleotide-based articles of manufacture or kits, the article of manufacture or kit can include, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a protein or (2) a pair of primers useful for amplifying a nucleic acid molecule. The article of manufacture or kit can also include, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The article of manufacture or kit can further include components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The article of manufacture or kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the article of manufacture or kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
Provided herein is a kit for identifying an individual having a cancer who may benefit from treatment with an anti-cancer therapy including an immunotherapy (e.g., a PD-L1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the kit including: (a) reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes set forth in Table 1 in a sample from the individual; and, optionally, (b) instructions for using the reagents to identify an individual having a cancer who may benefit from a treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In another example, provided herein is a kit for monitoring and/or assessing the response of an individual having a cancer to treatment with an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the kit including: (a) reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) of the genes set forth in Table 1 in a sample from the individual; and, optionally, (b) instructions for using the reagents to monitor and/or assess the response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
For example, provided herein is a kit for identifying an individual having a cancer who may benefit from treatment with an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the kit including: (a) reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2; and, optionally, (b) instructions for using the reagents to identify an individual having a cancer who may benefit from a treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In yet another example, provided herein is a kit for monitoring and/or assessing the response of an individual having a cancer to treatment with an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the kit including: (a) reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of the following genes in a sample from the individual: ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2; and, optionally, (b) instructions for using the reagents to monitor and/or assess the response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In any of the preceding embodiments, the kit may include reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, or 6) of ACTA2, ADAM19, COMP, CTGF, TGFB1, or TGFBR2. In some embodiments, the kit includes reagents for determining the expression level of at least two, at least three, at least four, at least five, or all six of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2. In some embodiments, the kit includes reagents for includes determining the expression level of two of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 2. In some embodiments, the kit includes reagents for includes determining the expression level of three of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 3. In some embodiments, the kit includes reagents for includes determining the expression level of four of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 4. In some embodiments, the kit includes reagents for includes determining the expression level of five of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2, for example, any of the exemplary combinations shown in Table 5. In some embodiments, the kit includes reagents for involves determining the expression level of ACTA2, ADAM19, COMP, CTGF, TGFB1, and TGFBR2.
In any of the preceding methods, the kit may include reagents determining the expression level of TGFB1 and/or TGFBR2. In any of the preceding methods, the kit may include reagents for determining the expression level of TGFB1 and TGFBR2.
In some embodiments of any of the preceding kits, the one or more genes includes at least ADAM19 or COMP. In some embodiments, the one or more genes includes ADAM19. In other embodiments, the one or more genes includes COMP. In still further embodiments, the one or more genes includes ADAM19 and COMP.
For example, provided herein is a kit for identifying an individual having a cancer who may benefit from treatment with an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist, e.g., an anti-TGF-β antibody), the kit including: (a) reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1; and, optionally, (b) instructions for using the reagents to identify an individual having a cancer who may benefit from a treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In yet another example, provided herein is a kit for monitoring and/or assessing the response of an individual having a cancer to treatment with an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-8 antagonist, e.g., an anti-TGF-β antibody), the kit including: (a) reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of the following genes in a sample from the individual: ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1; and, optionally, (b) instructions for using the reagents to monitor and/or assess the response of an individual having a cancer to treatment with an anti-cancer therapy comprising an immunotherapy and a suppressive stromal antagonist.
In any of the preceding methods, the kit may include reagents for determining the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19) of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the kit includes reagents for determining the expression level of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, or all nineteen of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1. In some embodiments, the kit includes reagents for determining the expression level of ACTA2, ACTG2, ADAM12, ADAM19, CNN1, COL4A1, CTGF, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, and TPM1.
In some embodiments of any of the preceding kits, the one or more genes includes at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of ADAM19, ACTG2, CNN1, CTPS1, FAM101B, FSTL3, HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A, TAGLN, TGFBI, TNS1, or TPM1.

VI. Examples

The following is an example of the methods of the invention. It is understood that various other embodiments may be practiced, given the general description provided above. The following examples are offered by way of illustration and not by way of limitation.

Materials and Methods

A. Study Design, Patient Cohort, PD-L1 Testing, Response Assessment
Samples for this analysis were collected from IMvigor210, which was a single-arm phase II study investigating atezolizumab (1200 mg every 3 weeks (q3w)) in metastatic urothelial carcinoma (mUC) patients (Clinical Trial Identifier: NCT02108652). The primary endpoint of the trial was to achieve a partial response (PR) rate of over 10% (Response Evaluation Criteria In Solid Tumors (RECIST) v1.1). Patients who had failed previous platinum-based chemotherapy or previously untreated patients who were not eligible for platinum-based chemotherapy were eligible. The primary endpoint was met for both previously treated and untreated populations. It was a requirement for all patients to have tumor tissue taken in the 2 years prior to study entry. The tissues were initially used for PD-L1 analysis (SP142 antibody using the Ventana platform). PD-L1 positivity was defined as more that 5% of cells staining in the immune component of the tumor tissue. Additional tissue was used for exploratory analysis. All patients had measurable disease at baseline facilitating response assessment. RECIST v1.1 was used to assess response to therapy. Cross-sectional imaging was performed every 6 weeks. All patients gave appropriate ethical approval for this analysis.
B. PD-L1 Immunohistochemistry (IHC)
PD-L1 IHC has been previously described in Powles et al. Nature 515:558-562, 2014. Briefly, the pre-screening biopsies were collected from archived paraffin-embedded tissue. Patients were required to have tissue sent to the central laboratory before study entry. Samples were processed at the time of screening. FFPE tumor tissue was stained prospectively for PD-L1 by immunohistochemistry using a proprietary diagnostic anti-human PD-L1 monoclonal antibody (SP142). Samples were scored for PD-L1 expression on tumor-infiltrating immune cells, which included macrophages, dendritic cells, and lymphocytes. Specimens were scored as immunohistochemistry IC 0, 1, 2, or 3 if <1%, ≥1% but <5%, ≥5% but <10%, or ≥10% of tumor-infiltrating immune cells were PD-L1-positive, respectively. PD-L1 scores in patients with multiple specimens from different time points or samples were based on the highest score. This assay was validated for investigational use in clinical trials at the IC1 and IC2 cutoff. An exploratory analysis of PD-L1 expression on tumor cells (TC) was conducted. Specimens were scored as immunohistochemistry TC0, TC1, TC2, or TC3 if <1%, ≥1% but <5%, ≥5% but <50%, or ≥50% of tumor cells were PD-L1-positive, respectively.
C. Nucleic Acid Sample Preparation
The pathologic diagnosis of each case was confirmed by review of H&E-stained slides and all samples that advanced to nucleic acid extraction contained a minimum of 20% tumor cells. H&E images were marked for macrodissection by a pathologist. RNA (High Pure FFPET RNA Isolation Kit, Roche) and DNA (QIAAMP® DNA FFPE Tissue Kit, Qiagen) were then extracted from the macro-dissected sections.
FIG. 5 shows the number of efficacy-evaluable patients that had a known PD-L1 IC status and at least one of the other molecular data points analyzed here: whole-exome sequencing, FMOne-based mutational profiling, RNA sequencing, and cancer-immune phenotyping. Table 7 provides demographic details of the biomarker-evaluable patient (BEP) population. Patients for which RNA sequencing data were generated were chosen as a representative BEP, and distribution of key clinical covariates are listed as compared to the intent-to-treat (ITT) population; efficacy evaluable patients only were assessed. Both the number of patients as well as percentages (in parentheses) are given. BCG: Bacille Calmette Guerin, ECOG: Eastern Cooperative Oncology Group.

TABLE 7

Demographics of ITT and BEP population

Covariate	ITT (%)	BEP (%)

Male sex	337	(79%)	233	(78%)
White race	390	(91%)	270	(91%)
Intravesical BCG administered	106	(25%)	67	(22%)

PD-L1	IC0	142	(33%)	84	(28%)
	IC1	155	(36%)	112	(38%)
	IC2+	132	(31%)	102	(34%)
ECOG	0	162	(38%)	121	(41%)
	1	243	(57%)	165	(55%)
	2	24	(6%)	12	(4%)
Tobacco use	current	42	(10%)	32	(11%)
	previous	245	(57%)	168	(56%)
	never	142	(33%)	98	(33%)
Metastatic sites	liver	121	(28%)	81	(27%)
at baseline	LN Only	74	(17%)	51	(17%)
	visceral	200	(47%)	139	(47%)
	NA	34	(8%)	27	(9%)

D. Mutational Profiling (Whole-Exome Sequencing (WES) and FOUNDATIONONE® (FMOne) Panel)
WES data were generated for 250 patients, sequencing DNA extracted from both tumor as well as peripheral blood mononuclear cells using the Agilent SURESELECT® v5 (51 MB) kit on a HISEQ® 2500 (ILLUMINA®) sequencer. FASTQ format reads were aligned to the human reference genome (NCBI Build 38) using GSNAP (Wu et al. Bioinformatics 26:873-881, 2010; Wu et al. Methods Mol. Biol. 1418:283-334, 2016) version ‘2013-10-10’ (parameters: ‘-M 2 -n 10 -B 2 -i 1 -pairmax-dna=1000 -terminal-threshold=1000 -gmap-mode=none -clip-overlap’). Duplicate reads in the resulting BAM file were marked using PicardTools, and indels realigned using the GATK IndelRealigner tool.
Somatic variants were called using a union of Lofreq v2.1.2 (Wilm et al. Nucleic Acids Res. 40:11189-11291, 2012) and Strelka (Saunders et al. Bioinformatics 28:1811-1817, 2012) calls. Indel qualities were assigned to the alignments using “lofreq indelqual -dindel,” and somatic mutations were called using “lofreq somatic” with the “-call-indels” option. Strelka-based somatic mutations were called using the Strelka-provided configuration file “strelka_config_bwa_default.ini,” with the only modification being the setting “isSkipDepthFilters=1” instead of “isSkipDepthFilters=0.” Somatic mutations were annotated for effects on transcripts using Ensembl Variant Effect Predictor (McLaren et al. Genome Biol. 17:122, 2016) on Refseq-based gene models.
In order to identify expressed mutations, RNAseq alignments were tallied for somatic mutations identified in the exome data using the tallyVariants function from the R package VariantTools (Lawrence et al. VariantTools: Tools for Working with Genetic Variants Version 1.12.0). Neoantigen potential of each mutation was predicted after identifying HLA genotypes of the subjects and assigning the optimal HLA-neoepitope pair across all HLA alleles and 8-11 mer peptides containing the mutation, based on minimum 1050 value predicted by NetMHCcons (Karosiene et al. Immunogenetics 64:177-186, 2012). HLA genotyping was done on whole exome data from peripheral blood mononuclear cells (PBMCs), using Polysolver (Shukla et al. Nat. Biotech. 33:1152-1158, 2015).
In addition, DNA was extracted from FFPE 10 micron sections from 293 patients and submitted to a Clinical Laboratory Improvement Amendments (CLIA)-certified, New York State-accredited, and College of American Pathologists (CAP)-accredited laboratory (Foundation Medicine, Cambridge, MA) for targeted next-generation sequencing (NGS)-based genomic profiling. Adaptor-ligated DNA underwent hybrid capture for all coding exons of 395 cancer-related genes plus select introns from 31 genes frequently rearranged in cancer (FMOne panel).
Captured libraries were sequenced to a median exon coverage depth of >500× (DNA) or ˜3M unique reads (RNA) using Agilent SURESELECT® v5 kit at 51 Mb on the ILLUMINA® HISEQ® 2500, and resultant sequences were analyzed for base substitutions, small insertions and deletions (indels), copy number alterations (focal amplifications and homozygous deletions), and gene fusions/rearrangements, as previously described (Frampton et al. Nat. Biotechnol. 31:1023-1031, 2013). Frequent germline variants from the 1000 Genomes Project (dbSNP142) were removed. To maximize mutation-detection accuracy (sensitivity and specificity) in impure clinical specimens, the test was previously optimized and validated to detect base substitutions at a ≥5% mutant allele frequency (MAF), indels with a ≥10% MAF with ≥99% accuracy, and fusions occurring within baited introns/exons with >99% sensitivity (Frampton et al. supra). Known confirmed somatic alterations deposited in the Catalog of Somatic Mutations in Cancer (COSMIC v62) are called at allele frequencies ≥1% (Forbes et al. Nucleic Acids Res. 39:D945-D950, 2011).
From the FMOne panel, tumor mutational burden was calculated from the number of short variants in coding regions (substitutions and indels, including synonymous alterations) that were detected. Known and predicted germline alterations as well as known somatic and likely somatic variants are not counted. The mutation burden per Mb is the number of mutations counted divided by the Mb of genomic target territory of the FMOne panel. Unless otherwise indicated, FMOne-based mutation burden was used for all analyses around mutation burden.
E. Association Between Mutations and Response to Atezolizumab or Mutation Burden
For each gene, a patient was called mutant if a known or likely mutation was reported for that gene. For pathway-based analyses, a patient was called mutant in a pathway if any gene within the pathway was reported to have a known or likely mutation. Otherwise, a patient was considered to be non-mutant. A Fisher's Exact Test was used to determine whether the number of mutant patients differed between responders (complete and partial responders) and non-responders (stable and progressive disease). An association between mutation status and mutation burden was tested using a Wilcoxon Rank Sum Test. Single gene p-values were corrected for the number of tests performed using Benjamini and Hochberg adjustment. For pathway-based analyses, nominal p-values are reported.
F. Gene Expression Profiling
Whole transcriptome profiles were generated for 368 patients using TRUSEQ® RNA Access technology (ILLUMINA®). RNAseq reads were first aligned to ribosomal RNA sequences to remove ribosomal reads. The remaining reads were aligned to the human reference genome (NCBI Build 38) using GSNAP (Wu et al. Bioinformatics 26:873-881, 2010; Wu et al. Methods Mol. Biol. 1418:283-334, 2016) version ‘2013-10-10’, allowing maximum of two mismatches per 75 base sequence (parameters: ‘-M 2 -n 10 - B 2 -i 1 -N 1 -w 200000 -E 1 -pairmax-rna=200000 -clip-overlap). To quantify gene expression levels, the number of reads mapped to the exons of each RefSeq gene was calculated in a strand-specific manner using the functionality provided by the R/Bioconductor package GenomicAlignments (Lawrence et al. PLoS Comput. Biol. 0:e1003118, 2013).
G. Differential Gene Expression and Association with Mutation Burden or PD-L1 IHC
After quality control using the R/Bioconductor package arrayQualityMetrics (Kauffmann et al. Genomics 95:138-142, 2010), count data was normalized using trimmed mean of M-values (TMM) and transformed with voom to log₂-counts per million with associated precision weights (Law et al. Genome Biol. 2014).
To identify genes associated with PD-L1 protein staining on immune cells (IC), a linear regression model was fitted, using each gene's normalized, log₂-transformed expression as the response variable and log₂-transformed percent of immune cells identified as PD-L1 positive (ICp) as independent variables. Reported effect sizes represent the coefficients from the model fit, scaled by twice the standard deviation of the log₂-transformed Icp multiplied by the slope of the regression. FIG. 1C plots effect size and adjusted p-value for the PD-L1 IC positivity term only.
To identify biologies associated with response to atezolizumab, we grouped patients into responders (complete and partial responders) and non-responders (stable and progressive disease). Differentially expressed genes between these two groups were determined using the R/Bioconductor package limma (Ritchie et al. Nucleic Acids Res. 43:e47, 2015), which implements an empirical Bayesian approach to estimate gene expression changes using moderated t-tests.
To identify genes associated with tumor mutational burden, a linear regression model was fitted, using each gene's normalized, log₂-transformed expression as the response variable and mutation burden, batch, as well as cohort as independent variables (as these latter two we found to be correlated with TMB). Using R's anova( ) function, F-test p-values were calculated. Reported effect sizes represent the coefficients from the model fit, scaled by twice the standard deviation of TMB.
H. Gene Set Enrichment Analysis Using Kyoto Encyclopedia of Genes and Genomes (KEGG)
Following association testing (gene expression with response or mutation burden), Fios Genomics analyzed the top 1,000 genes (ranked by p-value) for enrichment of KEGG pathway membership using a hypergeometric test (Falcon et al. Bioinformatics 23:257-258, 2007), assessing up- and down-regulated genes separately. Enrichment p-values were corrected for the number of pathways tested using the Benjamini and Hochberg procedure. Complete results from the enrichment analyses are reported in Table 8 (response) and Table 9 (mutation burden). Table 8 lists KEGG gene sets significantly (FDR<0.1) enriched in genes differentially expressed by response (CR/PR vs. SD/PD). “Direction” indicates whether the category was enriched in genes up- (“Up”) or down-regulated (“Down”) in responders. “Identified genes” lists all genes within a given category that were found to be associated with response. “S” indicates the number of these genes, “N” gives the total number of genes in a category, while “p (adj.)” holds the adjusted enrichment p-values (hypergeometric test). Table 9 lists KEGG gene sets significantly (FDR<0.05) enriched in genes correlated with TMB. “Direction” indicates whether the category was enriched in genes positively (“Up”) or negatively (“Down”) correlated with TMB. “Identified genes” lists all genes within a given category that were found to be correlated with TMB. “S,” “N,” and “p (adj.)” are as in Table 8.

TABLE 8

Pathways associated with response

Direction	Name	Identified Genes	S	N	p (adj.)

Up	DNA replication	DNA2, FEN1, LIG1, MCM2, MCM4, MCM6, MCM7,	20	36	0.0000
		PCNA, POLA2, POLE, POLE2, PRIM1, PRIM2,
		RFC2, RFC3, RFC4, RFC5, RNASEH2A, RPA1,
		RPA3
Up	Cell cycle	BUB1, BUB1B, CCNA2, CCNB2, CCNE1, CCNE2,	32	124	0.0000
		CDC20, CDC25A, CDC25C, CDC6, CDK1, CDK2,
		CDKN2A, DBF4, E2F1, E2F2, ESPL1, MAD2L1,
		MAD2L2, MCM2, MCM4, MCM6, MCM7, ORC1,
		ORC6, PCNA, PLK1, SKP2, SMC3, TFDP1, TTK,
		YWHAB
Up	Fanconi anemia	BLM, BRCA1, BRCA2, BRIP1, EME1, ERCC4,	19	53	0.0000
	pathway	FANCA, FANCB, FANCD2, FANCI, PALB2, RAD51,
		RAD51C, RMI1, RMI2, RPA1, RPA3, TOP3A,
		UBE2T
Up	Systemic lupus	H2AFV, H2AFZ, HIST1H2AB, HIST1H2AG,	27	134	0.0000
	erythematosus	HIST1H2AH, HIST1H2AI, HIST1H2AM,
		HIST1H2BC, HIST1H2BD, HIST1H2BF,
		HIST1H2BJ, HIST1H2BK, HIST1H2BL,
		HIST1H2BN, HIST1H2BO, HIST1H3B, HIST1H3D,
		HIST1H3H, HIST1H4A, HIST1H4B, HIST2H2AB,
		HIST2H2AC, HIST2H2BE, HIST2H2BF, HIST2H3D,
		HIST3H2A, IFNG
Up	Alcoholism	CREB3L4, H2AFV, H2AFZ, HIST1H2AB,	27	179	0.0000
		HIST1H2AG, HIST1H2AH, HIST1H2AI,
		HIST1H2AM, HIST1H2BC, HIST1H2BD,
		HIST1H2BF, HIST1H2BJ, HIST1H2BK,
		HIST1H2BL, HIST1H2BN, HIST1H2BO, HIST1H3B,
		HIST1H3D, HIST1H3H, HIST1H4A, HIST1H4B,
		HIST2H2AB, HIST2H2AC, HIST2H2BE,
		HIST2H2BF, HIST2H3D, HIST3H2A
Up	Homologous	BLM, BRCA2, EME1, RAD51, RAD51C, RAD54L,	11	28	0.0000
	recombination	RPA1, RPA3, TOP3A, XRCC2, XRCC3
Up	Mismatch repair	EXO1, LIG1, PCNA, RFC2, RFC3, RFC4, RFC5,	9	23	0.0000
		RPA1, RPA3
Up	Nucleotide	CETN2, ERCC4, LIG1, PCNA, POLE, POLE2,	12	47	0.0000
	excision repair	RFC2, RFC3, RFC4, RFC5, RPA1, RPA3
Up	Oocyte meiosis	AURKA, BUB1, CCNB2, CCNE1, CCNE2, CDC20,	17	113	0.0000
		CDC25C, CDK1, CDK2, ESPL1, FBXO5, MAD2L1,
		MAD2L2, PLK1, SGOL1, SMC3, YWHAB
Up	Viral	CCNA2, CCNE1, CCNE2, CDC20, CDK1, CDK2,	23	206	0.0000
	carcinogenesis	CDKN2A, CREB3L4, GTF2E1, HIST1H2BC,
		HIST1H2BD, HIST1H2BF, HIST1H2BJ,
		HIST1H2BK, HIST1H2BL, HIST1H2BN,
		HIST1H2BO, HIST1H4A, HIST1H4B, HIST2H2BE,
		HIST2H2BF, SKP2, YWHAB
Up	Base excision	FEN1, LIG1, NEIL3, PARP1, PARP2, PCNA, POLE,	9	33	0.0000
	repair	POLE2, UNG
Up	p53 signaling	CCNB2, CCNE1, CCNE2, CDK1, CDK2, CDKN2A,	10	68	0.0008
	pathway	GTSE1, PPM1D, RFWD2, RRM2
Up	Spliceosome	HNRNPM, LSM3, LSM4, LSM5, MAGOHB,	14	132	0.0013
		PRPF19, SF3B2, SF3B3, SF3B4, SNRNP40,
		SNRPA1, SNRPC, USP39, WBP11
Up	Proteasome	IFNG, PSMA4, PSMB2, PSMB4, PSMC4, PSMD4,	7	44	0.0045
		PSMD7
Up	Progesterone-	BUB1, CCNA2, CCNB2, CDC25A, CDC25C, CDK1,	10	88	0.0054
	mediated	CDK2, MAD2L1, MAD2L2, PLK1
	oocyte
	maturation
Up	MicroRNAs in	BRCA1, CCNE1, CCNE2, CDC25A, CDC25C,	14	186	0.0298
	cancer	CDCA5, CDKN2A, DNMT1, E2F1, E2F2, EZH2,
		KIF23, STMN1, TRIM71
Up	Pyrimidine	CTPS1, DTYMK, POLA2, POLE, POLE2, PRIM1,	9	104	0.0515
	metabolism	PRIM2, RRM2, TYMS
Up	RNA	CNOT10, EXOSC2, EXOSC8, LSM3, LSM4, LSM5,	7	76	0.0771
	degradation	PARN
Down	Cytokine-	ACVR1, CCL24, FLT4, IFNGR1, IL4R, IL6ST, KIT,	15	264	0.0835
	cytokine	LIF, PDGFA, PDGFRB, TGFB1, TGFBR2,
	receptor	TNFRSF10B, TNFRSF14, TNFRSF1A
	interaction

TABLE 9

Pathways associated with TMB

Direction	Name	Identified Genes	S	N	p (adj.)

Up	DNA replication	FEN1, LIG1, MCM2, MCM4, MCM7, POLD1,	11	36	0.0000
		POLE, POLE3, RFC2, RNASEH2A, RPA1
Up	Cell cycle	ANAPC2, CCNB1, CDC20, CDC25A, CDC45,	16	124	0.0001
		CDC6, CDKN2B, E2F1, E2F2, ESPL1,
		MCM2, MCM4, MCM7, PKMYT1, TFDP1,
		TTK
Up	Nucleotide	LIG1, POLD1, POLE, POLE3, RAD23A,	8	47	0.0039
	excision repair	RAD23B, RFC2, RPA1
Up	Huntington's	ATP5D, ATP5G1, ATP5O, BAX, COX5B,	15	193	0.0311
	disease	COX6B1, COX7B2, NDUFA13, NDUFA8,
		NDUFB7, NDUFS7, PLCB3, POLR2I,
		POLR2L, SLC25A5
Up	Fanconi anemia	ATRIP, EME1, FANCC, FANCG, RAD51,	7	53	0.0326
	pathway	RMI1, RPA1
Up	Homologous	EME1, POLD1, RAD51, RAD54L, RPA1	5	28	0.0361
	recombination
Up	Citrate cycle	ACLY, MDH1, MDH2, PCK2, SUCLG1	5	30	0.0418
	(TCA cycle)
Up	Non-alcoholic	BAX, COX5B, COX6B1, COX7B2, GSK3A,	12	151	0.0418
	fatty liver	NDUFA13, NDUFA8, NDUFB7, NDUFS7,
	disease	RXRA, SREBF1, TRAF2
	(NAFLD)
Up	Oxidative	ATP5D, ATP5G1, ATP5O, ATP6V1F,	11	133	0.0418
	phosphorylation	COX5B, COX6B1, COX7B2, NDUFA13,
		NDUFA8, NDUFB7, NDUFS7
Up	Base excision	FEN1, LIG1, POLD1, POLE, POLE3	5	33	0.0462
	repair
Down	Complement	CFH, F8, PROC, SERPINA5, SERPIND1,	6	69	0.0267
	and coagulation	TFPI
	cascades
Down	PI3K-Akt	DDIT4, EGF, FGF7, GNG11, ITGAV, ITGB3,	14	345	0.0267
	signaling	JAK1, KIT, LAMA4, LPAR1, LPAR6, NGFR,
	pathway	PTEN, RBL2
Down	Pathways in	AGTR1, EDNRA, EGF, FGF7, GNG11,	15	397	0.0267
	cancer	HIF1A, ITGAV, JAK1, KIT, LAMA4, LPAR1,
		LPAR6, PTEN, TGFBR2, TRAF5

I. Gene Sets Used for Signatures
Because KEGG pathways often include large numbers of genes with only loosely related functions, we constructed refined gene sets (Table 10) for the four core biologies called out in the text. Specifically:

- (i) for DNA damage repair genes, we used a gene set as described by Lange et al. Nat. Rev. Cancer 11:96-110, 2011;
- (ii) for cell cycle regulator genes, we used genes within the p53/Rb pathway that TOGA reported to be frequently reported in bladder cancer (The Cancer Genome Atlas Research Nature 507:315-322, 2014);
- (iii) for CD8 T-effector genes, we used our previously-published signature (Rosenberg et al. Lancet 387:1909-1920, 2016; Balar et al. Lancet 389:67-76, 2017); and
- (iv) for TGF-β, we considered three different signatures. First, for monitoring input of the pathway, we evaluated ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, and TGFBR3) for association with outcome in IMvigor210. TGFB1 and TGFBR2 were significantly associated with outcome (adj. p=8.956×10⁻³and adj. p=1.070×10⁻², respectively), and the two were used as our TGF-β two-gene signature. Second, for monitoring output of the pathway, we generated a pan-fibroblast TGF-β response signature (Pan-F-TBRS), as described below. Further, in FIG. 2A, we also show a larger set of genes reported to be involved in extracellular matrix organization (Sjödahl et al. Clin. Cancer. Res. 18:3377-3386, 2012).

J. Signature Score Computation
For gene expression analysis, the expression of each gene in a signature was first z-score transformed. Then, a principal component analysis was performed, and principal component 1 was extracted to serve as gene signature score. This approach has the advantage of focusing the score for the set on the largest block of well-correlated (or anti-correlated) genes in the set, while down-weighting contributions from genes that do not track with other set members.
K. Associations Between Gene Signature Scores or Tumor Mutation Burden and Traits of Interest
Below are the statistical methods that were used for the various tests throughout the Examples:

- (i) for associations with response, only efficacy evaluable patients were considered; these were grouped into responders (CR/PR) and non-responders (SD/PD), unless otherwise stated;
- (ii) for associations of binary traits, e.g., response, with normally distributed continuous traits, such gene expression scores, two sample t-tests were performed; if multiple pairwise tests were performed within one gene signature—trait combination, p-values were Bonferroni corrected;
- (iii) for associations of binary traits, e.g. response or mutation status, with TMB that showed a skewed distribution, Wilcoxon signed-rank tests were performed. TMB was log₂transformed; one outlier patient with 0 TMB was excluded from analysis;
- (iv) for assessment of associations between two categorical traits, e.g., response and IC level, Fisher's exact tests were performed;
- (v) for associations with immune phenotype, immune phenotype was treated as an ordered factor (desert <excluded <infiltrated) and likelihood ratio test p-values were calculated using ANOVA. An exception from this was testing the association with TMB, where a Kruskal Wallis H test was used; and
- (vi) likelihood ratio tests were also used to test for associations between a continuous trait, such as gene expression signature score and a categorical trait with more than 2 groups, such IC level. In addition, the same test was used to assess interaction between two traits, comparing a model where the two traits were included as two independent variables to a model including an interaction term between these two variables.

In visualizations of TMB on log₂scale, patients with no mutations were dropped (depending on the exact biomarker evaluable population, this was one or two patients only). In all graphs, the number of patients within each group is given at the top.
L. Explained Variance in Response
Generalized linear models were fit using binary response (CR/PR vs. SD/PD) as the dependent variable and scores from single/combinations of different signatures or TMB as independent variables. Logistic regression pseudo-R²was extracted as a measure of “explained variance” in patient response, i.e., the percent of variation in patient response that can be attributed to the contributions of the biological inputs (see Dobson et al. An Introduction to Generalized Linear Models. Chapman and Hall/CRC Press, 2008).
M. Molecular Subtyping
TOGA subtypes were assigned according to methodology described in Rosenberg et al. supra.
For assigning Lund subtypes, a data set of centroids of gene expression for 1038 genes was provided by Sjödahl et al. (Sjödahl et al. Clin. Cancer Res. 18:3377-3386, 2012; Sjödahl et al. Am. J. Pathol. 183:681-691, 2013). A reduced set of 884 centroids that overlapped with the genes detected in the reported RNAseq data was used to assign a molecular subtype (MS1a, MS1b, MS2a1, MS2a2, MS2b1, MS2b2.1 and MS2b2.2). Since these centroids were based on gene expression profiles from microarrays, which cannot be simply carried over to another platform like RNA sequencing, we chose to perform the molecular subtype assignment by calculating the Spearman correlation distance between the median-centered (log 2) expression levels of these 884 genes and the centroids. In using a rank-based approach, we aimed to minimize platform-specific effects (however, it is noteworthy that using Pearson instead of Spearman yielded very similar results). Each sample was assigned the molecular subtype with the shortest distance. Subsequently, clusters MS1a and MS1b were combined to urothelial A, MS2a1, and MS2a2 to genomically unstable subtypes, respectively; clusters MS2b1, MS2b2.1, and MS2b2.2 are equivalent to the infiltrated, urothelial B, and basal/SCC-like subtypes, respectively.
Note that “urothelial-like” has recently been suggested as a replacement for the original “urobasal” (Lerner et al. Bladder Cancer 2:37-47, 2016). We use the updated nomenclature here. Similarly, “basal/SCC-like” is now preferred over the original “SCC-like.”
N. Gene Sets Used for Subtype Heatmap
Lund subtype labels were assigned as described above. To better understand the relationship between these subtypes and various axes of biology considered in this manuscript, FIGS. 2A and 2H show gene expression values for additional genes (i.e., genes that were not necessarily used for subtype assignment) from 13 biologies. Displayed genes are representative members of larger gene sets constructed as follows:

- A: FGFR3 gene signature reported by Sjödahl et al. 2012, supra (representatives were selected);
- B: CD8 T-effector signature (Rosenberg et al. supra);
- C: antigen processing machinery reported by Şenbabao{hacek over (g)}lu et al. Genome Biol. 17:231, 2016;
- D: immune checkpoint signature;
- E: MKI67 and select cell cycle genes (KEGG) that contributed to gene set enrichment for association with response (Table 8);
- F: DNA replication-dependent histones (representatives of histones discovered in Table 8);
- G: select members of the DNA damage repair gene set, introduced above;
- H: extracellular matrix organization (ECM) set reported by Sjödahl et al. 2012 (representatives were selected);
- I: TGF-β two-gene signature;
- J: angiogenesis signature reported by Sjödahl et al. 2012 (representatives were selected);
- K: epithelial-mesenchymal transition (EMT) markers reported by Damrauer et al. Proc. Natl. Acad. Sci. USA 111:3110-3115, 2014;
- L: Pan-F-TBRS, determined as described below; and
- TCGA: genes expressed TOGA subtype-specifically, as reported by The Cancer Genome Atlas Research Nature, supra (FIG. 2H).

O. CD8/Trichrome Dual Staining
The combined CD8/trichrome dual staining was performed on 4 μm FFPE sections. The IHC procedure was performed first using the anti-human CD8 rabbit monoclonal antibody SP16 (Spring Bioscience; Cat. No. M3160) at 1:100 dilution for one hour at room temperature following antigen retrieval using Cell Conditioning 1 reagent (CC1, Ventana Medical Systems; Cat. No. 950-124). Specifically bound primary antibody was visualized using the OMNIMAP™ DAB kit (Ventana, Cat. No. 760-149). After completion of the IHC procedure and rinsing with distilled water, slides were fixed in Bouin's fixative at 60° C. for 1 h and then taken through a routine trichrome stain consisting of Weigert's hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and anilin blue as described previously (Laboratory Methods in Histotechnology, Armed Forces Institute of Pathology, 1992, p. 132). At the conclusion of the staining procedure, slides were dehydrated in increasing ethanol concentration, immersed in xylene, and then coverslipped using a synthetic mounting medium.
P. In Vivo Studies
The EMT6 murine mammary carcinoma cell line was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium plus 2 mM L-glutamine with 10% fetal bovine serum (HYCLONE®). Cells in log-phase growth were centrifuged, washed once with Hank's balanced salt solution (HBSS), counted, and resuspended in 50% HBSS and 50% MATRIGEL™ (BD Biosciences) at a concentration of 1×10⁶cells/mL for injection into mice. Female Balb/c mice were obtained from Charles River Laboratories (Hollister, CA). The mice were housed in standard rodent micro-isolator cages and were acclimated to study conditions for at least 3 days before tumor cell implantation. Animals were 8-10 weeks old. Only animals that appeared to be healthy and free of obvious abnormalities were used for the study. Mice were inoculated in the left mammary fat pad #5 with 1×10⁵EMT6 cells in 100 μl of HBSS MATRIGEL™ (1:1). When tumor reached a volume of 130-230 mm³(approximately 8 days after inoculation), animals were distributed into treatment groups based on tumor volume and treated with isotype control antibodies, anti-PD-L1 (10 mg/kg first dose followed by 5 mg/kg thereafter), anti-TGF beta (10 mg/kg), or a combination of anti-PD-L1 with anti-TGF beta. Seven days after treatment initiation, mice were euthanized and tumors collected for either IHC or flow cytometry analysis. For efficacy studies, antibodies were administered three times a week for 3 weeks (intravenously for the first dose and intraperitoneally thereafter). Tumors were measured two times per week by caliper, and tumor volumes were calculated using the modified ellipsoid formula, ½×(length×width²). When tumor volumes fell below 32 mm³(lowest limit of detection) they were considered complete regressions (CR) (100% tumor growth inhibition). Mice were euthanized if tumor volumes exceeded 2000 mm³. No mice met criteria for euthanasia because of body weight loss nor exhibited adverse clinical signs during the study. All animal studies herein were approved by the Genentech Institutional Animal Care and Use Committee. The group sizes used for this study were estimated to be the smallest necessary to generate meaningful data.
Q. Whole Tumor RNA and Protein Quantification from Mice
TGF-β isoform abundance in untreated EMT6 tumors was analyzed both at the RNA and protein levels, by RNAseq and ELISA, respectively. For RNAseq analysis, tumors of an average size of 300 mm³were collected. For ELISA quantification of TGF-β protein, tumors were collected 14 days after inoculation into the mammary fat pad, flash frozen, and subsequently lysed with radioimmunoprecipitation assay (RIPA) buffer on a TissueLyser disruption system (Qiagen). Total protein was quantified by bicinchoninic acid (BCA) assay, and total protein input was normalized across samples. The TGF-β in the samples was acid-activated and measured following the ELISA kit instructions (Mouse TGF-β1 and TGF-β2, R&D Systems; Mouse TGF-β3, LSBio). VEGF was quantified using Mouse VEGF QUANTIKINE® ELISA Kit (R&D Systems) in plasma collected seven days after treatment onset.
R. Preparation of Single Cell Suspension and Antibody Staining for Flow Cytometry
Tumors were collected seven days after treatment initiation. Tumors were weighed and enzymatically digested using a cocktail of dispase (Life Technologies), collagenase P, and DNasel (Roche) for 45 min at 37° C., to obtain a single cell suspension. Cells were counted using a VI-CELL® XR (Beckman Coulter).
For phospho-SMAD (pSMAD) staining, cells were fixed and permeabilized using BD PHOSFLOW™ Lyse/Fix Buffer and BD PHOSFLOW™ Perm Buffer III (BD Biosciences) following the manufacturer's instructions. The cells were stained with a phycoerythrin (PE)-conjugated antibody against the phosphorylated form of SMAD2 and SMAD3 for 1 h on ice at a concentration of 0.08 μg/ml (clone 072-670, BD Biosciences, San Jose, CA). For T-cell staining, cells were first incubated with mouse BD Fc block (clone 2.4G2, 5 μg/ml, BD Biosciences) and a viability stain (LIVE/DEAD® Aqua Fixable Dead Cell Stain, Invitrogen) for 30 min on ice. The cells were then stained with the following antibodies: CD45 (BV605, clone 30-F11, 0.67 μg/ml, BD Biosciences), TCRb (PE, clone H57-597, 2 μg/ml, Biolegend), CD8 (APC-Cy7, clone 53-6.7, 1 μg/ml, Biolegend) for 30 min on ice. Cells were fixed and permeabilized (EBIOSCIENCE™ Foxp3/Transcription Factor Staining Buffer Set, Thermo Fisher Scientific Inc.) to stain for Granzyme B (FITC, clone NGZB, 5 μg/ml, EBIOSCIENCE™, Thermo Fisher Scientific Inc.).
Flow cytometry data were collected with a BD LSRFORTESSA™ cell analyzer or FACSYMPHONY™ (BD Biosciences) and analyzed using FLOWJO® Software (Version 10.2, FlowJo, LLC).
S. Immunohistochemistry on Mouse Tissue
Tumors were collected seven days after treatment initiation. Tumors were fixed in 10% neutral buffered formalin (NBF) and paraffin embedded. IHC was performed on 4 μm thick paraffin-embedded tissue sections mounted on SUPERFROST™ Plus glass slides. Staining was performed on the LAB VISION™ Autostainer (ThermoFisher Scientific). Sections were de-paraffinized and rehydrated to deionized water. Antigen retrieval was performed with 1× DAKO Target Retrieval Solution (Agilent Technologies) for 20 min at 99° C. and cooled to 74° C. Subsequently, endogenous peroxidase was quenched by incubating in sections in 3% H₂O₂for 4 min at room temperature. CD3 (ThermoFisher Scientific, clone SP7, cat. no. RM-9107-S, 1:200 dilution) was detected using an anti-rabbit (7.5 μg/mL) secondary antibody with the ABC Peroxidase Elite Detection Kit (Vector Laboratories, cat. no. PK-6100). Sections were counterstained with Mayer's hematoxylin, dehydrated, mounted with permanent mounting medium, and coverslipped.
T. Generation of Pan-Fibroblast TGF-β Response Signature (Pan-F-TBRS)
Primary fibroblast cells from bladder (PHBR, primary normal bladder fibroblast cells, PCS-420-013™, ATCC), colon (CCD-18Co, CRL1459™, ATCC), breast (HMF, #7630, ScienCell Research Laboratories), lung (IMR90, CCL186™, ATCC), pancreas (HPaSteC, #3836, ScienCell Research Laboratories), and ovary (HOF, #7336, ScienCell Research Laboratories) were serum-starved overnight before treatment with (1) control, (2) TGF-μ1 (10 ng/mL, #), or (3) TGF-β1 (10 ng/mL)+the TGF-β inhibitor galunisertib (10 μM) in duplicates for 24 h. TGF-β-induced genes were identified by RNAseq transcriptome analysis comparing the above three conditions. The F-TBRS was determined using the following criteria: (1) expression level at least 2-fold higher in TGF-β1-treated group compared with controls (false discovery rate (FDR) 0.1); (2) expression level at least 2-fold lower in the TGF-β1+ inhibitor group compared to TGF-β1 treatment alone (FDR 0.1); (3) meet criteria (1) and (2) in all 6 fibroblast cell types. The genes that passed these three criteria (n=79) were ranked according to the strength of inhibition (control versus TGF-β1+TGF-β inhibitor); and filtered by at least 5 counts per million mean reduction to define a pan-tissue 19-gene F-TBRS.
U. Generation of Pan-Tissue 6-Gene F-TBRS (6-Gene Signature)
We developed a pan-tissue 6-gene F-TBRS based on the 19-gene Pan-F-TBRS data described above but further prioritizing TGF-β pathway components (TGFB1 and TGFBR2) and targets (CTGF, ADAM19, ACTA2, and COMP). To validate the predictive value of the 6-gene signature, we calculated hazard ratios for prognostic effects (using univariate continuous Cox proportional hazards regression models) and predictive effects, and calculated hazard ratios for predictive effects (using the log-rank test with median cutoff) in atezolizumab clinical trials. Our analysis demonstrated that high expression of the 6-gene signature is associated with lack of response to atezolimumab (atezo) monotherapy in UC, non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), and pancreatic ductal adenocarcinoma (PDAC) trials. See, e.g., FIGS. 6B and 6C, which show data from IMvigor210 and the POPLAR study (NCT01903993), respectively. In addition, in the TCGA colorectal cancer (CRC) data set, high expression of the 6-gene signature was also significantly associated with poor overall survival and enriched in CMS4 molecular subtype, which correlates with a poor survival subgroup of CRC patients (Guinney et al. Nat. Med. 21(11):1350-1356, 2015). See FIGS. 6A, 7A, and 7B.
V. Macrophage and T-Cell TGF-β Response Signatures (M- and T-Cell-TBRS)
The macrophage- and T-cell-specific TBRS were adopted from Calon et al. Nat. Genet. 47:320-329, 2015; 1178 and 69 genes, respectively. Signature scores were computed by (1) deriving the first principal component (PC1) of signature genes on the EMT6 RNAseq data, (2) obtaining the signature genes having at least 0.8 Spearman-rank correlation with PC1, and (3) taking the average of Z-scores for genes from step (2). The filtering in step (2) resulted in 101 and 10 genes for M-TBRS and T-cell-TBRS, respectively.
W. Statistical Analysis of T-Cell Infiltration
Bright field images were acquired by Hamamatsu NanoZoomer automated slide-scanning platforms at a final magnification of 200×. The images were analyzed with the 2016b version of the MATLAB® software package (MathWorks). Regions of interest (ROIs) were defined by a pathologist. Cells marked with CD3+ that lay within the ROI border were identified by intensity thresholding and simple morphological filtering. Immune cell infiltration was evaluated for each slide (i.e., each mouse) by calculating the mean nearest distance to the ROI border over all CD3+ marked cells within the ROI on that slide. Mean distances were then normalized per slide by dividing by the maximum distance from the ROI border to the ROI center. Normalized mean distances were then pooled across the three studies (666, 1430, 1436) and analyzed by linear regression with treatment group as a fixed categorical variable and ROI area and total number of CD3+ cells within the ROI as covariates. Covariate-adjusted means and 95% confidence intervals were reported for each treatment group. Pairwise comparisons among treatment groups were made using Tukey's honest significant difference (HSD) test. All analyses were performed using R, and distance calculations were made using the R package ‘spatstat’ (Baddeley et al. Spatial Point Patterns: Methodology and Applications with R. Chapman and Hall/CRC Press, 2015).

Example 1: Pre-Existing Tumor Immunity is Associated with Complete Responses in Inflamed Tumors

Most human solid tumors appear to exhibit one of three distinct immune phenotypes: the “immune inflamed” phenotype, characterized by robust CD8+ T-cell infiltration and PD-L1 expression; the “immune excluded” phenotype, in which T-cells accumulate in the extracellular matrix-rich stroma, and the “immune desert” phenotype, with a distinct paucity of infiltrating lymphocytes within the tumor or surrounding stroma. Inflamed tumors are thought to be more responsive to checkpoint blockade compared with immune excluded or immune desert phenotypes, which are predicted to be weakly or non-responsive. However, this association has not been systematically tested in the context of large clinical trials or within a single tumor type. To understand response to atezolizumab therapy in mUC, we combined integrated molecular and histological analyses with a reverse translation approach to test our findings functionally.
For investigation of the clinical activity of PD-L1 blockade with atezolizumab, 429 patients with advanced mUC were enrolled in a phase 2, single arm clinical trial (IMvigor210; NCT02108652). Objective response rate was assessed per RECIST v1.1 and served as a trial endpoint. Patients who achieved a complete response (CR) or partial response were categorized as responders and compared with non-responders, who displayed stable disease (SD) or progressive disease (PD). Assessment of the association of PD-L1 expression on tumor-infiltrating immune cells (IC) on baseline tumors with response (objective response rate) was a co-primary endpoint. PD-L1 IC status was defined by the percentage of PD-L1-positive immune cells in the tumor microenvironment (TME): IC0 (<1%), IC1 (≥1% but <5%) and IC2+ (≅5%).
As found previously in a smaller cohort of patients, PD-L1 expression on IC was significantly associated with response, and tumors with high PD-L1 expression (IC2+) displayed the highest CR rate (FIG. 1A). Improved OS benefit from atezolizumab was observed in patients with high PD-L1 IC scores (IC2/3) relative to patients with lower PD-L1 IC scores (IC0/1) (FIG. 8A). Whole transcriptome RNA sequencing was performed in pre-treatment tissues from 298 IMvigor210 participants to evaluate features associated with response to atezolizumab and identify genes that had expression levels associated with PD-L1 expression on IC (FIG. 1B). Interferon-γ (IFN-γ)-inducible genes were among the most highly correlated. Gene sets associated with CD8+T-effector (Teff) cells and immune checkpoint molecules (Table 10), many of which are IFN-γ-stimulated genes, showed the most differential expression (FIGS. 1B and 1C). The CD8+ Teff gene set was also significantly associated with response, particularly with CR (FIG. 1D). The CD8+ Teff score quartiles were associated with overall survival (FIG. 1U). This suggests that PD-L1 expression on IC marks tumor tissues that have pre-existing CD8+ T-cell immunity and adaptive immune suppression but are still poised to respond to anti-PD-L1 therapy.

TABLE 10

Gene sets used for signature analyses

Gene
Signature	Platform	Genes	Source

CD8 T-effector	Gene	CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG,	PMID:
(Teff)	expression	PRF1, TBX21	26952546
	profiling
DNA damage	Gene	ALKBH2, ALKBH3, APEX1, APEX2, APLF, ATM,	Lange et al.
repair	expression	ATR, ATRIP, BLM, BRCA1, BRCA2, BRIP1, CCNH,	supra
	profiling	CDK7, CETN2, CHAF1A, CHEK1, CHEK2, CLK2,
		DCLRE1C, DDB1, DDB2, DUT, ENDOV, ERCC1,
		ERCC2, ERCC3, ERCC4, ERCC5, ERCC6,
		ERCC8, FAN1, FANCA, FANCB, FANCC,
		FANCD2, FANCE, FANCF, FANCG, FANCL,
		FANCM, GTF2H1, GTF2H2, GTF2H3, GTF2H4,
		GTF2H5, H2AFX, HLTF, HUS1, LIG1, LIG3, LIG4,
		MBD4, MDC1, MGMT, MLH1, MLH3, MMS19,
		MNAT1, MPG, MSH2, MSH3, MSH4, MSH5, MSH6,
		MUTYH, NEIL1, NEIL2, NEIL3, NHEJ1, NTHL1,
		NUDT1, OGG1, PALB2, PARP1, PARP2, PARP3,
		PCNA, PER1, PMS1, PMS2, PNKP, POLB, POLD1,
		POLE, POLG, POLH, POLL, POLM, POLQ,
		PRKDC, RAD1, RAD17, RAD18, RAD23A,
		RAD23B, RAD51C, RAD9A, RECQL4, RECQL5,
		RIF1, RNF168, RNF4, RNF8, RPA1, RPA2, RPA3,
		RPA4, RRM2B, SETMAR, SHPRH, SMUG1, TDP1,
		TDP2, TOPBP1, TP53, TREX1, UBE2A, UBE2B,
		UBE2N, UBE2V2, UNG, UVSSA, WRN, XAB2,
		XPA, XPC, XRCC1, XRCC4, XRCC5, XRCC6
TGF-β 2-gene	Gene	TGFB1, TGFBR2	Single-gene
	expression		association
	profiling		with response
			in this study.
Pan-F-TBRS	Gene	ACTA2, ACTG2, ADAM12, ADAM19, CNN1,	Experimentally
	expression	COL4A1, CTGF, CTPS1, FAM101B, FSTL3,	determined
	profiling	HSPB1, IGFBP3, PXDC1, SEMA7A, SH3PXD2A,	pan-fibroblast
		TAGLN, TGFBI, TNS1, TPM1	TGF-β
			response
			signature.
Antigen	Gene	B2M, HLA-A, HLA-B, HLA-C, TAP1, TAP2	PMID:
processing	expression		27855702
machinery	profiling
Immune	Gene	CD274, CTLA4, HAVCR2, LAG3, PDCD1,
checkpoint	expression	PDCD1LG2, TIGIT
	profiling
EMT	Gene	CLDN3, CLDN7, CLDN4, CDH1, VIM, TWIST1,	PMID:
	expression	ZEB1, ZEB2	24520177
	profiling
ECM	Gene	ACTA2, COL1A2, COL3A1, COL5A1, COL6A1,	PMID:
	expression	DCN, LAMA4, LUM, PDGFRB, TGFBR3	22553347
	profiling
FGFR3-related	Gene	FGFR3, TP63, WNT7B	PMID:
genes	expression		22553347
	profiling
KEGG	Gene	HIST1H2AG, HIST1H2AI, HIST1H2BL, HIST2H2BF	This study.
discovered	expression
histones	profiling
Angiogenesis	Gene	CDH5, SOX17, SOX18, TEK	PMID:
	expression		22553347
	profiling
Fanconi	Gene	APITD1, ATR, ATRIP, BLM, BRCA1, BRCA2,	KEGG
anemia	expression	BRIP1, C17orf70, C19orf40, EME1, EME2, ERCC1,	hsa03460
	profiling	ERCC4, FAN1, FANCA, FANCB, FANCC,
		FANCD2, FANCE, FANCF, FANCG, FANCI,
		FANCL, FANCM, HES1, MLH1, MUS81, PALB2,
		PMS2, POLH, POLI, POLK, POLN, RAD51,
		RAD51C, REV1, REV3L, RMI1, RMI2, RPA1,
		RPA2, RPA3, RPA4, SLX4, STRA13, TELO2,
		TOP3A, TOP3B, UBE2T, USP1, WDR48
Cell cycle	Gene	ABL1, ANAPC1, ANAPC10, ANAPC11, ANAPC13,	KEGG
	expression	ANAPC2, ANAPC4, ANAPC5, ANAPC7, ATM, ATR,	hsa4110
	profiling	BUB1, BUB1B, BUB3, CCNA1, CCNA2, CCNB1,
		CCNB2, CCNB3, CCND1, CCND2, CCND3,
		CCNE1, CCNE2, CCNH, CDC14A, CDC14B,
		CDC16, CDC20, CDC23, CDC25A, CDC25B,
		CDC25C, CDC26, CDC27, CDC45, CDC6, CDC7,
		CDK1, CDK2, CDK4, CDK6, CDK7, CDKN1A,
		CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C,
		CDKN2D, CHEK1, CHEK2, CREBBP, CUL1, DBF4,
		E2F1, E2F2, E2F3, E2F4, E2F5, EP300, ESPL1,
		FZR1, GADD45A, GADD45B, GADD45G, GSK3B,
		HDAC1, HDAC2, MAD1L1, MAD2L1, MAD2L2,
		MCM2, MCM3, MCM4, MCM5, MCM6, MCM7,
		MDM2, MYC, ORC1, ORC2, ORC3, ORC4, ORC5,
		ORC6, PCNA, PKMYT1, PLK1, PRKDC, PTTG1,
		PTTG2, RAD21, RB1, RBL1, RBL2, RBX1, SFN,
		SKP1, SKP2, SMAD2, SMAD3, SMAD4, SMC1A,
		SMC1B, SMC3, STAG1, STAG2, TFDP1, TFDP2,
		TGFB1, TGFB2, TGFB3, TP53, TTK, WEE1,
		YWHAB, YWHAE, YWHAG, YWHAH, YWHAQ,
		YWHAZ, ZBTB17
DNA	Gene	DNA2, FEN1, LIG1, MCM2, MCM3, MCM4, MCM5,	KEGG
replication	expression	MCM6, MCM7, PCNA, POLA1, POLA2, POLD1,	hsa3030
	profiling	POLD2, POLD3, POLD4, POLE, POLE2, POLE3,
		POLE4, PRIM1, PRIM2, RFC1, RFC2, RFC3,
		RFC4, RFC5, RNASEH1, RNASEH2A,
		RNASEH2B, RNASEH2C, RPA1, RPA2, RPA3,
		RPA4, SSBP1
Nucleotide	Gene	CCNH, CDK7, CETN2, CUL4A, CUL4B, DDB1,	KEGG
excision	expression	DDB2, ERCC1, ERCC2, ERCC3, ERCC4, ERCC5,	hsa3420
repair	profiling	ERCC6, ERCC8, GTF2H1, GTF2H2, GTF2H3,
		GTF2H4, GTF2H5, LIG1, MNAT1, PCNA, POLD1,
		POLD2, POLD3, POLD4, POLE, POLE2, POLE3,
		POLE4, RAD23A, RAD23B, RBX1, RFC1, RFC2,
		RFC3, RFC4, RFC5, RPA1, RPA2, RPA3, RPA4,
		XPA, XPC
Homologous	Gene	BLM, BRCA2, EME1, MRE11A, MUS81, NBN,	KEGG
recombination	expression	POLD1, POLD2, POLD3, POLD4, RAD50, RAD51,	hsa3440
	profiling	RAD51B, RAD51C, RAD51D, RAD52, RAD54B,
		RAD54L, RPA1, RPA2, RPA3, RPA4, SHFM1,
		SSBP1, TOP3A, TOP3B, XRCC2, XRCC3
Mismatch	Gene	EXO1, LIG1, MLH1, MLH3, MSH2, MSH3, MSH6,	KEGG
repair	expression	PCNA, PMS2, POLD1, POLD2, POLD3, POLD4,	hsa3430
	profiling	RFC1, RFC2, RFC3, RFC4, RFC5, RPA1, RPA2,
		RPA3, RPA4, SSBP1
Cell cycle	Gene	ATM, CCND1, CCNE1, CDKN1A, CDKN2A, E2F3,	PMID:
regulators	expression	FBXW7, MDM2, RB1, TP53	24476821
	and
	mutational
	profiling
DNA damage	Mutational	ATM, ATR, BLM, BRCA1, BRCA2, BRIP1, CHEK1,	Lange et al.
repair	profiling	CHEK2, ERCC4, FANCA, FANCC, FANCD2,	supra
		FANCE, FANCF, FANCG, FANCL, FANCM, MLH1,
		MSH2, MSH6, MUTYH, NUDT1, PALB2, PARP1,
		PARP2, PARP3, PMS2, POLD1, POLE, PRKDC,
		RAD51C, RPA1, TP53

Example 2: Tumor Mutational Burden (TMB), Driven by Proliferation, APOBEC Expression, or DNA Damage Repair Deficiencies, is Associated with Response to Atezolizumab

Bladder cancer, along with melanoma and non-small cell lung cancer, is characterized by one of the highest somatic TMBs among human cancers. Response to atezolizumab was significantly associated with TMB in mUC, and this association was also significant in the complete IMvigor210 data set (FIG. 1E). The relevance of TMB is likely via increased potential for immunogenic neoantigens, and consistent with this, predicted tumor neoantigen burden (TNB) was also significantly associated with response (FIG. 1F). TMB and TNB were also associated with OS (FIGS. 1V and 1W). For example, improved OS benefit from atezolizumab was observed in patients with high TMB relative to patients with low TMB, using median TMB as a cutoff (FIG. 8B). Improved OS from atezolizumab was also observed in patients with both high TMB and high PD-L1 IC scores (FIG. 8C).
We next assessed the transcriptional and mutational correlates of TMB in mUC. We first performed differential gene expression analysis, followed by gene set enrichment analysis. The pathways most significantly associated with TMB were those involved in cell cycle, DNA replication and DNA damage, detection and repair (FIG. 1G, Table 9). Signatures for these pathways were correlated with MKI67 and, thus, with proliferation (FIG. 1H). Secondly, we observed that expression levels for apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A) and APOBEC3B, two cytidine deaminases up-regulated in bladder and other cancers, were weakly but significantly correlated with TMB and response (FIGS. 1I and 1J). Thirdly, we examined loss-of-function mutations in members of the DNA damage response (DDR) or, given the relevance of proliferation rate, cell cycle regulator gene sets for correlation with TMB. Tumors with one or more mutations in the DDR gene set showed significantly higher response rates and TMB, both with and without (“w/o”) inclusion of TP53 (FIGS. 1K and 1L, Table 11). Table 11 shows DNA repair (DDR) and cell cycle regulation gene sets were tested for association with response (CR/PR vs. SD/PD) and TMB (“category”), with or without inclusion of TP53. The number of patients with at least one mutation in the genes belonging to a gene set (“n mutant”), the effect size (“estimate”) as well as nominal p-values are reported. MKI67 expression and DNA replication score were also associated with response to atezolizumab (FIG. 1Z).

TABLE 11

Mutation status of DNA repair and cell cycle regulation
pathways and association with response and TMB

Pathway	n mutant	category	estimate	p

Cell cycle regulation	177	response	0.47948	0.038621
Cell cycle regulation	125	response	0.735606	0.31104
w/o TP53
DDR
	145	response	0.50265	0.0274968
DDR w/o TP53	51	response	0.426164	1.17E−02
Cell cycle regulation	214	TMB	−3.6	2.04E−06
Cell cycle regulation	150	TMB	−0.91003	4.76E−02
w/o TP53
DDR pathway	173	TMB	−3.59998	8.64E−07
DDR pathway w/o TP53	60	TMB	−5.39995	1.84E−06

Tumors with one or more mutations in the cell cycle regulator set also exhibited significantly higher TMB and response rates, although association with response here was driven by TP53 (FIGS. 1M and 1N, Tables 11 and 12). Together, these results demonstrate that multiple factors, including proliferation, APOBEC activity and defects in DDR or cell cycle regulation, contribute to TMB in bladder cancer, leading to increased responses to PD-L1 blockade. In Table 12, symbols and the number of mutant patients are given for each tested gene. Association with mutation status was tested for both response (CR/PR vs. SD/PD) and TMB (“category”). Effect size (“estimate”) as well as nominal (“p”) and adjusted p-values (“p (adj.)”) are reported. The last two columns indicate whether a given gene is member of the DDR and/or the Cell cycle regulator (“CCReg”) gene set.

TABLE 12

Mutation status of single genes and association with response and TMB

	n					In	In
Gene	mutant	category	estimate	p	p (adj.)	DDR	CCReg

ACVR1B	3	TMB	−0.900043	6.93E−01	0.900459333	FALSE	FALSE
ACVR1B	3	response	Inf	5.75E−01	1	FALSE	FALSE
AKT1	3	TMB	−1.79995	0.685694	0.900459333	FALSE	FALSE
AKT1	3	response	0.667846	1	1	FALSE	FALSE
AKT2	2	TMB	−7.20994	0.166424	0.453154506	FALSE	FALSE
AKT2	2	response	0.333324	0.439745	1	FALSE	FALSE
AKT3	2	TMB	−0.899936	0.886628	0.938081009	FALSE	FALSE
AKT3	2	response	Inf	1	1	FALSE	FALSE
APC	9	TMB	−2.70005	0.23619	0.550298351	FALSE	FALSE
APC	9	response	0.660537	0.69521	1	FALSE	FALSE
ARAF	1	TMB	−18.0201	0.137297	0.437029887	FALSE	FALSE
ARAF	1	response	0	0.250996	0.872612588	FALSE	FALSE
ARFRP1	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
ARFRP1	1	response	Inf	1	1	FALSE	FALSE
ARID1A	70	TMB	−4.50999	6.45E−07	0.000145851	FALSE	FALSE
ARID1A	62	response	0.559204	0.0904854	0.872612588	FALSE	FALSE
ARID2	7	TMB	−14.41	0.00512586	0.150903898	FALSE	FALSE
ARID2	5	response	0.496176	0.601863	1	FALSE	FALSE
ASXL1	5	TMB	−18.0199	0.0137686	0.207446907	FALSE	FALSE
ASXL1	5	response	0.216728	0.10244	0.872612588	FALSE	FALSE
ATM	13	TMB	−4.50002	0.0350601	0.3433731	TRUE	TRUE
ATM	11	response	0.261315	0.0317334	0.872612588	TRUE	TRUE
ATR	4	TMB	−13.51	0.00734488	0.150903898	TRUE	FALSE
ATR	3	response	0.164576	0.156242	0.872612588	TRUE	FALSE
ATRX	2	TMB	−15.3101	0.0395031	0.3433731	FALSE	FALSE
ATRX	2	response	0.333324	0.439745	1	FALSE	FALSE
AURKA	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
AURKA	1	response	Inf	1	1	FALSE	FALSE
AXIN1	2	TMB	−8.11002	0.155164	0.445262375	FALSE	FALSE
AXIN1	2	response	0.333324	0.439745	1	FALSE	FALSE
AXL	1	TMB	−27.03	0.113957	0.408798127	FALSE	FALSE
AXL	1	response	0	0.250996	0.872612588	FALSE	FALSE
BACH1	1	TMB	−27.03	0.113957	0.408798127	FALSE	FALSE
BACH1	0	response	0	1	1	FALSE	FALSE
BAP1	5	TMB	−0.900021	0.705022	0.905312341	FALSE	FALSE
BAP1	5	response	1.34632	1	1	FALSE	FALSE
BARD1	1	TMB	−1.80005	0.731325	0.922679337	FALSE	FALSE
BARD1	1	response	Inf	1	1	FALSE	FALSE
BCL2L1	4	TMB	1.80993	0.34711	0.68131722	FALSE	FALSE
BCL2L1	1	response	Inf	1	1	FALSE	FALSE
BCL2L2	2	TMB	−1.80991	0.548746	0.843650313	FALSE	FALSE
BCL2L2	2	response	0.333324	0.439745	1	FALSE	FALSE
BCOR	1	TMB	−25.22	0.119477	0.416119176	FALSE	FALSE
BCOR	1	response	0	0.250996	0.872612588	FALSE	FALSE
BCORL1	3	TMB	−13.51	0.065011	0.408798127	FALSE	FALSE
BCORL1	3	response	0.667846	1	1	FALSE	FALSE
BRAF	6	TMB	−2.11E−06	0.918449	0.956541355	FALSE	FALSE
BRAF	5	response	0.216728	0.10244	0.872612588	FALSE	FALSE
BRCA1	6	TMB	−7.21004	0.111409	0.408798127	FALSE	FALSE
BRCA1	4	response	0.329736	0.262556	0.872612588	FALSE	FALSE
BRCA2	10	TMB	−2.71	0.162778	0.452443732	TRUE	FALSE
BRCA2	9	response	0.660537	0.69521	1	TRUE	FALSE
BRD4	1	TMB	−27.93	0.107343	0.408798127	FALSE	FALSE
BRD4	0	response	0	1	1	FALSE	FALSE
BRIP1	3	TMB	0.900061	0.736787	0.922679337	TRUE	FALSE
BRIP1	2	response	0.333324	0.439745	1	TRUE	FALSE
CASP8	2	TMB	−21.6199	0.0254157	0.294538267	FALSE	FALSE
CASP8	1	response	0	0.250996	0.872612588	FALSE	FALSE
CCND1	32	TMB	2.35E−05	0.824791	0.92832651	FALSE	TRUE
CCND1	26	response	1.13041	1	1	FALSE	TRUE
CCND2	1	TMB	4.50997	0.222643	0.529786821	FALSE	FALSE
CCND2	0	response	0	1	1	FALSE	FALSE
CCND3	5	TMB	0.900012	0.73896	0.922679337	FALSE	FALSE
CCND3	5	response	1.34632	1	1	FALSE	FALSE
CCNE1	12	TMB	0.909984	0.417896	0.75467319	FALSE	TRUE
CCNE1	8	response	0.322416	0.111683	0.872612588	FALSE	TRUE
CD274	4	TMB	−0.234732	0.888785	0.938081009	FALSE	FALSE
CD274	2	response	0.333324	0.439745	1	FALSE	FALSE
CDC73	1	TMB	−2.35E−05	0.962226	0.968688231	FALSE	FALSE
CDC73	1	response	Inf	1	1	FALSE	FALSE
CDH1	6	TMB	−29.7301	0.00360566	0.135813193	FALSE	FALSE
CDH1	3	response	0.667846	1	1	FALSE	FALSE
CDH2	1	TMB	−27.03	0.113957	0.408798127	FALSE	FALSE
CDH2	0	response	0	1	1	FALSE	FALSE
CDK4	2	TMB	−3.59996	0.413533	0.753697242	FALSE	FALSE
CDK4	2	response	Inf	1	1	FALSE	FALSE
CDK6	5	TMB	−0.900046	0.808321	0.92832651	FALSE	FALSE
CDK6	4	response	Inf	0.574746	1	FALSE	FALSE
CDK8	2	TMB	−3.77E−06	1	1	FALSE	FALSE
CDK8	1	response	0	0.250996	0.872612588	FALSE	FALSE
CDKN1B	6	TMB	−3.60991	0.338032	0.68131722	FALSE	FALSE
CDKN1B	5	response	1.34632	1	1	FALSE	FALSE
CDKN2A	54	TMB	−0.899978	0.343403	0.68131722	FALSE	TRUE
CDKN2A	44	response	1.9556	0.130946	0.872612588	FALSE	TRUE
CDKN2B	41	TMB	−6.38E−05	0.795052	0.92832651	FALSE	FALSE
CDKN2B	34	response	2.11029	0.200236	0.872612588	FALSE	FALSE
CEBPA	2	TMB	−13.51	0.0482635	0.39863575	FALSE	FALSE
CEBPA	1	response	Inf	1	1	FALSE	FALSE
CHD2	2	TMB	−13.51	0.0626281	0.408798127	FALSE	FALSE
CHD2	1	response	Inf	1	1	FALSE	FALSE
CHD4	1	TMB	−7.21006	0.300198	0.646140457	FALSE	FALSE
CHD4	1	response	0	0.250996	0.872612588	FALSE	FALSE
CHEK2	3	TMB	−1.79997	0.510214	0.81554169	TRUE	FALSE
CHEK2	3	response	Inf	0.574746	1	TRUE	FALSE
CREBBP	30	TMB	−7.21	2.35E−05	0.002656438	FALSE	FALSE
CREBBP	27	response	0.441408	0.0598306	0.872612588	FALSE	FALSE
CRKL	2	TMB	3.60996	0.157615	0.445262375	FALSE	FALSE
CRKL	2	response	Inf	1	1	FALSE	FALSE
CTCF	2	TMB	−20.7201	0.0265376	0.294538267	FALSE	FALSE
CTCF	2	response	0.333324	0.439745	1	FALSE	FALSE
CTNNB1	8	TMB	−1.79998	0.612614	0.872346467	FALSE	FALSE
CTNNB1	7	response	0.436474	0.37208	1	FALSE	FALSE
CUL3	2	TMB	1.79994	0.74361	0.923383846	FALSE	FALSE
CUL3	2	response	Inf	1	1	FALSE	FALSE
CYLD	1	TMB	−27.03	0.113957	0.408798127	FALSE	FALSE
CYLD	1	response	0	0.250996	0.872612588	FALSE	FALSE
DAXX	1	TMB	−27.93	0.107343	0.408798127	FALSE	FALSE
DAXX	1	response	0	0.250996	0.872612588	FALSE	FALSE
DICER1	3	TMB	−5.40006	0.21188	0.529786821	FALSE	FALSE
DICER1	3	response	0.667846	1	1	FALSE	FALSE
DNMT3A	8	TMB	−1.79996	0.630572	0.872346467	FALSE	FALSE
DNMT3A	6	response	0.664231	0.642647	1	FALSE	FALSE
EGFR	11	TMB	0.900018	0.619994	0.872346467	FALSE	FALSE
EGFR	10	response	3.10714	0.458956	1	FALSE	FALSE
EMSY	1	TMB	1.79997	0.644251	0.872346467	FALSE	FALSE
EMSY	1	response	Inf	1	1	FALSE	FALSE
EP300	16	TMB	−2.70004	0.154497	0.445262375	FALSE	FALSE
EP300	11	response	0.889329	1	1	FALSE	FALSE
EPHA3	1	TMB	2.69992	0.462896	0.798583939	FALSE	FALSE
EPHA3	1	response	0	0.250996	0.872612588	FALSE	FALSE
EPHA6	2	TMB	−1.80005	0.785188	0.92832651	FALSE	FALSE
EPHA6	1	response	0	0.250996	0.872612588	FALSE	FALSE
EPHA7	1	TMB	−23.42	0.125204	0.416119176	FALSE	FALSE
EPHA7	0	response	0	1	1	FALSE	FALSE
EPHB1	2	TMB	0.90002	0.64461	0.872346467	FALSE	FALSE
EPHB1	2	response	Inf	1	1	FALSE	FALSE
ERBB2	31	TMB	−2.7	0.0624137	0.408798127	FALSE	FALSE
ERBB2	28	response	0.819104	0.647802	1	FALSE	FALSE
ERBB3	18	TMB	−1.80996	0.222186	0.529786821	FALSE	FALSE
ERBB3	16	response	0.403958	0.130846	0.872612588	FALSE	FALSE
ERBB4	3	TMB	−8.10995	0.0665214	0.408798127	FALSE	FALSE
ERBB4	2	response	0.333324	0.439745	1	FALSE	FALSE
ERRFI1	1	TMB	−40.54	0.0915622	0.408798127	FALSE	FALSE
ERRFI1	1	response	0	0.250996	0.872612588	FALSE	FALSE
EZH2	1	TMB	−13.51	0.1571	0.445262375	FALSE	FALSE
EZH2	1	response	Inf	1	1	FALSE	FALSE
FAM123B	1	TMB	−18.0201	0.137297	0.437029887	FALSE	FALSE
FAM123B	1	response	0	0.250996	0.872612588	FALSE	FALSE
FANCA	1	TMB	−29.73	0.0998104	0.408798127	TRUE	FALSE
FANCA	1	response	0	0.250996	0.872612588	TRUE	FALSE
FANCC	1	TMB	3.60996	0.319946	0.668246752	TRUE	FALSE
FANCC	1	response	Inf	1	1	TRUE	FALSE
FANCD2	1	TMB	−13.51	0.1571	0.445262375	TRUE	FALSE
FANCD2	1	response	Inf	1	1	TRUE	FALSE
FANCI	1	TMB	−6.30005	0.355732	0.68131722	FALSE	FALSE
FANCI	1	response	Inf	1	1	FALSE	FALSE
FAS	2	TMB	−10.81	0.0720309	0.408798127	FALSE	FALSE
FAS	1	response	Inf	1	1	FALSE	FALSE
FAT1	11	TMB	−0.900028	0.602166	0.872346467	FALSE	FALSE
FAT1	9	response	0.404735	0.234402	0.872612588	FALSE	FALSE
FBXW7	13	TMB	−12.61	0.000827519	0.046754824	FALSE	TRUE
FBXW7	12	response	0.220239	0.0125031	0.86158545	FALSE	TRUE
FGF10	17	TMB	−6.77E−05	0.872111	0.938081009	FALSE	FALSE
FGF10	12	response	0.450284	0.182862	0.872612588	FALSE	FALSE
FGF14	3	TMB	−3.59994	0.501442	0.81554169	FALSE	FALSE
FGF14	3	response	0.164576	0.156242	0.872612588	FALSE	FALSE
FGF19	20	TMB	0.89991	0.670271	0.894242118	FALSE	FALSE
FGF19	16	response	1.48364	0.767315	1	FALSE	FALSE
FGF23	1	TMB	4.50997	0.222643	0.529786821	FALSE	FALSE
FGF23	0	response	0	1	1	FALSE	FALSE
FGF3	22	TMB	0.900045	0.471973	0.802974602	FALSE	FALSE
FGF3	18	response	1.18599	1	1	FALSE	FALSE
FGF4	13	TMB	1.80002	0.256311	0.585114	FALSE	FALSE
FGF4	10	response	3.10714	0.458956	1	FALSE	FALSE
FGF6	2	TMB	4.51001	0.0638199	0.408798127	FALSE	FALSE
FGF6	0	response	0	1	1	FALSE	FALSE
FGFR1	7	TMB	−2.69998	0.311112	0.663314264	FALSE	FALSE
FGFR1	7	response	0.126038	0.0119594	0.86158545	FALSE	FALSE
FGFR2	3	TMB	−0.899953	0.797003	0.92832651	FALSE	FALSE
FGFR2	3	response	0.667846	1	1	FALSE	FALSE
FGFR3	56	TMB	−5.97E−05	0.84819	0.92832651	FALSE	FALSE
FGFR3	50	response	0.829608	0.588823	1	FALSE	FALSE
FLCN	3	TMB	−1.80006	0.458804	0.798583939	FALSE	FALSE
FLCN	3	response	0.667846	1	1	FALSE	FALSE
FLT3	1	TMB	1.79997	0.644251	0.872346467	FALSE	FALSE
FLT3	0	response	0	1	1	FALSE	FALSE
FOXP1	7	TMB	1.79994	0.395267	0.732215918	FALSE	FALSE
FOXP1	7	response	2.03921	0.683488	1	FALSE	FALSE
FRS2	23	TMB	−0.899976	0.639385	0.872346467	FALSE	FALSE
FRS2	19	response	0.42895	0.0970036	0.872612588	FALSE	FALSE
FUBP1	2	TMB	3.6	0.504942	0.81554169	FALSE	FALSE
FUBP1	2	response	Inf	1	1	FALSE	FALSE
GATA1	1	TMB	1.79997	0.644251	0.872346467	FALSE	FALSE
GATA1	1	response	Inf	1	1	FALSE	FALSE
GATA2	1	TMB	−3.60E+00	0.561803	0.852130725	FALSE	FALSE
GATA2	1	response	Inf	1	1	FALSE	FALSE
GATA3	3	TMB	1.80E+00	0.597378	0.872346467	FALSE	FALSE
GATA3	3	response	0.667846	1	1	FALSE	FALSE
GATA4	1	TMB	2.69992	0.462896	0.798583939	FALSE	FALSE
GATA4	1	response	0.00E+00	0.250996	0.872612588	FALSE	FALSE
GATA6	3	TMB	1.80001	0.51242	0.81554169	FALSE	FALSE
GATA6	2	response	0.333324	0.439745	1	FALSE	FALSE
GNA13	3	TMB	−3.59998	0.525757	0.825146403	FALSE	FALSE
GNA13	2	response	Inf	1	1	FALSE	FALSE
GNAS	6	TMB	1.80005	0.322296	0.668246752	FALSE	FALSE
GNAS	6	response	0.32612	0.168613	0.872612588	FALSE	FALSE
GPR124	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
GPR124	0	response	0.00E+00	1	1	FALSE	FALSE
GRIN2A	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
GRIN2A	1	response	Inf	1	1	FALSE	FALSE
HGF	1	TMB	−42.34	0.089309	0.408798127	FALSE	FALSE
HGF	0	response	0	1	1	FALSE	FALSE
HNF1A	2	TMB	−18.02	0.0335033	0.3433731	FALSE	FALSE
HNF1A	2	response	0.333324	0.439745	1	FALSE	FALSE
HRAS	12	TMB	0.899988	0.62817	0.872346467	FALSE	FALSE
HRAS	11	response	1.53E+00	0.735541	1	FALSE	FALSE
IGF1	2	TMB	−5.39992	0.284939	0.631335431	FALSE	FALSE
IGF1	2	response	Inf	1	1	FALSE	FALSE
IGF2R	1	TMB	−27.03	0.113957	0.408798127	FALSE	FALSE
IGF2R	1	response	0	0.250996	0.872612588	FALSE	FALSE
IKBKE	1	TMB	2.70E+00	0.462896	0.798583939	FALSE	FALSE
IKBKE	0	response	0	1	1	FALSE	FALSE
INPP4B	1	TMB	−5.05E+01	0.0871006	0.408798127	FALSE	FALSE
INPP4B	1	response	0.00E+00	0.250996	0.872612588	FALSE	FALSE
IRS2	5	TMB	−2.70008	0.367558	0.698051328	FALSE	FALSE
IRS2	5	response	4.96E−01	0.601863	1	FALSE	FALSE
JAK1	2	TMB	−2.43E+01	0.0232944	0.292474133	FALSE	FALSE
JAK1	2	response	0.00E+00	0.062247	0.872612588	FALSE	FALSE
JAK2	3	TMB	1.81E+00	0.519066	0.82034207	FALSE	FALSE
JAK2	2	response	3.33E−01	0.439745	1	FALSE	FALSE
JUN	1	TMB	−6.30E+00	0.355732	0.68131722	FALSE	FALSE
JUN	1	response	Inf	1	1	FALSE	FALSE
KDM5A	1	TMB	7.21E+00	0.0998104	0.408798127	FALSE	FALSE
KDM5A	1	response	Inf	1	1	FALSE	FALSE
KDM5C	4	TMB	−6.30E+00	0.260724	0.586228337	FALSE	FALSE
KDM5C	4	response	3.30E−01	0.262556	0.872612588	FALSE	FALSE
KDM6A	55	TMB	−1.8	0.121569	0.416119176	FALSE	FALSE
KDM6A	47	response	0.515273	0.0624163	0.872612588	FALSE	FALSE
KDR	2	TMB	0.899949	0.896573	0.938081009	FALSE	FALSE
KDR	1	response	0	0.250996	0.872612588	FALSE	FALSE
KEAP1	1	TMB	−27.03	0.113957	0.408798127	FALSE	FALSE
KEAP1	1	response	0	0.250996	0.872612588	FALSE	FALSE
KEL	2	TMB	−11.7101	0.088666	0.408798127	FALSE	FALSE
KEL	1	response	0	0.250996	0.872612588	FALSE	FALSE
KIT	3	TMB	3.51E−06	0.939854	0.961117665	FALSE	FALSE
KIT	2	response	0.333324	0.439745	1	FALSE	FALSE
KRAS	6	TMB	0.900048	0.571685	0.861338733	FALSE	FALSE
KRAS	5	response	1.34632	1	1	FALSE	FALSE
LRP1B	10	TMB	−9.01002	0.00902982	0.17006161	FALSE	FALSE
LRP1B	9	response	0.155516	0.00896804	0.86158545	FALSE	FALSE
LRP6	1	TMB	−27.03	0.113957	0.408798127	FALSE	FALSE
LRP6	1	response	0	0.250996	0.872612588	FALSE	FALSE
LYN	4	TMB	−5.79284	0.261987	0.586228337	FALSE	FALSE
LYN	4	response	Inf	0.574746	1	FALSE	FALSE
LZTR1	1	TMB	−18.0201	0.137297	0.437029887	FALSE	FALSE
LZTR1	1	response	0	0.250996	0.872612588	FALSE	FALSE
MAGI2	2	TMB	−0.899977	0.811092	0.92832651	FALSE	FALSE
MAGI2	2	response	Inf	1	1	FALSE	FALSE
MAP2K4	1	TMB	−12.61	0.171459	0.45461214	FALSE	FALSE
MAP2K4	1	response	Inf	1	1	FALSE	FALSE
MAP3K1	2	TMB	−9.91004	0.0878833	0.408798127	FALSE	FALSE
MAP3K1	2	response	0.333324	0.439745	1	FALSE	FALSE
MAP3K13	1	TMB	−12.61	0.171459	0.45461214	FALSE	FALSE
MAP3K13	1	response	Inf	1	1	FALSE	FALSE
MCL1	8	TMB	−4.49997	0.106906	0.408798127	FALSE	FALSE
MCL1	7	response	0.833937	1	1	FALSE	FALSE
MDM2	25	TMB	−0.89997	0.54233	0.843650313	FALSE	TRUE
MDM2	20	response	0.470372	0.114587	0.872612588	FALSE	TRUE
MDM4	1	TMB	−6.30005	0.355732	0.68131722	FALSE	FALSE
MDM4	1	response	Inf	1	1	FALSE	FALSE
MEN1	3	TMB	−1.80996	0.546091	0.843650313	FALSE	FALSE
MEN1	2	response	0	0.062247	0.872612588	FALSE	FALSE
MET	2	TMB	−1.80003	0.830658	0.92832651	FALSE	FALSE
MET	0	response	0	1	1	FALSE	FALSE
MITF	1	TMB	4.50997	0.222643	0.529786821	FALSE	FALSE
MITF	1	response	Inf	1	1	FALSE	FALSE
MLL2	63	TMB	−0.899944	0.501383	0.81554169	FALSE	FALSE
MLL2	53	response	1.18463	0.723338	1	FALSE	FALSE
MLL3	4	TMB	−21.6299	0.00550322	0.150903898	FALSE	FALSE
MLL3	3	response	0	0.0152493	0.86158545	FALSE	FALSE
MPL	1	TMB	−5.40005	0.420747	0.75467319	FALSE	FALSE
MPL	1	response	0	0.250996	0.872612588	FALSE	FALSE
MSH2	4	TMB	−25.2199	0.00171259	0.077409068	TRUE	FALSE
MSH2	3	response	0.667846	1	1	TRUE	FALSE
MSH6	6	TMB	−0.899991	0.720075	0.914252528	TRUE	FALSE
MSH6	5	response	1.34632	1	1	TRUE	FALSE
MST1R	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
MST1R	1	response	Inf	1	1	FALSE	FALSE
MTOR	3	TMB	−4.51002	0.183279	0.475640114	FALSE	FALSE
MTOR	2	response	0.333324	0.439745	1	FALSE	FALSE
MUTYH	3	TMB	−2.69997	0.460887	0.798583939	TRUE	FALSE
MUTYH	3	response	0.667846	1	1	TRUE	FALSE
MYC	13	TMB	−1.07E−05	0.894618	0.938081009	FALSE	FALSE
MYC	10	response	1.354	1	1	FALSE	FALSE
MYCL1	9	TMB	−1.8	0.472547	0.802974602	FALSE	FALSE
MYCL1	9	response	0.660537	0.69521	1	FALSE	FALSE
MYCN	5	TMB	−9.90994	0.0702785	0.408798127	FALSE	FALSE
MYCN	5	response	0.216728	0.10244	0.872612588	FALSE	FALSE
MYST3	7	TMB	−3.59996	0.185205	0.475640114	FALSE	FALSE
MYST3	6	response	0.664231	0.642647	1	FALSE	FALSE
NBN	1	TMB	−23.42	0.125204	0.416119176	FALSE	FALSE
NBN	1	response	0	0.250996	0.872612588	FALSE	FALSE
NCOR1	10	TMB	−7.20992	0.0119391	0.192731186	FALSE	FALSE
NCOR1	9	response	1.17881	1	1	FALSE	FALSE
NF1	6	TMB	−5.39999	0.0883754	0.408798127	FALSE	FALSE
NF1	5	response	0.496176	0.601863	1	FALSE	FALSE
NF2	6	TMB	−0.900005	0.71461	0.912439887	FALSE	FALSE
NF2	4	response	0.329736	0.262556	0.872612588	FALSE	FALSE
NFE2L2	2	TMB	1.80004	0.394637	0.732215918	FALSE	FALSE
NFE2L2	2	response	0.333324	0.439745	1	FALSE	FALSE
NFKBIA	2	TMB	0.90002	0.64461	0.872346467	FALSE	FALSE
NFKBIA	2	response	Inf	1	1	FALSE	FALSE
NKX2-1	6	TMB	0.899935	0.615534	0.872346467	FALSE	FALSE
NKX2-1	6	response	Inf	0.341617	1	FALSE	FALSE
NOTCH1	4	TMB	−1.79998	0.690117	0.900459333	FALSE	FALSE
NOTCH1	4	response	1.00538	1	1	FALSE	FALSE
NOTCH2	4	TMB	3.16E−06	0.884086	0.938081009	FALSE	FALSE
NOTCH2	3	response	0.667846	1	1	FALSE	FALSE
NOTCH3	2	TMB	1.79999	0.510315	0.81554169	FALSE	FALSE
NOTCH3	2	response	Inf	1	1	FALSE	FALSE
NOTCH4	5	TMB	−3.17E−05	0.934137	0.960632518	FALSE	FALSE
NOTCH4	3	response	0.667846	1	1	FALSE	FALSE
NRAS	2	TMB	−13.52	0.059729	0.408798127	FALSE	FALSE
NRAS	2	response	0	0.062247	0.872612588	FALSE	FALSE
NTRK1	1	TMB	−6.30005	0.355732	0.68131722	FALSE	FALSE
NTRK1	1	response	0	0.250996	0.872612588	FALSE	FALSE
NTRK3	1	TMB	6.30992	0.125204	0.416119176	FALSE	FALSE
NTRK3	1	response	Inf	1	1	FALSE	FALSE
NUP93	4	TMB	−1.79997	0.67266	0.894242118	FALSE	FALSE
NUP93	3	response	0.667846	1	1	FALSE	FALSE
PALB2	3	TMB	−1.80003	0.621392	0.872346467	TRUE	FALSE
PALB2	3	response	0.667846	1	1	TRUE	FALSE
PARK2	1	TMB	−13.51	0.1571	0.445262375	FALSE	FALSE
PARK2	1	response	Inf	1	1	FALSE	FALSE
PARP4	1	TMB	5.40997	0.164161	0.452443732	FALSE	FALSE
PARP4	1	response	Inf	1	1	FALSE	FALSE
PAX5	3	TMB	−2.78E−05	0.92622	0.960209725	FALSE	FALSE
PAX5	2	response	Inf	1	1	FALSE	FALSE
PBRM1	5	TMB	−9.01005	0.222102	0.529786821	FALSE	FALSE
PBRM1	4	response	0.108148	0.0499291	0.872612588	FALSE	FALSE
PDCD1LG2	4	TMB	0.910011	0.701112	0.905312341	FALSE	FALSE
PDCD1LG2	3	response	0.667846	1	1	FALSE	FALSE
PDGFRA	2	TMB	0.899949	0.896573	0.938081009	FALSE	FALSE
PDGFRA	1	response	0	0.250996	0.872612588	FALSE	FALSE
PIK3C2B	1	TMB	−6.30005	0.355732	0.68131722	FALSE	FALSE
PIK3C2B	1	response	Inf	1	1	FALSE	FALSE
PIK3CA	45	TMB	−0.900037	0.297028	0.646140457	FALSE	FALSE
PIK3CA	38	response	0.793515	0.547066	1	FALSE	FALSE
PIK3CB	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
PIK3CB	1	response	Inf	1	1	FALSE	FALSE
PIK3CG	1	TMB	−42.34	0.089309	0.408798127	FALSE	FALSE
PIK3CG	0	response	0	1	1	FALSE	FALSE
PIK3R1	4	TMB	−7.92E−05	0.964402	0.968688231	FALSE	FALSE
PIK3R1	4	response	0.329736	0.262556	0.872612588	FALSE	FALSE
PIK3R2	1	TMB	−4.50006	0.484823	0.81554169	FALSE	FALSE
PIK3R2	0	response	0	1	1	FALSE	FALSE
PMS2	2	TMB	−28.83	0.0208602	0.292474133	TRUE	FALSE
PMS2	1	response	0	0.250996	0.872612588	TRUE	FALSE
POLE	1	TMB	−27.03	0.113957	0.408798127	TRUE	FALSE
POLE	1	response	0	0.250996	0.872612588	TRUE	FALSE
PRKCI	4	TMB	4.80E−05	0.872354	0.938081009	FALSE	FALSE
PRKCI	3	response	0.667846	1	1	FALSE	FALSE
PRSS8	1	TMB	−9.91006	0.229453	0.540170604	FALSE	FALSE
PRSS8	1	response	0	0.250996	0.872612588	FALSE	FALSE
PTCH2	1	TMB	−29.73	0.0998104	0.408798127	FALSE	FALSE
PTCH2	1	response	0	0.250996	0.872612588	FALSE	FALSE
PTEN	11	TMB	−1.81007	0.404451	0.74313761	FALSE	FALSE
PTEN	8	response	0.322416	0.111683	0.872612588	FALSE	FALSE
PTPN11	1	TMB	−3.60006	0.561803	0.852130725	FALSE	FALSE
PTPN11	1	response	Inf	1	1	FALSE	FALSE
PTPRD	3	TMB	0.899972	0.791712	0.92832651	FALSE	FALSE
PTPRD	2	response	Inf	1	1	FALSE	FALSE
RAD50	3	TMB	−7.21005	0.0779225	0.408798127	FALSE	FALSE
RAD50	3	response	Inf	0.574746	1	FALSE	FALSE
RAD51	1	TMB	−2.35E−05	0.962226	0.968688231	FALSE	FALSE
RAD51	1	response	Inf	1	1	FALSE	FALSE
RAD51C	2	TMB	−1.81006	0.656689	0.88340306	TRUE	FALSE
RAD51C	1	response	0	0.250996	0.872612588	TRUE	FALSE
RAF1	16	TMB	0.900031	0.388914	0.732215918	FALSE	FALSE
RAF1	14	response	0.584639	0.349916	1	FALSE	FALSE
RB1	44	TMB	−1.80008	0.108637	0.408798127	FALSE	TRUE
RB1	38	response	0.683208	0.316049	1	FALSE	TRUE
RBM10	12	TMB	−4.50005	0.038652	0.3433731	FALSE	FALSE
RBM10	10	response	0.774318	0.715047	1	FALSE	FALSE
REL	2	TMB	−17.12	0.0379235	0.3433731	FALSE	FALSE
REL	2	response	0	0.062247	0.872612588	FALSE	FALSE
RICTOR	22	TMB	2.81E−05	0.830895	0.92832651	FALSE	FALSE
RICTOR	16	response	0.535247	0.242047	0.872612588	FALSE	FALSE
RNF43	2	TMB	−27.93	0.0220481	0.292474133	FALSE	FALSE
RNF43	2	response	0	0.062247	0.872612588	FALSE	FALSE
ROS1	2	TMB	−1.80997	0.687275	0.900459333	FALSE	FALSE
ROS1	2	response	Inf	1	1	FALSE	FALSE
RPA1	1	TMB	0.899972	0.854389	0.92832651	TRUE	FALSE
RPA1	1	response	Inf	1	1	TRUE	FALSE
RPTOR	1	TMB	−13.51	0.1571	0.445262375	FALSE	FALSE
RPTOR	1	response	Inf	1	1	FALSE	FALSE
RUNX1	2	TMB	−7.20997	0.139933	0.439234139	FALSE	FALSE
RUNX1	2	response	0.333324	0.439745	1	FALSE	FALSE
SDHA	1	TMB	−7.21006	0.300198	0.646140457	FALSE	FALSE
SDHA	1	response	Inf	1	1	FALSE	FALSE
SETD2	6	TMB	−6.30994	0.0273686	0.294538267	FALSE	FALSE
SETD2	6	response	1.69097	1	1	FALSE	FALSE
SF3B1	7	TMB	1.87E−05	0.935129	0.960632518	FALSE	FALSE
SF3B1	3	response	0.164576	0.156242	0.872612588	FALSE	FALSE
SLIT2	4	TMB	1.80005	0.610903	0.872346467	FALSE	FALSE
SLIT2	3	response	Inf	0.574746	1	FALSE	FALSE
SMAD2	1	TMB	−36.03	0.0950275	0.408798127	FALSE	FALSE
SMAD2	1	response	Inf	1	1	FALSE	FALSE
SMAD3	1	TMB	−2.35E−05	0.962226	0.968688231	FALSE	FALSE
SMAD3	1	response	Inf	1	1	FALSE	FALSE
SMAD4	3	TMB	4.50999	0.0493885	0.39863575	FALSE	FALSE
SMAD4	2	response	Inf	1	1	FALSE	FALSE
SMARCA4	9	TMB	−3.61005	0.172994	0.45461214	FALSE	FALSE
SMARCA4	8	response	0.547957	0.418026	1	FALSE	FALSE
SOX10	1	TMB	−2.70008	0.640011	0.872346467	FALSE	FALSE
SOX10	1	response	Inf	1	1	FALSE	FALSE
SOX2	1	TMB	3.60996	0.319946	0.668246752	FALSE	FALSE
SOX2	1	response	Inf	1	1	FALSE	FALSE
SOX9	3	TMB	−1.80001	0.49275	0.81554169	FALSE	FALSE
SOX9	3	response	Inf	0.574746	1	FALSE	FALSE
SPEN	4	TMB	−7.20994	0.0704492	0.408798127	FALSE	FALSE
SPEN	2	response	0.333324	0.439745	1	FALSE	FALSE
SPTA1	5	TMB	−0.900026	0.806257	0.92832651	FALSE	FALSE
SPTA1	5	response	Inf	0.335003	1	FALSE	FALSE
SRC	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
SRC	1	response	Inf	1	1	FALSE	FALSE
STAG2	21	TMB	−2.69996	0.222698	0.529786821	FALSE	FALSE
STAG2	17	response	0.591772	0.383584	1	FALSE	FALSE
STAT4	2	TMB	−0.899977	0.811092	0.92832651	FALSE	FALSE
STAT4	2	response	0.333324	0.439745	1	FALSE	FALSE
STK11	2	TMB	0.899989	0.772323	0.92832651	FALSE	FALSE
STK11	1	response	Inf	1	1	FALSE	FALSE
TBX3	3	TMB	−7.21003	0.0706912	0.408798127	FALSE	FALSE
TBX3	2	response	0	0.062247	0.872612588	FALSE	FALSE
TERT	186	TMB	−1.80005	0.00617139	0.150903898	FALSE	FALSE
TERT	159	response	0.991699	1	1	FALSE	FALSE
TET2	5	TMB	−11.71	0.0113234	0.192731186	FALSE	FALSE
TET2	3	response	0.667846	1	1	FALSE	FALSE
TNFRSF14	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
TNFRSF14	1	response	Inf	1	1	FALSE	FALSE
TNKS	2	TMB	1.79999	0.629643	0.872346467	FALSE	FALSE
TNKS	2	response	Inf	1	1	FALSE	FALSE
TOP1	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
TOP1	1	response	Inf	1	1	FALSE	FALSE
TOP2A	3	TMB	−22.52	0.00709567	0.150903898	FALSE	FALSE
TOP2A	3	response	0.667846	1	1	FALSE	FALSE
TP53	145	TMB	−2.70001	0.000173155	0.013044343	TRUE	TRUE
TP53	120	response	0.720106	0.307979	1	TRUE	TRUE
TRRAP	1	TMB	−13.51	0.1571	0.445262375	FALSE	FALSE
TRRAP	1	response	Inf	1	1	FALSE	FALSE
TSC1	19	TMB	3.68E−06	0.82796	0.92832651	FALSE	FALSE
TSC1	19	response	1.27769	0.789154	1	FALSE	FALSE
TSC2	1	TMB	0.899972	0.854389	0.92832651	FALSE	FALSE
TSC2	1	response	Inf	1	1	FALSE	FALSE
VEGFA	2	TMB	1.81002	0.504942	0.81554169	FALSE	FALSE
VEGFA	2	response	Inf	1	1	FALSE	FALSE
WT1	2	TMB	−6.30991	0.254046	0.585114	FALSE	FALSE
WT1	1	response	0	0.250996	0.872612588	FALSE	FALSE
ZNF217	2	TMB	0.89999	0.791643	0.92832651	FALSE	FALSE
ZNF217	2	response	Inf	1	1	FALSE	FALSE
ZNF703	17	TMB	−0.900011	0.354452	0.68131722	FALSE	FALSE
ZNF703	16	response	0.535247	0.242047	0.872612588	FALSE	FALSE

Example 3: The TGF-β Axis is Associated with Primary Immune Escape

Next, we sought to identify any additional features beyond CD8+ T-cell immunity and TMB that were associated with response. Consistent with a positive relationship between proliferation and TMB, gene sets associated with DNA replication, cell cycle, and histones were significantly enriched in responders (FIG. 1O, Table 8). Gene set enrichment analysis also identified the cytokine-cytokine receptor gene set as the sole feature associated with non-response (FIG. 1O, Table 8). Importantly, the most significantly associated genes within this pathway were IFNGR1 and genes involved in the TGF-β signaling pathway, including TGFB1, ACVR1, and TGFBR2. While IFN-γ is known to have favorable effects on anti-tumor immunity, this cytokine is also emerging as a key player in primary resistance to checkpoint therapy and acquired immune escape. In our large cohort of patients with mUC, we observed significantly enhanced expression of IFN-γ in responders, whereas IFNGR1 expression was enriched in non-responders. This is in line with recent studies suggesting that IFN-γ signaling promotes expression of multiple T-cell inhibitory ligands and induces epigenetic mechanisms that suppress T-cells.
TGF-β is a pleiotropic cytokine associated with poor prognosis in multiple tumor types. TGF-β signaling is generally thought to play a pro-tumorigenic role in advanced cancers by promoting immunosuppression, angiogenesis, metastasis, epithelial to mesenchymal transition (EMT), fibroblast activation, and desmoplasia. In IMvigor210, a signature based on the two top-scoring TGF-β pathway genes comprised of TGFB1 and TGFBR2 showed increased mean expression in non-responders (FIG. 1P). TGFB1 expression was associated with poor objective response, and, when split by quartiles, was also associated with reduced OS (FIGS. 1X and 1Y). Given that elevated TGF-β expression could indicate increased T regulatory cell function, EMT, and/or stromal-associated immunosuppression, it is likely to play a significant role in restricting response to atezolizumab.

Example 4: Three Core Pathways Drive Response and Resistance to Anti-PD-L1 Therapy

Collectively, these data suggest that response to checkpoint inhibition in bladder cancer is dictated by the combination of three core biological axes: pre-existing T-cell immunity and TMB are positively associated with response to atezolizumab, whereas TGF-β pathway activity is associated with disease progression and lack of response (FIG. 1Q).
To better understand how these three pathways relate to one another, and to reveal their relative importance in predicting outcome, a statistical analysis of competing models was performed. Logistic regression pseudo-R²was used as a measure of “explained variance” in overall patient response (i.e., the percent of variation in patient response that can be attributed to the biological inputs) (see, e.g., Dobson et al. supra). When single inputs were considered, the CD8+ Teff signature explained 5% of variability in patient response, while the two-gene TGF-β signature explained 16% and TMB explained 30% (FIG. 1R). We next asked whether these three biological inputs are interchangeable or if they independently contribute to predicting response. A model combining all three axes increased explained variance in patient response to 42% and improved over each single-axis model (FIG. 1R). The model combining all three axes also significantly improved on all two-axis models (FIG. 1S), indicating that the information provided by each axis is at least partially independent of the other two.

Example 5: Molecular Taxonomy Reveals Relationships Among Drivers of Response and Resistance

To highlight the power of the present framework of three pathways described above (i.e., pre-existing T-cell immunity, TMB, and TGF-β pathway activity) and illustrate the relevance of TGF-β signaling, we explored the relationship between established tumor molecular subtyping and the three pathways. Multiple taxonomic classification methods based on gene expression were recently described (Lerner et al. supra). We previously applied The Cancer Genome Atlas (TCGA) taxonomy (Caton et al. Nat. Genet. 47:320-329, 2015) to IMvigor210 samples but also explored the Lund taxonomy (Sjödahl et al. Clin. Cancer Res. 18:3377-3386, 2012; Sjödahl et al. Am. J. Pathol. 183:681-691, 2013), which further classifies mUC to include a genomically unstable (GU) subtype (FIG. 2A). Given the demonstrated importance of TMB, we hypothesized that the GU subtype would significantly enrich for responders. Indeed, GU enriched for high-TMB tumors (FIGS. 2A-2C) and had a much higher response rate (47.2%) than the other Lund subtypes (17.6%) (FIG. 2D). The boost in response rate in the GU subtype exceeded differences observed among TCGA subtypes (FIG. 2E), but, unexpectedly, this could not be attributed to mutation burden, as the TCGA luminal II subtype similarly enriched for high-TMB tumors (FIGS. 2B and 2C). Instead, we identified the source of the difference by separately evaluating patients classified as TCGA luminal II only, Lund GU only, or both (FIG. 2F). While luminal II tumors had higher CD8+ Teff gene expression regardless of their Lund label, only those luminal II tumors that were also classified as GU had a low TGF-β signature. Thus, an unfavorable TGF-β signature, leading to an unfavorable CD8 Teff to TGF-β ratio, trumped high TMB in this patient group, resulting in poor response.
An association between expression subtypes defined by The Cancer Genome Atlas (TCGA) and clinical response to atezolizumab monotherapy in individual cohorts of IMvigor210 has been identified. We have found that TMB is strongly related to response and, further, at least partially captured by patterns of gene expression. With this in mind, we explored an alternative molecular classification for UC, the Lund taxonomy (Sjödahl et al. Clin. Cancer Res. 18:3377-3386, 2012; Sjödahl et al. Am. J. Pathol. 183:681-691, 2013), which includes a genomically-unstable (GU) subtype (FIG. 2A).
The Lund GU subtype had a dramatically higher response rate than the other Lund subtypes (FIG. 2D), and strongly enriched for high-TMB tumors (FIGS. 2B and 2C). The Lund GU subtype also had the highest mutation rates for TP53 (34%) and RB1 (43%), consistent with an association between proliferation and mutation (FIG. 2A).
The basal/SCC-like (SCCL) and Urothelial-like B (UroB) subtypes were associated with high expression of CD8+ T-effector genes and their correlates, including high PD-L1 expression on IC (FIG. 2A). While these tumors showed similar CR rates to GU tumors, the overall response rate was lower (FIG. 2D). This could in part be due to the low TMB and the relative enrichment of the TGF-β signature (FIGS. 2A and 2G).
Urothelial-like A (UroA) tumors shared many molecular features with the luminal I subtype by the TCGA classification, e.g., high FGFR3, TP63, and WNT7B expression (FIG. 2H). The minimal response rate observed in the UroA subtype is likely a reflection of low TMB and limited pre-existing immunity.
The Lund Infiltrated subtype was named as such due to the presence of both T-cells and myofibroblasts. In line with this classification, infiltrated tumors had elevated expression of CD8+T-effector genes, but also the highest relative expression of genes associated with pro-tumorigenic and immunosuppressive pathways, including TGF-β, stromal activation and angiogenesis, extracellular matrix, and EMT, leading to an unfavorable ratio of CD8+T-effector to TGF-β signature (FIGS. 2A and 2G). The Infiltrated subtype also had the lowest median TMB (FIGS. 2B and 2C), and this, in combination with the unfavorable CD8+ T-effector to TGF-β ratio, is consistent with the infiltrated subtype showing the poorest response rate (FIG. 2D).

Example 6: TGF-β Pathway Activation Restricts Responses to Anti-PD-L1 in Immune Excluded Tumors

Given the association of TGF-β signaling with stromal components and the negative impact that stromal cells can have on CD8+ T-cells, we next examined the relationship between tumor-immune phenotype and response to atezolizumab. We used immunohistochemistry to evaluate CD8+ T-cell infiltration and classified tumors into immune desert, immune excluded, or immune inflamed phenotypes based on the presence and location of CD8+ T-cells in the TME (FIG. 3A). A significant proportion of bladder tumors (approximately 48%) exhibited the immune excluded phenotype, whereas immune desert and immune inflamed tumors constituted approximately 26% each. The immune excluded phenotype was characterized by the localization of CD8+ T-cells primarily in the peri-tumoral stromal compartment, juxtaposed to collagen fibers (revealed by trichrome staining; FIG. 3B).
The proximity of CD8 T-cells to desmoplastic stroma in immune excluded tumors (FIGS. 3A and 3B) and the association of TGF-β ligand and receptor gene expression with lack of response (FIG. 1O) both implicate the TGF-β pathway. To better understand this pathway's role, we considered two different gene signatures. The first is a two-gene signature (TGFB1 and TGFBR2, described above) that represents input to the pathway. The two-gene input signature showed no difference in expression level across immune phenotypes, but it was elevated in non-responders in immune excluded tumors only (FIG. 3C). The second signature was developed to measure TGF-β pathway output specifically in fibroblasts from different tissues. The “pan-fibroblast TGF-β response signature” (Pan-F-TBRS) includes 19 genes that are strongly and specifically induced by TGFB1 across a panel of human primary fibroblast cell lines derived from six tissues. As with the two-gene signature, lack of response in immune excluded tumors was significantly associated with higher Pan-F-TBRS scores (FIG. 3D). Unlike the two-gene signature, the Pan-F-TBRS score was elevated in both immune inflamed and immune excluded tumors. Consistent with the 2-gene TGF-β signature, however, the Pan-F-TBRS stratified non-responders and responders only in immune excluded tumors, with a significant enrichment in patients who progressed on therapy. These results suggest that the observed association between TGF-β pathway genes and non-response can be at least in part attributed to this pathway's impact on fibroblasts in the tissue microenvironment (TME).

Example 7: TGF-β Inhibition Improves T-Cell Infiltration and Therapeutic Responses to Anti-PD-L1

Perhaps the most unexpected implication of the biomarker analysis is the association of the TGF-β pathway with a lack of response to atezolizumab in IMvigor210 (FIGS. 1O, 1P, 3C, and 3D). TGF-β signaling can have many complex effects on both tumors and the immune system, such as directly impeding tumor growth at early stage, promoting tumor EMT and metastasis, favoring the development of T regulatory cells, and promoting stromal investment of tumors. Observing elevated Pan-F-TBRS levels in the immune excluded phenotype suggested that the effects of TGF-β signaling on activated stroma may lead to the restriction of both T-cell influx and efficacy. Indeed, activated CD8+ T-cells were not absent from immune excluded tumors (as opposed to immune desert tumors; FIG. 3A), and they did not exhibit low TMB (FIG. 3E), suggesting that the physical exclusion of T-cells from the tumor parenchyma by the stromal barrier was an important feature limiting response to therapy in this group.
We therefore established a mouse model to investigate a barrier role for a TGF-β-activated stroma. For this purpose, we utilized the EMT6 mouse mammary carcinoma model because it recapitulates the immune excluded phenotype in which T-cells are localized to the periphery of the tumor in association with the collagen fibers (FIG. 4A) and all TGF-β isoforms and PD-L1 are expressed in the TME (FIGS. 4B and 4C). EMT6 tumor cells were orthotopically implanted into BALB/c mice. Once tumors reached approximately 160 mm³, treatment with anti-PD-L1 and/or 1 D11, a pan-specific TGF-β-blocking antibody, was initiated. 1 D11 treatment reduced TGF-β signaling, as demonstrated by reduced levels of phosphorylated SMAD2/3 in the tumor (FIG. 4L) and VEGF-A in the plasma (FIG. 4M). Dual blockade of PD-L1 and TGF-β in mice with established tumors led to complete tumor regression in the majority of mice, whereas anti-PD-L1 alone led to fewer complete regressions (FIGS. 4D-4F). Blockade of TGF-β alone had no effect on tumor growth in vivo (FIGS. 4D-4F). Dual antibody blockade also led to an increase in the abundance of tumor infiltrating T-cells (FIGS. 4G-4I and 4O). Granzyme B levels also increased on a per CD8+ T-cell basis in the combination therapy arm (FIGS. 4J and 4N). Notably, combined PD-L1 and TGF-β blockade altered the distribution of T-cells from a largely peri-tumoral localization to a more intratumoral pattern (FIGS. 4G, 4K, and 4O). Thus, dual blockade of TGF-β and PD-L1 promoted T-cell localization to tumor islets and improved therapeutic responses,
To better understand the mechanisms underlying response to this dual blockade, we performed RNA sequencing on tumors from each of the four treatment conditions. The CD8+ Teff signature used to analyze human tumors was elevated in mouse tumors treated with a combination of anti-PD-L1 and anti-TGF-β (FIGS. 4P and 4T). Despite anti-PD-L1 monotherapy increasing expression of the Teff gene signature, the effect was not significant (FIG. 4P). These results suggested that TGF-β inhibition potentiated the effect of anti-PD-L1 in a significant way to enhance anti-tumor immunity. We then evaluated TGF-β expression signature in different stromal cell populations within the tumor. The Pan-F-TBRS was significantly reduced in the combination treatment (FIG. 4Q). In contrast, no reduction was observed in the TBRS associated with T-cells or macrophages (FIG. 4R). Consistent with these results, phosphoflow analysis in EMT6 tumors demonstrated that TGF-β signaling, as reflected by pSMAD2/3, was significantly reduced in CD45− but not in CD45+ cells following combination therapy (FIG. 4L). The competing effects of the Teff signature and Pan-F-TBRS in the TME can be integrated with the Teff to Pan-F-TBRS ratio. This ratio score showed an increase in the combination treatment compared with control and single antibody treatments (FIG. 4S). Expression of CD8+ Teff genes was elevated in mouse tumors treated with a combination of anti-PD-L1 and anti-TGF-β, while expression of cancer-associated fibroblast (CAF) remodeling genes was decreased (FIGS. 4T and 4U). For example, expression of IFNG, GZMB, and Zeta-chain associated protein kinase 70 (ZAP70) was elevated in mouse tumors treated with a combination of anti-PD-L1 and anti-TGF-β, and expression of lysyl oxidase homolog 2 (LOXL2), tenascin C (TNC), and periostin (POSTN) was decreased in mouse tumors treated with a combination of anti-PD-L1 and anti-TGF-β (FIG. 4U). Without wishing to be bound by theory, dual therapeutic blockade of TGF-β and PD-L1 may switch an immune excluded tumor to an inflamed tumor by reprogramming CAFs and modifying the stromal architecture.
In sum, blockade of TGF-β can synergize with anti-PD-L1 antibodies to increase CD8+ Teff cell counts in the tumor bed and drive robust anti-tumor immunity. Although elevated TGF-β signaling may have additional consequences, these results are consistent with the idea that the TGF-β-activated stroma in the immune excluded group acts to dampen Teff function and physically restrict T-cell entry into the tumor itself.

Pan-Cancer Signatures

We performed a comprehensive evaluation of the molecular, cellular, and tumor genetic factors associated with response and primary immune escape to checkpoint blockade in patients enrolled in a phase 2, single arm study testing the efficacy of atezolizumab in mUC. Given the large size of the cohort used for this analysis, we reached several important, functional conclusions regarding tumor characteristics that govern the likelihood of response. Indeed, three biological axes were found to play distinct and independent roles: (1) pre-existing immunity, as represented by PD-L1 expression on IC and gene expression or histological correlates of CD8+ Teff activity; (2) TMB, measured directly but also reflected in molecular signatures of proliferation and DNA damage response or in loss-of-function mutations impacting these processes; and (3) TGF-β pathway signalling. While each contributes significantly to the multifactorial basis of response, TGF-β signalling and TMB together provide the greatest explanatory power for bladder cancer.
Based on the enrichment of the TGF-β signature in non-responding immune excluded mUC tumors, as well as a possible role in offsetting high TMB and/or pre-existing immunity in Lund subtypes enriched for non-response, we hypothesized that TGF-β signalling may counteract anti-tumor immunity. To test this hypothesis and better understand the functional role of TGF-β in restraining response to immune checkpoint blockade, we used a mouse model that emulates several aspects of immune excluded tumors in patients. In this model, simultaneous inhibition of TGF-β and PD-L1 signalling converted tumors from an excluded phenotype to an inflamed phenotype, resulting in enhanced tumor infiltration by CD8+ Teff cells and a marked increase in tumor regression.
The multifactorial basis of response to immunotherapy described herein is expected to be applicable to other tumor types beyond bladder cancer, and pan-cancer response rates to immune checkpoint blockade may be improved further by taking all three axes into account. Likewise, the determinants of response and resistance to anti-PD-L1 therapy are expected to extend to other immune checkpoint inhibitors.
In total, these data support use of the biomarkers and signatures described herein (e.g., the pan-F-TBRS), for example, as predictive and pharmacodynamic biomarkers for predicting and monitoring response of cancer patients (e.g., bladder cancer patients) to anti-cancer therapy that includes an immunotherapy (e.g., a PD-L1 axis binding antagonist such as an anti-PD-L1 antibody (e.g., atezolizumab)) and a suppressive stromal antagonist (e.g., a TGF-β antagonist such as an anti-TGF-β antibody).

VII. Sequences

Table 13 shows sequences that are described herein.

TABLE 13

Sequence Table

SEQ ID
NO.	Sequence

1	accucccucc gcggagcagc cagacagcga gggccccggc cgggggcagg ggggacgccc
	cguccggggc acccccccgg cucugagccg cccgcggggc cggccucggc ccggagcgga
	ggaaggaguc gccgaggagc agccugaggc cccagagucu gagacgagcc gccgccgccc
	ccgccacugc ggggaggagg gggaggagga gcgggaggag ggacgagcug gucgggagaa
	gaggaaaaaa acuuuugaga cuuuuccguu gccgcuggga gccggaggcg cggggaccuc
	uuggcgcgac gcugccccgc gaggaggcag gacuugggga ccccagaccg ccucccuuug
	ccgccgggga cgcuugcucc cucccugccc ccuacacggc gucccucagg cgcccccauu
	ccggaccagc ccucgggagu cgccgacccg gccucccgca aagacuuuuc cccagaccuc
	gggcgcaccc ccugcacgcc gccuucaucc ccggccuguc uccugagccc ccgcgcaucc
	uagacccuuu cuccuccagg agacggaucu cucuccgacc ugccacagau ccccuauuca
	agaccaccca ccuucuggua ccagaucgcg cccaucuagg uuauuuccgu gggauacuga
	gacacccccg guccaagccu ccccuccacc acugcgcccu ucucccugag gaccucagcu
	uucccucgag gcccuccuac cuuuugccgg gagaccccca gccccugcag gggcggggcc
	uccccaccac accagcccug uucgcgcucu cggcagugcc ggggggcgcc gccuccccca
	ugccgcccuc cgggcugcgg cugcugccgc ugcugcuacc gcugcugugg cuacuggugc
	ugacgccugg ccggccggcc gcgggacuau ccaccugcaa gacuaucgac auggagcugg
	ugaagcggaa gcgcaucgag gccauccgcg gccagauccu guccaagcug cggcucgcca
	gccccccgag ccagggggag gugccgcccg gcccgcugcc cgaggccgug cucgcccugu
	acaacagcac ccgcgaccgg guggccgggg agagugcaga accggagccc gagccugagg
	ccgacuacua cgccaaggag gucacccgcg ugcuaauggu ggaaacccac aacgaaaucu
	augacaaguu caagcagagu acacacagca uauauauguu cuucaacaca ucagagcucc
	gagaagcggu accugaaccc guguugcucu cccgggcaga gcugcgucug cugaggcuca
	aguuaaaagu ggagcagcac guggagcugu accagaaaua cagcaacaau uccuggcgau
	accucagcaa ccggcugcug gcacccagcg acucgccaga gugguuaucu uuugauguca
	ccggaguugu gcggcagugg uugagccgug gaggggaaau ugagggcuuu cgccuuagcg
	cccacugcuc cugugacagc agggauaaca cacugcaagu ggacaucaac ggguucacua
	ccggccgccg aggugaccug gccaccauuc auggcaugaa ccggccuuuc cugcuucuca
	uggccacccc gcuggagagg gcccagcauc ugcaaagcuc ccggcaccgc cgagcccugg
	acaccaacua uugcuucagc uccacggaga agaacugcug cgugcggcag cuguacauug
	acuuccgcaa ggaccucggc uggaagugga uccacgagcc caagggcuac caugccaacu
	ucugccucgg gcccugcccc uacauuugga gccuggacac gcaguacagc aagguccugg
	cccuguacaa ccagcauaac ccgggcgccu cggcggcgcc gugcugcgug ccgcaggcgc
	uggagccgcu gcccaucgug uacuacgugg gccgcaagcc caagguggag cagcugucca
	acaugaucgu gcgcuccugc aagugcagcu gaggucccgc cccgccccgc cccgccccgg
	caggcccggc cccaccccgc cccgcccccg cugccuugcc caugggggcu guauuuaagg
	acacccgugc cccaagccca ccuggggccc cauuaaagau ggagagagga cugcggaucu
	cugugucauu gggcgccugc cuggggucuc caucccugac guucccccac ucccacuccc
	ucucucuccc ucucugccuc cuccugccug ucugcacuau uccuuugccc ggcaucaagg
	cacaggggac caguggggaa cacuacugua guuagaucua uuuauugagc accuugggca
	cuguugaagu gccuuacauu aaugaacuca uucagucacc auagcaacac ucugagaugc
	agggacucug auaacaccca uuuuaaaggu gaggaaacaa gcccagagag guuaagggag
	gaguuccugc ccaccaggaa ccugcuuuag ugggggauag ugaagaagac aauaaaagau
	aguaguucag gccaggcggg guggcucacg ccuguaaucc uagcacuuuu gggaggcaga
	gaugggagga uuacuugaau ccaggcauuu gagaccagcc uggguaacau agugagaccc
	uaucucuaca aaacacuuuu aaaaaaugua caccuguggu cccagcuacu cuggaggcua
	aggugggagg aucacuugau ccugggaggu caaggcugca g

2	MPPSGLRLLLLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLA
	SPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRVLMVETHNEI
	YDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQHVELYQKYSNNSWR
	YLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLSAHCSCDSRDNTLQVDINGFT
	TGRRGDLATIHGMNRPFLLLMATPLERAQHLQSSRHRRALDTNYCFSSTEKNCCVRQLYI
	DFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASAAPCCVPQA
	LEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

3	ggagagggag aaggcucucg ggcggagaga gguccugccc agcuguuggc gaggaguuuc
	cuguuucccc cgcagcgcug aguugaaguu gagugaguca cucgcgcgca cggagcgacg
	acacccccgc gcgugcaccc gcucgggaca ggagccggac uccugugcag cuucccucgg
	ccgccggggg ccuccccgcg ccucgccggc cuccaggccc ccuccuggcu ggcgagcggg
	cgccacaucu ggcccgcaca ucugcgcugc cggcccggcg cgggguccgg agagggcgcg
	gcgcggaggc gcagccaggg guccgggaag gcgccguccg cugcgcuggg ggcucggucu
	augacgagca gcggggucug ccaugggucg ggggcugcuc aggggccugu ggccgcugca
	caucguccug uggacgcgua ucgccagcac gaucccaccg cacguucaga agucggaugu
	ggaaauggag gcccagaaag augaaaucau cugccccagc uguaauagga cugcccaucc
	acugagacau auuaauaacg acaugauagu cacugacaac aacggugcag ucaaguuucc
	acaacugugu aaauuuugug augugagauu uuccaccugu gacaaccaga aauccugcau
	gagcaacugc agcaucaccu ccaucuguga gaagccacag gaagucugug uggcuguaug
	gagaaagaau gacgagaaca uaacacuaga gacaguuugc caugacccca agcuccccua
	ccaugacuuu auucuggaag augcugcuuc uccaaagugc auuaugaagg aaaaaaaaaa
	gccuggugag acuuucuuca uguguuccug uagcucugau gagugcaaug acaacaucau
	cuucucagaa gaauauaaca ccagcaaucc ugacuuguug cuagucauau uucaagugac
	aggcaucagc cuccugccac cacugggagu ugccauaucu gucaucauca ucuucuacug
	cuaccgcguu aaccggcagc agaagcugag uucaaccugg gaaaccggca agacgcggaa
	gcucauggag uucagcgagc acugugccau cauccuggaa gaugaccgcu cugacaucag
	cuccacgugu gccaacaaca ucaaccacaa cacagagcug cugcccauug agcuggacac
	ccuggugggg aaaggucgcu uugcugaggu cuauaaggcc aagcugaagc agaacacuuc
	agagcaguuu gagacagugg cagucaagau cuuucccuau gaggaguaug ccucuuggaa
	gacagagaag gacaucuucu cagacaucaa ucugaagcau gagaacauac uccaguuccu
	gacggcugag gagcggaaga cggaguuggg gaaacaauac uggcugauca ccgccuucca
	cgccaagggc aaccuacagg aguaccugac gcggcauguc aucagcuggg aggaccugcg
	caagcugggc agcucccucg cccgggggau ugcucaccuc cacagugauc acacuccaug
	ugggaggccc aagaugccca ucgugcacag ggaccucaag agcuccaaua uccucgugaa
	gaacgaccua accugcugcc ugugugacuu ugggcuuucc cugcgucugg acccuacucu
	gucuguggau gaccuggcua acagugggca ggugggaacu gcaagauaca uggcuccaga
	aguccuagaa uccaggauga auuuggagaa uguugagucc uucaagcaga ccgaugucua
	cuccauggcu cuggugcucu gggaaaugac aucucgcugu aaugcagugg gagaaguaaa
	agauuaugag ccuccauuug guuccaaggu gcgggagcac cccugugucg aaagcaugaa
	ggacaacgug uugagagauc gagggcgacc agaaauuccc agcuucuggc ucaaccacca
	gggcauccag auggugugug agacguugac ugagugcugg gaccacgacc cagaggcccg
	ucucacagcc cagugugugg cagaacgcuu cagugagcug gagcaucugg acaggcucuc
	ggggaggagc ugcucggagg agaagauucc ugaagacggc ucccuaaaca cuaccaaaua
	gcucuucugg ggcaggcugg gccaugucca aagaggcugc cccucucacc aaagaacaga
	ggcagcagga agcugccccu gaacugauge uuccuggaaa accaaggggg ucacuccccu
	cccuguaagc uguggggaua agcagaaaca acagcagcag ggagugggug acauagagca
	uucuaugccu uugacauugu cauaggauaa gcuguguuag cacuuccuca ggaaaugaga
	uugauuuuua caauagccaa uaacauuugc acuuuauuaa ugccuguaua uaaauaugaa
	uagcuauguu uuauauauau auauauauau cuauauaugu cuauagcucu auauauauag
	ccauaccuug aaaagagaca aggaaaaaca ucaaauauuc ccaggaaauu gguuuuauug
	gagaacucca gaaccaagca gagaaggaag ggacccauga cagcauuagc auuugacaau
	cacacaugca gugguucucu gacuguaaaa cagugaacuu ugcaugagga aagaggcucc
	augucucaca gccagcuaug accacauugc acuugcuuuu gcaaaauaau cauucccugc
	cuagcacuuc ucuucuggcc auggaacuaa guacaguggc acuguuugag gaccaguguu
	cccgggguuc cugugugccc uuauuucucc uggacuuuuc auuuaagcuc caagccccaa
	aucugggggg cuaguuuaga aacucucccu caaccuaguu uagaaacucu accccaucuu
	uaauaccuug aauguuuuga accccacuuu uuaccuucau ggguugcaga aaaaucagaa
	cagauguccc cauccaugcg auugccccac caucuacuaa ugaaaaauug uucuuuuuuu
	caucuuuccc cugcacuuau guuacuauuc ucugcuccca gccuucaucc uuuucuaaaa
	aggagcaaau ucucacucua ggcuuuaucg uguuuacuuu uucauuacac uugacuugau
	uuucuaguuu ucuauacaaa caccaauggg uuccaucuuu cugggcuccu gauugcucaa
	gcacaguuug gccugaugaa gaggauuuca acuacacaau acuaucauug ucaggacuau
	gaccucaggc acucuaaaca uauguuuugu uuggucagca cagcguuuca aaaagugaag
	ccacuuuaua aauauuugga gauuuugcag gaaaaucugg auccccaggu aaggauagca
	gaugguuuuc aguuaucucc aguccacguu cacaaaaugu gaaggugugg agacacuuac
	aaagcugccu cacuucucac uguaaacauu agcucuuucc acugccuacc uggaccccag
	ucuaggaauu aaaucugcac cuaaccaagg ucccuuguaa gaaaugucca uucaagcagu
	cauucucugg guauauaaua ugauuuugac uaccuuaucu gguguuaaga uuugaaguug
	gccuuuuauu ggacuaaagg ggaacuccuu uaagggucuc aguuagccca aguuucuuuu
	gcuuauaugu uaauaguuuu acccucugca uuggagagag gagugcuuua cuccaagaag
	cuuuccucau gguuaccguu cucuccauca ugccagccuu cucaaccuuu gcagaaauua
	cuagagagga uuugaaugug ggacacaaag gucccauuug caguuagaaa auuugugucc
	acaaggacaa gaacaaagua ugagcuuuaa aacuccauag gaaacuuguu aaucaacaaa
	gaaguguuaa ugcugcaagu aaucucuuuu uuaaaacuuu uugaagcuac uuauuuucag
	ccaaauagga auauuagaga gggacuggua gugagaauau cagcucuguu uggauggugg
	aaggucucau uuuauugaga uuuuuaagau acaugcaaag guuuggaaau agaaccucua
	ggcacccucc ucaguguggg ugggcugaga guuaaagaca guguggcugc aguagcauag
	aggcgccuag aaauuccacu ugcaccguag ggcaugcuga uaccauccca auagcuguug
	cccauugacc ucuaguggug aguuucuaga auacuggucc auucaugaga uauucaagau
	ucaagaguau ucucacuucu ggguuaucag cauaaacugg aauguagugu cagaggauac
	uguggcuugu uuuguuuaug uuuuuuuuuc uuauucaaga aaaaagacca aggaauaaca
	uucuguaguu ccuaaaaaua cugacuuuuu ucacuacuau acauaaaggg aaaguuuuau
	ucuuuuaugg aacacuucag cuguacucau guauuaaaau aggaauguga augcuauaua
	cucuuuuuau aucaaaaguc ucaagcacuu auuuuuauuc uaugcauugu uugucuuuua
	cauaaauaaa auguuuauua gauugaauaa agcaaaauac ucaggugagc auccugccuc
	cuguucccau uccuaguagc uaaa

4	GRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTC
	DNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKC
	IMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAIS
	VIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTEL
	LPIELDTLVGKGRFAEVYKAKLKQNTSEQFETVAVKIFPYEEYASWKTEKDIFSDINLKH
	ENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRHVISWEDLRKLGSSLARGIAHL
	HSDHTPCGRPKMPIVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTLSVDDLANSGQVGT
	ARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGSKVREH
	PCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSEL
	EHLDRLSGRSCSEEKIPEDGSLNTTK

5	gcaggcucuc uccccgcccc cgcggggcgg cgcgcacuca cccacccgcg ccggagcgga
	ccuuuggcuu ggcuugucag ggcuugucca ggaguuccgc uccucucucc aaccgggguc
	ccccuccagc gacccuaaag cuucccagac uuccgcuuca auuccugucc gcaccccacg
	cccaccucaa cguggagcgc aguggucucc gaggagcgcc ggagcugccc cgccugccca
	gcggggucag cacuucgcau caaggcccaa gaaaagcaag uccuccagcg uucugagcac
	ccgggccuga gggaaggucc uaacagcccc cgggagccag ucuccaacgc cucccgcagc
	agcccgccgc ucccaggugc ccgcgugcgc cgcugccgcc gcaaucccgc acgcgucccg
	cgcccgcccc acuuugccua uccccgggac uaagacggga auccugugaa gcagcuccag
	cuauguguga agaagaggac agcacugccu ugguguguga caauggcucu gggcucugua
	aggccggcuu ugcuggggac gaugcuccca gggcuguuuu cccauccauu gugggacguc
	ccagacauca gggggugaug gugggaaugg gacaaaaaga cagcuacgug ggugacgaag
	cacagagcaa aagaggaauc cugacccuga aguacccgau agaacauggc aucaucacca
	acugggacga cauggaaaag aucuggcacc acucuuucua caaugagcuu cguguugccc
	cugaagagca ucccacccug cucacggagg caccccugaa ccccaaggcc aaccgggaga
	aaaugacuca aauuauguuu gagacuuuca augucccagc cauguaugug gcuauccagg
	cggugcuguc ucucuaugcc ucuggacgca caacuggcau cgugcuggac ucuggagaug
	gugucaccca caaugucccc aucuaugagg gcuaugccuu gccccaugcc aucaugcguc
	uggaucuggc uggccgagau cucacugacu accucaugaa gauccugacu gagcguggcu
	auuccuucgu uacuacugcu gagcgugaga uuguccggga caucaaggag aaacuguguu
	auguagcucu ggacuuugaa aaugagaugg ccacugccgc auccucaucc ucccuugaga
	agaguuacga guugccugau gggcaaguga ucaccaucgg aaaugaacgu uuccgcugcc
	cagagacccu guuccagcca uccuucaucg ggauggaguc ugcuggcauc caugaaacca
	ccuacaacag caucaugaag ugugauauug acaucaggaa ggaccucuau gcuaacaaug
	uccuaucagg gggcaccacu auguacccug gcauugccga ccgaaugcag aaggagauca
	cggcccuagc acccagcacc augaagauca agaucauugc cccuccggag cgcaaauacu
	cugucuggau cgguggcucc auccuggccu cucuguccac cuuccagcag auguggauca
	gcaaacagga auacgaugaa gccgggccuu ccauugucca ccgcaaaugc uucuaaaaca
	cuuuccugcu ccucucuguc ucuagcacac aacugugaau guccugugga auuaugccuu
	caguucuuuu ccaaaucauu ccuagccaaa gcucugacuc guuaccuaug uguuuuuuaa
	uaaaucugaa auaggcuacu gguaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
	aaaaa

6	MCEEEDSTALVCDNGSGLCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEA
	QSKRGILTLKYPIEHGIITNWDDMEKIWHHSFYNELRVAPEEHPTLLTEAPLNPKANREK
	MTQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRL
	DLAGRDLTDYLMKILTERGYSFVTTAEREIVRDIKEKLCYVALDFENEMATAASSSSLEK
	SYELPDGQVITIGNERFRCPETLFQPSFIGMESAGIHETTYNSIMKCDIDIRKDLYANNV
	LSGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWIS
	KQEYDEAGPSIVHRKCF

7	gccucugggg uuuuauauug cucugguauu caugccaaag acacaccagc ccucagucac
	ugggagaaga accucucaua cccucggugc uccagucccc agcucacuca gccacacaca
	ccauguguga agaggagacc accgcgcucg ugugugacaa uggcucuggc cugugcaagg
	caggcuucgc aggagaugau gccccccggg cugucuuccc cuccauugug ggccgcccuc
	gccaccaggg ugugauggug ggaaugggcc agaaagacag cuaugugggg gaugaggcuc
	agagcaagcg agggauccua acucucaaau accccauuga acacggcauc aucaccaacu
	gggaugacau ggagaagauc uggcaccacu ccuucuacaa ugagcugcgu guagcaccug
	aagagcaccc cacccugcuc acagaggcuc cccuaaaucc caaggccaac agggaaaaga
	ugacccagau cauguuugaa accuucaaug ucccugccau guacgucgcc auucaagcug
	ugcucucccu cuaugccucu ggccgcacga caggcaucgu ccuggauuca ggugauggcg
	ucacccacaa uguccccauc uaugaaggcu augcccugcc ccaugccauc augcgccugg
	acuuggcugg ccgugaccuc acggacuacc ucaugaagau ccucacagag agaggcuauu
	ccuuugugac cacagcugag agagaaauug ugcgagacau caaggagaag cugugcuaug
	uggcccugga uuuugagaau gagauggcca cagcagcuuc cucuuccucc cuggagaaga
	gcuaugagcu gccagauggg cagguuauca ccauuggcaa ugagcgcuuc cgcugcccug
	agacccucuu ccagccuucc uuuauuggca uggaguccgc uggaauucau gagacaaccu
	acaauuccau caugaagugu gacauugaca uccguaagga cuuauaugcc aacaaugucc
	ucucuggggg caccaccaug uacccuggca uugcugacag gaugcagaag gagaucacag
	cccuggcccc cagcaccaug aagaucaaga uuauugcucc cccagagcgg aaguacucag
	ucuggaucgg gggcucuauc cuggccucuc ucuccaccuu ccagcagaug uggaucagca
	agccugagua ugaugaggca gggcccucca uuguccacag gaagugcuuc uaaagucaga
	acagguucuc caaggauccc cucgagacua cucuguuacc agucaugaaa cauuaaaacc
	uacaagccuu aaaaaaaaaa aaaaa

8	MCEEETTALVCDNGSGLCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQ
	SKRGILTLKYPIEHGIITNWDDMEKIWHHSFYNELRVAPEEHPTLLTEAPLNPKANREKM
	TQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLDSGDGVTHNVPIYEGYALPHAIMRLD
	LAGRDLTDYLMKILTERGYSFVTTAEREIVRDIKEKLCYVALDFENEMATAASSSSLEKS
	YELPDGQVITIGNERFRCPETLFQPSFIGMESAGIHETTYNSIMKCDIDIRKDLYANNVL
	SGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISK
	PEYDEAGPSIVHRKCF

9	acgccgccaa cuggcggggg ugcgggggag acaauaauuu guuccgcggu aauaagaacg
	gugacugcug gccguggauc cauuucacag gccugccuuc ucucacuaac gcucuuccua
	guccccgggc caacucggac aguuugcuca uuuauugcaa cggucaaggc uggcuugugc
	cagaacggcg cgcgcgcgcg cacgcacgca cacacacggg gggaaacuuu uuuaaaaaug
	aaaggcuaga agagcucagc ggcggcgcgg gcgcugcgcg agggcuccgg agcugacucg
	ccgaggcagg aaaucccucc ggucgcgacg cccggccccg gcucggcgcc cgcgugggau
	ggugcagcgc ucgccgccgg gcccgagagc ugcugcacug aaggccggcg acgauggcag
	cgcgcccgcu gcccgugucc cccgcccgcg cccuccugcu cgcccuggcc ggugcucugc
	ucgcgcccug cgaggcccga ggggugagcu uauggaacca aggaagagcu gaugaaguug
	ucagugccuc uguugggagu ggggaccucu ggaucccagu gaagagcuuc gacuccaaga
	aucauccaga agugcugaau auucgacuac aacgggaaag caaagaacug aucauaaauc
	uggaaagaaa ugaaggucuc auugccagca guuucacgga aacccacuau cugcaagacg
	guacugaugu cucccucgcu cgaaauuaca cgguaauucu gggucacugu uacuaccaug
	gacauguacg gggauauucu gauucagcag ucagucucag cacguguucu ggucucaggg
	gacuuauugu guuugaaaau gaaagcuaug ucuuagaacc aaugaaaagu gcaaccaaca
	gauacaaacu cuucccagcg aagaagcuga aaagcguccg gggaucaugu ggaucacauc
	acaacacacc aaaccucgcu gcaaagaaug uguuuccacc acccucucag acaugggcaa
	gaaggcauaa aagagagacc cucaaggcaa cuaaguaugu ggagcuggug aucguggcag
	acaaccgaga guuucagagg caaggaaaag aucuggaaaa aguuaagcag cgauuaauag
	agauugcuaa ucacguugac aaguuuuaca gaccacugaa cauucggauc guguugguag
	gcguggaagu guggaaugac auggacaaau gcucuguaag ucaggaccca uucaccagcc
	uccaugaauu ucuggacugg aggaagauga agcuucuacc ucgcaaaucc caugacaaug
	cgcagcuugu cagugggguu uauuuccaag ggaccaccau cggcauggcc ccaaucauga
	gcaugugcac ggcagaccag ucugggggaa uugucaugga ccauucagac aauccccuug
	gugcagccgu gacccuggca caugagcugg gccacaauuu cgggaugaau caugacacac
	uggacagggg cuguagcugu caaauggcgg uugagaaagg aggcugcauc augaacgcuu
	ccaccgggua cccauuuccc augguguuca gcaguugcag caggaaggac uuggagacca
	gccuggagaa aggaaugggg gugugccugu uuaaccugcc ggaagucagg gagucuuucg
	ggggccagaa gugugggaac agauuugugg aagaaggaga ggagugugac uguggggagc
	cagaggaaug uaugaaucgc ugcugcaaug ccaccaccug uacccugaag ccggacgcug
	ugugcgcaca ugggcugugc ugugaagacu gccagcugaa gccugcagga acagcgugca
	gggacuccag caacuccugu gaccucccag aguucugcac aggggccagc ccucacugcc
	cagccaacgu guaccugcac gaugggcacu caugucagga uguggacggc uacugcuaca
	auggcaucug ccagacucac gagcagcagu gugucacgcu cuggggacca ggugcuaaac
	cugccccugg gaucugcuuu gagagaguca auucugcagg ugauccuuau ggcaacugug
	gcaaagucuc gaagaguucc uuugccaaau gcgagaugag agaugcuaaa uguggaaaaa
	uccaguguca aggaggugcc agccggccag ucauugguac caaugccguu uccauagaaa
	caaacauccc ccugcagcaa ggaggccgga uucugugccg ggggacccac guguacuugg
	gcgaugacau gccggaccca gggcuugugc uugcaggcac aaagugugca gauggaaaaa
	ucugccugaa ucgucaaugu caaaauauua gugucuuugg gguucacgag ugugcaaugc
	agugccacgg cagaggggug ugcaacaaca ggaagaacug ccacugcgag gcccacuggg
	caccucccuu cugugacaag uuuggcuuug gaggaagcac agacageggc cccauccggc
	aagcagauaa ccaagguuua accauaggaa uucuggugac cauccugugu cuucuugcug
	ccggauuugu gguuuaucuc aaaaggaaga ccuugauacg acugcuguuu acaaauaaga
	agaccaccau ugaaaaacua aggugugugc gcccuucccg gccaccccgu ggcuuccaac
	ccugucaggc ucaccucggc caccuuggaa aaggccugau gaggaagccg ccagauuccu
	acccaccgaa ggacaauccc aggagauugc ugcaguguca gaauguugac aucagcagac
	cccucaacgg ccugaauguc ccucagcccc agucaacuca gcgagugcuu ccuccccucc
	accgggcucc acgugcaccu agcgucccug ccagaccccu gccagccaag ccugcacuua
	ggcaggccca ggggaccugu aagccaaacc ccccucagaa gccucugccu gcagauccuc
	uggccagaac aacucggcuc acucaugccu uggccaggac cccaggacaa ugggagacug
	ggcuccgccu ggcaccccuc agaccugcuc cacaauaucc acaccaagug cccagaucca
	cccacaccgc cuauauuaag ugagaagccg acaccuuuuu ucaacaguga agacagaagu
	uugcacuauc uuucagcucc aguuggaguu uuuuguacca acuuuuagga uuuuuuuuaa
	uguuuaaaac aucauuacua uaagaacuuu gagcuacugc cgucagugcu gugcugugcu
	auggugcucu gucuacuugc ucagguacuu guaaauuauu aauuuaugca gaauguugau
	uacagugcag ugcgcuguag uaggcauuuu uaccaucacu gaguuuucca uggcaggaag
	gcuuguugug cuuuuaguau uuuagugaac uugaaauauc cugcuugaug ggauucugga
	caggaugugu uugcuuucug aucaaggccu uauuggaaag caguccccca acuaccccca
	gcugugcuua ugguaccaga ugcagcucaa gagaucccaa guagaaucuc aguugauuuu
	cuggauuccc caucucaggc cagagccaag gggcuucagg uccaggcugu guuuggcuuu
	cagggaggcc cugugccccu ugacaacugg caggcaggcu cccagggaca ccugggagaa
	aucuggcuuc uggccaggaa gcuuugguga gaaccugggu ugcagacagg aaucuuaagg
	uguagccaca ccaggauaga gacuggaaca cuagacaagc cagaacuuga cccugagcug
	accagccgug agcauguuug gaaggggucu guagugucac ucaaggcggu gcuugauaga
	aaugccaagc acuucuuuuu cucgcugucc uuucuagagc acugccacca guagguuauu
	uagcuuggga aagguggugu uucuguaaga aaccuacugc ccaggcacug caaaccgcca
	ccucccuaua cugcuuggag cugagcaaau caccacaaac uguaauacaa ugauccugua
	uucagacaga ugaggcuuuc caugggacca caacuauuuu cagaugugaa ccauuaacca
	gaucuaguca aucaagucug uuuacugcaa gguucaacuu auuaacaauu aggcagacuc
	uuuaugcuug caaaaacuac aaccaaugga augugauguu cauggguaua guucaugucu
	gcuaucauua uucguagaua uuggacaaag aaccuucucu auggggcauc cucuuuuucc
	aacuuggcug caggaaucuu uaaaagaugc uuuuaacaga gucugaaccu auuucuuaaa
	cacuugcaac cuaccuguug agcaucacag aaugugauaa ggaaaucaac uugcuuauca
	acuuccuaaa uauuaugaga ugcuggcuug ggcagcaucc ccuugaacuc uucacucuuc
	aaaugccuga cuagggagcc auguuucaca aggucuuuaa agugacuaau ggcaugagaa
	auacaaaaau acucagauaa gguaaaaugc caugaugccu cugucuucug gacugguuuu
	cacauuagaa gacaauugac aacaguuaca uaauucacuc ugaguguuuu augagaaagc
	cuucuuuugg gggucaacag uuuuccuaug cuuugaaaca gaaaaauaug uaccaagaau
	cuugguuugc cuuccagaaa acaaaacugc auuucacuuu cccgguguuc cccacuguau
	cuaggcaaca uaguauucau gacuauggau aaacuaaaca cgugacacaa acacacacaa
	aagggaaccc agcucuaaua cauuccaacu cguauagcau gcaucuguuu auucuauagu
	uauuaaguuc uuuaaaaugu aaagccaugc uggaaaauaa uacugcugag auacauacag
	aauuacugua acugauuaca cuugguaauu guacuaaagc caaacauaua uauacuauua
	aaaagguuua cagaauuuua uggugcauua cgugggcauu gucuuuuuag augcccaaau
	ccuuagaucu ggcauguuag cccuuccucc aauuauaaga ggauaugaac ugaguuuuuc
	uuuuguuguu uguucuuagc uguaauuccu augcuucuau uucagagagc caggagaguu
	ugauauuaaa ggagguuaaa acugugaucu uaugccaugu caucaauggc cacuuagggg
	ccauggcuga ugacacauuc uuaucucuac aguacuaaug uguuauuaua gagccaugca
	uuuuauuucu gaauaagaac auauuuaaac uaauauuccc uuacaauaug gacaguauua
	auccuuccaa gaugcaguau uuaucaagug aagcauauuu agcagcaaau uccauuuuaa
	cauaacuuag gaaccaauaa ccaggguguu uugugguugg gggaggcacg ggguggagua
	uucuuuuuua uauccucaaa acaaaaaaaa ucaauacuua uauuucaaug gcaaucuagu
	auuuuuuuaa aagacuguau aggcaugaau aauagaggug guuugaguuu uguagggcca
	ucaccuggaa agucaaugug acuagacaca aaguagccca gaggcuacuu uucuuccuac
	agcuuauuau aguuguaggu ucuaugaccu cacuucaugg guuccaggca auuccgcuga
	aagguuuguc uccugaaauu uuuuaaguuu guuuuccuga cacauguaau cagaugugua
	gcaaccgagg gaaacgaagc cuaacauucu ccauugugga aauacacaca ggagguuaca
	uuucacagcg uggauuuuuc cagcuuacac augugggaug acaucacaga aaccacaaaa
	gcagcaaauu aaacuguagg agagucaaua cuccugacga gucucggggg gggggcauuu
	uuaugccuuc uuaacuuuau gagaauucuc aggcugaacu auaggccauu guucccaggc
	aaaucaauac aucaaugcau ccucaaaaaa aaaaaaaaaa aaaaaaaacc ggcuaaaacu
	gugucaaaau guucuuaagg agccuauggu cuccacggug cuaaaaagag ccuggugcug
	ggccgacugg cagggcugag cauccuccug cccccucgcc acugauguuu acuaagcacu
	cugagccaau gagaccccca gcagcagaaa gggcacaagg uggcgccagg gcagcagggc
	cagaucuuuc ucaugcaccu cgaccucuug cagacuuucu ucgugagaug uacuacucau
	uucaaaacug cuuugcaggg cuccccuaug uauucggggg gcccacggca cacucaggcu
	ggagauccuu ccucacugcg cucaagaugg ccucagccag acaccaguua cccagcugaa
	agucacaauc ccucccagaa gucucccaac acuagugcug accagaggug gggcucucag
	gcuaggaguu ucacacacaa ugacaggcug cugggggaca uugcaggacc ccuuuuccuc
	uccucuccau gcuagaagcc agcccuaggc agcugcaguu acucccugug acucagcagc
	aggcugauuc aacacagcug cccacacaaa gccaguggcu aauacaucug uuuaccuuuc
	ccuaucaccc agacacaagc cccuuuccca ggucaaacca caggccgaug caucuccagu
	uugacaguca aaucacuacu uccauugcua cuuuagauca gccaaagugg ugacugcugc
	aguguguggc uaucccuaca aggcccaccc aagggaugcc caaagcccaa ccuucuccag
	ggcugcagcc cagagcaacc ccaccagccu aaguccagca gaggaccucc cacccaaugu
	cuuguucuaa uuagaagggg aaguuagcca cagaaaauca acuuaucuau aauuacaaaa
	uucucuugac ucaccuuaaa guuccuauug acaucuacug cuuuuaaacc uauuugaaaa
	cucugauacu aaaacaaaug acacucuaag aaaguuuggg agccccaugc ugagaaccau
	uucugugcag ugaggauguu uccagaagcu acuuaccuac augugaaugu gccauuuucu
	uuccuuuugu agagaaaauc cccuuuacuu uuuggaacag uaauggcagc uucuaguaca
	gccauuacag uuucauauga gaaaaauuaa gaauaacuau aaaauuguua aaauauccaa
	uaauggauaa ugauggccag aagauuuaac auacaaagua auucucaaug uaaagcuauu
	cagcucuucc agguugaaug cccuguaacc cacccugacc uuccacauca ucuucaaaaa
	gcaguuucuc uguuccccau gauucuccua uaagguaacu cuuuaguccu ccauuuagca
	cauuuuaaau ccuccaaaga auaaguauca ugugauuauu uuagcuuuac aaaaaaaaag
	uugaauggcg uuuuauuuuc auggccuaua agcagguacc uuaguagggc agauauagga
	aaaacaaauu agagcaaaac aaauccucua caaauccaag gcaggaaaag ugguggcaga
	gugacucauu cuccuguccc ucccaucagg ucaaaucagg aggcugcagu gaaugccugu
	ucuuugaaug uguagcaguu guuccuguaa cucuuuaaaa cuuggcuaua ggcuguuuag
	cacaguacag auuaaagaua caguuacqua aacagcaaag uaauuuuaua gugcuucauc
	cauuuaucau gcuuugguuu gcuaauuuuu ucacauaccu uuuucuauca cagucuguug
	cuuuuguaca cauuucucau auugggguuc gacagguaaa cacaaacugc uauuucagua
	gaaaaaguua uuguuaugaa uauuaaaccc aauaaauugu auaaagguaa auaucaaaaa
	aaaaaaaaaa auaaagguaa auaucaaaaa

10	MAARPLPVSPARALLLALAGALLAPCEARGVSLWNQGRADEVVSASVGSGDLWIPVKSFD
	SKNHPEVLNIRLQRESKELIINLERNEGLIASSFTETHYLQDGTDVSLARNYTVILGHCY
	YHGHVRGYSDSAVSLSTCSGLRGLIVFENESYVLEPMKSATNRYKLFPAKKLKSVRGSCG
	SHHNTPNLAAKNVFPPPSQTWARRHKRETLKATKYVELVIVADNREFQRQGKDLEKVKQR
	LIEIANHVDKFYRPLNIRIVLVGVEVWNDMDKCSVSQDPFTSLHEFLDWRKMKLLPRKSH
	DNAQLVSGVYFQGTTIGMAPIMSMCTADQSGGIVMDHSDNPLGAAVTLAHELGHNFGMNH
	DTLDRGCSCQMAVEKGGCIMNASTGYPFPMVFSSCSRKDLETSLEKGMGVCLFNLPEVRE
	SFGGQKCGNRFVEEGEECDCGEPEECMNRCCNATTCTLKPDAVCAHGLCCEDCQLKPAGT
	ACRDSSNSCDLPEFCTGASPHCPANVYLHDGHSCQDVDGYCYNGICQTHEQQCVTLWGPG
	AKPAPGICFERVNSAGDPYGNCGKVSKSSFAKCEMRDAKCGKIQCQGGASRPVIGTNAVS
	IETNIPLQQGGRILCRGTHVYLGDDMPDPGLVLAGTKCADGKICLNRQCQNISVFGVHEC
	AMQCHGRGVCNNRKNCHCEAHWAPPFCDKFGFGGSTDSGPIRQADNQGLTIGILVTILCL
	LAAGFVVYLKRKTLIRLLFTNKKTTIEKLRCVRPSRPPRGFQPCQAHLGHLGKGLMRKPP
	DSYPPKDNPRRLLQCQNVDISRPLNGLNVPQPQSTQRVLPPLHRAPRAPSVPARPLPAKP
	ALRQAQGTCKPNPPQKPLPADPLARTTRLTHALARTPGQWETGLRLAPLRPAPQYPHQVP
	RSTHTAYIK

11	gagggcuggg agccgggugg ggaggcgcgg ggcgagccgg gggguuccag acgcgccucc
	accgccgggc agugggcagg uauggcugag ggcgugugag cgccgagcgc uaagggccgc
	cgccaccaug ccagggggcg caggcgccgc ccggcucugc uugcuggcgu uugcccugca
	gccccuccgg ccgcgggcgg cgcgggagcc uggauggaca agaggaagug aggaaggcag
	ccccaagcug cagcaugaac uuaucauacc ucaguggaag acuucagaaa gccccgugag
	agaaaagcau ccacucaaag cugagcucag gguaauggcu gaggggcgag aacugauccu
	ggaccuggag aagaaugagc aacuuuuugc uccuuccuac acagaaaccc auuauacuuc
	aagugguaac ccucaaacca ccacacggaa auuggaggau cacugcuuuu accacggcac
	ggugagggag acagaacugu ccagcgucac gcucagcacu ugccgaggaa uuagaggacu
	gauuacggug agcagcaacc ucagcuacgu caucgagccc cucccugaca gcaagggcca
	acaccuuauu uacagaucug aacaucucaa gccgcccccg ggaaacugug gguucgagca
	cuccaagccc accaccaggg acugggcucu ucaguuuaca caacagacca agaagcgacc
	ucgcaggaug aaaagggaag auuuaaacuc caugaaguau guggagcuuu accucguggc
	ugauuauuua gaguuucaga agaaucgacg agaccaggac gccaccaaac acaagcucau
	agagaucgcc aacuauguug auaaguuuua ccgauccuug aacauccgga uugcucucgu
	gggcuuggaa guguggaccc acgggaacau gugugaaguu ucagagaauc cauauucuac
	ccucuggucc uuucucaguu ggaggcgcaa gcugcuugcc cagaaguacc augacaacgc
	ccaauuaauc acgggcaugu ccuuccacgg caccaccauc ggccuggccc cccucauggc
	caugugcucu guguaccagu cuggaggagu caacauggac cacuccgaga augccauugg
	cguggcugcc accauggccc acgagauggg ccacaacuuu ggcaugaccc augauucugc
	agauugcugc ucggccagug cggcugaugg ugggugcauc auggcagcug ccacugggca
	ccccuuuccc aaaguguuca auggaugcaa caggagggag cuggacaggu aucugcaguc
	agguggugga augugucucu ccaacaugcc agacaccagg auguuguaug gaggccggag
	gugugggaac ggguaucugg aagaugggga agagugugac uguggagaag aagaggaaug
	uaacaacccc ugcugcaaug ccucuaauug uacccugagg ccgggggcgg agugugcuca
	cggcuccugc ugccaccagu guaagcuguu ggcuccuggg acccugugcc gcgagcaggc
	caggcagugu gaccucccgg aguucuguac gggcaagucu ccccacugcc cuaccaacuu
	cuaccagaug gaugguaccc ccugugaggg cggccaggcc uacugcuaca acggcaugug
	ccucaccuac caggagcagu gccagcagcu guggggaccc ggagcccgac cugccccuga
	ccucugcuuc gagaagguga auguggcagg agacaccuuu ggaaacugug gaaaggacau
	gaauggugaa cacaggaagu gcaacaugag agaugcgaag ugugggaaga uccaguguca
	gagcucugag gcccggcccc uggaguccaa cgcggugccc auugacacca cuaucaucau
	gaaugggagg cagauccagu gccggggcac ccacgucuac cgagguccug aggaggaggg
	ugacaugcug gacccagggc uggugaugac uggaaccaag uguggcuaca accauauuug
	cuuugagggg cagugcagga acaccuccuu cuuugaaacu gaaggcugug ggaagaagug
	caauggccau ggggucugua acaacaacca gaacugccac ugccugccgg gcugggcccc
	gcccuucugc aacacaccgg gccacggggg caguaucgac agugggccua ugcccccuga
	gagugugggu ccugugguag cuggaguguu gguggccauc uuggugcugg cgguccucau
	gcugauguac uacugcugca gacagaacaa caaacuaggc caacucaagc ccucagcucu
	cccuuccaag cugaggcaac aguucaguug ucccuucagg guuucucaga acagcgggac
	uggucaugcc aacccaacuu ucaagcugca gacgccccag ggcaagcgaa aggugaucaa
	cacuccggaa auccugegga agcccuccca gccuccuccc cggcccccuc cagauuaucu
	gcgugguggg uccccaccug caccacugcc agcucaccug agcagggcug cuaggaacuc
	cccagggccc gggucucaaa uagagaggac ggagucgucc aggaggccuc cuccaagccg
	gccaauuccc cccgcaccaa auugcaucgu uucccaggac uucuccaggc cucggccgcc
	ccagaaggca cucccggcaa acccagugcc aggccgcagg agccucccca ggccaggagg
	ugcaucccca cugcggcccc cuggugcugg cccucagcag ucccggccuc uggcagcacu
	ugccccaaag uuuccagaau acagaucaca gagggcugga gggaugauua gcucgaaaau
	cuagaccugu ccaaggggcu ucucccuuuc cuugagcucu cuggacacug cagaggaccc
	auggccaugg aacccugaag aagcaugucu ggccgccucu gagcuccucc cacccuccuc
	caggaaccuc cacaucucca aaaaucuccc uguugacuca gugccuccuc ggcuuccuug
	gaagcccaga gggacuauga ucugauggcc ucuagguguu guuuugugca auauacagcc
	ccagguaggg aggggagagu augaggaggg ugacuggcag cuucuccucc agacuccuag
	ccccgaggug cugauggaga ugcucaaggc cagcaagccc cucaggccag cacuucgcuu
	gcagaagcca uccauucacu ccuggggugc agggcacgca agagagcuuc ccauugcuuc
	ugcucuccuc agaggucccg ggcuggaugg aggcugguac uuacccaccc cuuuuagcuu
	uuagggauua aggaaggguc aagccagcca cugcuguggc ccugcccagg gcuugguuga
	gggaacggcu ucuggcugua uggcugcaug ugacaagcca cguccccucc caccucuccc
	caaaccccug caucccugua uucacacggg ucacucugac ucagacaggu acuauucgua
	ggcaguguag acagcaggag gagcaccggg cuugggcuuc cucugagccg ugaugccaaa
	gguugcgacu ccugacucug gauaauuuuu aguugcucuu uguuuucucu gccgcacuuu
	ccuggugccc cacgcuuuuc ucucuuccuu ccccucucau ucucccucua auguguggug
	cuuuggugag caaacccuca gcaguccuga ccuucgggug accaggugcu ugugaccuac
	aagucagagu ccucucucac agucggccac uggauuuccc ucacuggcuc ucaggagugu
	gaccagagua gacuuggggc auggccauug gggucauaug uuuauuuuuc auuguguuuu
	gugaccucag cagggugggg gucuuccucc uuacucuaag cuaaaucuag gugagguuuc
	cccuuaggga gcccagcuau uuacaaagua cacacgaggg agcaggcugg ucauugacuu
	cgggcuggac cguugcccuc ugagcagaga acagacccau uucugggagc ugcccgagau
	cacuggagaa ggcagccagc agcagcugca cuggaacagu cagagcaggg agccucuucc
	ucaacccagc uuuuugucau ucacuuccuu uuguucucuc ucuggucacu gcccuuaccu
	gacccucaca gaaagagagc ucugagcagg ugaggggguc ugcgguggcu ccugucuucc
	cugcagcagg gaaggagggc cguguggugc uuugcuagau aggacgguuu uugcaaagca
	ccuggagaug uuugcuggga gauagacucc cacuccacaa aggugcuggg uggcucuccg
	gacaggagcu ggccugacuc ucacuccucu gaggcuuucc uggggccucc ucccauccug
	ccaugagcaa uuguuugcuc uugaaaaccu cacugcaagg cugaggcuga gcuucugauu
	caccacccca gggccuccuu auaguucucu gcacacaaua ggugcuucuu ggauguucuu
	ggguuuggaa auaaguggaa aauacgggau guaccccugg gggaaaagcc uggguugggu
	uuagaaagau cucaggaaaa ugaguuucuc uucccucagg guggcuguga uacagguucc
	ccauguccuu gccguggguc auccuugcug ugggucaucc uugcugugga gauccauucc
	ccaccuuucc uguggcccaa ccuuuuauuu aaaugugcua cccucugccu caaggcuugg
	uuccuggaaa guaaagguga aaacaucccc uuucaccccu cugcaaaaca aacaagcaac
	auccucaaaa cccaacccca ugccucacag agcuuccugu ggcuucucca gccuuucucc
	cucacaucag gagguagaua gcucugaaau gacagcgcca cagccauagu gacugcauga
	gccaucugaa ccugcagucc acccucccug gaaccacacc agaaagagac cuggguuguc
	guuuucuugc uuuuuguuuu guuuuguuuu auuauuuuca uaucaccucc aucccauaaa
	guuguacugu gaacuggaag augguggaau guuuuggaau uugauagacu uucggcaacc
	aguucuacua augcuucacu ccuggcucug uucagggagg cugcccagga ggaagacugg
	ccauuaugca uccccuuuuc uuuccagugc ccaguaugcu guuuugaggu gucaaauaca
	aauaaaucug ggcuuaggga aggagagacc uuauuccaaa gcacgauugc agaaggggaa
	agggaauauu gcaaaaggga gaggaagggg ccuuauggga auagugaaaa ggcucagacc
	gaccgauggc aagaucugca agcgucucaa agcccaggca gaaaaggacu uuucuuuuau
	uggaagaagu aaacauggcu agaaagaacc acguucaggg aaugacguug ugcccagccu
	uuuuuuuuuu uuuuuuuuuu uugucuccag gggaggggcu guuugcuggc ucaggcugag
	gauggcccaa aguccagggu cuggugggga ggagggaagc uuaacucaag uuuggguuag
	ugaguuagca agcucuuugu gcagaugggg auguagguaa aucuuuuuaa aagugaaauu
	aaccuccugc caauuuuaca acccaagaau uuuuuuuuaa gggccuugga gccaucucua
	aaacaaaccu caagggauuu agugcccugu cucccugucu cuagaagccu uagccugggc
	accuggcuca aucuuguaac ugccugcuag ccauagauuc cuuucagccu ugcugacuuc
	ucccuauaaa aguaaagccu uuuucugccc cagcucugag acacuugcag aucuuaaggu
	cugagacuug cugauuuucu gguuggagug uuuuuuugua uugccauagu cccuuccccc
	ugaagcaaua gccccucccc accuccugca auacgccuuu ccaaucuuua uuggaagucu
	cucccugccu acuuccuaau uuauucuuau uugacagagg guauggaaga cuugcaauuu
	gaaaacuggg gaccaguucc aaagucagua auuguguuaa ccacguguau aacagcucug
	cuggacaccc aagaaagcca ugggaacgcc aacuggaaag guccccuucc ccaggggagc
	cugcgaagga gagguucugu agaauccaag cccacauuuc caaagucacc cccaacgcgu
	ccucucacac cguccacugu gcguuuguau gugucuggga uccagggcaa ugugaauuuu
	cuuuuuauuu gggagauugu ucacggaaaa cagaucuucu ucucucuugu ccaccuauua
	auuguuuaca auauuuguac aucuaugcaa aauacuugaa ugggccaugg ugccuuuuuu
	ccuuguuagu auuuaauuaa aaaugaauug uuugucauuu gcaauguuaa aaaaaaaaaa
	aaaaaaaaaa aaaa

12	MPGGAGAARLCLLAFALQPLRPRAAREPGWTRGSEEGSPKLQHELIIPQWKTSESPVREK
	HPLKAELRVMAEGRELILDLEKNEQLFAPSYTETHYTSSGNPQTTTRKLEDHCFYHGTVR
	ETELSSVTLSTCRGIRGLITVSSNLSYVIEPLPDSKGQHLIYRSEHLKPPPGNCGFEHSK
	PTTRDWALQFTQQTKKRPRRMKREDLNSMKYVELYLVADYLEFQKNRRDQDATKHKLIEI
	ANYVDKFYRSLNIRIALVGLEVWTHGNMCEVSENPYSTLWSFLSWRRKLLAQKYHDNAQL
	ITGMSFHGTTIGLAPLMAMCSVYQSGGVNMDHSENAIGVAATMAHEMGHNFGMTHDSADC
	CSASAADGGCIMAAATGHPFPKVFNGCNRRELDRYLQSGGGMCLSNMPDTRMLYGGRRCG
	NGYLEDGEECDCGEEEECNNPCCNASNCTLRPGAECAHGSCCHQCKLLAPGTLCREQARQ
	CDLPEFCTGKSPHCPTNFYQMDGTPCEGGQAYCYNGMCLTYQEQCQQLWGPGARPAPDLC
	FEKVNVAGDTFGNCGKDMNGEHRKCNMRDAKCGKIQCQSSEARPLESNAVPIDTTIIMNG
	RQIQCRGTHVYRGPEEEGDMLDPGLVMTGTKCGYNHICFEGQCRNTSFFETEGCGKKCNG
	HGVCNNNQNCHCLPGWAPPFCNTPGHGGSIDSGPMPPESVGPVVAGVLVAILVLAVLMLM
	YYCCRQNNKLGQLKPSALPSKLRQQFSCPFRVSQNSGTGHANPTFKLQTPQGKRKVINTP
	EILRKPSQPPPRPPPDYLRGGSPPAPLPAHLSRAARNSPGPGSQIERTESSRRPPPSRPI
	PPAPNCIVSQDFSRPRPPQKALPANPVPGRRSLPRPGGASPLRPPGAGPQQSRPLAALAP
	KVSPREALKVKAGTRGLQGGRCRVEKTKQFMLLVVWTELPEQKPRAKHSCFLVPA

13	gggaggggaa ggcagcugag guuguggggg gaggugcccc gccccuuggc aggccccuac
	agccaaugga acggcccugg aagagacccg ggucgccucc ggagcuucaa aaacauguga
	ggagggaaga gugugcagac ggaacuucag ccgcugccuc uguucucagc gucagugccg
	ccacugcccc cgccagagcc caccggccag cauguccucu gcucacuuca accgaggccc
	ugccuacggg cugucagccg agguuaagaa caagcuggcc cagaaguaug accaccagcg
	ggagcaggag cugagagagu ggaucgaggg ggugacaggc cgucgcaucg gcaacaacuu
	cauggacggc cucaaagaug gcaucauucu uugcgaauuc aucaauaagc ugcagccagg
	cuccgugaag aagaucaaug agucaaccca aaauuggcac cagcuggaga acaucggcaa
	cuucaucaag gccaucacca aguauggggu gaagccccac gacauuuuug aggccaacga
	ccuguuugag aacaccaacc auacacaggu gcaguccacc cuccuggcuu uggccagcau
	ggcgaagacg aaaggaaaca aggugaacgu gggagugaag uacgcagaga agcaggagcg
	gaaauucgag ccggggaagc uaagagaagg gcggaacauc auugggcugc agaugggcac
	caacaaguuu gccagccagc agggcaugac ggccuauggc acccggcgcc accucuacga
	ccccaagcug ggcacagacc agccucugga ccaggcgacc aucagccugc agaugggcac
	caacaaagga gccagccagg cuggcaugac ugcgccaggg accaagcggc agaucuucga
	gccggggcug ggcauggagc acugcgacac gcucaauguc agccugcaga ugggcagcaa
	caagggcgcc ucgcagcggg gcaugacggu guaugggcug ccacgccagg ucuacgaccc
	caaguacugu cugacucccg aguacccaga gcugggugag cccgcccaca accaccacgc
	acacaacuac uacaauuccg ccuagggcca caaggccuuc ccuguuuucc ccccaaggga
	ggcugcugcu gcucuuggcu ggacccagcc aggcccagcc gacccccucu cccugcaugg
	cauccuccag ccccuguaga acucaaccuc uacaggguua gaguuuggag agagcagacu
	ggcggggggc ccauuggggg gaaggggacc cuccgcucug uagugcuaca ggguccaaca
	uagagccggg uguccccaac agcgcccaaa ggacgcacug agcaacgcua uuccagcugu
	ccccccacuc ccucacaagu ggguaccccc aggaccagaa gcucccccag caaagccccc
	agagcccagg cucggccugc ccccacccca uucccgcagu gggagcaaac ugcaugccca
	gagacccagc ggacacacgc gguuugguuu gcagcgacug gcauacuaug uggaugugac
	aguggcguuu guaaugagag cacuuucuuu uuuuucuauu ucacuggagc acaauaaaug
	gcuguaaaau cucaaaaaaa aaaaaa

14	MSSAHFNRGPAYGLSAEVKNKLAQKYDHQREQELREWIEGVTGRRIGNNFMDGLKDGIIL
	CEFINKLQPGSVKKINESTQNWHQLENIGNFIKAITKYGVKPHDIFEANDLFENTNHTQV
	QSTLLALASMAKTKGNKVNVGVKYAEKQERKFEPGKLREGRNIIGLQMGTNKFASQQGMT
	AYGTRRHLYDPKLGTDQPLDQATISLQMGTNKGASQAGMTAPGTKRQIFEPGLGMEHCDT
	LNVSLQMGSNKGASQRGMTVYGLPRQVYDPKYCLTPEYPELGEPAHNHHAHNYYNSA

15	aggucuccgc uuggagccgc cgcacccggg acggugcgua gcgcuggaag uccggccuuc
	cgagagcuag cuguccgccg cggcccccgc acgccgggca gccgucccuc gccgccucgg
	gcgcgccacc auggggcccc ggcucagcgu cuggcugcug cugcugcccg ccgcccuucu
	gcuccacgag gagcacagcc gggccgcugc gaaggguggc ugugcuggcu cuggcugugg
	caaaugugac ugccauggag ugaagggaca aaagggugaa agaggccucc cgggguuaca
	aggugucauu ggguuuccug gaaugcaagg accugagggg ccacagggac caccaggaca
	aaagggugau acuggagaac caggacuacc uggaacaaaa gggacaagag gaccuccggg
	agcaucuggc uacccuggaa acccaggacu ucccggaauu ccuggccaag acggcccgcc
	aggcccccca gguauuccag gaugcaaugg cacaaagggg gagagagggc cgcucgggcc
	uccuggcuug ccugguuucg cuggaaaucc cggaccacca ggcuuaccag ggaugaaggg
	ugauccaggu gagauacuug gccaugugcc cgggaugcug uugaaaggug aaagaggauu
	ucccggaauc ccagggacuc caggcccacc aggacugcca gggcuucaag guccuguugg
	gccuccagga uuuaccggac caccaggucc cccaggcccu cccggcccuc caggugaaaa
	gggacaaaug ggcuuaaguu uucaaggacc aaaaggugac aagggugacc aaggggucag
	ugggccucca ggaguaccag gacaagcuca aguucaagaa aaaggagacu ucgccaccaa
	gggagaaaag ggccaaaaag gugaaccugg auuucagggg augccagggg ucggagagaa
	aggugaaccc ggaaaaccag gacccagagg caaacccgga aaagauggug acaaagggga
	aaaagggagu cccgguuuuc cuggugaacc cggguaccca ggacucauag gccgccaggg
	cccgcaggga gaaaagggug aagcaggucc uccuggccca ccuggaauug uuauaggcac
	aggaccuuug ggagaaaaag gagagagggg cuacccugga acuccggggc caagaggaga
	gccaggccca aaagguuucc caggacuacc aggccaaccc ggaccuccag gccucccugu
	accugggcag gcuggugccc cuggcuuccc uggugaaaga ggagaaaaag gugaccgagg
	auuuccuggu acaucucugc caggaccaag uggaagagau gggcucccgg guccuccugg
	uuccccuggg cccccugggc agccuggcua cacaaaugga auuguggaau gucagcccgg
	accuccaggu gaccaggguc cuccuggaau uccagggcag ccaggauuua uaggcgaaau
	uggagagaaa ggucaaaaag gagagaguug ccucaucugu gauauagacg gauaucgggg
	gccucccggg ccacagggac ccccgggaga aauagguuuc ccagggcagc caggggccaa
	gggcgacaga gguuugccug gcagagaugg uguugcagga gugccaggcc cucaagguac
	accagggcug auaggccagc caggagccaa gggggagccu ggugaguuuu auuucgacuu
	gcggcucaaa ggugacaaag gagacccagg cuuuccagga cagcccggca ugccagggag
	agcggguucu ccuggaagag auggccaucc gggucuuccu ggccccaagg gcucgccggg
	uucuguagga uugaaaggag agcguggccc cccuggagga guuggauucc caggcagucg
	uggugacacc ggccccccug ggccuccagg auaugguccu gcugguccca uuggugacaa
	aggacaagca ggcuuuccug gaggcccugg auccccaggc cugccagguc caaaggguga
	accaggaaaa auuguuccuu uaccaggccc cccuggagca gaaggacugc cggggucccc
	aggcuuccca gguccccaag gagaccgagg cuuucccgga accccaggaa ggccaggccu
	gccaggagag aagggcgcug ugggccagcc aggcauugga uuuccagggc cccccggccc
	caaagguguu gacggcuuac cuggagacau ggggccaccg gggacuccag gucgcccggg
	auuuaauggc uuaccuggga acccaggugu gcagggccag aagggagagc cuggaguugg
	ucuaccggga cucaaagguu ugccaggucu ucccggcauu ccuggcacac ccggggagaa
	ggggagcauu gggguaccag gcguuccugg agaacaugga gcgaucggac ccccugggcu
	ucaggggauc agaggugaac cgggaccucc uggauugcca ggcuccgugg ggucuccagg
	aguuccagga auaggccccc cuggagcuag gggucccccu ggaggacagg gaccaccggg
	guugucaggc ccuccuggaa uaaaaggaga gaaggguuuc cccggauucc cuggacugga
	caugccgggc ccuaaaggag auaaaggggc ucaaggacuc ccuggcauaa cgggacaguc
	ggggcucccu ggccuuccug gacagcaggg ggcuccuggg auuccugggu uuccagguuc
	caagggagaa augggcguca uggggacccc cgggcagccg ggcucaccag gaccaguggg
	ugcuccugga uuaccgggug aaaaagggga ccauggcuuu ccgggcuccu caggacccag
	gggagacccu ggcuugaaag gugauaaggg ggaugucggu cucccuggca agccuggcuc
	cauggauaag guggacaugg gcagcaugaa gggccagaaa ggagaccaag gagagaaagg
	acaaauugga ccaauuggug agaagggauc ccgaggagac ccugggaccc caggagugcc
	uggaaaggac gggcaggcag gacagccugg gcagccagga ccuaaaggug auccagguau
	aaguggaacc ccaggugcuc caggacuucc gggaccaaaa ggaucuguug guggaauggg
	cuugccagga acaccuggag agaaaggugu gccuggcauc ccuggcccac aagguucacc
	uggcuuaccu ggagacaaag gugcaaaagg agagaaaggg caggcaggcc caccuggcau
	aggcauccca gggcugcgag gugaaaaggg agaucaaggg auagcggguu ucccaggaag
	cccuggagag aagggagaaa aaggaagcau ugggauccca ggaaugccag gguccccagg
	ccuuaaaggg ucucccggga guguuggcua uccaggaagu ccugggcuac cuggagaaaa
	aggugacaaa ggccucccag gauuggaugg caucccuggu gucaaaggag aagcaggucu
	uccugggacu ccuggcccca caggcccagc uggccagaaa ggggagccag gcagugaugg
	aaucccgggg ucagcaggag agaaggguga accaggucua ccaggaagag gauucccagg
	guuuccaggg gccaaaggag acaaagguuc aaagggugag guggguuucc caggauuagc
	cgggagccca ggaauuccug gauccaaagg agagcaagga uucauggguc cuccggggcc
	ccagggacag ccgggguuac cgggaucccc aggccaugcc acggaggggc ccaaaggaga
	ccgcggaccu cagggccagc cuggccugcc aggacuuccg ggacccaugg ggccuccagg
	gcuuccuggg auugauggag uuaaagguga caaaggaaau ccaggcuggc caggagcacc
	cgguguccca gggcccaagg gagacccugg auuccagggc augccuggua uugguggcuc
	uccaggaauc acaggcucua agggugauau ggggccucca ggaguuccag gauuucaagg
	uccaaaaggu cuuccuggcc uccagggaau uaaaggugau caaggcgauc aaggcguccc
	gggagcuaaa ggucucccgg guccuccugg ccccccaggu ccuuacgaca ucaucaaagg
	ggagcccggg cucccugguc cugagggccc cccagggcug aaagggcuuc agggacugcc
	aggcccgaaa ggccagcaag guguuacagg auuggugggu auaccuggac cuccagguau
	uccuggguuu gacggugccc cuggccagaa aggagagaug ggaccugccg ggccuacugg
	uccaagagga uuuccagguc caccaggccc cgauggguug ccaggaucca uggggccccc
	aggcacccca ucuguugauc acggcuuccu ugugaccagg cauagucaaa caauagauga
	cccacagugu ccuucuggga ccaaaauucu uuaccacggg uacucuuugc ucuacgugca
	aggcaaugaa cgggcccaug gccaggacuu gggcacggcc ggcagcugcc ugcgcaaguu
	cagcacaaug cccuuccugu ucugcaauau uaacaacgug ugcaacuuug caucacgaaa
	ugacuacucg uacuggcugu ccaccccuga gcccaugccc augucaaugg cacccaucac
	gggggaaaac auaagaccau uuauuaguag gugugcugug ugugaggcgc cugccauggu
	gauggccgug cacagccaga ccauucagau cccaccgugc cccagcgggu gguccucgcu
	guggaucggc uacucuuuug ugaugcacac cagcgcuggu gcagaaggcu cuggccaagc
	ccuggcgucc cccggcuccu gccuggagga guuuagaagu gcgccauuca ucgaguguca
	cggccguggg accugcaauu acuacgcaaa cgcuuacagc uuuuggcucg ccaccauaga
	gaggagcgag auguucaaga agccuacgcc guccaccuug aaggcagggg agcugcgcac
	gcacgucagc cgcugccaag ucuguaugag aagaacauaa ugaagccuga cucagcuaau
	gucacaacau ggugcuacuu cuucuucuuu uuguuaacag caacgaaccc uagaaauaua
	uccuguguac cucacugucc aauaugaaaa ccguaaagug ccuuauagga auuugcguaa
	cuaacacacc cugcuucauu gaccucuacu ugcugaagga gaaaaagaca gcgauaagcu
	uucaauagug gcauaccaaa uggcacuuuu gaugaaauaa aauaucaaua uuuucugcaa
	uccaaugcac ugauguguga agugagaacu ccaucagaaa accaaagggu gcuaggaggu
	gugggugccu uccauacugu uugcccauuu ucauucuugu auuauaauua auuuucuacc
	cccagagaua aauguuuguu uauaucacug ucuagcuguu ucaaaauuua ggucccuugg
	ucuguacaaa uaauagcaau guaaaaaugg uuuuuugaac cuccaaaugg aauuacagac
	ucaguagcca uaucuuccaa ccccccagua uaaauuucug ucuuucugcu auguguggua
	cuuugcagcu gcuuuugcag aaaucacaau uuuccugugg aauaaagaug guccaaaaau
	agucaaaaau uaaauauaua uauauauuag uaauuuauau agaugucagc aauuaggcag
	aucaagguuu aguuuaacuu ccacuguuaa aauaaagcuu acauaguuuu cuuccuuuga
	aagacugugc uguccuuuaa cauagguuuu uaaagacuag gauauugaau gugaaacauc
	cguuuucauu guucacuucu aaaccaaaaa uuauguguug ccaaaaccaa acccagguuc
	augaauaugg ugucuauuau agugaaacau guacuuugag cuuauuguuu uuauucugua
	uuaaauauuu ucaggguuuu aaacacuaau cacaaacuga augacuugac uucaaaagca
	acaaccuuaa aggccgucau uucauuagua uuccucauuc ugcauccugg cuugaaaaac
	agcucuguug aaucacagua ucaguauuuu cacacquaag cacauucggg ccauuuccgu
	gguuucucau gagcuguguu cacagaccuc agcagggcau cgcauggacc gcaggagggc
	agauucggac cacuaggccu gaaaugacau uucacuaaaa gucuccaaaa cauuucuaag
	acuacuaagg ccuuuuaugu aauuucuuua aauguguauu ucuuaagaau ucaaauuugu
	aauaaaacua uuuguauaaa aauuaagcuu uuauuaauuu guugcuagua uugccacaga
	cgcauuaaaa gaaacuuacu gcacaagcug cuaauaaauu uguaagcuuu gcauaccuua
	gauua

16	MGPRLSVWLLLLPAALLLHEEHSRAAAKGGCAGSGCGKCDCHGVKGQKGERGLPGLQGVI
	GFPGMQGPEGPQGPPGQKGDTGEPGLPGTKGTRGPPGASGYPGNPGLPGIPGQDGPPGPP
	GIPGCNGTKGERGPLGPPGLPGFAGNPGPPGLPGMKGDPGEILGHVPGMLLKGERGFPGI
	PGTPGPPGLPGLQGPVGPPGFTGPPGPPGPPGPPGEKGQMGLSFQGPKGDKGDQGVSGPP
	GVPGQAQVQEKGDFATKGEKGQKGEPGFQGMPGVGEKGEPGKPGPRGKPGKDGDKGEKGS
	PGFPGEPGYPGLIGRQGPQGEKGEAGPPGPPGIVIGTGPLGEKGERGYPGTPGPRGEPGP
	KGFPGLPGQPGPPGLPVPGQAGAPGFPGERGEKGDRGFPGTSLPGPSGRDGLPGPPGSPG
	PPGQPGYTNGIVECQPGPPGDQGPPGIPGQPGFIGEIGEKGQKGESCLICDIDGYRGPPG
	PQGPPGEIGFPGQPGAKGDRGLPGRDGVAGVPGPQGTPGLIGQPGAKGEPGEFYFDLRLK
	GDKGDPGFPGQPGMTGRAGSPGRDGHPGLPGPKGSPGSVGLKGERGPPGGVGFPGSRGDT
	GPPGPPGYGPAGPIGDKGQAGFPGGPGSPGLPGPKGEPGKIVPLPGPPGAEGLPGSPGFP
	GPQGDRGFPGTPGRPGLPGEKGAVGQPGIGFPGPPGPKGVDGLPGDMGPPGTPGRPGFNG
	LPGNPGVQGQKGEPGVGLPGLKGLPGLPGIPGTPGEKGSIGVPGVPGEHGAIGPPGLQGI
	RGEPGPPGLPGSVGSPGVPGIGPPGARGPPGGQGPPGLSGPPGIKGEKGFPGFPGLDMPG
	PKGDKGAQGLPGITGQSGLPGLPGQQGAPGIPGFPGSKGEMGVMGTPGQPGSPGPVGAPG
	LPGEKGDHGFPGSSGPRGDPGLKGDKGDVGLPGKPGSMDKVDMGSMKGQKGDQGEKGQIG
	PIGEKGSRGDPGTPGVPGKDGQAGQPGQPGPKGDPGISGTPGAPGLPGPKGSVGGMGLPG
	TPGEKGVPGIPGPQGSPGLPGDKGAKGEKGQAGPPGIGIPGLRGEKGDQGIAGFPGSPGE
	KGEKGSIGIPGMPGSPGLKGSPGSVGYPGSPGLPGEKGDKGLPGLDGIPGVKGEAGLPGT
	PGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSP
	GIPGSKGEQGFMGPPGPQGQPGLPGSPGHATEGPKGDRGPQGQPGLPGLPGPMGPPGLPG
	IDGVKGDKGNPGWPGAPGVPGPKGDPGFQGMPGIGGSPGITGSKGDMGPPGVPGFQGPKG
	LPGLQGIKGDQGDQGVPGAKGLPGPPGPPGPYDIIKGEPGLPGPEGPPGLKGLQGLPGPK
	GQQGVTGLVGIPGPPGIPGFDGAPGQKGEMGPAGPTGPRGFPGPPGPDGLPGSMGPPGTP
	SVDHGFLVTRHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTM
	PFLFCNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAV
	HSQTIQIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRG
	TCNYYANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRT

17	aaacucacac aacaacucuu ccccgcugag aggagacagc cagugcgacu ccacccucca
	gcucgacggc agccgccccg gccgacagcc ccgagacgac agcccggcgc gucccggucc
	ccaccuccga ccaccgccag cgcuccaggc cccgccgcuc cccgcucgcc gccaccgcgc
	ccuccgcucc gcccgcagug ccaaccauga ccgccgccag uaugggcccc guccgcgucg
	ccuucguggu ccuccucgcc cucugcagcc ggccggccgu cggccagaac ugcagcgggc
	cgugccggug cccggacgag ccggcgccgc gcugcccggc gggcgugagc cucgugcugg
	acggcugcgg cugcugccgc gucugcgcca agcagcuggg cgagcugugc accgagcgcg
	accccugcga cccgcacaag ggccucuucu gugacuucgg cuccccggcc aaccgcaaga
	ucggcgugug caccgccaaa gauggugcuc ccugcaucuu cggugguacg guguaccgca
	gcggagaguc cuuccagagc agcugcaagu accagugcac gugccuggac ggggcggugg
	gcugcaugcc ccugugcagc auggacguuc gucugcccag cccugacugc cccuucccga
	ggagggucaa gcugcccggg aaaugcugcg aggagugggu gugugacgag cccaaggacc
	aaaccguggu ugggccugcc cucgcggcuu accgacugga agacacguuu ggcccagacc
	caacuaugau uagagccaac ugccuggucc agaccacaga guggagcgcc uguuccaaga
	ccugugggau gggcaucucc acccggguua ccaaugacaa cgccuccugc aggcuagaga
	agcagagccg ccugugcaug gucaggccuu gcgaagcuga ccuggaagag aacauuaaga
	agggcaaaaa gugcauccgu acucccaaaa ucuccaagcc uaucaaguuu gagcuuucug
	gcugcaccag caugaagaca uaccgagcua aauucugugg aguauguacc gacggccgau
	gcugcacccc ccacagaacc accacccugc cgguggaguu caagugcccu gacggcgagg
	ucaugaagaa gaacaugaug uucaucaaga ccugugccug ccauuacaac ugucccggag
	acaaugacau cuuugaaucg cuguacuaca ggaagaugua cggagacaug gcaugaagcc
	agagagugag agacauuaac ucauuagacu ggaacuugaa cugauucaca ucucauuuuu
	ccguaaaaau gauuucagua gcacaaguua uuuaaaucug uuuuucuaac ugggggaaaa
	gauucccacc caauucaaaa cauugugcca ugucaaacaa auagucuauc aaccccagac
	acugguuuga agaauguuaa gacuugacag uggaacuaca uuaguacaca gcaccagaau
	guauauuaag guguggcuuu aggagcagug ggaggguacc agcagaaagg uuaguaucau
	cagauagcau cuuauacgag uaauaugccu gcuauuugaa guguaauuga gaaggaaaau
	uuuagcgugc ucacugaccu gccuguagcc ccagugacag cuaggaugug cauucuccag
	ccaucaagag acugagucaa guuguuccuu aagucagaac agcagacuca gcucugacau
	ucugauucga augacacugu ucaggaaucg gaauccuguc gauuagacug gacagcuugu
	ggcaagugaa uuugccugua acaagccaga uuuuuuaaaa uuuauauugu aaauauugug
	ugugugugug uguguguaua uauauauaua uguacaguua ucuaaguuaa uuuaaaguug
	uuugugccuu uuuauuuuug uuuuuaaugc uuugauauuu caauguuagc cucaauuucu
	gaacaccaua gguagaaugu aaagcuuguc ugaucguuca aagcaugaaa uggauacuua
	uauggaaauu cugcucagau agaaugacag uccgucaaaa cagauuguuu gcaaagggga
	ggcaucagug uccuuggcag gcugauuucu agguaggaaa ugugguagcc ucacuuuuaa
	ugaacaaaug gccuuuauua aaaacugagu gacucuauau agcugaucag uuuuuucacc
	uggaagcauu uguuucuacu uugauaugac uguuuuucgg acaguuuauu uguugagagu
	gugaccaaaa guuacauguu ugcaccuuuc uaguugaaaa uaaaguguau auuuuuucua
	uaaaaaaaaa aaaaaaaa

18	MTAASMGPVRVAFVVLLALCSRPAVGQNCSGPCRCPDEPAPRCPAGVSLVLDGCGCCRVC
	AKQLGELCTERDPCDPHKGLFCHFGSPANRKIGVCTAKDGAPCIFGGTVYRSGESFQSSC
	KYQCTCLDGAVGCMPLCSMDVRLPSPDCPFPRRVKLPGKCCEEWVCDEPKDQTVVGPALA
	AYRLEDTFGPDPTMIRANCLVQTTEWSACSKTCGMGISTRVTNDNASCRLEKQSRLCMVR
	PCEADLEENIKKGKKCIRTPKISKPIKFELSGCTSMKTYRAKFCGVCTDGRCCTPHRTTT
	LPVEFKCPDGEVMKKNMMFIKTCACHYNCPGDNDIFESLYYRKMYGDMA

19	acuuccgucu uggaggccag gguggcgcug ccgccguccc ucgcccggag gcagagaugc
	gcuggcgcau caccgccagg agcccacagu gaaagaccau cggauggaag gcgacgccca
	gaacuccagg agaccgcugu gggaggacgc gaggccaggu gacgaauagg ccaggcguga
	guucccaaac agccuccucc ccuucaagag aguaagcuug ggccacaggc ugggacggaa
	gcagaggggc agacacgcca ccacccgccc ggccucgaac cuacggcggc acaguucagc
	ggaggcggcc cagcgguccu gucccgcgcc ugcgcacucc aggccccgcc ccgccccgcg
	cccuccaggc ccggcccgcc cuccaaccuc ugcgugcgca cagccuagag cccgccuccg
	ugaaagacug ccgggcgcau gcggucgggg uuguucacug gcuguccggg gcuccgcgcg
	cgucgccggc ccagcucugu cgcugacggg aggaucugaa gccggccgca ggucaaagag
	uaaaaugaag uacauucugg uuacuggugg uguuauauca ggaauuggaa aaggaaucau
	ugccagcagu gugggcacaa uacucaaguc augugguuua cauguaacuu caaucaaaau
	ugaccccuac auuaacauug augcaggaac auucucuccu uaugagcaug gugagguuuu
	ugugcuggau gauggugggg aaguagaccu ugaccugggu aacuaugagc gguuccuuga
	cauccgccuc accaaggaca auaaucugac cacuggaaag auauaccagu augucauuaa
	caaggaacgg aaaggagauu acuuggggaa aacuguccaa guugucccuc auaucacaga
	ugcaauccag gaguggguga ugagacaggc guuaauaccu guagaugaag auggccugga
	accucaagug uguguuauug agcuuggugg aaccgugggg gacauagaaa gcaugcccuu
	uauugaggcc uuccgucagu uccaauucaa ggucaaaaga gagaacuuuu guaacaucca
	cgucagucua guuccccagc caaguucaac aggggaacag aagacuaaac cuacccagaa
	uaguguucgg gaacuuagag gacuugggcu uuccccagau cugguuguau gcaggugcuc
	aaauccacuu gacacaucag ugaaggagaa aauaucaaug uucugccaug uugagccuga
	acaagugauc uguguccacg augucucauc caucuaccga guccccuugu uguuagagga
	gcaagggguu guagauuauu uucuucgaag acuugaccuu ccuauugaga ggcagccaag
	aaaaaugcug augaaaugga aagagauggc ugacagauau gaucgcuugc uggagaccug
	cucuauugcc cuugugggca aauacacgaa guucucagac uccuaugccu cugucauuaa
	ggcucuggag cauucugcac uggccaucaa ccacaaauug gaaaucaagu acauagauuc
	ugcggacuug gagcccauca ccucgcaaga agagcccgug cgcuaccacg aagcuuggca
	gaagcucugu agugcucaug gagugcuggu uccaggagga uuugguguuc gaggaacaga
	aggaaaaauc caagcaauug ccugggcucg gaaucagaaa aagccuuuuu ugggcgugug
	cuuagggaug caguuggcag ugguugaauu cucaagaaac gugcugggau ggcaagaugc
	caauucuaca gaguuugacc cuacgaccag ucaucccgug gucguagaca ugccagaaca
	caacccaggg cagaugggcg gaaccaugag gcugggcaag aggagaaccc uguuccagac
	caagaacuca gucaugagga aacucuaugg agacgcagac uacuuggaag agaggcaccg
	ccaccgauuu gaggugaauc cagucuggaa aaaguguuug gaagaacaag gcuugaaguu
	uguuggccaa gauguugaag gagagagaau ggaaauugug gaguuagaag aucaucccuu
	uuuuguuggg guucaguacc acccugaguu ccuguccagg ccuaucaagc ccuccccacc
	auacuuuggc cuccuccugg ccucuguggg gcggcucuca cauuaccucc agaaaggcug
	caggcucuca cccagggaca ccuauaguga caggagugga agcagcuccc cugacucuga
	aaucaccgaa cugaaguuuc caucaauaaa ucaugacuga ucuuguagcg gaugauucuu
	caagagaccc uucaaacuug gguagaguuu acagcucuga cuuuacacuc ggcuuuggag
	acuuucuuua aauuauguuu uuauuaagau uauuuuauua ugcggaaagg uauuugggaa
	acuugucacu ugcauguccc aucacgugua cuggcuccuc uguggugucu gccuguugcg
	ugacacucuc cuugcaguuc uugaguugcg gcagaacauc gcgaugggaa ccgauggugg
	guggggcugc agagugcccc aucggucacc uuguuucuca acuaccucgc aucauugcag
	augcuagcgc guugccuguc gcuuucccuu ggauaccuag accguuauaa agugugccac
	auggacuuac cgagcaugga gagaggauuu uagcuaggau uugaacacuu ggugcuggga
	accucagggu auugcuugcc acuaagccau gaaaccagag acaaaaucuc uauacugccc
	ugaguugggg ggaauucuca gugccaacug uggcuggucc ucauucaaag ggacggucag
	uuugguguca acaugaaaca ccaagauguc ugucucugaa gcgugauuuu aaaaucccca
	ugccuguggc ugcgcuuccu auuucuaggg cugggaaaca cuccuugcau caagggguca
	cuuacagaac aaagaaucuu uugggggaaa cuuccucuaa aacccucuca uauauagaca
	gcuuugacug gaggguccau uuuucuucca ggaugguguu acugcaguug aaagggcaau
	augaaguuac uuucuuaaug ugaccuagca auaggcauag cuacguggca cuauauucug
	gccagacucg auguguacuc uaacuuaaga aauaaaucag
	aaaaaaaa

20	MKYILVTGGVISGIGKGIIASSVGTILKSCGLHVTSIKIDPYINIDAGTFSPYEHGEVFV
	LDDGGEVDLDLGNYERFLDIRLTKDNNLTTGKIYQYVINKERKGDYLGKTVQVVPHITDA
	IQEWVMRQALIPVDEDGLEPQVCVIELGGTVGDIESMPFIEAFRQFQFKVKRENFCNIHV
	SLVPQPSSTGEQKTKPTQNSVRELRGLGLSPDLVVCRCSNPLDTSVKEKISMFCHVEPEQ
	VICVHDVSSIYRVPLLLEEQGVVDYFLRRLDLPIERQPRKMLMKWKEMADRYDRLLETCS
	IALVGKYTKFSDSYASVIKALEHSALAINHKLEIKYIDSADLEPITSQEEPVRYHEAWQK
	LCSAHGVLVPGGFGVRGTEGKIQAIAWARNQKKPFLGVCLGMQLAVVEFSRNVLGWQDAN
	STEFDPTTSHPVVVDMPEHNPGQMGGTMRLGKRRTLFQTKNSVMRKLYGDADYLEERHRH
	RFEVNPVWKKCLEEQGLKFVGQDVEGERMEIVELEDHPFFVGVQYHPEFLSRPIKPSPPY
	FGLLLASVGRLSHYLQKGCRLSPRDTYSDRSGSSSPDSEITELKFPSINHD

21	gucugcgccc cucgcugucc cgcgaccccg gcgcgggugc cucgggcccc ccucgcgcgc
	cgccauggug ggccggcuga gccuacagga ugugcccgag cucguggacg cgaagaagaa
	gggcgacggc guccuggaca gcccggacuc ggggcugccc cccagcccca gccccagcca
	cugggggcuc gcggcgggcg gaggcggcgg agagcgcgcg gcggcaccgg ggacgcugga
	gcccgacgeg gcggcggcga cccccgcggc uccgagucca gcgucucucc cccuggcucc
	cggcugugcg cugaggcuuu guccccuguc cuuuggcgaa ggaguggagu uugaccccuu
	accaccaaag gaaguaaggu acaccuccuu ggucaaguac gacuccgaga ggcacuucau
	cgacgacgug cagcugcccc ugggccuggc gguggccucc ugcagccaga cggucaccug
	uguccccaau ggcacguggc gcaacuacaa ggccgaggug cgcuucgagc cacgccacag
	gcccacccgc uuccucagua ccaccaucgu guaccccaag uaccccaagg ccgucuacac
	caccacccug gauuacaacu gccgcaagac gcugaggagg uuucugucca gcguggagcu
	cgaagccgcg gagcucccgg gcagcgacga ccucucugau gaaugcugac ccagcaggcu
	gggcuggggu cggaccggcu cucccgcugu ccugcccccg acugcccugg ccaagcuggg
	cgaccuuacc cuucacguca uuccagcccc agaggagucu aaccuagaga uaccuccaag
	cgcggccggg ggaagagaaa aaaaagaaaa aaucacugca ucucauaauu auugagaucu
	uuguguugua auuuucaagc uguuuuuaga gggaauauau guccugguuu gcugcugugu
	uguuucccca aagucucaau cagaugaggc aacaaaaaga ccaccagaau ugcaggaaag
	cagcagcagu cugggauagg gaggggggag gagagcucuc ccuccguggu caguuuguca
	gaagaaaagc auggaaaaag ugagauuuaa gaaaucucag ccacgggugu cuucacugca
	caaaaguggu gcuaauucag uaaacaguga caccugugug gguucaaucu guggagaguu
	gaguuccauu ccuuuguuuu uaaauuucca ccuccauuug uguuuccuau uaaccuaaac
	ucuuguaauu cucuaaaucu uuuuucauga uauaaaaaaa aaagaaaauc ccaaaccaca
	auuuaagaug ccuuuuuuuu uuaaaaaaaa agguucuccg uguugccacc ugcuccggga
	aggccaggcc gaugggcggc cagguuuggg agcagcggcu ucggcuccga uugcuuccug
	acggucaguu ccaagcacag gcucugagaa acgccucgaa cccuugguau cuggugaccg
	ucuccgcagg cuuccgugag ucuccgauag guguuuuggg cuuuuggaau acuuucagaa
	uacuuuccca uuuuuucaua aggcaaaacg gaguggacgg ccuuuuuuug agagaaacag
	cauucuaaga gugugcuugg aaaacacacu uuuugggagu ccacgugggg aguggacacu
	caggacagga agcgguggcc cuaagaccug cggccacccg gaggaggcag acgcaggcga
	ugaccugguu caucaugaga ggagcuagag uugacucagu cuuaggaucu cgauggcguc
	agaauugcag aggguuucug ccagugccuu aggucuaauu cagaaagcaa ggaaggcugg
	gcacgguggc uggcgccugu agucucagcu acuuggaaac ugaggcagga ggaucucuug
	agcccaagaa uucgagaccg gucuggcaac auagugagcc uccaucucua aaaaaaagug
	gcaaagcuga ggaaaucugu gcccccaucc cccgacacca gacaagcuaa ggugcauuca
	gaguagggaa cgaugucaaa accuggccgc cgugguggcg cgcauuccuc ucacaccacc
	guacggaggc aggugacagu caacugaaau guccccaagg accgugugaa uucggcuggc
	guggcccagc agcggggugg accagggugc gguggcuuca gaccugccuc ccgccacccc
	guccuccccu aaccccuucu ugggaccucc ugccaauccc ugugguguuu gaguaaagaa
	aguggcugau gguuuuacuu uuuucuucca auauuggaaa gaacaaacua gauauucccc
	uugaacacau ccaaauuauu uccagugccu gaggguuacc gagcacugua agcucagcuc
	ggcuguagcu ccggguuucu gccgauggcc uccaucugcg ccgaaagcug aacugagccc
	cgcggugccc cggagccaca gcagcaggug cagcgacaau cgccgucgug cgcagucgcu
	ggaguccugg agagccacgc uguccucugc agacaacagc aucaguccgc ugugccccaa
	ugcaucgacg cccuguggac ggggccugca guccacccgu ggucugagcu ugccgggcug
	aagcucggcu gggacccagg gaggggccac ccugggaccc acagucuccu cagucacgug
	cagagcaaaa aaugcuuccu guguuuggaa gagcaaguga cgcagcauuu auugauaaaa
	auggauuuuu cugucugauu ucaugcugug gucguuuaag uggucaaugu cuuuuuuuuu
	uuuuuuuuug acauggaguc ucgcucuguu gcccaggcug aguggaaugg cgccaacuca
	gcucacugca accuucaccu cccaggcuca agcgacucuc cugccucagc cuccugagca
	gcugggauua caggugccca ccaccacgcc cagcuaauuu uuguauuuuu aguagagacg
	ggguuucacc auguuggcca ggcuggucuc gaacaccuga ccuugugauc cacccgccuc
	ggccucccaa uaauuuguaa guuauguuag cgggauccuc aaggccuugc uuugccccgu
	ggagacgcuu gcucggauga gcucaggaaa caguaccggc ugcguggcag gucugggugu
	ugugugcgag gacguggccu uugaacaccg cuguguucuc agagguccuu aggagauauu
	uuuuuuuguc uuagggggac uguguuaagu ucagacaaau caugcugggu guguagagag
	ugugaaauac gucagugaag uaaguagcag ugagcgauug ugaaugugua auguaaaugg
	aaaaccgggu uuuaccgugu uaaguuauuc acuagggagc cagucguagu ucuuuguaau
	ccucuuucuu ccaaaccugc uuugcugaaa guugcagaaa aggaagugug uggagagaaa
	cagaacccuu cagggugggu cagaggacgc cauccacagu ggauucgugu ucguuugcag
	guggaagcag ugauuuuuag gacccacuga uuaaaaacaa acauucccaa gugucucuga
	gagaugcugu uuauuuguua auuaaaaagc uuuuuucucu gucuuuuaaa uuauggcuuu
	cauguaauaa ggauauuuuu agugaaaaau uguuuuccuu ucaaauuaca gaccuuuuaa
	aaaaacuuaa uuugagcgag uaccuuuuca uuugacacuu uuccuguuuc uaaccuuagg
	aaaccagaau agcguuuggc agacacgacg uuuucaguuu accuuugaca ccugccccac
	uccauuuugc uuugugaugu cuucauuuaa caauaaauua ucugaaaaaa caaaa

22	MVGRLSLQDVPELVDAKKKGDGVLDSPDSGLPPSPSPSHWGLAAGGGGGERAAAPGTLEP
	DAAAATPAAPSPASLPLAPGCALRLCPLSFGEGVEFDPLPPKEVRYTSLVKYDSERHFID
	DVQLPLGLAVASCSQTVTCVPNGTWRNYKAEVRFEPRHRPTRFLSTTIVYPKYPKAVYTT
	TLDYNCRKTLRRFLSSVELEAAELPGSDDLSDEC

23	gggaagucgg ugccgcugcc gucucugcgu ucgccaugcg ucccggggcg ccagggccac
	ucuggccucu gcccuggggg gcccuggcuu gggccguggg cuucgugagc uccaugggcu
	cggggaaccc cgcgcccggu gguguuugcu ggcuccagca gggccaggag gccaccugca
	gccuggugcu ccagacugau gucacccggg ccgagugcug ugccuccggc aacauugaca
	ccgccugguc caaccucacc cacccgggga acaagaucaa ccuccucggc uucuugggcc
	uuguccacug ccuucccugc aaagauucgu gcgacggcgu ggagugcggc ccgggcaagg
	cgugccgcau gcuggggggc cgcccgcgcu gcgagugcgc gcccgacugc ucggggcucc
	cggcgcggcu gcaggucugc ggcucagacg gcgccaccua ccgcgacgag ugcgagcugc
	gcgccgcgcg cugccgcggc cacccggacc ugagcgucau guaccggggc cgcugccgca
	aguccuguga gcacguggug ugcccgcggc cacagucgug cgucguggac cagacgggca
	gcgcccacug cguggugugu cgagcggcgc ccugcccugu gcccuccagc cccggccagg
	agcuuugcgg caacaacaac gucaccuaca ucuccucgug ccacaugcgc caggccaccu
	gcuuccuggg ccgcuccauc ggcgugcgcc acgcgggcag cugcgcaggc accccugagg
	agccgccagg uggugagucu gcagaagagg aagagaacuu cgugugagcc ugcaggacag
	gccugggccu ggugcccgag gccccccauc auccccuguu auuuauugcc acagcagagu
	cuaauuuaua ugccacggac acuccuuaga gcccggauuc ggaccacuug gggaucccag
	aaccucccug acgauauccu ggaaggacug aggaagggag gccugggggc cggcuggugg
	gugggauaga ccugcguucc ggacacugag cgccugauuu agggcccuuc ucuaggaugc
	cccagccccu acccuaagac cuauugccgg ggaggauucc acacuuccgc uccuuugggg
	auaaaccuau uaauuauugc uacuaucaag agggcugggc auucucugcu gguaauuccu
	gaagaggcau gacugcuuuu cucagcccca agccucuagu cugggugugu acggaggguc
	uagccugggu guguacggag ggucuagccu gggugaguac ggagggucua gccuggguga
	guacggaggg ucuagccugg gugaguacgg agggucuagc cugggugugu auggaggauc
	uagccugggu gaguauggag ggucuagccu gggugaguau ggagggucua gccugggugu
	guauggaggg ucuagccugg gugaguaugg agggucuagc cugggugugu auggaggguc
	uagccugggu gaguauggag ggucuagccu ggguguguac ggagggucua gucugagugc
	guguggggac cucagaacac ugugaccuua gcccagcaag ccaggcccuu caugaaggcc
	aagaaggcug ccaccauucc cugccagccc aagaacucca gcuuccccac ugccucugug
	ugccccuuug cguccuguga aggccauuga gaaaugccca gugugccccc ugggaaaggg
	cacggccugu gcuccugaca cgggcugugc uuggccacag aaccacccag cgucuccccu
	gcugcugucc acgucaguuc augaggcaac gucgcguggu cucagacgug gagcagccag
	cggcagcuca gagcagggca cuguguccgg cggagccaag uccacucugg gggagcucug
	gcggggacca cgggccacug cucacccacu ggccccgagg gggguguaga cgccaagacu
	cacgcaugug ugacauccgg aguccuggag ccgggugucc caguggcacc acuaggugcc
	ugcugccucc acaguggggu ucacacccag ggcuccuugg ucccccacaa ccugccccgg
	ccaggccugc agacccagac uccagccaga ccugccucac ccaccaaugc agccggggcu
	ggcgacacca gccaggugcu ggucuugggc caguucuccc acgacggcuc acccuccccu
	ccaucugcgu ugaugcucag aaucgccuac cugugccugc guguaaacca cagccucaga
	ccagcuaugg ggagaggaca acacggagga uauccagcuu ccccggucug gggugaggaa
	uguggggagc uugggcaucc uccuccagcc uccuccagcc cccaggcagu gccuuaccug
	uggugcccag aaaagugccc cuagguuggu gggucuacag gagccucagc caggcagccc
	accccacccu ggggcccugc cucaccaagg aaauaaagac ucaagccauu uaaaaaaaaa
	aaaaa

24	MRPGAPGPLWPLPWGALAWAVGFVSSMGSGNPAPGGVCWLQQGQEATCSLVLQTDVTRAE
	CCASGNIDTAWSNLTHPGNKINLLGFLGLVHCLPCKDSCDGVECGPGKACRMLGGRPRCE
	CAPDCSGLPARLQVCGSDGATYRDECELRAARCRGHPDLSVMYRGRCRKSCEHVVCPRPQ
	SCVVDQTGSAHCVVCRAAPCPVPSSPGQELCGNNNVTYISSCHMRQATCFLGRSIGVRHA
	GSCAGTPEEPPGGESAEEEENFV

25	gcauggggag gggcggcccu caaacggguc auugccauua auagagaccu caaacaccgc
	cugcuaaaaa uacccgacug gaggagcaua aaagcgcagc cgagcccagc gccccgcacu
	uuucugagca gacguccaga gcagagucag ccagcaugac cgagcgccgc guccccuucu
	cgcuccugcg gggccccagc ugggaccccu uccgcgacug guacccgcau agccgccucu
	ucgaccaggc cuucgggcug ccccggcugc cggaggagug gucgcagugg uuaggcggca
	gcagcuggcc aggcuacgug cgcccccugc cccccgccgc caucgagagc cccgcagugg
	ccgcgcccgc cuacagccgc gegcucagcc ggcaacucag cagcgggguc ucggagaucc
	ggcacacugc ggaccgcugg cgcguguccc uggaugucaa ccacuucgcc ccggacgagc
	ugacggucaa gaccaaggau ggcguggugg agaucaccgg caagcacgag gagcggcagg
	acgagcaugg cuacaucucc cggugcuuca cgcggaaaua cacgcugccc cccggugugg
	accccaccca aguuuccucc ucccuguccc cugagggcac acugaccgug gaggccccca
	ugcccaagcu agccacgcag uccaacgaga ucaccauccc agucaccuuc gagucgcggg
	cccagcuugg gggcccagaa gcugcaaaau ccgaugagac ugccgccaag uaaagccuua
	gcccggaugc ccaccccugc ugccgccacu ggcugugccu cccccgccac cuguguguuc
	uuuugauaca uuuaucuucu guuuuucuca aauaaaguuc aaagcaacca ccugucaaaa
	aaaaaaaaaa aaaa

26	MTERRVPFSLLRGPSWDPFRDWYPHSRLFDQAFGLPRLPEEWSQWLGGSSWPGYVRPLPP
	AAIESPAVAAPAYSRALSRQLSSGVSEIRHTADRWRVSLDVNHFAPDELTVKTKDGVVEI
	TGKHEERQDEHGYISRCFTRKYTLPPGVDPTQVSSSLSPEGTLTVEAPMPKLATQSNEIT
	IPVTFESRAQLGGPEAAKSDETAAK

27	agaugcgagc acugcggcug ggcgcugagg aucagccgcu uccugccugg auuccacagc
	uucgcgccgu guacugucgc cccaucccug cgcgcccagc cugccaagca gcgugccccg
	guugcaggcg ucaugcagcg ggcgcgaccc acgcucuggg ccgcugcgcu gacucugcug
	gugcugcucc gcgggccgcc gguggcgcgg gcuggcgcga gcucggcggg cuuggguccc
	guggugcgcu gcgagccgug cgacgcgcgu gcacuggccc agugcgcgcc uccgcccgcc
	gugugcgcgg agcuggugcg cgagccgggc ugcggcugcu gccugacgug cgcacugagc
	gagggccagc cgugcggcau cuacaccgag cgcuguggcu ccggccuucg cugccagccg
	ucgcccgacg aggcgcgacc gcugcaggcg cugcuggacg gccgcgggcu cugcgucaac
	gcuagugccg ucagccgccu gcgcgccuac cugcugccag cgccgccagc uccaggugag
	ccgcccgcgc caggaaaugc uagugagucg gaggaagacc gcagcgccgg caguguggag
	agcccguccg ucuccagcac gcaccgggug ucugauccca aguuccaccc ccuccauuca
	aagauaauca ucaucaagaa agggcaugcu aaagacagcc agcgcuacaa aguugacuac
	gagucucaga gcacagauac ccagaacuuc uccuccgagu ccaagcggga gacagaauau
	ggucccugcc guagagaaau ggaagacaca cugaaucacc ugaaguuccu caaugugcug
	agucccaggg guguacacau ucccaacugu gacaagaagg gauuuuauaa gaaaaagcag
	ugucgcccuu ccaaaggcag gaagcggggc uucugcuggu guguggauaa guaugggcag
	ccucucccag gcuacaccac caaggggaag gaggacgugc acugcuacag caugcagagc
	aaguagacgc cugccgcaag guuaaugugg agcucaaaua ugccuuauuu ugcacaaaag
	acugccaagg acaugaccag cagcuggcua cagccucgau uuauauuucu guuuguggug
	aacugauuuu uuuuaaacca aaguuuagaa agagguuuuu gaaaugccua ugguuucuuu
	gaaugguaaa cuugagcauc uuuucacuuu ccaguaguca gcaaagagca guuugaauuu
	ucuugucgcu uccuaucaaa auauucagag acucgagcac agcacccaga cuucaugcgc
	ccguggaaug cucaccacau guuggucgaa gcggccgacc acugacuuug ugacuuaggc
	ggcuguguug ccuauguaga gaacacgcuu cacccccacu ccccguacag ugcgcacagg
	cuuuaucgag aauaggaaaa ccuuuaaacc ccggucaucc ggacauccca acgcaugcuc
	cuggagcuca cagccuucug uggugucauu ucugaaacaa gggcguggau cccucaacca
	agaagaaugu uuaugucuuc aagugaccug uacugcuugg ggacuauugg agaaaauaag
	guggaguccu acuuguuuaa aaaauaugua ucuaagaaug uucuagggca cucugggaac
	cuauaaaggc agguauuucg ggcccuccuc uucaggaauc uuccugaaga cauggcccag
	ucgaaggccc aggauggcuu uugcugcggc cccguggggu aggagggaca gagagacagg
	gagagucagc cuccacauuc agaggcauca caaguaaugg cacaauucuu cggaugacug
	cagaaaauag uguuuuguag uucaacaacu caagacgaag cuuauuucug aggauaagcu
	cuuuaaaggc aaagcuuuau uuucaucucu caucuuuugu ccuccuuagc acaauguaaa
	aaagaauagu aauaucagaa caggaaggag gaauggcuug cuggggagcc cauccaggac
	acugggagca cauagagauu cacccauguu uguugaacuu agagucauuc ucaugcuuuu
	cuuuauaauu cacacauaua ugcagagaag auauguucuu guuaacauug uauacaacau
	agccccaaau auaguaagau cuauacuaga uaauccuaga ugaaauguua gagaugcuau
	augauacaac uguggccaug acugaggaaa ggagcucacg cccagagacu gggcugcucu
	cccggaggcc aaacccaaga aggucuggca aagucaggcu cagggagacu cugcccugcu
	gcagaccucg guguggacac acgcugcaua gagcucuccu ugaaaacaga ggggucucaa
	gacauucugc cuaccuauua gcuuuucuuu auuuuuuuaa cuuuuugggg ggaaaaguau
	uuuugagaag uuugucuugc aauguauuua uaaauaguaa auaaaguuuu uaccauuaaa
	aaaauaucuu ucccuuuguu auugaccauc ucugggcuuu guaucacuaa uuauuuuauu
	uuauuauaua auaauuauuu uauuauaaua aaauccugaa aggggaaaau aaaaaaaa

28	MQRARPTLWAAALTLLVLLRGPPVARAGASSAGLGPVVRCEPCDARALAQCAPPPAVCAE
	LVREPGCGCCLTCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVNASAV
	SRLRAYLLPAPPAPGNASESEEDRSAGSVESPSVSSTHRVSDPKFHPLHSKIIIIKKGHA
	KDSQRYKVDYESQSTDTQNFSSESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNC
	DKKGFYKKKQCRPSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK

29	agucaccgac cugcggcucc ggggccgcga gggaggaggc gcggggggcg cgaggcgggg
	gcgagcgcuu gggacucggc ccggcucccg gcuccggggg uucucguggc cgcggcagcg
	cggucucugc ggaggcggcg ggggcgcggc cagccggacc ucuuccuuuc cagagcggcc
	cgcggcgccc guuccgcggg aggcgggcgg gaggcggacg cggccuaacc ucgacgucga
	cuaccgcgcc gcccgcgaug ggaagcgccu uauaaagccg cgcccggccg gcccgagcca
	cucgccgccg cgccgccccg cugccccgaa cgcgggccau acgcagccuc cuuggaguga
	cgggccgacc ccggacgacc ccggccacgg acagacccgg gacgaccccg gccggggcgc
	gccuccugcg ggcgggcggg cggcggggcu ggggagcccu uggcgggggc augcgugcga
	cauggccucg gcgguguuug agggcacguc gcucgugaac auguucgugc gcggcugcug
	ggugaacggc auccgcaggc ucaucgucag ccggcgcggc gacgaagagg aguucuucga
	gauccgcacg gaguggucgg accgcagcgu gcucuaccug caccgcagcc uggcggaccu
	gggccgccug uggcagegcc ugcgcgacgc cuuucccgag gaccgguccg aacuggcgca
	ggggccgcug cggcaaggac ugguugccau aaaggaagcc cacgacauag agaccaggcu
	uaaugaggug gagaagcugc ugaagacgau cauaagcaug cccuguaaau auucuagauc
	ggaaguugug cucaccuucu ucgaaagauc uccucuggau cagguguuaa aaaaugauaa
	ugugcauaaa auucaaccca gcuuucaaag uccagucaaa auaucagaaa ucaugagguc
	caauggauuu uguuuagcaa auaccgaaac aauaguuauu gaccacagua uaccaaaugg
	aagagaccag cagcugggcg uggacccaac agagcauuua uuugagaaug gcagugaguu
	ucccucagag cuggaggacg gggacgaccc agcagccuac gucaccaacc ugucauauua
	ccaccugguc cccuucgaga cagacauuug ggacugaacc ucucuaucag gccucccccg
	ccucagcugu ucacugccag cucugaugcu guccacugug cuggccccca uggucacagc
	ugcugcccca gcucuggaac ucccagacgu cccaggcccc acugacgcca ggaggccugg
	guguccugca aguggagcag caggggcucc uagugggcca ggcugaccgc agagucucca
	gcaagcugag gucacagccg gaggccacag ggccuguguu cugagggucu ucacucccug
	cuuggugccc acagggcugc gcacugccag ggcagggaca guuccugugg ggcucgcucu
	cacugcuacu gcuuccuggg aggugcaucc cucuugguga aguggccuag ugccccccag
	ccacccugac uccccaccau ucccucccac ggcaaacgca caaaacgucu ugagggagau
	uuugacaagu cacccauucc aggugccaca ggcaggggau guuuaguaau ccaggauuuc
	ucugagaacu gggauaugug gccuuuuuuu uuuucuuuuu aucuuuuauc uggaaacugg
	guuuggguca accccaggag uauuugcaga aggcccagca cagugggggg uauuggcugc
	agggcaggga aggcauugcc gacuagauaa ccgugugagc uuggacugag cguugguggu
	ucuucccaaa ggaaaggaau uucuucccgg ccugccaggu cucugggccu ucagcgcggg
	uccuggugcu gcggccacac cacccugggg ugcucauuga cagagcugcc auaaugaacu
	ugaaaggacg ggaaucacga gggaagcugg ggcuccccug cccacaggag aggauccccg
	uucuucaagc uucucugcuc agugucuacu aacgaccgac auuugcuaau guaaauaaua
	guaaauuauu gagaauucua auucuuuuac acagucuguu uuuaaucuau uuuaauuaaa
	uaaaaucuau gacucuacuu ugaucugucc aggaaaaaaa aaaaaaaaaa a

30	MASAVFEGTSLVNMFVRGCWVNGIRRLIVSRRGDEEEFFEIRTEWSDRSVLYLHRSLADL
	GRLWQRLRDAFPEDRSELAQGPLRQGLVAIKEAHDIETRLNEVEKLLKTIISMPCKYSRS
	EVVLTFFERSPLDQVLKNDNVHKIQPSFQSPVKISEIMRSNGFCLANTETIVIDHSIPNG
	RDQQLGVDPTEHLFENGSEFPSELEDGDDPAAYVTNLSYYHLVPFETDIWD

31	cagucucggc ugauugccgc ugucgcuccc ggggccacgg gaugacgccu ccuccgcccg
	gacgugccgc ccccagcgca ccgcgcgccc gcgucccugg cccgccggcu cgguuggggc
	uuccgcugcg gcugcggcug cugcugcugc ucugggcggc cgccgccucc gcccagggcc
	accuaaggag cggaccccgc aucuucgccg ucuggaaagg ccauguaggg caggaccggg
	uggacuuugg ccagacugag ccgcacacgg ugcuuuucca cgagccaggc agcuccucug
	uguggguggg aggacguggc aaggucuacc ucuuugacuu ccccgagggc aagaacgcau
	cugugcgcac ggugaauauc ggcuccacaa agggguccug ucuggauaag cgggacugcg
	agaacuacau cacucuccug gagaggcgga gugaggggcu gcuggccugu ggcaccaacg
	cccggcaccc cagcugcugg aaccugguga auggcacugu ggugccacuu ggcgagauga
	gaggcuacgc ccccuucagc ccggacgaga acucccuggu ucuguuugaa ggggacgagg
	uguauuccac cauccggaag caggaauaca augggaagau cccucgguuc cgccgcaucc
	ggggcgagag ugagcuguac accagugaua cugucaugca gaacccacag uucaucaaag
	ccaccaucgu gcaccaagac caggcuuacg augacaagau cuacuacuuc uuccgagagg
	acaauccuga caagaauccu gaggcuccuc ucaauguguc ccguguggcc caguugugca
	ggggggacca ggguggggaa aguucacugu cagucuccaa guggaacacu uuucugaaag
	ccaugcuggu augcagugau gcugccacca acaagaacuu caacaggcug caagacgucu
	uccugcuccc ugaccccagc ggccagugga gggacaccag ggucuauggu guuuucucca
	accccuggaa cuacucagcc gucugugugu auucccucgg ugacauugac aaggucuucc
	guaccuccuc acucaagggc uaccacucaa gccuucccaa cccgcggccu ggcaagugcc
	ucccagacca gcagccgaua cccacagaga ccuuccaggu ggcugaccgu cacccagagg
	uggcgcagag gguggagccc auggggccuc ugaagacgcc auuguuccac ucuaaauacc
	acuaccagaa aguggccguc caccgcaugc aagccagcca cggggagacc uuucaugugc
	uuuaccuaac uacagacagg ggcacuaucc acaagguggu ggaaccgggg gagcaggagc
	acagcuucgc cuucaacauc auggagaucc agcccuuccg ccgcgcggcu gccauccaga
	ccaugucgcu ggaugcugag cggaggaagc uguaugugag cucccagugg gaggugagcc
	aggugccccu ggaccugugu gaggucuaug gcgggggcug ccacgguugc cucauguccc
	gagaccccua cugcggcugg gaccaaggcc gcugcaucuc caucuacagc uccgaacggu
	cagugcugca auccauuaau ccagccgagc cacacaagga gugucccaac cccaaaccag
	acaaggcccc acugcagaag guuucccugg ccccaaacuc ucgcuacuac cugagcugcc
	ccauggaauc ccgccacgcc accuacucau ggcgccacaa ggagaacgug gagcagagcu
	gcgaaccugg ucaccagagc cccaacugca uccuguucau cgagaaccuc acggcgcagc
	aguacggcca cuacuucugc gaggcccagg agggcuccua cuuccgcgag gcucagcacu
	ggcagcugcu gcccgaggac ggcaucaugg ccgagcaccu gcugggucau gccugugccc
	uggccgccuc ccucuggcug ggggugcugc ccacacucac ucuuggcuug cugguccacu
	agggccuccc gaggcugggc augccucagg cuucugcagc ccagggcacu agaacgucuc
	acacucagag ccggcuggcc cgggagcucc uugccugcca cuucuuccag gggacagaau
	aacccagugg aggaugccag gccuggagac guccagccgc aggcggcugc ugggccccag
	guggcgcacg gauggugagg ggcugagaau gagggcaccg acugugaagc uggggcaucg
	augacccaag acuuuaucuu cuggaaaaua uuuuucagac uccucaaacu ugacuaaaug
	cagcgaugcu cccagcccaa gagcccaugg gucggggagu ggguuuggau aggagagcug
	ggacuccauc ucgacccugg ggcugaggcc ugaguccuuc uggacucuug guacccacau
	ugccuccuuc cccucccucu cucauggcug gguggcuggu guuccugaag acccagggcu
	acccucuguc cagcccuguc cucugcagcu cccucucugg uccugggucc cacaggacag
	ccgccuugca uguuuauuga aggauguuug cuuuccggac ggaaggacgg aaaaagcucu
	auuuuuaugu uaggcuuauu ucauguauag cuacuuccga cugcaucugu augaaaauac
	caaaacuaca ugcggggggg ugggugggaa agggaggggc ugggaaggga uggguugggg
	agcgggggug aucccagucu gaggcucccg gggaugagau aagagucugg agacgggcau
	ggguucuugg agaguggcau gagcuggcuc ugcccuggga gcccggucug agggggacgu
	uguuggagcc ccuaguguug ggggugguua ugggaggggg uggggugagg gaaacgggag
	aaugaaggag aaaacugagc ccuaguuuca ccguguucau uuggaaggac gagccggguc
	cucaggggga gguuccagga cucugcccuu ggcguugagg guuggggggc ggggggccuc
	cucccuuccu cucagccccc uuccccaggg gcugugcuuc caugcuccua gccucccacc
	uucgcucagg acauguuaua acuuaggcua aacugugaaa auuccggugg ggauggccug
	ggccgagcuc uccaggcagg cggcccugcc cccagcccug uccauccauu ucagggggga
	gcugggcccu ucuccggcug ugucuggcca cccagggcag uggcuggggc caguggccuu
	ccagcuuugg ccccugcacc ucuucucaau gcacuuuaau aauguaacau auuacuaaua
	aacaagcuau uuauuuaccu gcaaaaaaa

32	MTPPPPGRAAPSAPRARVPGPPARLGLPLRLRLLLLLWAAAASAQGHLRSGPRIFAVWKG
	HVGQDRVDFGQTEPHTVLFHEPGSSSVWVGGRGKVYLFDFPEGKNASVRTVNIGSTKGSC
	LDKRDCENYITLLERRSEGLLACGTNARHPSCWNLVNGTVVPLGEMRGYAPFSPDENSLV
	LFEGDEVYSTIRKQEYNGKIPRFRRIRGESELYTSDTVMQNPQFIKATIVHQDQAYDDKI
	YYFFREDNPDKNPEAPLNVSRVAQLCRGDQGGESSLSVSKWNTFLKAMLVCSDAATNKNF
	NRLQDVFLLPDPSGQWRDTRVYGVFSNPWNYSAVCVYSLGDIDKVFRTSSLKGYHSSLPN
	PRPGKCLPDQQPIPTETFQVADRHPEVAQRVEPMGPLKTPLFHSKYHYQKVAVHRMQASH
	GETFHVLYLTTDRGTIHKVVEPGEQEHSFAFNIMEIQPFRRAAAIQTMSLDAERRKLYVS
	SQWEVSQVPLDLCEVYGGGCHGCLMSRDPYCGWDQGRCISIYSSERSVLQSINPAEPHKE
	CPNPKPDKAPLQKVSLAPNSRYYLSCPMESRHATYSWRHKENVEQSCEPGHQSPNCILFI
	ENLTAQQYGHYFCEAQEGSYFREAQHWQLLPEDGIMAEHLLGHACALAASLWLGVLPTLT
	LGLLVH

33	ccgggagucc gggcagcguu cggcgcgccg ggccggggug gcgggcggcc ccgggacccc
	ggcagcugga gaaggagccg gagcccggcc gggaugagaa ggugacgccg ccgggggcgc
	cacucgcuuu gugggggaag augcucgccu acugcgugca ggaugccacc gugguggacg
	uggagaagcg gaggaacccc uccaagcacu acguauacau aaucaaugug accuggucug
	acuccaccuc ccagacuauc uaccggaggu acagcaaguu cuuugaccug cagaugcagc
	uuuuggauaa guuucccauu gaagguggcc agaaggaccc caagcaaagg aucauccccu
	uccucccagg caagauccuc uuccgcagaa gccacauccg ggacquagcu gugaagagac
	ugaagcccau cgaugaauac ugccgggcac uuguccggcu gcccccccac aucucacagu
	gugacgaagu cuuccgguuc uucgaggcuc gacccgagga ugucaacccu ccaaaagagg
	acuauggcag uuccaagagg aaaucagugu ggcuguccag cugggcugag ucgcccaaga
	aggacgugac aggugccgac gccaccgccg agcccaugau ccuggaacag uacguggugg
	uguccaacua uaagaagcag gagaacucgg agcugagccu ccaggccggg gagguggugg
	augucaucga gaagaacgag agcggcuggu gguucgugag cacuucugag gagcagggcu
	gggucccugc caccuaccug gaggcccaga augguacucg ggaugacucc gacaucaaca
	ccucuaagac uggagaagag gagaaguaug ucaccgugca gccuuacacc agccaaagca
	aggacgagau uggcuuugag aagggcguca caguggaggu gauccggaag aaucuggaag
	gcugguggua uaucagauac cugggcaaag agggcugggc gccagcaucc uaccugaaga
	aggccaagga ugaccugcca acccggaaga agaaccuggc cggcccagug gagaucauug
	ggaacaucau ggagaucagc aaccugcuga acaagaaggc gucuggggac aaggaaacuc
	caccagccga aggcgagggc caugaggccc ccauugccaa gaaggagauc agccugccca
	uccucugcaa ugccuccaau ggcagugccg ugggcguucc ugacaggacu gucuccaggc
	uggcccaggg cucuccagcu guggccagga uugccccuca gcgggcccag aucagcuccc
	cgaaccuacg gacaagaccu ccaccacgca gagaauccag ccugggguuc caacugccaa
	agccaccaga gcccccuucu guugaggugg aguacuacac cauugccgaa uuccagucgu
	gcauuuccga uggcaucagc uuucggggug gacagaaggc agaggucauu gauaagaacu
	cagguggcug gugguacgug cagaucggug agaaggaggg cugggccccc gcaucauaca
	ucgauaagcg caagaagccc aaccugagcc gccgcacaag cacgcugacc cggcccaagg
	ugcccccgcc agcacccccc agcaagccca aggaggccga ggagggcccu acgggggcca
	gugagagcca ggacuccccg cggaagcuca aguaugagga gccugaguau gacaucccug
	cauucggcuu ugacucagag ccugagcuga gcgaggagcc cguggaggac agagccucag
	gggagaggcg gccugcccag ccccaccggc ccucgccggc cucuucucug cagcgggccc
	gcuucaaggu gggugagucu ucagaggaug uggcccugga agaggagacc aucuaugaga
	augagggcuu ccggccauau gcagaggaca cccugucagc cagaggcucc uccggggaca
	gcgacucccc aggcagcucc ucgcuguccc ugaccaggaa aaacuccccc aaaucaggcu
	cccccaaguc aucaucacuc cuaaagcuca aggcagagaa gaaugcccag gcagaaaugg
	ggaagaacca cuccucagcc uccuuuuccu cauccaucac caucaacacc acuugcugcu
	ccuccucuuc cuccuccucc ucuuccuugu ccaaaaccag uggcgaccug aagccccgcu
	cugcuucgga cgcaggcauc cgcggcacuc ccaaggucag ggcaaagaag gaugcugaug
	cgaacgcugg gcugaccucc uguccccggg ccaagccauc gguccggccc aagccauucc
	uaaaccgagc agagucgcag agccaagaga agauggacau cagcacuuua cggcgccagc
	ugagacccac aggccagcuc cguggagggc ucaagggcuc caagagugag gauucggagc
	ugcccccgca gacggccucc gaggcuccca gugagggguc uaggagaagc ucauccgacc
	ucaucacccu cccagccacc acucccccau gucccaccaa gaaggaaugg gaagggccag
	ccaccucgua caugacaugc agcgccuacc agaaggucca ggacucggag aucagcuucc
	ccgcgggcgu ggaggugcag gugcuggaga agcaggagag cggguggugg uaugugaggu
	uuggggagcu ggagggcugg gccccuuccc acuauuuggu gcuggaugag aacgagcaac
	cugaccccuc uggcaaagag cuggacacag ugcccgccaa gggcaggcag aacgaaggca
	aaucagacag ccuggagaag aucgagaggc gcguccaagc acugaacacc gucaaccaga
	gcaagaaggc cacgcccccc auccccucca aaccucccgg gggcuucggc aagaccucag
	gcacuccagc ggugaagaug aggaacggag ugcggcaggu ggcggucagg ccccagucgg
	uguuuguguc cccgccaccc aaggacaaca accuguccug cgcccugcgg aggaaugagu
	cacucacggc cacugauggc cuccgaggcg uccgacggaa cuccuccuuu agcacugcuc
	gcuccgcugc cgccgaggcc aagggccgcc uggccgaacg ggcugccagc caggguucag
	acucaccccu acugcccgcc cagcgcaaca gcauacccgu guccccugug cgccccaagc
	ccaucgagaa gucucaguuc auccacaaua accucaaaga uguguacguc ucuaucgcag
	acuacgaggg ggaugaggag acagcaggcu uccaggaggg gguguccaug gagguucugg
	agaggaaccc uaauggcugg ugguacugcc agauccugga uggugugaag cccuucaaag
	gcugggugcc uuccaacuac cuugagaaaa agaacuagca gagggccugg gcucuuccag
	ccucagugug ccucucuggc cgcccacugg augagcggug agacgaacaa aagggaaagg
	aaaaaauggg gguggggggu ggggggugga caacauucaa cacugcagaa ugggugaccu
	caaagaugcc cccuguccaa gccaucccac agcuggaagg uaggggaugg gggugcccac
	acugagugag gaagggaaug gaccagggag ucccaggccu gggacccaga gccaagaaag
	cugagauauc cugugcacca uagggacuuc accaauggau uacaugccau cugggacagg
	ccauguggga gaccccaguu gugccuuugc uacagaucug gaaaagacaa ggucaugggg
	gccuccagug uccugccccu gcuuggccca guuuugauug cuggcaucuu gccaccccag
	guaucccugg uauuguccua agcuguauuu gugaauugug cugguuuccu gggcauugcc
	acgccuacca caggugggua cauuagaagc caccacuggc uuucaggcuu gggggugucu
	ucugagcuca agccugcuuc ugggccaggc cauugucacu guuaguugaa gaaaaagcag
	uucccaggug ccagcaaaga ccaucuuuca uaacugucac ugucuuggcc uugagaagag
	agcccgcucu ccguggggca ccccauggag gacacaguac cagaguuuac agagagggug
	ggcgaagcca ccggucucuu ccuaaucugc acagacuauu uuggguauuu cugggcgggc
	aguuccuuug cauguuucgg gagagguuug uugauuuggg gcuuauaugu caggccuuug
	guuugcgucu uauuuuaggg guuguuuggg ggccugggug gucggccuca caugggaagg
	ggauggguag uggauggggu uucuguugua ucuugugggc gggugauuuu gcuuuuguuu
	uuguuucaca uucuuccccc uccacaagcc aaagucguuu cauuugguuu ccacugugug
	gacugugcug gagcuuggcg ccugccagaa aaauuugggg cuaggcaagc cccagguugc
	agacauggug aagcagagaa acuguucuuc ugguuccugc acaaccucag aggggcaaaa
	acccucccca ggaaggagga ggguguucag gagccagacu uuuggagaga aggcagcucc
	cagccugcug ggugaccgcc auucugcgug uguuccccag cugggcaggg cuggaagccu
	uacguaugaa gcauggagaa gcagccauug uccccacuau gggcagaggg gggacccggc
	uggccccuug ggucagacug gagccaacac cgccagccac ccccucuggc cugcuggcaa
	ugccacaggu gcccaagaag auggaggauc ccugugccag gagccaaccu ggucuucccg
	agggucagug ccccagugaa gacagaagcg agagaauaaa guucccugua gguccucugu
	caccuuuggg uuguguuuuu caauuguuga cauuucagag gggacccucc agaagcccag
	ccggcuuccc ccaaggacuc ccccuucgcu gggaguggau uuccacacgu gccuuugauu
	ucggacagau ugggccucac agccaccgau ucagcugcca gggucccugg acuggggguu
	gguguuuucu auagaggagg aaaggcccuc ccucacccug cuccccaccc aggcagggca
	gcaugggacc cagugucuca gugccuucaa aacccacccc caccccuacc cuaccccacc
	acaccccauc ccagaggccu ugccugggca acccuaagcc ccugucccuc gccauacacu
	gaugccuggc agcuagagca aauggcucgu guucuuuguc gaaggccugu ggugagauug
	uuuuguuucc uuuuguuuug ugaguuuguu uaaaauugaa auuaguuauu uucuucugcu
	ggacaguauu aaauagagca ggauguugag uuaaucugcu agauugcagu acuaauggua
	gugguuuagu gucuucaugu uaauauuauu uguacuuauu ugaacaauaa ugauaaagaa
	gugguucauu auuuuuuaau uaaugcacuu uaaauaaggu agaauggaaa aaacccagag
	agcaaagugc auuacuuaaa gaugcaguau auacuuuucu cauuuuuaaa cagcacauau
	uuauuaagag aaaaaaagua auuuaugacu auuuaaaaua aaauuuaaaa guagagugac
	ugucagguaa agaaccuuca auguagcuau cuuccagggg gagggcccug cagccucgcu
	ccucagaugu cugcacugag ccaguucagu cacuagugcg ccagccaggc caggagggag
	ugcagagcau gucugccaag cacagagcau cucaguugga cuggaccaca gugcucccga
	gagccugccu uuccugcccu uccccaccac cugcacugcc ccccacauuc ucccagcccc
	ccccaaggac ccccucuccu ugacccccag ugcuguaagu aauagaguca uuaaaaugca
	ggacugaaag agaccucaga ggucucuauc ccaucccuuc cuguuacugc ggcccaagga
	caggaaacaa cuuuuccaaa aucccacagc uaguuaauga cagaucuccc cacuacauua
	aucccgugcu ccuccucacu gccauuauag uuuauugugu ggucauguuu acauugugac
	auccagcccc aaggauggcu gggaguugcu caggcaucca ggaaaagugg ggaaugcugc
	caucugguga cagcauguag auuugggcag ugaacaggga uugccaugcc ccagucuucg
	caccucgccc ccucaccccc uaacucacag cacaaacccu gcaaacccaa agagaauauu
	aauacuugaa gcaagaaggg ugcaugccag uccuucuccg acauggccag gggguaccag
	gccuugugcg ccuggccucc uagcccuagc cagggcgaga gggccuuucc cuugagcuac
	ugagcugcuu ugugaugcug aaggauacug ccggcagcag gcagaggagg gagggggccu
	agggcucuga cccaucugga ggaaaaccag aucggacuaa aaaaggaaaa agaaaaacaa
	aaucuccgca guguccaaac cuaguuuccc augaguugcg acugaaaaug ugaaacgaca
	cuguauugcu guauacugca acuuuaacca uaaugagccc uucugagguc auuauugagg
	uucauauaau gcccgaagau gcagcauuug gugcuuuguu uuccuuuccg uuucaagacc
	uuuuucuuuu auugcaaggg gaaaaaaaug ucaucucccc ccaccucauc ccucuuugug
	acagcagacc uggcuguguu gccccucucc uccucacuau ucucaccagg ccaugggacu
	uugggggcag ugagagagag cuggaaaggg gcaacccuga gcagcaggug ugccccaugc
	ccuguggguu uccccuaugu ccaaugacug uagacgggcu gucuugccag gccucagccc
	cgucuacccu aagccuggcc ccaaagguuu cucuuucuuc cuccuugauu uuccaucuug
	acgaaggcau uauauagaau cauuuuaauc acuuucagau guuagagcag cguauuuuac
	aggcauuuau auccauugcu uuccucagca aggcauguua cuacuuuuau caucuaagga
	aaaaagacaa gggaauucca gucaggcauu auuuuccuau uacuaguguu ugcagaauag
	guguaggacu auuuaaguuu agaccuuggu uugguaguuc uuguuuuuaa uaaggggaaa
	aagauaaaau aaccccuauu uuuccuguua uuguauuuaa cuaauauauu auuucuuuaa
	gguuacucac uuccccuacc ccuccaaaua ccuugcauuc ucaaucaaaa auggaaacaa
	ucugagagac aggaaaagug caauauuacc aagauggaug ccagggcuca uuggggacaa
	uggagggaau accaguggcg cucagagagc aagaggcagg gagcggggug cugaaggaau
	ccuagcugug gaacaggugg guggguuggu ggaguuugau cuuguggcgu ucuccucucc
	cccuucuuug ggaagaugau aggggucccu gccagaucca cccagaagaa agggauucag
	gcauggggcc cuugaccucu aggccccagu cccuggagca gaggcaggcc cucgggagcu
	guuccuuguu uugauuucug uuguggugca gccagcugcu cagagagacc uggccuaaaa
	augacuccca gcagcccucu cucaccccag uguccugaua uuugggcugu gauccuucug
	guguauguuu gaaucuuucu aaaacugggu gcccucaguu caguuucuag gcaggaagcc
	uagaagucac cagaucuuuu ugggggaugu gagaaccuug agccgcgcac acccugguga
	gacaccaauu cccacaagcc ugcagcaggg ccuggggcug agccugggcu gcccauucau
	cucagcgacu ucagccugag aagugagccc ugccugggcu ccacacccag agaguccaua
	caaauucugc uccgggaaga gucggggggu cuauucaagu uucucugcag acaaaacuuc
	ccacaacagg uaccaaucug gccuccuucc ucagcaccgg uagagaaagc aacagaaugg
	gaaguuuccu cuggguugga gccucagagc ucugccccuc aaggugacag ggacgucccu
	guggcuuguu cccuccaccu ccaguacugu augcuugcua cuucaacccc cuauuuggug
	aauuucugca cagacacaga ucucugugcc uggaauggga cugugcccug ugcgggucuc
	ucccuuggcg uauauccauc uagauauuua gucuuugaga aucucaaagc agagcucucu
	gggaagagaa cuguccacau ugcuaaauaa uuaagauucc cucacuuuuu ugagggccau
	guguugugag agagagagag agagagagag ugugugugug ugugugugug uguguguguc
	uguguaugca aguguuggua acuucccacu ugaacuaaau aacauggggu uagagaaaaa
	aaaauaccag gcaagcuguc uccauugaac aaguccuugg caaugggcag gucccaaggg
	acucacagcu ucuggcagca agugugucau ucacacacau cauucuggcu ggagagugca
	augugucauu uuuuuuucuu uuuguaauua uuuuauuaag uauuuaguug gaaauuucac
	acuggcauua acaggucuag cauaaguggc cuaggcaguc aucccaggcu ccaaaaugaa
	gaugugcaaa agagaugcca cugggaauag aaacacugag uugguucagu uaggucaucc
	ccugcagacg ugucaucgag caggcugacu cccaccccuc agccaugcca uggguaugag
	aagccccuua uaaugaaagc ugccagcccu uucguccuug uuucagaggg ugggucaggu
	gguuggggug agaacuugcu cacggugcac ccaacaagac cugcaggugc auauaaguuu
	agucccaacu gcagggccag accaaacacu uccugggaag uguguggagg gcugugcuag
	accuuccuga guuucuggcu aaaucaucag cccuguuugg ugcagucuca ugucucugug
	guucccaagc ugcaugauca gagccaguga gaagacagga ucagugaccc acagcuuugg
	ggaaaaacag ccccacuguu aacuucccuc cugcaaaccu ggguccccag gccauaaggu
	gggcacacug gugcuuacag acugggugga gagcccuacc uuccaagguc uugaucccag
	ccugccuaua agguugggau uagcaugcaa ucccccuucc ccaauccugu cuuuuuaaaa
	ucucaaguuu gcacuuaacc uugacaacag cacccucucc uacuccaguc cuagaacuca
	guggccuuag agaauggggu ccccugcacu gaaggucccc gccuugcucc caguuccauc
	cuggccaaua ggcugcgccu caagagguga aagagaaaaa agggagggag ggaggaagaa
	uuauuuagaa caaaaggaug gcucgagcac guuagaggca agugagaggc acguugguga
	gaagagcaug ugcauguuug ggguagcugg ggccuacugu cccuucauua gggaaggagg
	cuuccagaag cggaugucuu cuagaaagaa aaauugugug aaggcugaaa aggggcuugg
	aguuuugucu uuguugauua gaaagaagga agaagucagc ucugaguguu ucaggaagaa
	gagagcaggu agaaagggaa uuuagugauu uaacacccaa ggguccagcc auagcagguu
	ggaaaauccu ccaaauuugg ccacagaaac uggcuaggaa aaaacugcca cucauugggc
	cacacgcugg guccccauca guucucaaug aauggucauu gauuuacuua gcagagagaa
	gucaccagcc acaaaccaau cuuugaguuu gcaggcccug auuccagaau auaugcaucc
	agcucccggg uucucagcug guuuugccca cuucccuuug acuguccaau ccaaagccag
	ucucucaagu uguauggcuc aaagagcagu gaccacaaug ggucauacag uagggaccca
	ccuccacaaa uuagaaccag aguucagacu ccauugggca caucugggag gaaggcaacc
	uccuuugucg ucuuguuggu accagucauu cucaaguauc ucugacaccu guggugguuc
	aguuugcuga gccugccacc ugguaugaau uagacugggu gugaugaaca uucauccaug
	gauauacccu accauuuugc guugccuuau aaccaaggca cacuccccau aagaguuuac
	ugcagagaaa gaacagcaaa acagccaccc uccuugaauu uacaacucau uaucugcaac
	agguuuucuu uaaauccaag acacaggaug ggaaaugggu uuccccacca gguacucaga
	ggucugcagg aagugacucc cgggcaaggc agacuucagu aaucccugaa gcgugagcau
	guggacugca uggcugggug gggacuggug gaugucucug gagcuccaga accuuggaga
	auuccucaug gaauuccccu cccagcucuu agugggcucu guggggucag gaggagcccu
	uccuccaggu uuuccuucuu uccuccucag cagagaaacu ggagaaagga cauuaaacuc
	agugcagucg auuugagugc ugaaauauuu ccagaaucaa ugguggugcu aaacuaucuc
	cauguuucua gcauuuuuaa uaguggaguu gguuuguuuu uaaucucauc acaaaaaugc
	agugcccuug gggaagggac cagccccuug gccugccacu uuccaggugu ccuuuaucac
	uuugacggga cucuuugguc ugcagaaaau gcucugucuu ggcaugcuuc uagacuguaa
	gauuuggguu uuguuuugua uuuuauguuu acaugcaucu uauauuuccc ugaaaacuaa
	auaaaguuuu gggccuuuuu aaccgaaaaa aaaaaaaaaa aaaa

34	MLAYCVQDATVVDVEKRRNPSKHYVYIINVTWSDSTSQTIYRRYSKFFDLQMQLLDKFPI
	EGGQKDPKQRIIPFLPGKILFRRSHIRDVAVKRLKPIDEYCRALVRLPPHISQCDEVFRF
	FEARPEDVNPPKEDYGSSKRKSVWLSSWAESPKKDVTGADATAEPMILEQYVVVSNYKKQ
	ENSELSLQAGEVVDVIEKNESGWWFVSTSEEQGWVPATYLEAQNGTRDDSDINTSKTGEV
	SKRRKAHLRRLDRRWTLGGMVNRQHSREEKYVTVQPYTSQSKDEIGFEKGVTVEVIRKNL
	EGWWYIRYLGKEGWAPASYLKKAKDDLPTRKKNLAGPVEIIGNIMEISNLLNKKASGDKE
	TPPAEGEGHEAPIAKKEISLPILCNASNGSAVGVPDRTVSRLAQGSPAVARIAPQRAQIS
	SPNLRTRPPPRRESSLGFQLPKPPEPPSVEVEYYTIAEFQSCISDGISFRGGQKAEVIDK
	NSGGWWYVQIGEKEGWAPASYIDKRKKPNLSRRTSTLTRPKVPPPAPPSKPKEAEEGPTG
	ASESQDSPRKLKYEEPEYDIPAFGFDSEPELSEEPVEDRASGERRPAQPHRPSPASSLQR
	ARFKVGESSEDVALEEETIYENEGFRPYAEDTLSARGSSGDSDSPGSSSLSLTRKNSPKS
	GSPKSSSLLKLKAEKNAQAEMGKNHSSASFSSSITINTTCCSSSSSSSSSLSKTSGDLKP
	RSASDAGIRGTPKVRAKKDADANAGLTSCPRAKPSVRPKPFLNRAESQSQEKMDISTLRR
	QLRPTGQLRGGLKGSKSEDSELPPQTASEAPSEGSRRSSSDLITLPATTPPCPTKKEWEG
	PATSYMTCSAYQKVQDSEISFPAGVEVQVLEKQESGWWYVRFGELEGWAPSHYLVLDENE
	QPDPSGKELDTVPAKGRQNEGKSDSLEKIERRVQALNTVNQSKKATPPIPSKPPGGFGKT
	SGTPAVKMRNGVRQVAVRPQSVFVSPPPKDNNLSCALRRNESLTATDGLRGVRRNSSFST
	ARSAAAEAKGRLAERAASQGSDSPLLPAQRNSIPVSPVRPKPIEKSQFIHNNLKDVYVSI
	ADYEGDEETAGFQEGVSMEVLERNPNGWWYCQILDGVKPFKGWVPSNYLEKKN

35	ucaccacggc ggcagcccuu uaaaccccuc acccagccag cgccccaucc ugucuguccg
	aacccagaca caagucuuca cuccuuccug cgagcccuga ggaagccuug ugagugcauu
	ggcuggggcu uggagggaag uugggcugga gcuggacagg agcagugggu gcauuucagg
	caggcucucc ugagguccca ggcgccagcu ccagcucccu ggcuagggaa acccacccuc
	ucagucagca ugggggccca agcuccaggc agggugggcu ggaucacuag cguccuggau
	cucucucaga cugggcagcc ccgggcucau ugaaaugccc cggaugacuu ggcuagugca
	gaggaauuga uggaaaccac cggggugaga gggaggcucc ccaucucagc cagccacauc
	cacaaggugu guguaagggu gcaggcgccg gccgguuagg ccaaggcucu acugucuguu
	gccccuccag gagaacuucc aaggagcuuu ccccagacau ggccaacaag gguccuuccu
	auggcaugag ccgcgaagug caguccaaaa ucgagaagaa guaugacgag gagcuggagg
	agcggcuggu ggaguggauc auagugcagu guggcccuga ugugggccgc ccagaccgug
	ggcgcuuggg cuuccagguc uggcugaaga auggcgugau ucugagcaag cuggugaaca
	gccuguaccc ugauggcucc aagccgguga aggugcccga gaacccaccc uccauggucu
	ucaagcagau ggagcaggug gcucaguucc ugaaggcggc ugaggacuau ggggucauca
	agacugacau guuccagacu guugaccucu uugaaggcaa agacauggca gcagugcaga
	ggacccugau ggcuuugggc agcuuggcag ugaccaagaa ugaugggcac uaccguggag
	aucccaacug guuuaugaag aaagcgcagg agcauaagag ggaauucaca gagagccagc
	ugcaggaggg aaagcauguc auuggccuuc agaugggcag caacagaggg gccucccagg
	ccggcaugac aggcuacgga cgaccucggc agaucaucag uuagagcgga gagggcuagc
	ccugagcccg gcccuccccc agcuccuugg cugcagccau cccgcuuagc cugccucacc
	cacacccgug ugguaccuuc agcccuggcc aagcuuugag gcucugucac ugagcaaugg
	uaacugcacc ugggcagcuc cucccugugc ccccagccuc agcccaacuu cuuacccgaa
	agcaucacug ccuuggcccc ucccucccgg cugcccccau caccucuacu gucuccuccc
	ugggcuaagc aggggagaag cgggcugggg guagccugga ugugggccaa guccacuguc
	cuccuuggcg gcaaaagccc auugaagaag aaccagccca gccugccccc uaucuugucc
	uggaauauuu uugggguugg aacucaaaaa aaaaaaaaaa aaaucaaucu uuucucaaaa
	aaaaaaaaaa aaaa

36	MANKGPSYGMSREVQSKIEKKYDEELEERLVEWIIVQCGPDVGRPDRGRLGFQVWLKNGV
	ILSKLVNSLYPDGSKPVKVPENPPSMVFKQMEQVAQFLKAAEDYGVIKTDMFQTVDLFEG
	KDMAAVQRTLMALGSLAVTKNDGHYRGDPNWFMKKAQEHKREFTESQLQEGKHVIGLQMG
	SNRGASQAGMTGYGRPRQIIS

37	cuccuugcac gggccggccc agcuuccccg ccccuggcgu ccgcucccuc ccgcucgcag
	cuuacuuaac cuggcccggg cggcggaggc gcucucacuu cccuggagcc gcccgcuugc
	ccgucggucg cuagcucgcu cggugcgcgu cgucccgcuc cauggcgcuc uucgugcggc
	ugcuggcucu cgcccuggcu cuggcccugg gccccgccgc gacccuggcg ggucccgcca
	agucgcccua ccagcuggug cugcagcaca gcaggcuccg gggccgccag cacggcccca
	acgugugugc ugugcagaag guuauuggca cuaauaggaa guacuucacc aacugcaagc
	agugguacca aaggaaaauc uguggcaaau caacagucau cagcuacgag ugcuguccug
	gauaugaaaa ggucccuggg gagaagggcu guccagcagc ccuaccacuc ucaaaccuuu
	acgagacccu gggagucguu ggauccacca ccacucagcu guacacggac cgcacggaga
	agcugaggcc ugagauggag gggcccggca gcuucaccau cuucgccccu agcaacgagg
	ccugggccuc cuugccagcu gaagugcugg acucccuggu cagcaauguc aacauugagc
	ugcucaaugc ccuccgcuac cauauggugg gcaggcgagu ccugacugau gagcugaaac
	acggcaugac ccucaccucu auguaccaga auuccaacau ccagauccac cacuauccua
	augggauugu aacugugaac ugugcccggc ugcugaaagc cgaccaccau gcaaccaacg
	ggguggugca ccucaucgau aaggucaucu ccaccaucac caacaacauc cagcagauca
	uugagaucga ggacaccuuu gagacccuuc gggcugcugu ggcugcauca gggcucaaca
	cgaugcuuga agguaacggc caguacacgc uuuuggcccc gaccaaugag gccuucgaga
	agaucccuag ugagacuuug aaccguaucc ugggcgaccc agaagcccug agagaccugc
	ugaacaacca caucuugaag ucagcuaugu gugcugaagc caucguugcg gggcugucug
	uagagacccu ggagggcacg acacuggagg ugggcugcag cggggacaug cucacuauca
	acgggaaggc gaucaucucc aauaaagaca uccuagccac caacggggug auccacuaca
	uugaugagcu acucauccca gacucagcca agacacuauu ugaauuggcu gcagagucug
	auguguccac agccauugac cuuuucagac aagccggccu cggcaaucau cucucuggaa
	gugagcgguu gacccuccug gcuccccuga auucuguauu caaagaugga accccuccaa
	uugaugccca uacaaggaau uugcuucgga accacauaau uaaagaccag cuggccucua
	aguaucugua ccauggacag acccuggaaa cucugggcgg caaaaaacug agaguuuuug
	uuuaucguaa uagccucugc auugagaaca gcugcaucgc ggcccacgac aagaggggga
	gguacgggac ccuguucacg auggaccggg ugcugacccc cccaaugggg acugucaugg
	auguccugaa gggagacaau cgcuuuagca ugcugguagc ugccauccag ucugcaggac
	ugacggagac ccucaaccgg gaaggagucu acacagucuu ugcucccaca aaugaagccu
	uccgagcccu gccaccaaga gaacggagca gacucuuggg agaugccaag gaacuugcca
	acauccugaa auaccacauu ggugaugaaa uccugguuag cggaggcauc ggggcccugg
	ugcggcuaaa gucucuccaa ggugacaagc uggaagucag cuugaaaaac aaugugguga
	gugucaacaa ggagccuguu gccgagccug acaucauggc cacaaauggc gugguccaug
	ucaucaccaa uguucugcag ccuccagcca acagaccuca ggaaagaggg gaugaacuug
	cagacucugc gcuugagauc uucaaacaag caucagcguu uuccagggcu ucccagaggu
	cugugcgacu agccccuguc uaucaaaagu uauuagagag gaugaagcau uagcuugaag
	cacuacagga ggaaugcacc acggcagcuc uccgccaauu ucucucagau uuccacagag
	acuguuugaa uguuuucaaa accaaguauc acacuuuaau guacaugggc cgcaccauaa
	ugagauguga gccuugugca ugugggggag gagggagaga gauguacuuu uuaaaucaug
	uucccccuaa acauggcugu uaacccacug caugcagaaa cuuggauguc acugccugac
	auucacuucc agagaggacc uaucccaaau guggaauuga cugccuaugc caagucccug
	gaaaaggagc uucaguauug uggggcucau aaaacaugaa ucaagcaauc cagccucaug
	ggaaguccug gcacaguuuu uguaaagccc uugcacagcu ggagaaaugg caucauuaua
	agcuaugagu ugaaauguuc ugucaaaugu gucucacauc uacacguggc uuggaggcuu
	uuauggggcc cuguccaggu agaaaagaaa ugguauguag agcuuagauu ucccuauugu
	gacagagcca ugguguguuu guaauaauaa aaccaaagaa acaua

38	MALFVRLLALALALALGPAATLAGPAKSPYQLVLQHSRLRGRQHGPNVCAVQKVIGTNRK
	YFTNCKQWYQRKICGKSTVISYECCPGYEKVPGEKGCPAALPLSNLYETLGVVGSTTTQL
	YTDRTEKLRPEMEGPGSFTIFAPSNEAWASLPAEVLDSLVSNVNIELLNALRYHMVGRRV
	LTDELKHGMTLTSMYQNSNIQIHHYPNGIVTVNCARLLKADHHATNGVVHLIDKVISTIT
	NNIQQIIEIEDTFETLRAAVAASGLNTMLEGNGQYTLLAPTNEAFEKIPSETLNRILGDP
	EALRDLLNNHILKSAMCAEAIVAGLSVETLEGTTLEVGCSGDMLTINGKAIISNKDILAT
	NGVIHYIDELLIPDSAKTLFELAAESDVSTAIDLFRQAGLGNHLSGSERLTLLAPLNSVF
	KDGTPPIDAHTRNLLRNHIIKDQLASKYLYHGQTLETLGGKKLRVFVYRNSLCIENSCIA
	AHDKRGRYGTLFTMDRVLTPPMGTVMDVLKGDNRFSMLVAAIQSAGLTETLNREGVYTVF
	APTNEAFRALPPRERSRLLGDAKELANILKYHIGDEILVSGGIGALVRLKSLQGDKLEVS
	LKNNVVSVNKEPVAEPDIMATNGVVHVITNVLQPPANRPQERGDELADSALEIFKQASAF
	SRASQRSVRLAPVYQKLLERMKH

39	cucaguugcu cccagcagug gcccugggac cagcucugcu ccuugcaccc cgcucccugc
	cuggacacag gcucacucgc ugccuucuuc ugggggaaac cagcuucuug ccagccacag
	cugcugccuc cgccacuggc caccgccccu guccugggag ucccuuggcc cagacaccca
	ccugacuuag uggcuccucu gcaggaaagg uggcugcccc cugcguuccu ccauccaacc
	augagcuggu gcccaucacc acugagaaug caccaaagaa uguaguggac aagggagaag
	gagccucccg ggguggaaac acacggaaaa gccucgagga caacggcucc accaggguca
	ccccgagugu ccagccccac cuccagccca ucagaaacau gagugugagc cggaccaugg
	aggacagcug ugagcuggac cugguguacg ucacagagag gaucaucgcu gucuccuucc
	ccagcacagc caaugaggag aacuuccgga gcaaccuccg ugagguggcg cagaugcuca
	aguccaaaca uggaggcaac uaccugcugu ucaaccucuc ugagcggaga ccugacauca
	cgaagcucca ugccaaggua cuggaauuug gcuggcccga ccuccacacc ccagcccugg
	agaagaucug cagcaucugu aaggccaugg acacauggcu caaugcagac ccucacaaug
	ucguuguucu acacaacaag ggaaaccgag gcaggauagg aguugucauc gcggcuuaca
	ugcacuacag caacauuucu gccagugcgg accaggcucu ggaccgguuu gcaaugaagc
	gguucuauga ggauaagauu gugcccauug gccagccauc ccaaagaagg uacgugcauu
	acuucagugg ccugcucucc ggcuccauca aaaugaacaa caagcccuug uuucugcacc
	acgugaucau gcacggcauc cccaacuuug agucuaaagg aggaugucgg ccauuucucc
	gcaucuacca ggccaugcaa ccuguguaca caucuggcau cuacaacauc ccaggagaca
	gccagacuag cgucugcauc accaucgagc caggacugcu cuugaaggga gacaucuugc
	ugaagugcua ccacaagaag uuccgaagcc cagcccgaga cgucaucuuc cgugugcagu
	uccacaccug ugccauccau gaccuggggg uugucuuugg gaaggaggac cuugaugaug
	cuuucaaaga ugaucgauuu ccagaguaug gcaaagugga guuuguauuu ucuuaugggc
	cagagaaaau ucaaggcaug gagcaccugg agaacgggcc gagcgugucu guggacuaua
	acaccuccga cccccucauc cgcugggacu ccuacgacaa cuucaguggg caucgagaug
	acggcaugga ggagguggug ggacacacgc aggggccacu agaugggagc cuguaugcua
	aggugaagaa gaaagacucc cugcacggca gcaccggggc uguuaaugcc acacguccua
	cacugucggc cacccccaac cacguggaac acacgcuuuc ugugagcagc gacucgggca
	acuccacagc cuccaccaag accgacaaga ccgacgagcc uguccccggg gccuccagug
	ccacugcugc cuugaguccc caggagaagc gggagcugga ccgccugcug aguggcuuug
	gcuuagagcg agagaagcaa ggcgccaugu accacaccca gcaccucagg ucccgcccag
	cagggggcuc ggcugugccc uccucuggac gccacguugu cccagcccag guucauguca
	augguggggc guuagcaucu gagcgggaga cagacauccu ggacgaugaa uugccaaacc
	aggaugguca cagugcgggc agcaugggca cacucucuuc ucuggacggg gucaccaaca
	ccagugaggg gggcuaccca gaggcccugu ccccacugac caacggucug gacaaguccu
	accccaugga gccuaugguc aauggaggag gcuaccccua cgagucugcc agccgggcgg
	ggccugccca ugcuggccac acggccccca ugcggcccuc cuacucugca caggaggguu
	uagcuggcua ccagagggag gggccccacc cagccuggcc acagccagug accaccuccc
	acuaugccca ugaccccagc gguauguucc gcucucaauc cuuuucggaa gcugaacccc
	agcugccccc agcuccgguc cgagggggaa gcagccggga ggcugugcaa aggggacuga
	auucguggca gcagcagcag cagcagcagc agcagccucg cccaccucca cgccagcagg
	aaagagccca cuuggagagu cuuguagcca gcaggcccag cccucagcca uuggcagaga
	cccccauccc cagucucccu gaguucccgc gagcagccuc ccagcaggag auugaacagu
	ccaucgaaac acucaauaug cugaugcugg accuggagcc agccuccgcu gcugccccac
	uacacaaguc ccagaguguc cccggggccu ggccaggggc uucuccacuc uccucccagc
	cccucucugg auccucccgu cagucccauc cacugaccca guccagaucu ggcuauaucc
	ccagugggca uucguuggga accccugagc cagccccacg ggccucucug gagucugucc
	cuccuggcag gucuuacuca ccuuaugacu aucagccaug uuuggcuggg ccuaaccagg
	auuuccauuc aaagagccca gccucuuccu ccuugccugc cuuccuuccg accacccaca
	gcccuccagg gccucagcaa cccccagccu cucucccugg ccucacugcu cagccucugc
	ucucaccaaa ggaagcgacu ucagaccccu cccggacucc agaggaggag ccauugaauu
	uagaagggcu gguggcccac aggguagcag ggguacaggc ucgggagaag cagccugcag
	agcccccagc cccucugcgg aggcgggcgg ccagugaugg acaguaugag aaccagucuc
	cagaagccac auccccucgu agcccugggg uucgcucccc uguccagugu gucuccccgg
	agcuggcucu uaccaucgcu cucaauccug gagggcggcc caaagagccc cauuugcaca
	gcuacaagga ggccuucgag gagauggagg gaaccucccc gagcagccca ccacccagug
	gggugcgguc ccccccgggu cuggccaaga caccccuguc ugcucugggc cugaaaccuc
	acaacccagc ggacauccug uugcacccca caggaguuac cagaagacgg auccagccag
	aggaagauga ggggaaggug guggucaggc ugucagaaga gccccggagc uauguggagu
	cuguggcacg gacagcggug gcuggacccc gagcucagga cucugagccc aagagcuuua
	gugcuccagc cacccaggcc uauggccaug agauaccccu gaggaacggg acccugggug
	gcuccuuugu cucccccagc ccccucucca ccagcagccc cauccucagu gcugacagca
	cuucaguggg gaguuucccg ucgggagaga gcagugacca ggguccccgg acgcccaccc
	agccucuguu ggagucuggc uuccgcucag gcagccuggg acagcccagc ccgucugccc
	agagaaacua ccagagcucu ucuccucucc cgacuguggg caguagcuac agcagccccg
	acuacucacu ucagcauuuc agcuccucuc cggaaagcca ggcucgagcu caguucagug
	uggcuggcgu ccacacggug ccugggagcc cucaggegcg ccacagaaca gugggcacca
	acacuccccc uaguccuggc uucggcuggc gggccaucaa ucccagcaug gcugccccca
	gcagucccag uuugagccau caccagauga uggguccacc aggcacuggc uuccauggua
	gcacugucuc cagcccccag agcagugcag cgaccacccc ggggagcccc agccuguguc
	ggcacccagc aggggucuac cagguuucug gccuccacaa caaaguggcc accaccccgg
	ggagucccag ccugggccgg cacccugggg cucaccaagg caaccuggcc uccggucuuc
	auagcaaugc aauagccagc ccuggaagcc ccagccuggg ccgucaccuc ggagggucug
	gaucuguggu ucccggcagc cccugcuugg accggcaugu ggccuauggu ggcuauucua
	ccccggagga ucggagaccc acacuguccc ggcagagcag ugccucuggc uaccaggcuc
	cuuccacgcc cuccuucccu gucuccccug ccuacuaccc uggccugagc agcccugcca
	ccuccccguc accagacucc gcagccuucc ggcaagggag cccaacacca gccuugccag
	agaagcgaag gaugucagug ggagaccggg caggcagccu ccccaacuau gccaccauca
	augggaaggu gucuucgccu gucgccagcg gcauguccag ucccaguggg ggcagcaccg
	ucuccuucuc ccacacucug cccgacuucu ccaaguacuc caugccagac aacagcccgg
	agacgcgggc uaaagugaag uuuguccagg acacuucuaa guauugguac aagccugaga
	ucuccaggga gcaggccauc gcgcuccuca aggaccagga gccgggggcc uucaucaucc
	gcgacaguca cuccuuccga ggcgcguacg ggcuggccau gaaggugucu ucgccaccuc
	caaccaucau gcagcagaau aaaaaaggag acaugaccca ugagcugguc aggcauuuuc
	ugauagagac uggccccaga ggagucaagc ucaagggcug ccccaaugag ccaaacuucg
	gaucgcuguc ugcccugguc uaccagcacu ccaucauccc auuggcccug ccuugcaagc
	uggucauucc aaaccgagac cccacagaug aaucgaaaga uagcuccggc ccugccaacu
	caacugcaga ccugcugaaa caaggggcag ccugcaaugu gcucuucguc aacucugugg
	acauggaguc acucacuggg ccacaggcca ucucuaaagc cacaucugag acguuggcug
	cagaccccac gccagcugcc accaucguuc acuucaaagu cucugcccag ggaaucacuc
	ugacugacaa ccagagaaag cucuuuuuca gacgccacua cccucucaac acugucaccu
	ucugugaccu ggauccacag gaaagaaagu ggaugaaaac agaggguggu gccccugcua
	agcucuucgg cuucguggcc cggaagcagg gcagcaccac ggacaacgcc ugccaccucu
	uugcugagcu ugaccccaac cagccggccu cugccaucgu caacuucguc uccaagguca
	ugcugaaugc cggccaaaag agaugaaccc ugccccuugc ccagggccag ugccaugggg
	aaggggcuug uggggagggg acccaugaau ccugaccacu cuugaaccca gaaggaggac
	uuugggccaa uuucggagga gagaagaaag ugcaacgugg ggagagggaa gugaauugca
	gaggggaggg ggaaaagaga gagagagaga gagagagaga gagagagaaa gauggaggag
	aagaacuugg auuccccugg guagauggaa acugcaaaaa cccaaagccu ccaaaacuaa
	ccagguccac cuaacacccc cucccucccc uaagaagaug gauguccuca aaagagaagg
	aacaaaccuc cuugggaauc cacauuuuuu gggggaaugg aaaagcucug ucucccuaac
	ucaacugcuu ugcaagggga aaucaagcug ggagaaucuu uuucuggcca ccuguggggu
	agguugucaa accaaacaga gccaccgugg gacaucaagu ggaagaacuu guuugcuuga
	aaguaucuca gacccaaggc accucagguc ucuuugcugu gccuccacua uauugucgug
	ugggugugug ucugcaccca cauccucaca cauugaucua gaucugccuu uauccacucg
	aauuauaaac agcucggcuu guccuugucc cauguguuug uagacacaca ugcauacugu
	ccaaagauua ggguuggugg uggcagugca gcaggggagg gacaaacaac caagcuaugg
	gugacagagg cucucuccug gugccugcac cugcacucua gugacccugg gugccgccag
	acccuucucu ucuacaaaga ccccagcagg agugggaggg ucugcaaugg caucgcccug
	uccugccuug gccagaagcc uggagcuuug guuugaggag guagagauau guguauccau
	aggaagagau cugucagaac aggcagcugu ugagcucggg gugucuuccc caaggcaugu
	ggcucagcag caagaaaggc aaguugcucc ugcuggggcc cuggacucug ccuuagcucc
	caccucucag ccuuguuauu ggguuucaug ccccuggacc agccuuaucu cagaccugcu
	uaccugcaug augccuuuuu gggggcuggg gauugagucu ugcugcucug cccagcccug
	uucuauucug cagggucccu guguuggaau ucucccuggg gaaccuacuu ucugcucagu
	gaggcuccgg ccagaaaccu ggaguccuua uccuccccuc uguaaguguu uuagggucug
	gcuuuugcag gcacccucug accucagcag agcuccuggg ccugcugccu gcacaccaca
	ucgccuaccu acaaugccaa agccucacug ucacccuuuc ugccuugguu ucccuagcug
	agccacgcug cccaugcagc agagggcaga aggcuugcac uugggccaaa gggccuaagg
	uccacuggac aguugggaaa acaccugacc accauuuaag gacucuaagc cagaauggaa
	aauucaccag gacuccauuc uuaagccuau gcgagucccc uagagagagg cauuguacug
	auauauaaau auuauauaau auauacauga gacauacuga cagaaucugu aagcuaauaa
	aauguaagaa aagguuaaaa aaagaauagg uaaauugaca agaaguauuu auuguuuuuc
	cauauugcuu uauugccuuc cuuggggaua aaccaauucc uauccuuuuu uauaugugua
	aguaaagccu gaaguguagg gggccuuugu ucuugaagca gccagggucu ccuugcccug
	gccuuggccu ucccuagacu guguggggcu cagcauuggg agggguugca caugucccag
	ccuuuggccc ccuuacuuuu cagcaagcca ggggcccagc agucagcucc caggaugugu
	ggggagcugu cccugacucu gcaggccuga gcgagugugu gagcaugcgg ggacaugggu
	guguauggca cacauaggug cguguguguc uuuuguauuu uuucuccucc aaggagcugu
	gucagugugg acguucuguu ucagggaguu ggaaaggagg gugucugcag aagguggaga
	gcaggggcag aggccccacu ggccaccccc ugcuucccag agugaaaccu ugugccuggu
	gaccaaaguc ccuccaaagu gcucuuccuu cuggguuauu caagccaaau aucuggguuu
	cccccucucc ucauucccua gcaaacccca auuaucuucc aagauaggag auauuuccca
	uccccuuccu uuguaaauau cucaucuccc acuggagagc ccaggagccu auuccuggca
	uggauguucu guccacacuu gaggcugggc gguguaucag acccuucaag cagccuggcu
	ggggcccagg acugagucug gggucagcuu ucacggucgc uuuucccuuc cucaccaccc
	accacagccc accuugcaug cauggccagc cccuccacuc cagccugagc caugugugcc
	ccugcgggag gacccauuca ugccagaaag cugguaacuc ccucccagca ucccugcgga
	aggagucagu uucugagagu gugacuuuuc aaggcgaaug auggggaagg guuccccagu
	ccccacagug gccccaccuc ugggcccugc accagagccc uucuguguca cggcgggcug
	ugcacccaug cacacaccua cgcacacaca acacuccgca cugcaguaua uucuugccaa
	agauuuccuu uaaaagcaag cacuuuuacu aauuauuauu uuguaaaugu uuaucuucuu
	cugucuucuc ccucccugaa ucuauuuuac uguuguuuau uguugaaucu gugugucagc
	caggagagcg cugucuggcc uugaacaugg gcugggaugg gaaagggucu gggagaagau
	gggcaacaaa gagccaggga gucauggaca ucgcagcgac gcagacccca gcagguucag
	ucccgugcug ccaccagcug uccagcuggg ugucuggagg gaagagggca gaggaggguc
	augucccuuc agcuggggga ggggcccagu gagcuccacg uggcuuuuuc ccaaagggag
	caagagggaa ggauugggcg agaaaacaau ggagagggga ccugcgaagg aaaacaggga
	ggaagugagc gguuugauca gccugcuauc acgguguucu ggcucucuua uuuagccagg
	cgcuuaaggg acagauacau cacauccuaa guuugggaaa ggccuuugac ccaugucauc
	ugagcgucuc cuccaguagc ucugaaagcu guggacacca auggccagga uuccuucucc
	ccugguuuuu gaggaucccu gggucuucug agacuggcca ggagagggau gguggggcca
	gugguugugu gaaagcagga ggggcagccc uccuggacaa gugugauccc ccuauaaacg
	gcucucagga gguuagugag uaggagauuc ugccuuguuc ugaugagccu gugcaggggc
	uccaggggag caugcugucc agggggcaca gaaggguggu gagugugauc aaaucuaguc
	ucacucccac uuuuuagucu cacuccuacu uuuguccacc accccugccu ccuggaucuu
	cucccacuuu uuuuuucagc uuuaggaccu ggggagaucc ugugagucaa ggcagacacc
	caauccugcc cccacacucg ggguccucca agagguuggg gggcagaguc ccagagcagc
	ccuuuacccc agguccaggc ccuggaaucc ugagacucgc guuuccuugg ccagugguaa
	cacaggacgu gugugcgcau gugcaagugu ggauguaugu gugugcgugu guuuugcuca
	uuucuuuagg gaacuuggga gucgggguug gaggugcugg gcaauggaac uucaaauuca
	augucgccca gcagugaggg gagucgggag gugaggccug uaggccaacc aauuggugga
	gucucagcga uagcccaggu gagaaguggu ucacccagag gggcagggug ggggccucgg
	gcagaucugu cccucuuggc accucugucc ucaaaugucc aaaauguugg aggaccucug
	uucauauccc acgccugggc ucuugccagc aguggaguua cuguagaggg augucccaag
	cuuguuuucc aaucaguguu aagcuguuug aaacucuccu gugucugugu uuuguuugug
	cgugugugug agagcacauc agugugugca ggcuguguuu ccccauuucu cuccucccuu
	cagacccauc auugagaaca aauguaagaa aucccuuccc accacccucc cugccuccca
	ggcccucugc gggggaaaca agaucaccca gcauccuucc ccaccccagc uguguauuua
	uauagaugga aauauacuuu auauuuugua ucaucgugcc uauagccgcu gccaccgugu
	auaaauccug guguaugcuc cuuauccugg acaugaaugu auuguacacu gacgcguccc
	cacuccugua cagcugcuuu guuucuuugc aaugcauugu auggcuuuau aaaugauaaa
	guuaaagaaa acucugaaaa aaaaaaaaaa aaaa

40	MSVSRTMEDSCELDLVYVTERIIAVSFPSTANEENFRSNLREVAQMLKSKHGGNYLLFNL
	SERRPDITKLHAKVLEFGWPDLHTPALEKICSICKAMDTWLNADPHNVVVLHNKGNRGRI
	GVVIAAYMHYSNISASADQALDRFAMKRFYEDKIVPIGQPSQRRYVHYFSGLLSGSIKMN
	NKPLFLHHVIMHGIPNFESKGGCRPFLRIYQAMQPVYTSGIYNIPGDSQTSVCITIEPGL
	LLKGDILLKCYHKKFRSPARDVIFRVQFHTCAIHDLGVVFGKEDLDDAFKDDRFPEYGKV
	EFVFSYGPEKIQGMEHLENGPSVSVDYNTSDPLIRWDSYDNFSGHRDDGMEEVVGHTQGP
	LDGSLYAKVKKKDSLHGSTGAVNATRPTLSATPNHVEHTLSVSSDSGNSTASTKTDKTDE
	PVPGASSATAALSPQEKRELDRLLSGFGLEREKQGAMYHTQHLRSRPAGGSAVPSSGRHV
	VPAQVHVNGGALASERETDILDDELPNQDGHSAGSMGTLSSLDGVTNTSEGGYPEALSPL
	TNGLDKSYPMEPMVNGGGYPYESASRAGPAHAGHTAPMRPSYSAQEGLAGYQREGPHPAW
	PQPVTTSHYAHDPSGMFRSQSFSEAEPQLPPAPVRGGSSREAVQRGLNSWQQQQQQQQQP
	RPPPRQQERAHLESLVASRPSPQPLAETPIPSLPEFPRAASQQEIEQSIETLNMLMLDLE
	PASAAAPLHKSQSVPGAWPGASPLSSQPLSGSSRQSHPLTQSRSGYIPSGHSLGTPEPAP
	RASLESVPPGRSYSPYDYQPCLAGPNQDFHSKSPASSSLPAFLPTTHSPPGPQQPPASLP
	GLTAQPLLSPKEATSDPSRTPEEEPLNLEGLVAHRVAGVQAREKQPAEPPAPLRRRAASD
	GQYENQSPEATSPRSPGVRSPVQCVSPELALTIALNPGGRPKEPHLHSYKEAFEEMEGTS
	PSSPPPSGVRSPPGLAKTPLSALGLKPHNPADILLHPTGVTRRRIQPEEDEGKVVVRLSE
	EPRSYVESVARTAVAGPRAQDSEPKSFSAPATQAYGHEIPLRNGTLGGSFVSPSPLSTSS
	PILSADSTSVGSFPSGESSDQGPRTPTQPLLESGFRSGSLGQPSPSAQRNYQSSSPLPTV
	GSSYSSPDYSLQHFSSSPESQARAQFSVAGVHTVPGSPQARHRTVGTNTPPSPGFGWRAI
	NPSMAAPSSPSLSHHQMMGPPGTGFHGSTVSSPQSSAATTPGSPSLCRHPAGVYQVSGLH
	NKVATTPGSPSLGRHPGAHQGNLASGLHSNAIASPGSPSLGRHLGGSGSVVPGSPCLDRH
	VAYGGYSTPEDRRPTLSRQSSASGYQAPSTPSFPVSPAYYPGLSSPATSPSPDSAAFRQG
	SPTPALPEKRRMSVGDRAGSLPNYATINGKVSSPVASGMSSPSGGSTVSFSHTLPDFSKY
	SMPDNSPETRAKVKFVQDTSKYWYKPEISREQAIALLKDQEPGAFIIRDSHSFRGAYGLA
	MKVSSPPPTIMQQNKKGDMTHELVRHFLIETGPRGVKLKGCPNEPNFGSLSALVYQHSII
	PLALPCKLVIPNRDPTDESKDSSGPANSTADLLKQGAACNVLFVNSVDMESLTGPQAISK
	ATSETLAADPTPAATIVHFKVSAQGITLTDNQRKLFFRRHYPLNTVTFCDLDPQERKWMK
	TEGGAPAKLFGFVARKQGSTTDNACHLFAELDPNQPASAIVNFVSKVMLNAGQKR

41	ggaagggggg caggagaaaa aagcuuuucc aaaaaaguau uggcugucuu gaggaaugcg
	gucgcccccu ugggaaagua cauaucuggg agaagcaggc ggcuccgcgc ucgcacuccc
	gcuccuccgc ccgaccgcgc gcucgccccg ccgcuccugc ugcagcccca gggccccucg
	ccgccgccac cauggacgcc aucaagaaga agaugcagau gcugaagcuc gacaaggaga
	acgccuugga ucgagcugag caggcggagg ccgacaagaa ggcggcggaa gacaggagca
	agcagcugga agaugagcug gugucacugc aaaagaaacu caagggcacc gaagaugaac
	uggacaaaua cucugaggcu cucaaagaug cccaggagaa gcuggagcug gcagagaaaa
	aggccaccga ugcugaagcc gacguagcuu cucugaacag acgcauccag cugguugagg
	aagaguugga ucgugcccag gagcgucugg caacagcuuu gcagaagcug gaggaagcug
	agaaggcagc agaugagagu gagagaggca ugaaagucau ugagagucga gcccaaaaag
	augaagaaaa aauggaaauu caggagaucc aacugaaaga ggccaagcac auugcugaag
	augccgaccg caaauaugaa gagguggccc guaagcuggu caucauugag agcgaccugg
	aacgugcaga ggagcgggcu gagcucucag aaggcaaaug ugccgagcuu gaagaagaau
	ugaaaacugu gacgaacaac uugaagucac uggaggcuca ggcugagaag uacucgcaga
	aggaagacag auaugaggaa gagaucaagg uccuuuccga caagcugaag gaggcugaga
	cucgggcuga guuugcggag aggucaguaa cuaaauugga gaaaagcauu gaugacuuag
	aagacgagcu guacgcucag aaacugaagu acaaagccau cagcgaggag cuggaccacg
	cucucaacga uaugacuucc auauaaguuu cuuugcuuca cuucucccaa gacucccucg
	ucgagcugga ugucccaccu cucugagcuc ugcauuuguc uauucuccag cugacccugg
	uucucucucu uagcauccug ccuuagagcc aggcacacac ugugcuuucu auuguacaga
	agcucuucgu uucaguguca aauaaacacu guguaagcua aaaaaa

42	MDAIKKKMQMLKLDKENALDRAEQAEADKKAAEDRSKQLEDELVSLQKKLKGTEDELDKY
	SEALKDAQEKLELAEKKATDAEADVASLNRRIQLVEEELDRAQERLATALQKLEEAEKAA
	DESERGMKVIESRAQKDEEKMEIQEIQLKEAKHIAEDADRKYEEVARKLVIIESDLERAE
	ERAELSEGKCAELEEELKTVTNNLKSLEAQAEKYSQKEDRYEEEIKVLSDKLKEAETRAE
	FAERSVTKLEKSIDDLEDELYAQKLKYKAISEELDHALNDMTSI

43	acuuuccccc cucggcgccc caccggcucc cgcgcgccuc cccucgcgcc cgagcuucga
	gccaagcagc guccugggga gcgcgucaug gccuuaccag ugaccgccuu gcuccugccg
	cuggccuugc ugcuccacgc cgccaggccg agccaguucc gggugucgcc gcuggaucgg
	accuggaacc ugggcgagac aguggagcug aagugccagg ugcugcuguc caacccgacg
	ucgggcugcu cguggcucuu ccagccgcgc ggcgccgccg ccagucccac cuuccuccua
	uaccucuccc aaaacaagcc caaggcggcc gaggggcugg acacccagcg guucucgggc
	aagagguugg gggacaccuu cguccucacc cugagcgacu uccgccgaga gaacgagggc
	uacuauuucu gcucggcccu gagcaacucc aucauguacu ucagccacuu cgugccgguc
	uuccugccag cgaagcccac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc
	aucgcgucgc agccccuguc ccugcgccca gaggcgugcc ggccagcggc ggggggcgca
	gugcacacga gggggcugga cuucgccugu gauaucuaca ucugggcgcc ccuggccggg
	acuugugggg uccuucuccu gucacugguu aucacccuuu acugcaacca caggaaccga
	agacguguuu gcaaaugucc ccggccugug gucaaaucgg gagacaagcc cagccuuucg
	gcgagauacg ucuaacccug ugcaacagcc acuacauuac uucaaacuga gauccuuccu
	uuugagggag caaguccuuc ccuuucauuu uuuccagucu uccucccugu guauucauuu
	ucaugauuau uauuuuagug ggggcggggu gggaaagauu acuuuuucuu uauguguuug
	acgggaaaca aaacuaggua aaaucuacag uacaccacaa gggucacaau acuguugugc
	gcacaucgcg guagggcgug gaaaggggca ggccagagcu acccgcagag uucucagaau
	caugcugaga gagcuggagg cacccaugcc aucucaaccu cuuccccgcc cguuuuacaa
	agggggaggc uaaagcccag agacagcuug aucaaaggca cacagcaagu caggguugga
	gcaguagcug gagggaccuu gucucccagc ucagggcucu uuccuccaca ccauucaggu
	cuuucuuucc gaggccccug ucucagggug aggugcuuga gucuccaacg gcaagggaac
	aaguacuucu ugauaccugg gauacugugc ccagagccuc gaggagguaa ugaauuaaag
	aagagaacug ccuuuggcag aguucuauaa uguaaacaau aucagacuuu uuuuuuuuau
	aaucaagccu aaaauuguau agaccuaaaa uaaaaugaag uggugagcuu aacccuggaa
	aaugaauccc ucuaucucua aagaaaaucu cugugaaacc ccuacgugga ggcggaauug
	cucucccagc ccuugcauug cagaggggcc caugaaagag gacaggcuac cccuuuacaa
	auagaauuug agcaucagug agguuaaacu aaggcccucu ugaaucucug aauuugagau
	acaaacaugu uccugggauc acugaugacu uuuuauacuu uguaaagaca auuguuggag
	agccccucac acagcccugg ccuccgcuca acuagcagau acagggauga ggcagaccug
	acucucuuaa ggaggcugag agcccaaacu gcugucccaa acaugcacuu ccuugcuuaa
	gguaugguac aagcaaugcc ugcccauugg agagaaaaaa cuuaaguaga uaaggaaaua
	agaaccacuc auaauucuuc accuuaggaa uaaucuccug uuaauauggu guaca

44	MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQP
	RGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSN
	SIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA
	CDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

45	auccaauaca ggagugacuu ggaacuccau ucuaucacua ugaagaaaag ugguguucuu
	uuccucuugg gcaucaucuu gcugguucug auuggagugc aaggaacccc aguagugaga
	aagggucgcu guuccugcau cagcaccaac caagggacua uccaccuaca auccuugaaa
	gaccuuaaac aauuugcccc aagcccuucc ugcgagaaaa uugaaaucau ugcuacacug
	aagaauggag uucaaacaug ucuaaaccca gauucagcag augugaagga acugauuaaa
	aagugggaga aacaggucag ccaaaagaaa aagcaaaaga augggaaaaa acaucaaaaa
	aagaaaguuc ugaaaguucg aaaaucucaa cguucucguc aaaagaagac uacauaagag
	accacuucac caauaaguau ucuguguuaa aaauguucua uuuuaauuau accgcuauca
	uuccaaagga ggauggcaua uaauacaaag gcuuauuaau uugacuagaa aauuuaaaac
	auuacucuga aauuguaacu aaaguuagaa aguugauuuu aagaauccaa acguuaagaa
	uuguuaaagg cuaugauugu cuuuguucuu cuaccaccca ccaguugaau uucaucaugc
	uuaaggccau gauuuuagca auacccaugu cuacacagau guucacccaa ccacauccca
	cucacaacag cugccuggaa gagcagcccu aggcuuccac guacugcagc cuccagagag
	uaucugaggc acaugucagc aaguccuaag ccuguuagca ugcuggugag ccaagcaguu
	ugaaauugag cuggaccuca ccaagcugcu guggccauca accucuguau uugaaucagc
	cuacaggccu cacacacaau gugucugaga gauucaugcu gauuguuauu ggguaucacc
	acuggagauc accagugugu ggcuuucaga gccuccuuuc uggcuuugga agccauguga
	uuccaucuug cccgcucagg cugaccacuu uauuucuuuu uguuccccuu ugcuucauuc
	aagucagcuc uucuccaucc uaccacaaug cagugccuuu cuucucucca gugcaccugu
	cauaugcucu gauuuaucug agucaacucc uuucucaucu uguccccaac accccacaga
	agugcuuucu ucucccaauu cauccucacu caguccagcu uaguucaagu ccugccucuu
	aaauaaaccu uuuuggacac acaaauuauc uuaaaacucc uguuucacuu gguucaguac
	cacaugggug aacacucaau gguuaacuaa uucuugggug uuuauccuau cucuccaacc
	agauugucag cuccuugagg gcaagagcca caguauauuu cccuguuucu uccacagugc
	cuaauaauac uguggaacua gguuuuaaua auuuuuuaau ugauguuguu augggcagga
	uggcaaccag accauugucu cagagcaggu gcuggcucuu uccuggcuac uccauguugg
	cuagccucug guaaccucuu acuuauuauc uucaggacac ucacuacagg gaccagggau
	gaugcaacau ccuugucuuu uuaugacagg auguuugcuc agcuucucca acaauaagaa
	gcacguggua aaacacuugc ggauauucug gacuguuuuu aaaaaauaua caguuuaccg
	aaaaucauau aaucuuacaa ugaaaaggac uuuauagauc agccagugac caaccuuuuc
	ccaaccauac aaaaauuccu uuucccgaag gaaaagggcu uucucaauaa gccucagcuu
	ucuaagaucu aacaagauag ccaccgagau ccuuaucgaa acucauuuua ggcaaauaug
	aguuuuauug uccguuuacu uguuucagag uuuguauugu gauuaucaau uaccacacca
	ucucccauga agaaagggaa cggugaagua cuaagcgcua gaggaagcag ccaagucggu
	uaguggaagc augauuggug cccaguuagc cucugcagga uguggaaacc uccuuccagg
	ggagguucag ugaauugugu aggagagguu gucuguggcc agaauuuaaa ccuauacuca
	cuuucccaaa uugaaucacu gcucacacug cugaugauuu agagugcugu ccgguggaga
	ucccacccga acgucuuauc uaaucaugaa acucccuagu uccuucaugu aacuucccug
	aaaaaucuaa guguuucaua aauuugagag ucugugaccc acuuaccuug caucucacag
	guagacagua uauaacuaac aaccaaagac uacauauugu cacugacaca cacguuauaa
	ucauuuauca uauauauaca uacaugcaua cacucucaaa gcaaauaauu uuucacuuca
	aaacaguauu gacuuguaua ccuuguaauu ugaaauauuu ucuuuguuaa aauagaaugg
	uaucaauaaa uagaccauua aucag

46	MKKSGVLFLLGIILLVLIGVQGTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEK
	IEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSR
	QKKTT

47	gagacauucc ucaauugcuu agacauauuc ugagccuaca gcagaggaac cuccagucuc
	agcaccauga aucaaacugc gauucugauu ugcugccuua ucuuucugac ucuaaguggc
	auucaaggag uaccucucuc uagaaccgua cgcuguaccu gcaucagcau uaguaaucaa
	ccuguuaauc caaggucuuu agaaaaacuu gaaauuauuc cugcaagcca auuuugucca
	cguguugaga ucauugcuac aaugaaaaag aagggugaga agagaugucu gaauccagaa
	ucgaaggcca ucaagaauuu acugaaagca guuagcaagg aaaugucuaa aagaucuccu
	uaaaaccaga ggggagcaaa aucgaugcag ugcuuccaag gauggaccac acagaggcug
	ccucucccau cacuucccua cauggaguau augucaagcc auaauuguuc uuaguuugca
	guuacacuaa aaggugacca augaugguca ccaaaucagc ugcuacuacu ccuguaggaa
	gguuaauguu caucauccua agcuauucag uaauaacucu acccuggcac uauaauguaa
	gcucuacuga ggugcuaugu ucuuagugga uguucugacc cugcuucaaa uauuucccuc
	accuuuccca ucuuccaagg guacuaagga aucuuucugc uuugggguuu aucagaauuc
	ucagaaucuc aaauaacuaa aagguaugca aucaaaucug cuuuuuaaag aaugcucuuu
	acuucaugga cuuccacugc cauccuccca aggggcccaa auucuuucag uggcuaccua
	cauacaauuc caaacacaua caggaaggua gaaauaucug aaaauguaug uguaaguauu
	cuuauuuaau gaaagacugu acaaaguaua agucuuagau guauauauuu ccuauauugu
	uuucagugua cauggaauaa cauguaauua aguacuaugu aucaaugagu aacaggaaaa
	uuuuaaaaau acagauagau auaugcucug cauguuacau aagauaaaug ugcugaaugg
	uuuucaaaua aaaaugaggu acucuccugg aaauauuaag aaagacuauc uaaauguuga
	aagaucaaaa gguuaauaaa guaauuauaa cu

48	MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRV
	EIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP

49	augaggaacu ccuauagauu ucuggcaucc ucucucucag uugucguuuc ucuccugcua
	auuccugaag augucuguga aaaaauuauu ggaggaaaug aaguaacucc ucauucaaga
	cccuacaugg uccuacuuag ucuugacaga aaaaccaucu gugcuggggc uuugauugca
	aaagacuggg uguugacugc agcucacugu aacuugaaca aaagguccca ggucauucuu
	ggggcucacu caauaaccag ggaagagcca acaaaacaga uaaugcuugu uaagaaagag
	uuucccuauc caugcuauga cccagccaca cgcgaaggug accuuaaacu uuuacagcug
	acggaaaaag caaaaauuaa caaauaugug acuauccuuc aucuaccuaa aaagggggau
	gaugugaaac caggaaccau gugccaaguu gcaggguggg ggaggacuca caauagugca
	ucuugguccg auacucugag agaagucaau aucaccauca uagacagaaa agucugcaau
	gaucgaaauc acuauaauuu uaacccugug auuggaauga auaugguuug ugcuggaagc
	cuccgaggug gaagagacuc gugcaaugga gauucuggaa gcccuuuguu gugcgagggu
	guuuuccgag gggucacuuc cuuuggccuu gaaaauaaau gcggagaccc ucgugggccu
	ggugucuaua uucuucucuc aaagaaacac cucaacugga uaauuaugac uaucaaggga
	gcaguuuaaa uaaccguuuc cuuucauuua cuguggcuuc uuaaucuuuu cacaaauaaa
	aucaauuug


50	MRNSYRFLASSLSVVVSLLLIPEDVCEKIIGGNEVTPHSRPYMVLLSLDRKTICAGALIA
	KDWVLTAAHCNLNKRSQVILGAHSITREEPTKQIMLVKKEFPYPCYDPATREGDLKLLQL
	MEKAKINKYVTILHLPKKGDDVKPGTMCQVAGWGRTHNSASWSDTLREVNITIIDRKVCN
	DRNHYNFNPVIGMNMVCAGSLRGGRDSCNGDSGSPLLCEGVFRGVTSFGLENKCGDPRGP
	GVYILLSKKHLNWIIMTIKGAV

51	ugagaagaug caaccaaucc ugcuucugcu ggccuuccuc cugcugccca gggcagaugc
	aggggagauc aucgggggac augaggccaa gccccacucc cgccccuaca uggcuuaucu
	uaugaucugg gaucagaagu cucugaagag gugcgguggc uuccugauac aagacgacuu
	cgugcugaca gcugcucacu guuggggaag cuccauaaau gucaccuugg gggcccacaa
	uaucaaagaa caggagccga cccagcaguu uaucccugug aaaagaccca ucccccaucc
	agccuauaau ccuaagaacu ucuccaacga caucaugcua cugcagcugg agagaaaggc
	caagcggacc agagcugugc agccccucag gcuaccuagc aacaaggccc aggugaagcc
	agggcagaca ugcagugugg ccggcugggg gcagacggcc ccccugggaa aacacucaca
	cacacuacaa gaggugaaga ugacagugca ggaagaucga aagugcgaau cugacuuacg
	ccauuauuac gacaguacca uugaguugug cgugggggac ccagagauua aaaagacuuc
	cuuuaagggg gacucuggag gcccucuugu guguaacaag guggcccagg gcauugucuc
	cuauggacga aacaauggca ugccuccacg agccugcacc aaagucucaa gcuuuguaca
	cuggauaaag aaaaccauga aacgcuacua acuacaggaa gcaaacuaag cccccgcugu
	aaugaaacac cuucucugga gccaagucca gauuuacacu gggagaggug ccagcaacug
	aauaaauacc ucucccagug uaaaucugga gccaagucca gauuuacacu gggagaggug
	ccagcaacug aauaaauacc ucuuagcuga gugg

52	MQPILLLLAFLLLPRADAGEIIGGHEAKPHSRPYMAYLMIWDQKSLKRCGGFLIRDDFVL
	TAAHCWGSSINVTLGAHNIKEQEPTQQFIPVKRPIPHPAYNPKNFSNDIMLLQLERKAKR
	TRAVQPLRLPSNKAQVKPGQTCSVAGWGQTAPLGKHSHTLQEVKMTVQEDRKCESDLRHY
	YDSTIELCVGDPEIKKTSFKGDSGGPLVCNKVAQGIVSYGRNNGMPPRACTKVSSFVHWI
	KKTMKRY

53	auggcagccc gucugcuccu ccugggcauc cuucuccugc ugcugccccu gcccgucccu
	gccccgugcc acacagccgc acgcucagag ugcaagcgca gccacaaguu cgugccuggu
	gcauggcugg ccggggaggg uguggacgug accagccucc gccgcucggg cuccuuccca
	guggacacac aaagguuccu gcggcccgac ggcaccugca cccucuguga aaaugcccua
	caggagggca cccuccagcg ccugccucug gogcucacca acuggcgggc ccagggcucu
	ggcugccagc gccauguaac cagggccaaa gucagcucca cugaagcugu ggcccgggau
	gcggcucgua gcauccgcaa cgacuggaag gucgggcugg acgugacucc uaagcccacc
	agcaaugugc augugucugu ggccggcuca cacucacagg cagccaacuu ugcagcccag
	aagacccacc aggaccagua cagcuucagc acugacacgg uggagugccg cuucuacagu
	uuccaugugg uacacacucc cccgcugcac ccugacuuca agagggcccu cggggaccug
	ccccaccacu ucaacgccuc cacccagccc gccuaccuca ggcuuaucuc caacuacggc
	acccacuuca uccgggcugu ggagcugggu ggccgcauau cggcccucac ugcccugcgc
	accugcgagc uggcccugga agggcucacg gacaacgagg uggaggacug ccugacuguc
	gaggcccagg ucaacauagg cauccacggc agcaucucug ccgaagccaa ggccugugag
	gagaagaaga agaagcacaa gaugacggcc uccuuccacc aaaccuaccg ggagcgccac
	ucggaagugg uuggcggcca ucacaccucc auuaacgacc ugcuguucgg gauccaggcc
	gggcccgagc aguacucagc cuggguaaac uccgugcccg gcagcccugg ccugguggac
	uacacccugg aaccccugca cgugcugcug gacagccagg acccgcggcg ggaggcacug
	aggagggccc ugagucagua ccugacggac agggcucgcu ggagggacug cagccggccg
	ugcccaccag ggcggcagaa gagcccccga gacccaugcc agugugugug ccauggcuca
	gcggucacca cccaggacug cugcccucgg cagaggggcc uggcccagcu ggaggugacc
	uucauccaag cauggagccu guggggggac ugguucacug ccacggaugc cuaugugaag
	cucuucuuug guggccagga gcugaggacg agcaccgugu gggacaauaa caaccccauc
	uggucagugc ggcuggauuu uggggaugug cuccuggcca caggggggcc ccugagguug
	caggucuggg aucaggacuc uggcagggac gaugaccucc uuggcaccug ugaucaggcu
	cccaagucug guucccauga ggugagaugc aaccugaauc auggccaccu aaaauuccgc
	uaucaugcca ggugcuugcc ccaccuggga ggaggcaccu gccuggacua ugucccccaa
	augcuucugg gggagccucc aggaaaccgg aguggggccg ugugguga

54	MAARLLLLGILLLLLPLPVPAPCHTAARSECKRSHKFVPGAWLAGEGVDVTSLRRSGSFP
	VDTQRFLRPDGTCTLCENALQEGTLQRLPLALTNWRAQGSGCQRHVTRAKVSSTEAVARD
	AARSIRNDWKVGLDVTPKPTSNVHVSVAGSHSQAANFAAQKTHQDQYSFSTDTVECRFYS
	FHVVHTPPLHPDFKRALGDLPHHFNASTQPAYLRLISNYGTHFIRAVELGGRISALTALR
	TCELALEGLTDNEVEDCLTVEAQVNIGIHGSISAEAKACEEKKKKHKMTASFHQTYRERH
	SEVVGGHHTSINDLLFGIQAGPEQYSAWVNSLPGSPGLVDYTLEPLHVLLDSQDPRREAL
	RRALSQYLTDRARWRDCSRPCPPGRQKSPRDPCQCVCHGSAVTTQDCCPRQRGLAQLEVT
	FIQAWGLWGDWFTATDAYVKLFFGGQELRTSTVWDNNNPIWSVRLDFGDVLLATGGPLRL
	QVWDQDSGRDDDLLGTCDQAPKSGSHEVRCNLNHGHLKFRYHARCLPHLGGGTCLDYVPQ
	MLLGEPPGNRSGAVW

55	ugaagaucag cuauuagaag agaaagauca guuaaguccu uuggaccuga ucagcuugau
	acaagaacua cugauuucaa cuucuuuggc uuaauucucu cggaaacgau gaaauauaca
	aguuauaucu uggcuuuuca gcucugcauc guuuuggguu cucuuggcug uuacugccag
	gacccauaug uaaaagaagc agaaaaccuu aagaaauauu uuaaugcagg ucauucagau
	guagcggaua auggaacucu uuucuuaggc auuuugaaga auuggaaaga ggagagugac
	agaaaaauaa ugcagagcca aauugucucc uuuuacuuca aacuuuuuaa aaacuuuaaa
	gaugaccaga gcauccaaaa gaguguggag accaucaagg aagacaugaa ugucaaguuu
	uucaauagca acaaaaagaa acgagaugac uucgaaaagc ugacuaauua uucgguaacu
	gacuugaaug uccaacgcaa agcaauacau gaacucaucc aagugauggc ugaacugucg
	ccagcagcua aaacagggaa gcgaaaaagg agucagaugc uguuucaagg ucgaagagca
	ucccaguaau gguuguccug ccugcaauau uugaauuuua aaucuaaauc uauuuauuaa
	uauuuaacau uauuuauaug gggaauauau uuuuagacuc aucaaucaaa uaaguauuua
	uaauagcaac uuuuguguaa ugaaaaugaa uaucuauuaa uauauguauu auuuauaauu
	ccuauauccu gugacugucu cacuuaaucc uuuguuuucu gacuaauuag gcaaggcuau
	gugauuacaa ggcuuuaucu caggggccaa cuaggcagcc aaccuaagca agaucccaug
	gguugugugu uuauuucacu ugaugauaca augaacacuu auaagugaag ugauacuauc
	caguuacugc cgguuugaaa auaugccugc aaucugagcc agugcuuuaa uggcauguca
	gacagaacuu gaauguguca ggugacccug augaaaacau agcaucucag gagauuucau
	gccuggugcu uccaaauauu guugacaacu gugacuguac ccaaauggaa aguaacucau
	uuguuaaaau uaucaauauc uaauauauau gaauaaagug uaaguucaca acu

56	MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWK
	EESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN
	YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ

57	cggcccgcug gagaggaagc ccgagagcug ccgcgcgccu gccggacgag ggcguagaag
	ccaggcguca gagcccgggc uccggugggg ucccccaccc ggcccucggg ucccccgccc
	ccugcucccu gcccauccca gcccacgcga cccucucgcg cgcggagggg cggguccucg
	acggcuacgg gaaggugcca gcccgccccg gaugggcauc guggagccgg guugcggaga
	caugcugacg ggcaccgagc cgaugccggg gagcgacgag ggccgggcgc cuggcgccga
	cccgcagcac cgcuacuucu acccggagcc gggcgcgcag gacgcggacg agcgucgcgg
	gggcggcagc cuggggucuc ccuacccggg gggcgccuug gugcccgccc cgccgagccg
	cuuccuugga gccuacgccu acccgccgeg accccaggcg gccggcuucc ccggcgcggg
	cgaguccuuc ccgccgcccg cggacgccga gggcuaccag ccgggcgagg gcuacgccgc
	cccggacccg cgcgccgggc ucuacccggg gccgcgugag gacuacgcgc uacccgcggg
	acuggaggug ucggggaaac ugagggucgc gcucaacaac caccuguugu gguccaaguu
	uaaucagcac cagacagaga ugaucaucac caagcaggga cggcggaugu ucccauuccu
	gucauuuacu guggccgggc uggagcccac cagccacuac aggauguuug uggacguggu
	cuugguggac cagcaccacu ggcgguacca gagcggcaag ugggugcagu guggaaaggc
	cgagggcagc augccaggaa accgccugua cguccacccg gacuccccca acacaggagc
	gcacuggaug cgccaggaag uuucauuugg gaaacuaaag cucacaaaca acaagggggc
	guccaacaau gugacccaga ugauugugcu ccagucccuc cauaaguacc agccccggcu
	gcauaucguu gaggugaacg acggagagcc agaggcagcc ugcaacgcuu ccaacacgca
	uaucuuuacu uuccaagaaa cccaguucau ugccgugacu gccuaccaga augccgagau
	uacucagcug aaaauugaua auaaccccuu ugccaaagga uuccgggaga acuuugaguc
	cauguacaca ucuguugaca ccagcauccc cuccccgccu ggacccaacu gucaauuccu
	ugggggagau cacuacucuc cucuccuacc caaccaguau ccuguuccca gccgcuucua
	ccccgaccuu ccuggccagg cgaaggaugu gguuccccag gcuuacuggc ugggggcccc
	ccgggaccac agcuaugagg cugaguuucg agcagucagc augaagccug cauucuugcc
	cucugccccu gggcccacca uguccuacua ccgaggccag gagguccugg caccuggagc
	uggcuggccu guggcacccc aguacccucc caagaugggc ccggccagcu gguuccgccc
	uaugcggacu cugcccaugg aacccggccc uggaggcuca gagggacggg gaccagagga
	ccaggguccc cccuuggugu ggacugagau ugcccccauc cggccggaau ccagugauuc
	aggacugggc gaaggagacu cuaagaggag gcgcgugucc cccuauccuu ccagugguga
	cagcuccucc ccugcugggg ccccuucucc uuuugauaag gaagcugaag gacaguuuua
	uaacuauuuu cccaacugag cagaugacau gaugaaagga acagaaacag uguuauuagg
	uuggaggaca ccgacuaauu ugggaaacgg augaaggacu gagaaggccc ccgcucccuc
	uggcccuucu cuguuuagua guugguuggg gaaguggggc ucaagaagga uuuugggguu
	caccagaugc uuccuggccc acgaugaaac cugagagggg uguccccuug ccccauccuc
	ugcccuaacu acagucguuu accuggugcu gcgucuugcu uuugguuucc agcuggagaa
	aagaagacaa gaaagucuug ggcaugaagg agcuuuuugc aucuaguggg ugggaggggu
	cagguguggg acaugggagc aggagacucc acuuucuucc uuuguacagu aacuuucaac
	cuuuucguug gcaugugugu uaaucccuga uccaaaaaga acaaauacac guauguuaua
	accaucagcc cgccaggguc agggaaagga cucaccugac uuuggacagc uggccugggc
	ucccccugcu caaacacagu ggggaucaga gaaaaggggc uggaaagggg ggaauggccc
	acaucucaag aagcaagaua uuguuugugg ugguugugug ugggugugug uuuuuucuuu
	uucuuucuuu uuauuuuuuu ugaauggggg aggcuauuua uuguacugag aguggugucu
	ggauauauuc cuuuugucuu caucacuuuc ugaaaauaaa cauaaaacug uuaaaaaaaa
	aaaaaaaaa

58	MGIVEPGCGDMLTGTEPMPGSDEGRAPGADPQHRYFYPEPGAQDADERRGGGSLGSPYPG
	GALVPAPPSRFLGAYAYPPRPQAAGFPGAGESFPPPADAEGYQPGEGYAAPDPRAGLYPG
	PREDYALPAGLEVSGKLRVALNNHLLWSKFNQHQTEMIITKQGRRMFPFLSFTVAGLEPT
	SHYRMFVDVVLVDQHHWRYQSGKWVQCGKAEGSMPGNRLYVHPDSPNTGAHWMRQEVSFG
	KLKLTNNKGASNNVTQMIVLQSLHKYQPRLHIVEVNDGEPEAACNASNTHIFTFQETQFI
	AVTAYQNAEITQLKIDNNPFAKGFRENFESMYTSVDTSIPSPPGPNCQFLGGDHYSPLLP
	NQYPVPSRFYPDLPGQAKDVVPQAYWLGAPRDHSYEAEFRAVSMKPAFLPSAPGPTMSYY
	RGQEVLAPGAGWPVAPQYPPKMGPASWFRPMRTLPMEPGPGGSEGRGPEDQGPPLVWTEI
	APIRPESSDSGLGEGDSKRRRVSPYPSSGDSSSPAGAPSPFDKEAEGQFYNYFPN

59	ggcgcaacgc ugagcagcug gcgcgucccg cgcggcccca guucugcgca gcuucccgag
	gcuccgcacc agccgcgcuu cuguccgccu gcagggcauu ccagaaagau gaggauauuu
	gcugucuuua uauucaugac cuacuggcau uugcugaacg cauuuacugu cacgguuccc
	aaggaccuau augugguaga guaugguagc aauaugacaa uugaaugcaa auucccagua
	gaaaaacaau uagaccuggc ugcacuaauu gucuauuggg aaauggagga uaagaacauu
	auucaauuug ugcauggaga ggaagaccug aagguucagc auaguagcua cagacagagg
	gcccggcugu ugaaggacca gcucucccug ggaaaugcug cacuucagau cacagaugug
	aaauugcagg augcaggggu guaccgcugc augaucagcu augguggugc cgacuacaag
	cgaauuacug ugaaagucaa ugccccauac aacaaaauca accaaagaau uuugguugug
	gauccaguca ccucugaaca ugaacugaca ugucaggcug agggcuaccc caaggccgaa
	gucaucugga caagcaguga ccaucaaguc cugaguggua agaccaccac caccaauucc
	aagagagagg agaagcuuuu caaugugacc agcacacuga gaaucaacac aacaacuaau
	gagauuuucu acugcacuuu uaggagauua gauccugagg aaaaccauac agcugaauug
	gucaucccag aacuaccucu ggcacauccu ccaaaugaaa ggacucacuu gguaauucug
	ggagccaucu uauuaugccu ugguguagca cugacauuca ucuuccguuu aagaaaaggg
	agaaugaugg augugaaaaa auguggcauc caagauacaa acucaaagaa gcaaagugau
	acacauuugg aggagacgua auccagcauu ggaacuucug aucuucaagc agggauucuc
	aaccuguggu uuagggguuc aucggggcug agcgugacaa gaggaaggaa ugggcccgug
	ggaugcaggc aaugugggac uuaaaaggcc caagcacuga aaauggaacc uggcgaaagc
	agaggaggag aaugaagaaa gauggaguca aacagggagc cuggagggag accuugauac
	uuucaaaugc cugaggggcu caucgacgcc ugugacaggg agaaaggaua cuucugaaca
	aggagccucc aagcaaauca uccauugcuc auccuaggaa gacggguuga gaaucccuaa
	uuugaggguc aguuccugca gaagugcccu uugccuccac ucaaugccuc aauuuguuuu
	cugcaugacu gagagucuca guguuggaac gggacaguau uuauguauga guuuuuccua
	uuuauuuuga gucugugagg ucuucuuguc augugagugu gguugugaau gauuucuuuu
	gaagauauau uguaguagau guuacaauuu ugucgccaaa cuaaacuugc ugcuuaauga
	uuugcucaca ucuaguaaaa cauggaguau uuguaaggug cuuggucucc ucuauaacua
	caaguauaca uuggaagcau aaagaucaaa ccguugguug cauaggaugu caccuuuauu
	uaacccauua auacucuggu ugaccuaauc uuauucucag accucaagug ucugugcagu
	aucuguucca uuuaaauauc agcuuuacaa uuauguggua gccuacacac auaaucucau
	uucaucgcug uaaccacccu guugugauaa ccacuauuau uuuacccauc guacagcuga
	ggaagcaaac agauuaagua acuugcccaa accaguaaau agcagaccuc agacugccac
	ccacuguccu uuuauaauac aauuuacagc uauauuuuac uuuaagcaau ucuuuuauuc
	aaaaaccauu uauuaagugc ccuugcaaua ucaaucgcug ugccaggcau ugaaucuaca
	gaugugagca agacaaagua ccuguccuca aggagcucau aguauaauga ggagauuaac
	aagaaaaugu auuauuacaa uuuaguccag ugucauagca uaaggaugau gcgaggggaa
	aacccgagca guguugccaa gaggaggaaa uaggccaaug uggucuggga cgguuggaua
	uacuuaaaca ucuuaauaau cagaguaauu uucauuuaca aagagagguc gguacuuaaa
	auaacccuga aaaauaacac uggaauuccu uuucuagcau uauauuuauu ccugauuugc
	cuuugccaua uaaucuaaug cuuguuuaua uagugucugg uauuguuuaa caguucuguc
	uuuucuauuu aaaugccacu aaauuuuaaa uucauaccuu uccaugauuc aaaauucaaa
	agaucccaug ggagaugguu ggaaaaucuc cacuucaucc uccaagccau ucaaguuucc
	uuuccagaag caacugcuac ugccuuucau ucauauguuc uucuaaagau agucuacauu
	uggaaaugua uguuaaaagc acquauuuuu aaaauuuuuu uccuaaauag uaacacauug
	uaugucugcu guguacuuug cuauuuuuau uuauuuuagu guucuuuaua uagcagaugg
	aaugaauuug aaguucccag ggcugaggau ccaugccuuc uuuguuucua aguuaucuuu
	cccauagcuu uucauuaucu uucauaugau ccaguauaug uuaaauaugu ccuacauaua
	cauuuagaca accaccauuu guuaaguauu ugcucuagga cagaguuugg auuuguuuau
	guuugcucaa aaggagaccc augggcucuc cagggugcac ugagucaauc uaguccuaaa
	aagcaaucuu auuauuaacu cuguaugaca gaaucauguc uggaacuuuu guuuucugcu
	uucugucaag uauaaacuuc acuuugaugc uguacuugca aaaucacauu uucuuucugg
	aaauuccggc aguguaccuu gacugcuagc uacccugugc cagaaaagcc ucauucguug
	ugcuugaacc cuugaaugcc accagcuguc aucacuacac agcccuccua agaggcuucc
	uggagguuuc gagauucaga ugcccuggga gaucccagag uuuccuuucc cucuuggcca
	uauucuggug ucaaugacaa ggaguaccuu ggcuuugcca caugucaagg cugaagaaac
	agugucucca acagagcucc uuguguuauc uguuuguaca ugugcauuug uacaguaauu
	ggugugacag uguucuuugu gugaauuaca ggcaagaauu guggcugagc aaggcacaua
	gucuacucag ucuauuccua aguccuaacu ccuccuugug guguuggauu uguaaggcac
	uuuaucccuu uugucucaug uuucaucgua aauggcauag gcagagauga uaccuaauuc
	ugcauuugau ugucacuuuu uguaccugca uuaauuuaau aaaauauucu uauuuauuuu
	guuacuuggu acaccagcau guccauuuuc uuguuuauuu uguguuuaau aaaauguuca
	guuuaacauc ccaguggaga aaguuaaaaa a

60	MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEME
	DKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGG
	ADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTT
	TTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTH
	LVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET


61	QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYY
	ADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPS
	VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
	VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP
	KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
	VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC
	LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
	MHEALHNHYTQKSLSLSLGK

62	EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA
	RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPP
	SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
	LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

63	GFTFSDSWIH

64	AWISPYGGSTYYADSVKG

65	RHWPGGFDY

66	RASQDVSTAVA

67	SASFLYS

68	QQYLYHPAT

69	EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY
	ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS

70	DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS
	RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR

71	EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYY
	ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAS
	TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL
	YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
	VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST
	YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT
	KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
	GNVFSCSVMHEALHNHYTQKSLSLSPG

72	DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS
	RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPP
	SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
	LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

73	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFY
	ADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS
	ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
	GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
	PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
	STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE
	LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
	QQGNVFSCSVMHEALHNHYTQKSLSLSPG

74	QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGV
	SNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVT
	LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASS
	YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

75	EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYY
	VDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVS
	SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
	SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEG
	GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
	NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRE
	EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
	WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

76	EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIP
	DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFP
	PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
	TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

77	QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYY
	ADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPS
	VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
	VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP
	KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT
	VLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTC
	LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV
	MHEALHNHYTQKSLSLSLGK

78	EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA
	RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPP
	SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT
	LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

79	QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNF
	NEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS
	ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
	GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV
	FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY
	RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK
	NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG
	NVFSCSVMHEALHNHYTQKSLSLSLGK


80	EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES
	GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF
	IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
	STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

81	agaaagcgag cagccaccca gcuccccgcc accgccaugg uccccgacac cgccugcguu
	cuucugcuca cccuggcugc ccucggegcg uccggacagg gccagagccc guugggcuca
	gaccugggcc cgcagaugcu ucgggaacug caggaaacca acgcggcgcu gcaggacgug
	cgggagcugc ugcggcagca ggucagggag aucacguucc ugaaaaacac ggugauggag
	ugugacgcgu gcgggaugca gcagucagua cgcaccggcc uacccagcgu gcggccccug
	cuccacugcg cgcccggcuu cugcuucccc ggcguggccu gcauccagac ggagagcggc
	gcgcgcugcg gccccugccc cgcgggcuuc acgggcaacg gcucgcacug caccgacguc
	aacgagugca acgcccaccc cugcuucccc cgaguccgcu guaucaacac cagcccgggg
	uuccgcugcg aggcuugccc gccgggguac agcggcccca cccaccaggg cguggggcug
	gcuuucgcca aggccaacaa gcagguuugc acggacauca acgaguguga gaccgggcaa
	cauaacugcg uccccaacuc cgugugcauc aacacccggg gcuccuucca gugcggcccg
	ugccagcccg gcuucguggg cgaccaggcg uccggcugcc agcggcgcgc acagcgcuuc
	ugccccgacg gcucgcccag cgagugccac gagcaugcag acugcguccu agagcgcgau
	ggcucgcggu cgugcgugug ugccguuggc ugggccggca acgggauccu cuguggucgc
	gacacugacc uagacggcuu cccggacgag aagcugcgcu gcccggagcg ccagugccgu
	aaggacaacu gcgugacugu gcccaacuca gggcaggagg auguggaccg cgauggcauc
	ggagacgccu gcgauccgga ugccgacggg gacggggucc ccaaugaaaa ggacaacugc
	ccgcuggugc ggaacccaga ccagcgcaac acggacgagg acaagugggg cgaugcgugc
	gacaacugcc ggucccagaa gaacgacgac caaaaggaca cagaccagga cggccggggc
	gaugcgugcg acgacgacau cgacggcgac cggauccgca accaggccga caacugcccu
	aggguaccca acucagacca gaaggacagu gauggcgaug guauagggga ugccugugac
	aacugucccc agaagagcaa cccggaucag gcggaugugg accacgacuu ugugggagau
	gcuugugaca gcgaucaaga ccaggaugga gacggacauc aggacucucg ggacaacugu
	cccacggugc cuaacagugc ccaggaggac ucagaccacg auggccaggg ugaugccugc
	gacgacgacg acgacaauga cggagucccu gacagucggg acaacugccg ccuggugccu
	aaccccggcc aggaggacgc ggacagggac ggcgugggcg acgugugcca ggacgacuuu
	gaugcagaca aggugguaga caagaucgac guguguccgg agaacgcuga agucacgcuc
	accgacuuca gggccuucca gacagucgug cuggacccgg agggugacgc gcagauugac
	cccaacuggg uggugcucaa ccagggaagg gagaucgugc agacaaugaa cagcgaccca
	ggccuggcug uggguuacac ugccuucaau ggcguggacu ucgagggcac guuccaugug
	aacacgguca cggaugacga cuaugcgggc uucaucuuug gcuaccagga cagcuccagc
	uucuacgugg ucauguggaa gcagauggag caaacguauu ggcaggcgaa ccccuuccgu
	gcuguggccg agccuggcau ccaacucaag gcugugaagu cuuccacagg ccccggggaa
	cagcugcgga acgcucugug gcauacagga gacacagagu cccaggugcg gcugcugugg
	aaggacccgc gaaacguggg uuggaaggac aagaaguccu aucguugguu ccugcagcac
	cggccccaag ugggcuacau cagggugcga uucuaugagg gcccugagcu gguggccgac
	agcaacgugg ucuuggacac aaccaugcgg gguggccgcc ugggggucuu cugcuucucc
	caggagaaca ucaucugggc caaccugcgu uaccgcugca augacaccau cccagaggac
	uaugagaccc aucagcugcg gcaagccuag ggaccagggu gaggacccgc cggaugacag
	ccacccucac cgcggcugga ugggggcucu gcacccagcc ccaaggggug gccguccuga
	gggggaagug agaagggcuc agagaggaca aaauaaagug ugugugcagg gaaaaaaaaa
	aaaaaaaaaa a

82	MVPDTACVLLLTLAALGASGQGQSPLGSDLGPQMLRELQETNAALQDVRELLRQQVREIT
	FLKNTVMECDACGMQQSVRTGLPSVRPLLHCAPGFCFPGVACIQTESGARCGPCPAGFTG
	NGSHCTDVNECNAHPCFPRVRCINTSPGFRCEACPPGYSGPTHQGVGLAFAKANKQVCTD
	INECETGQHNCVPNSVCINTRGSFQCGPCQPGFVGDQASGCQRRAQRFCPDGSPSECHEH
	ADCVLERDGSRSCVCAVGWAGNGILCGRDTDLDGFPDEKLRCPERQCRKDNCVTVPNSGQ
	EDVDRDGIGDACDPDADGDGVPNEKDNCPLVRNPDQRNTDEDKWGDACDNCRSQKNDDQK
	DTDQDGRGDACDDDIDGDRIRNQADNCPRVPNSDQKDSDGDGIGDACDNCPQKSNPDQAD
	VDHDFVGDACDSDQDQDGDGHQDSRDNCPTVPNSAQEDSDHDGQGDACDDDDDNDGVPDS
	RDNCRLVPNPGQEDADRDGVGDVCQDDFDADKVVDKIDVCPENAEVTLTDFRAFQTVVLD
	PEGDAQIDPNWVVLNQGREIVQTMNSDPGLAVGYTAFNGVDFEGTFHVNTVTDDDYAGFI
	FGYQDSSSFYVVMWKQMEQTYWQANPFRAVAEPGIQLKAVKSSTGPGEQLRNALWHTGDT
	ESQVRLLWKDPRNVGWKDKKSYRWFLQHRPQVGYIRVRFYEGPELVADSNVVLDTTMRGG
	RLGVFCFSQENIIWANLRYRCNDTIPEDYETHQLRQA

VIII. Other Embodiments

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

1-139. (canceled)

140. A method of identifying and treating an individual having a cancer who may benefit from treatment with an anti-cancer therapy comprising a PD-L1 binding antagonist and a TGF-β antagonist, the method comprising:

(i) determining the expression level of TGFB1 and/or TGFBR2 in a sample from the individual obtained prior to treatment with the PD-L1 binding antagonist and the TGF-β antagonist, wherein the expression level of TGFB1 and/or TGFBR2 in the sample is at or above a reference expression level of TGFB1 and/or TGFBR2, thereby identifying the individual as one who may benefit from treatment with an anti-cancer therapy comprising a PD-L1 binding antagonist and a TGF-β antagonist; and

(ii) administering an effective amount of an anti-cancer therapy comprising a PD-L1 binding antagonist and a TGF-β antagonist to the individual identified in step (i) as being one who may benefit from treatment with an anti-cancer therapy comprising an anti-PD-L1 antibody and a TGF-β antagonist, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

141. The method of claim 140, wherein the cancer is urothelial carcinoma (UC), non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC), or pancreatic ductal adenocarcinoma (PDAC).

142. The method of claim 140, wherein the anti-PD-L1 antibody comprises the following hypervariable regions (HVRs):

(a) an HVR-H1 sequence of (SEQ ID NO: 63) GFTFSDSWIH; (b) an HVR-H2 sequence of (SEQ ID NO: 64) AWISPYGGSTYYADSVKG; (c) an HVR-H3 sequence of (SEQ ID NO: 65) RHWPGGFDY; (d) an HVR-L1 sequence of (SEQ ID NO: 66) RASQDVSTAVA; (e) an HVR-L2 sequence of (SEQ ID NO: 67) SASFLYS; and (f) an HVR-L3 sequence of (SEQ ID NO: 68) QQYLYHPAT

143. The method of claim 141, wherein the anti-PD-L1 antibody comprises the following HVRs:

144. A method of treating an individual having a cancer, the method comprising administering to the individual an anti-cancer therapy comprising a PD-L1 binding antagonist and a TGF-β antagonist, wherein prior to treatment the expression level of TGFB1 and/or TGFBR2 in a sample obtained from the individual has been determined to be at or above a reference expression level of TGFB1 and/or TGFBR2, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

145. The method of claim 144, wherein the cancer is UC, NSCLC, TNBC, or PDAC.

146. The method of claim 144, wherein the anti-PD-L1 antibody comprises the following HVRs:

147. The method of claim 145, wherein the anti-PD-L1 antibody comprises the following HVRs:

148. The method of claim 144, further comprising:

(i) determining the expression level in the sample of one or more additional genes selected from PD-L1, CD8A, CXCL10, CXCL9, GZMA, GZMB, IFNG, PRF1, and TBX21;

(ii) determining a tumor mutational burden (TMB) score in a tumor sample from the individual; and/or

(iii) administering an additional therapeutic agent to the individual.

149. The method of claim 148, wherein the additional therapeutic agent is an immunotherapy agent, a cytotoxic agent, a growth inhibitory agent, a radiation therapy agent, an anti-angiogenic agent, or a combination thereof.

150. The method of claim 144, wherein:

(i) a tumor from the individual has an immune excluded phenotype characterized by the localization of CD8+ T-cells in the peri-tumoral stromal compartment;

(ii) the reference expression level is determined from a population of individuals having a cancer;

(iii) the expression level is a nucleic acid expression level;

(iv) the expression level is a protein expression level; and/or

(v) the sample is a tissue sample, a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof.

151. The method of claim 150, wherein the reference expression level is a median expression level or is determined by principal component analysis of Z-score-transformed expression levels.

152. The method of claim 144, wherein:

(i) the TGF-β antagonist is a polypeptide, a small molecule, or a nucleic acid; and/or

(ii) the anti-PD-L1 antibody comprises:

(a) a VH domain comprising the amino acid sequence of SEQ ID NO: 69; and

(b) a VL domain comprising the amino acid sequence of SEQ ID NO: 70.

153. The method of claim 152, wherein:

(i) the polypeptide is an anti-TGF-β antibody, a soluble TGF-β receptor, or a peptide; the small molecule is galunisertib (LY2157299), LY2382770, LY3022859, SB-431542, SD208, SM16, tranilast, pirfenidone, TEW-7197, PF-03446962, or pyrrole-imidazole polyamide; or the nucleic acid is trabedersen (AP12009) or belagenpumatucel-L; and/or

(ii) the anti-PD-L1 antibody is atezolizumab.

154. The method of claim 153, wherein:

(i) the anti-TGF-β antibody is fresolimumab, metelimumab, lerdelimumab, 1 D11, 2G7, or a derivative thereof; or

(ii) the peptide is disitertide (P144).

155. The method of claim 141, wherein the UC is a metastatic UC.

156. The method of claim 145, wherein the UC is a metastatic UC.

157. The method of claim 140, wherein:

(i) the anti-PD-L1 antibody is atezolizumab; and/or

(ii) the reference expression level is determined from a population of individuals having the cancer.

158. The method of claim 157, wherein the reference expression level is a median expression level or is determined by principal component analysis of Z-score-transformed expression levels.

159. A method of treating an individual having a cancer, the method comprising administering to the individual an anti-cancer therapy comprising a PD-L1 binding antagonist and a TGF-β antagonist, wherein prior to treatment the expression level of TGFB1 and/or TGFBR2 in the individual is at or above a reference expression level of TGFB1 and/or TGFBR2, wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.